A Besri

X Margueron

J-P Keradec

B Delinchant

Keywords:

Lumped element equivalent circuits that accurately represent the electrical behavior of H.F. nwinding transformers are now available. These circuits suit for time domain simulation but evaluation of their parameters relies on measurements. In order to avoid prototype manufacturing, engineers need to deduce same parameters from geometrical and physical data and, to carry out optimization, they prefer analytical approach to fem one. This paper shows how to do this for planar transformers which have simple geometry. Comparison of results with experimental data is given and analyzed.

I.

INTRODUCTION

For a long time, representation of the electrical behavior of a H.F. transformer has been an unsolved problem. After several successive improvements, equivalent circuits able to do this accurately are now available, even for nwinding-components [START_REF] Margueron | Design of equivalent circuits and characterization strategy for n-input coupled inductors[END_REF]. As long as transformers behave linearly, magnetic and capacitive couplings are correctly described, as well as losses, by lumped elements circuits.

Owing to the lumped elements nature of these circuits, they suit for time domain electronic simulation of the whole electronic circuit, with any circuit simulator like PSpice [2]. All the values of the components included in these circuits can be deduced from well chosen impedance measurements, according to methods which have been described elsewhere [START_REF] Cogitore | Two winding ferrite core transformer: an experimental method to obtain a wide frequency range equivalent circuit[END_REF][START_REF] Margueron | Identifying the magnetic part of the equivalent circuit of n-winding transformer[END_REF][START_REF] Keradec | Identification expérimentale progressive des circuits équivalents de transformateurs HF. Les techniques de l'ingénieur (F)[END_REF].

Of course, using this way to know the parameters of the equivalent circuit supposes that a prototype has been built. Because recent technology prototypes are expensive and their realization is time consuming, engineers wish to reduce the number of such prototypes. With this in mind, methods to deduce the values of equivalent circuit parameters from the physical and geometrical description of the component are expected. In view of entering an optimization process, it is more desirable to do this by analytical calculation rather than by fem simulation.

Analytical studies related to this wide field are still in progress but, for simple shape planar transformers, needed theoretical material to do this is already available [START_REF] Schellmanns | Equivalent circuit for transformers based on one-dimensional propagation: accounting for multilayer structure of windings and ferrite losses[END_REF][START_REF] Heinemann | Modelling and design of high frequency planar transformers[END_REF]. In this paper, we apply this theoretical work to deduce the equivalent circuit of such a planar transformer. Nowadays, planar transformers are widely used in H.F. converters because they are mechanically robust (due to the PCB structure of their windings), their electrical characteristics are reproducible (especially for capacitive aspects) and their thermal draining is efficient owing to their slim shape. Moreover, if carefully designed, they offer high power efficiency that make them very attractive for H.F. systems embedded in aircrafts (Fig. 1).

In section II-A, we precise geometrical assumptions on which our theoretical approach is based. Then (section II-B), we present this theory. According to it, each layer (conductive, insulating or magnetic) gone through inside the transformer is represented by an electrical circuit which, includes frequency dependant impedances. Equivalent circuit of the whole transformer is obtained by connecting all together these elementary circuits.

In section II-C, a transformation of this circuit that avoids presence of negative real part impedances is presented. Then, (section II-D) frequency dependant impedances are replaced by sets of lumped elements in small numbers in order to ease time domain simulations.

Section II-E is dedicated to the capacitive aspects representation. The above mentioned model doesn't take this into account. For this reason, an independent and simple model is added.

Finally, in section III, an industrial transformer (Fig. 1) is studied. We catch this opportunity to explain how to use specifications of the ferrite core to account its losses, the effect of grinding and the introduction of an air gap. For this component, measurements are compared to model predictions. The fit is quite good and most of observed disagreements are explained. Some of these explanations are experimentally checked.

II. THEORETICAL BASIS

A. Geometry of the Concerned Transformers

A large part of planar transformer appears as a stack of different types of layers which are either conductive or insulating or, also, magnetic. In such a physical structure, energy exchanges between conductive layers intervene mainly through plane waves perpendicular to the layers.

Derivation of the theoretical results supposes that each layer of the transformer is a finite width, finite length, piece of an infinite plane layer. Moreover, it is assumed that all layers of the stack have the same widths and lengths and that they are all aligned face to face.

According to these assumptions, two extensions can be considered. First, a conductive layer can be made of several parallel and identical wires connected in series. In this case, because the current is the same in all strands, current distribution through the width of the layer is quite uniform and results are close to that of a homogeneous layer. Second, as long as curvature radius is much larger than layer thickness, curvature can be neglected. This is why some transformers having windings made of cylindrically wound foil can be studied this way.

B. Equivalent Circuit Representing a Conductive Plate

Let us consider (Fig. 2) a parallelepiped piece of a homogenous, isotropic material. We also assume that this material behaves linearly, so, for harmonic studies, its electrical properties are described by a few complex parameters (permittivity, permeability and conductivity) which can vary with frequency. Indeed, as shown by Maxwell-Ampere equation ( 1), when conductivity is not null, it gives a major contribution to the imaginary part of permittivity and, in every case, it can be grouped with that parameter, so the material is completely described, at each frequency, by only two complex constants: permittivity and permeability .

E j E j E rotH d if: j d (1)
Now, have a look to the device shown in figure 2. This piece of an infinite conductive plate of thickness a, normal to Oz, has a width b and a length c. The plate is exposed to incident and reflected plane waves on both its faces. Magnetic field is, anywhere, parallel to Oy. An electric power supply, which forces a current J per unit of width and a voltage V per unit of length, is also connected to this plate. This external circuit adds a uniform electric field E s inside the plate and it allows conduction current to flow inside. Attenuation of a plane wave going through a homogenous and isotropic infinite plate is a well-known problem. It is shown [START_REF] Lorain | Electromagnetic fields and waves[END_REF] that, while going from one plate side to the other, complex amplitude of magnetic field is multiplied by A, given by (2). The same attenuation holds for the reverse direction of propagation. Accounting for continuity of electric and magnetic fields on both surfaces, it becomes possible to express (3) total electric field on left and right sides (E l and E r ) and electric field E s in terms of total magnetic field on both sides (H l and H r ) and current per unit of width J.

J A A Z H A A Z E E c l c s l 2 1 2 1 1 r c c s r H A A Z J A A Z E E 1 1 1 2 2
where: c Z

(3)

This linear system, which connects three power sources (an electromagnetic field on each face and an electric power supply), behaves as a three port circuit. However, considering that total magnetic field on both faces and total current circulating inside the layer are linked by Ampere's theorem, the three inputs of this circuit are not independent so its representation is simpler than expected (Fig. 3). Input-output symmetry of this circuit results of corresponding symmetry of the layer and the two included impedances are given by (4).

2 1

1 1 tan Z j A A Z Z c c f sin Z j A A Z Z c c f 2 2 1 2 (4)
A simple change of circuit shown in figure 3 brings electrical variables linked to the external circuit into light. Indeed, replacing J by I (total current supplied by the source) needs only a multiplication by the plate width b. In the same way, electric fields can be replaced by voltage owing to a multiplication by the plate length c. So, after multiplication of all currents by b and all electrical fields by c, circuit of figure 4 is consistent with that of figure 3 if Z f1 and Z f2 are both multiplied by c/b to gives Z 1 and Z 2 [START_REF] Keradec | Identification expérimentale progressive des circuits équivalents de transformateurs HF. Les techniques de l'ingénieur (F)[END_REF].

2 1 tan b c Z j Z c sin b c Z j Z c 1 2 (5)
At last, if a conducting layer is made of N strands connected in series, input current source must be multiplied by N to give the total current going through the layer while input voltage must be divided by N to obtain the voltage across one strand. This is easily done by introducing an N-ratio coupler (Fig. 4). In practice, we keep this coupler in the circuit, even when its ratio is 1, to insure electrical insulation of each electrical input. To understand the meaning of impedance Z1 and Z2, it is in interesting to look at their low frequency limits [START_REF] Schellmanns | Equivalent circuit for transformers based on one-dimensional propagation: accounting for multilayer structure of windings and ferrite losses[END_REF].

2 2 1 1 a b c j a b c j Z ab c j a b c j Z 1 1 2 (6)
In the low frequency side, Z 1 is the inductance given by a reluctance of permeability (equal to 0 for copper), length b and section ca/2. It looks like the leakage inductance introduced by half the thickness of the plate (magnetic field is parallel to dimension b). In the same range of frequency, Z 2 is the resistance of the plate because, in low frequency, second term of (1) dominates the value of , so first fraction of Z 2 in ( 6) is the reverse of . We must admit that, despite their complicated expressions that account for their frequency variations, these impedances appear quite natural.

For a non supplied plate (insulator, magnetic material) vertical branch of circuit of fig. 4 is not relevant and circuit reduces to unique impedance equal to 2 Z 1 . So, according to this, an insulating layer of thickness a, is introduced, in the electrical circuit, as an inductance l a given by [START_REF] Heinemann | Modelling and design of high frequency planar transformers[END_REF]. b ac l a 0 [START_REF] Heinemann | Modelling and design of high frequency planar transformers[END_REF] Same reasoning leads to represent a magnetic layer by an inductance l m (having losses) given by [START_REF] Lorain | Electromagnetic fields and waves[END_REF]. Because theory suppose magnetic field is mull outside the core [START_REF] Keradec | Power transfer in a two winding transformer: from 1D propagation to an equivalent circuit[END_REF], electric field is null on the external surface of this plate and one terminal of related impedance is at the reference voltage. So, it is useful to remind these impedances supply the return path for all currents injected as shown in Fig. 5. b ac l m [START_REF] Lorain | Electromagnetic fields and waves[END_REF] This value is tightly linked to the al factor (inductance per square turn) given by core manufacturers. Generally it equals twice this value because two plates are needed to represent the whole magnetic core. Despite core permeability is given on a wide frequency range, only low frequency value of the inductance per square turn is commonly specified. Its value in H.F. is calculated as follows [START_REF] Keradec | Power transfer in a two winding transformer: from 1D propagation to an equivalent circuit[END_REF].

( ) ( ) (0) (0) f al f al (9)

C. Representation of a Plate Using Passive Impedances

Frequency dependant impedances are not easily taken into account by circuit simulation software and using them for time domain simulation is quite impossible. For this reason, we now look for lumped element circuits that can accurately replace circuits found above.

Because permeability of an insulating layer is equal to that of vacuum on the whole frequency range, its representation only require a constant inductance. Capacitive aspects will be studied in II-E.

Representation of a magnetic layer must account for all aspects of permeability variation with frequency, including losses. The needed impedance is designed, in

c.E l c.E r N V I b.H l b.H r rdc A A 4B c.E l c.E r N V V I b.H l b.H r rdc A A 4B
Fig. 5. Final equivalent l circuit associated to a conductive plate each case, by looking at manufacturer data. An example will be given in section III. The roughest approximation is given by an inductance paralleled by a resistance often called "iron losses resistor".

Conductive plate is more complicated to represent (several impedances to design) and, due to eddy current effects, its impedances begin varying at rather low frequency so these variations can't be neglected at all.

In a first try, we kept the resistor rdc found as the low frequency limit of Z 2 [START_REF] Schellmanns | Equivalent circuit for transformers based on one-dimensional propagation: accounting for multilayer structure of windings and ferrite losses[END_REF] in series and we remarked that the remaining part of Z 2 was negative. That led us to introduce a second coupler in the circuit (Fig. 5) to naturally account for this negative part on the mutual impedance. Identification of circuits of figures 4 and 5 gives expressions of A and B.

B rdc Z

where:

1 c rdc ab [START_REF] Brezinski | Extrapolation Methods. Theory and Practice[END_REF] Frequency dependence of these impedances ( 11) is clarified by returning to (2), (3), ( 5) and (6).

1 2 1 1 f j fs A rdc f tan j fs 2 1 1 2 1 f j fs B rdc f sin j fs (11)
In these expressions, fs (we call skin frequency) is the frequency for which skin depth (12) equals half the plate thickness a [START_REF] Margueron | Current sharing between parallel turns of a planar transformer: prediction and improvement using a circuit simulation software[END_REF]. Its value is close to 1 MHz for a 125 m thick copper layer. 0 2 0 4 2 a f s [START_REF] Margueron | Current sharing between parallel turns of a planar transformer: prediction and improvement using a circuit simulation software[END_REF] Owing to this change, involved impedances A and B have positive real parts and their frequency behaviors are consistent with that of passive impedances (Fig 6). Impedances B shows strongly damped resonances that are related to propagation through the plate. Fig. 7 shows that these resonances appear when thickness is a half integer multiple of the wavelength (= 2 in this material). Both impedances look inductive in the low frequency side. For B, a Lp-Rp approximation suits pretty well (dotted line) but HF asymptote of A is of half order so representation by a finite number of components will have, necessarily, a bounded frequency range of validity.

D. Lumped Element Representation of Impedances

Now we begin looking for lumped element equivalent circuits for A and B, expecting modulus inaccuracy to be under 1 % up till, at least, 30 fs.

To reach this result for B, we use Pade approximations [START_REF] Brezinski | Extrapolation Methods. Theory and Practice[END_REF] three times. We first develop admittance in Mac Laurin series of the frequency and we keep the first complex term (which corresponds to a resistor paralleled by an inductor). Then, the remaining part of the admittance is inverted and the result is also developed in Mac Laurin series. We keep the first complex term that gives a resistor and a capacitor in series. One more time, developing remaining part gives an inductor paralleled by a resistor.

At this point, obtained equivalent circuit (Fig. 8) shows impedance variation very close (+2.15 %, -1.2 %) to that 9). Component expressions are grouped in table 1. . Now, we focus on impedance A. On the low frequency side, this impedance behaves as an inductance equal to ldc / 6. Beyond .77 fs this approximation is not accurate enough (fig. 6). To extend the frequency range of validity, we alternatively divide and multiply this initial impedance, by a degree 1 polynomial as shown by [START_REF] Prieto | Using parallel windings in planar magnetic components[END_REF]. We note this impedance Am (m like model).

1 LB 2 RB 3 R C LB 1 RB 1 RB 2 CB 1 LB 2 RB 3 R C LB 1 RB 1 RB 2 CB
2 1 3 1 1 1 2 6 1 1 f j fc Am j ldc f f f j j fc fc ... (13)
In practice, to adjust parameters fc1 to fc3, we introduce related terms one by one. For one extra term, we adjust fc 1 to keep the modulus of the ratio: Am / A, in the allowed interval (.99 to 1.01) up till a frequency as high as possible. Then, we add the term that depends on fc 2 and, starting from the previous value of fc 1 we adjust fc1 and fc2 to extend the frequency limit of accuracy as high as possible. We find fc 3 using the same method so parameters of expression ( 13) are now all known.

To finish that process, we express Am/ldc s as a function of s = j2 f and we expand this rational fraction of As indicated before, validity of equivalent circuit for A has, contrary to B, a high frequency limit. However, let us remind that, for a 125 m thick copper layer, fs is located at about 1 MHz so, with usual thickness used in planar technology, we can be confident in proposed circuits up till, at least, 20 MHz. At this frequency, we have no manufacturer data regarding our magnetic core….

E. Representation of Capacitive Behavior

Theory presented above relies on the propagation of plane waves normally to all plates. For this reason, the only component of electric field taken into account is parallel to the plates. Obviously, this component is not the most significant for the study of capacitive properties. To compensate this lack, another model is now joined to former one.

In studied transformers, metallic plates of length c and width b are separated by dielectric material of thickness a. This suggests introducing a simple capacitor between each pair of metallic plates but, contrary to plane capacitors, metallic plates are not equipotential. Indeed, electrostatic field in these regions depends on 3 independent voltages. If we suppose, for each metallic layer (= one turn), that potential varies linearly when going from one terminal to the opposite, electric field in each dielectric layer is easily computed and, according to [START_REF] Laveuve | Electrostatic of wound components: analytical results, simulation and experimental validation of the parasitic capacitance[END_REF], 6 capacitors (Fig. 12), placed between the four terminals of the two surrounding conductive layers, suit to fully account for electrostatic properties.

In figure 12, C 0 equals the capacitance of a plane capacitor (= bc/a). This value is seen between the two layers when they both have a null voltage across them. Two negatives values appear in this circuit but all directly measurable capacitances are positive.

III. COMPARISON WITH MEASUREMNTS

A. Component Short Description

Tested component is that shown in figure 1. It is a 3windings device made of 38 copper layers among which are 14 screens. All these layers are 105 m thick and they are separated by same insulating material of various thickness. Magnetic core comes from TDK. It is a PQ59 core that has been machined as described below. It is made of PC40 ferrite.

B. Circuit Introduced in PSpice

To begin, every copper layer is represented by circuits shown in figures 5, 9 and 10. For a thickness of 105 m, skin frequency fs equals 1.5 MHz. Looking at figures 9 and 11, one can know the expected accuracy of this representation.

Used insulating material is a composite one, the relative permittivity of which is close to 4.5. All insulating layer, including electrostatic screens, have been represented according to figure 12. These circuits are connected to floating sides of input couplers belonging to adjacent conductive layers.

Representation of ferrite layers needs some extra explanations. First, PQ 59 core has been grinded in order to first reduce its thickness of 5 mm, second, introduce an .37 mm thick air gap air gap. This reduces inductance per square turn Al to Alm as follows. Initial length of mean field line equals 80 mm and 10.37 mm is subtracted of this length so: In practice, this inductance is not pure, it slightly varies with frequency and it introduces losses. This is directly linked to complex permeability variation (Fig. 13). This modification leads to replace the pure inductance by a four component equivalent circuit (Fig. 14). This simple model agrees with manufacturer data with less than 10 % of discrepancy from d.c. to 8 MHz.

To end, we now focus on air gap. Knowing its thickness e and the surface Sm of the magnetic core, calculating its reluctance is easy. Related inductance (the reverse of the reluctance) is a pure inductance given by ( 16) which intervenes in parallel with impedance linked to ferrite as usual for reluctances in series. We are now able to complete the introduction of our circuit in PSpice. Because elements are numerous, this is a little bit long but all metallic layers being identical, that piece of circuit is repeated 38 times. Final circuit includes 479 resitors, 542 inductors, 262 capacitors, 76 couplers… for a total of about 1500 components.

Sm

. L R 1 R 2 C L = 14.7 H R 1 = 689 R 2 = 138 C = 392 pF L R 1 R 2 C L R 1 R 2 C L = 14.7 H R 1 = 689 R 2 = 138 C = 392 pF L = 14.7 H R 1 = 689 R 2 = 138 C = 392 pF
As a first step, we use it with sinusoidal excitation in order to compare the results with impedances measured habitually to identify such a component. Despite this transformer owns three windings, to clarify this presentation, we focus on a pair of them. Other one and internal screens are kept floating.

Fig. 16 shows measured and simulated results. Zp0 and Zpcc are impedances seen from primary side with, respectively, secondary open and shorted. In the same manner, Zs0 and Zscc are seen from the secondary side.

To reach a so good agreement, only one component has been added to the circuit described above: a weak inductance (6 nH) has been added in series with secondary winding. Indeed, a first simulation showed good agreement for many things but simulated short circuit impedances were about 10 times too weak. After a short moment, it appeared to us that, close to secondary terminals, the structure of our transformer doesn't respect our model requirements.

Large conductors with screws seen on figure 1 are connected to circular turn by parallel conductors which don't face each over. So, we supposed this pair of parallel conductors, 2 cm long, to be responsible for a small (6 nH) but significant inductance, not taken into account by our approach.

To check that hypothesis, we replaced our initial shortcircuit by a U-shaped one (Fig. 17) in order to prolong incriminated conductors by 2 cm. This, of course, increased the inductance seen from primary side and we deduced the inductance experimentally added to the secondary side. Because conductors responsible for this increase had about the same dimensions that those initially present in the transformer and which are not taken into account, we thought found inductance value also characterized to the guilty part of the transformer. As a conclusion, we introduced an inductance of the same value (6 nH) in series with the secondary winding of our equivalent circuit. Result of this is the agreement seen in Fig. 16.

Before concluding, we wish to share our opinion about the relative places of equivalent circuit introduced in this paper and global circuit deduced from measurements. As mentioned before, equivalent circuit studied in this paper can include more than two thousand elements. This can be heavy to run time domain simulation of a complete circuit. Fortunately, there is an alternate way to proceed. Using curves like those of figure 16, we have developed methods [START_REF] Cogitore | Two winding ferrite core transformer: an experimental method to obtain a wide frequency range equivalent circuit[END_REF][START_REF] Margueron | Identifying the magnetic part of the equivalent circuit of n-winding transformer[END_REF][START_REF] Keradec | Identification expérimentale progressive des circuits équivalents de transformateurs HF. Les techniques de l'ingénieur (F)[END_REF] that lead to a global equivalent circuit which suffices to forecast all the external behavior of a transformer, even if submitted to complex waveforms. This global circuit only includes a small number of components: generally twenty to fifty. After the simulation of the entire circuit with the global equivalent circuit, all input waveforms of the transformer are known. If necessary, these waveforms can be applied to the transformer alone, depicted by its detailed equivalent circuit, in order to reach fine internal behavior.

IV. CONCLUSION

A new way of forecasting electrical behavior of planar transformer has been presented. It applies for transformers having simple but common geometries. Assuming they only have a circuit simulator like PSpice, engineers can now deduce, from physical and geometrical description of their component, its complete electrical behavior, from d.c. to several tens of MHz.

Linear H.F. properties of the core, eddy current effects in conductors and stray capacitive effects are taken into account. Owing to the use of a circuit simulator, after the description of the component, the simulation lasts a few seconds. Notice that, with a fem simulator, these problems are very difficult to solve because, at 10 MHz, skin depth equals 20 m so meshing must be very fine.

Because our equivalent circuit only includes lumped elements, it suits for harmonic studies and complex waveforms as well. Electrical description of the device is complete enough to forecast the results of measurements generally carried out to characterize these components. In a second step, a reduced equivalent circuit, which only includes 20 to 50 lumped elements, can be deduced from these simulated measurements.

Inter-winding impedances, current sharing between parallel turns [START_REF] Laveuve | Electrostatic of wound components: analytical results, simulation and experimental validation of the parasitic capacitance[END_REF][START_REF] Margueron | Current sharing between parallel turns of a planar transformer: prediction and improvement using a circuit simulation software[END_REF], impact of electrical screens on behavior are, among many others, properties that can be studied this way for a fixed topology transformer. More interesting is the possibility of quickly compare several designs for a given component. Following this approach, comparing several interleaving of windings, checking impact of a screen introduction or of a dielectric thickness is done very fast.

Fig. 1 .

 1 Fig. 1. A 9 kVA, 290 g, 3-winding planar transformer

Fig. 2 .Fig. 3 .

 23 Fig. 2.Elementary device composing a planar transformer Zf

Fig. 4 .

 4 Fig. 4. Equivalent electrical circuit associated to a conductive plate

Fig. 6 .Fig. 7 .

 67 Fig. 6. Variation of impedances modulus (A and B) vs. frequency

Fig. 8 .Fig. 9 .

 89 Fig. 8. Equivalent circuit for impedance B

Fig. 11 .Fig. 12 .

 1112 Fig. 11. Accuracy of representation of A by lumped elements

Fig. 13 .

 13 Fig. 13. Permeability vs. frequency, as given by TDK Accounting for this is simple (15) because inductance is proportional to permeability.

  inductance in parallel, circuit of Fig.14must be used with an inductance value equal to 1.41 H

Fig. 14 .

 14 Fig. 14. Impedance per square turn: lumped element equivalent circuit

Fig. 16 .

 16 Fig. 16. Measured and simulated impedances curves

Fig. 17 .

 17 Fig. 17. Two different short-circuits for output impedance evaluation

TABLE 1 .

 1 EXPRESSIONS OF COMPONENT VALUES FOR B.

TABLE 2 .

 2 EXPRESSIONS OF COMPONENT VALUES FOR A.

			LA 1 LA 1	LA 2 LA 2
			RA 1 RA 1	RA 2 RA 2
		Fig. 10 . Equivalent circuit for impedance A
	s in simple elements. Each of them is easily identified to a
	Lp-Rp cell. Corresponding circuit is represented in
	figure 10 and component values are given in table 2.
	Accuracy of modulus and phase is checked in figure 11. It
	reaches our target from d.c. up till 46.2 fs.
	1 LA	115 .	ldc	2 LA	51 4 10 .	-3	ldc
	1 RA	4 93 .	rdc	2 RA	15 92 .	rdc

Authorized licensed use limited to: IMAG -Universite Joseph Fourrier. Downloaded on February 10, 2009 at 05:51 from IEEE Xplore. Restrictions apply.

ACKNOWLEDGMENT

We want to warmly thank Thales Airborne Systems for their long term interest in our work and their financial support.


Kui Zhai

Fabien Hubert

Valérie Nicolas

Guangju Ji

Rodolphe Fischmeister

Véronique Leblais
email: veronique.leblais@u-psud.fr

-Véronique Leblais

β-Adrenergic cAMP Signals are Predominantly Regulated by Phosphodiesterase Type 4 in Cultured Adult Rat Aortic Smooth Muscle Cells

Keywords:

Background:

We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMC).

Methodology/Principal findings:

The rank order of PDE families contributing to global cAMP-PDE activity was PDE4 > PDE3 = PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β1and β2-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β1-and β2-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after -AR stimulation.

Conclusion/Significance

: Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

Introduction

In the vascular system, cAMP is a key physiological second messenger, which inhibits contraction, proliferation and migration of the smooth muscle cells (SMCs) [START_REF] Koyama | Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation[END_REF][START_REF] Mcdaniel | Cyclic nucleotide dependent relaxation in vascular smooth muscle[END_REF]. Intracellular concentration of cAMP is determined by the balance of its production by adenylyl cyclase and its degradation by specific enzymes, the 3',5'-cyclic nucleotide phosphodiesterases (PDEs). PDEs are classified in 11 families based on structural similarity and enzymatic properties, including substrate specificity (cAMP versus cGMP), kinetic properties and regulation [START_REF] Omori | Overview of PDEs and their regulation[END_REF]. Within these PDE families, multiple isoforms are expressed, either as products of different genes or multiple transcriptional products of one gene. It is usually admitted that vascular SMCs express three dominant cAMP-PDE families (PDE1, PDE3 and PDE4), with a pattern of activity depending on the species, the vascular bed and the phenotype of the cell [START_REF] Maurice | Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system[END_REF]. However, the expression/activity of more recently identified cAMP-PDEs (PDE7 to PDE11) has been poorly investigated. By comparing the mRNA expression of PDE1 to PDE10 in rat pulmonary and systemic vascular SMCs, Phillips et al. showed that PDE7 mRNA was expressed in all studied cells but PDE10 mRNA was never detected, whereas PDE8 and PDE9 mRNAs were differentially expressed depending on the vascular bed [START_REF] Phillips | cAMP phosphodiesterase inhibitors potentiate effects of prostacyclin analogs in hypoxic pulmonary vascular remodeling[END_REF]. PDE11 was not examined in this study. Such a multiplicity of PDE isoforms might seem functionally redundant. However, it is now well-accepted that cAMP is not uniformly distributed within cells so that its action may be restricted to subcellular domains of the cells, and that different signaling pathway components, including PDEs and cAMP-dependent protein kinase (PKA), may contribute to this phenomenon. This concept has been extensively developed in cardiac myocytes: the different cardiac PDE isoforms are targeted to distinct subcellular microdomains and contribute to the intracellular compartmentation of cAMP by limiting its diffusion to the entire cell, generating specific cardiac responses at discrete intracellular loci [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases[END_REF][START_REF] Xiang | Compartmentalization of β-adrenergic signals in cardiomyocytes[END_REF][START_REF] Mika | PDEs create local domains of cAMP signaling[END_REF]. A similar picture of cardiac cyclic nucleotide compartmentation is also proposed for cGMP [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases[END_REF][START_REF] Castro | Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes[END_REF]. By contrast, this concept has been poorly investigated in SMCs. In the case of cAMP signaling, Delpy et al. showed that, in rat endothelium-denuded aorta, PDE3 inhibition potentiates both the increase in intracellular cAMP level and the cAMP-dependent vasorelaxation elicited by -adrenergic stimulation, whereas PDE4 inhibition only potentiates the former response without modifying the latter one [START_REF] Delpy | Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3[END_REF].

The lack of correlation between the cAMP concentration and the functional response during PDE inhibition suggests the presence of distinct intracellular cAMP pools in vascular SMCs controlled by different PDE isoforms. However, these data obtained in an integrated tissue were never confirmed at the cellular level. In the case of cGMP signaling, recent studies provided evidence that in isolated vascular SMCs, nitric oxide and natriuretic peptides induce distinct cGMP signals, partly due to a differential regulation by PDE5 [START_REF] Piggott | Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments[END_REF][START_REF] Nausch | Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors[END_REF]. Thus, the main objective of this study was to investigate the role of the different PDE families in the intracellular cAMP compartmentation of vascular SMCs. For this purpose, we took advantage of the Fluorescence Resonance Energy Transfer (FRET)-based imaging technique, using Epac-based sensors which allow a spatiotemporal monitoring of cAMP concentrations in intact living cells [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF][START_REF] Nikolaev | Monitoring of cAMP synthesis and degradation in living cells[END_REF]. As cellular model of vascular SMCs, we used rat aorta SMCs (RASMCs) maintained in culture. During cell culture, vascular SMCs undergo a phenotypic switch from a contractile/quiescent to a proliferative/synthetic phenotype, miming the phenotype of a cell isolated from an injured vessel [START_REF] Thyberg | Regulation of differentiated properties and proliferation of arterial smooth muscle cells[END_REF]. It is known that this phenotypic switch is associated with modifications of PDE levels and activity, which depend on the species and may vary between culture conditions [START_REF] Dunkerley | Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues[END_REF][START_REF] Rybalkin | Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype[END_REF][START_REF] Liu | Expression of phosphodiesterase 4D (PDE4D) is regulated by both the cyclic AMP-dependent protein kinase and mitogen-activated protein kinase signaling pathways. A potential mechanism allowing for the coordinated regulation of PDE4D activity and expression in cells[END_REF]. Thus, the first aim of this study was to characterize the expression and activity pattern of cAMP-PDE isoforms in cultured RASMCs. For this purpose, we performed reverse transcription-polymerase chain reaction (RT-PCR) experiments and a biochemical PDE activity assay coupled to a pharmacological approach by using selective PDE inhibitors. The second aim of this study was to delineate the role of PDEs in modulating the spatiotemporal dynamic of cAMP signals elicited in living RASMCs, by using FRET-based sensors targeted to either the cytosolic or the plasma membrane compartments. Isoproterenol (Iso), a -adrenoceptor (-AR) agonist, was used here as a physiological cAMP-elevating stimulus.

Materials and Methods

All

Cell isolation and culture

RASMCs were prepared as previously described with minor modifications [START_REF] Vallot | Intracellular Ca 2+ handling in vascular smooth muscle cells is affected by proliferation[END_REF].

Briefly, RASMCs were isolated from the thoracic medial layer of adult male Wistar rat (180-200g) by an enzymatic digestion with collagenase type 2 (60 U/mL; Worthington Biochemical Corporation, Lakewood, NJ, USA) and elastase (0.3 mg/mL; MP Biomedicals, Solon, Ohio, USA) for 3 hours at 37°C, with continuous slow shaking. After periods of 30 minutes, the suspension was centrifuged at 1300 rpm for 3 minutes, and the cells were collected and placed in Dulbecco's Modified Eagle Medium (DMEM; GIBCO, Invitrogen, Cergy Pontoise, France) containing antibiotics/antimycotic (100 U/mL penicillin, 100 µg/mL streptomycine, 0.25 µg/mL amphotericin B; GIBCO) and supplemented with 20% Fetal Bovine Serum "Gold" (FBS; PAA Laboratories, Les Mureaux, France). Cells obtained during the first period were discarded. Those obtained in the subsequent cycles were pooled, centrifuged, suspended in DMEM containing 20% FBS, seeded in a flask coated with collagen І (rat tail, BD Biosciences, Le Pont de Claix, France) and maintained at 37°C in a 95% air-5% CO2 humidified atmosphere. The medium was changed every 2 days with DMEM containing 10% FBS. At confluence, cells were detached with 0.05% trypsin containing 0.53 mM EDTA (GIBCO) and seeded in coated flasks (passage 1). All experiments were performed on cells cultured between passages 2 and 6. Cells were plated on collagen-coated Petri dishes for RT-PCR experiments and PDE activity assay or on collagen-coated glass coverslips for FRET experiments, at a density of 3.10 3 or 10 4 cells/cm 2 for experiments performed at 24 or 48 hours, respectively. As shown in Figure S1, markers characteristic of cells with a synthetic phenotype were expressed in cultured RASMCs.

Quantitative RT-PCR

Cells, maintained in DMEM containing 10% FBS for 48 hours after plating, were washed twice with cold PBS, immediately frozen and kept at -80°C. Cells were scrapped and homogenized using a tissue homogenizer (Bertin Technologies) in ice-cold TRI reagent (Molecular Research center, Cincinnati, USA). Total RNA was extracted using standard procedure. cDNA was synthesized from 1 µg total RNA using iSCRIPT cDNA synthesized (Biorad, Marnes-la-Coquette, France). Negative controls were performed without the reverse transcriptase. Then, qPCR was performed using the SYBR  -Green method on a CFX96 real-time PCR detection system (Biorad). Reactions were carried out in SYBR-Green master mix with 12.5 ng cDNA and 0.5 µM sense and anti-sense primers for subtypes of PDE 1 to 8 and housekeeping genes (TBP: Tata Box Binding Protein, RPL32: Ribosomal protein L32 and Ywhaz: 14-3-3 protein zeta/delta) (Table 1). Negative controls were performed without cDNA template to check for exogenous contamination. The cycling conditions were 30 seconds at 95°C, following by 35 cycles with one step at 95°C for 5 seconds and another step at 60-62°C for 20 seconds. For each target gene, a standard curve was constructed from the analysis of serial dilution of cDNA and was used to determine efficiency (E). Threshold cycle (Ct) for target was subtracted from the geometric mean of Ct for housekeeping genes to calculate (1+E) Ct according to the 2 Ct method. PCR products were analyzed by electrophoresis on a 3% agarose gel and visualized with GelRed  (Biotium, Hayward, USA) under UV light.

Cyclic AMP-PDE activity assay

Cells, maintained in DMEM containing 10% FBS for 48 hours after plating, were washed twice with cold PBS, homogenized using a tissue homogenizer (Bertin Technologies) in ice-cold lysis buffer (containing: NaCl 150 mM, HEPES 20 mM, EDTA 2 mM, glycerol 10%, NP40 0.5% and microcystin 1 µM) and centrifuged at 12,000 g for 10 min at 4°C. Protein concentration was determined using the bicinchoninic acid protein assay, according to the manufacturer's protocol (Pierce, Thermo Fisher Scientific, Brebières, France). The cAMP-PDE activity was measured in the supernatant according to a modification of the two-step assay procedure method described by Thompson and Appleman [START_REF] Thompson | Characterization of cyclic nucleotide phosphodiesterases of rat tissues[END_REF], using 20-30 µg of RASMCs proteins in a total volume of 200 µl including 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 5 mM -mercaptoethanol and 1 µM cAMP and supplemented with 10 5 cpm [ 3 H]-cAMP, as detailed previously [START_REF] Saucerman | Cardiac β-adrenergic signaling: from subcellular microdomains to heart failure[END_REF]. To evaluate PDE families-specific activities, the assay was performed in the absence or presence of either one or a combination of several selective PDE inhibitors: 10 or 50 µM MIMX for PDE1, 100 nM BAY for PDE2, 1 µM Cil for PDE3, 10 µM Ro for PDE4, 50 µM BRL for PDE7 and 1 mM IBMX as a non-selective PDE inhibitor. The residual hydrolytic activity observed in the presence of PDE inhibitors was expressed as a percentage of the total cAMP-PDE activity, corresponding to the cAMP-PDE activity in the absence of inhibitor.

FRET imaging

Two different FRET-based cAMP sensors were used: Epac1-camps, containing the single cAMP-binding domain of Epac1 fused to an enhanced yellow fluorescent protein (YFP) and an enhanced cyan fluorescent protein (CFP) [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF]; and the plasma membrane-targeted cAMP sensor Epac2-camps, called pm-Epac2-camps, corresponding to Epac2-camps (containing the cAMP-binding domain B of Epac2 fused to YFP and CFP) which was N-terminally-modified with the SH4 motif of Lyn kinase [START_REF] Wachten | Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells[END_REF]. FRET between CFP and YFP appears in the absence of cAMP. Upon increase in cAMP concentration, cAMP binding to its Epac domain promotes reversible conformational changes of the sensor, resulting in a decrease in FRET between CFP and YFP [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF].

RASMCs were transfected with a pcDNA3 plasmid encoding Epac1-camps by using Lipofectamine TM 2000 (Invitrogen) diluted in Opti-MEM medium  (Invitrogen) according to the manufacturer's instructions. After 6 hours, the transfection medium was replaced with DMEM containing 10% FBS. In some experiments, the RASMCs were infected with an adenovirus encoding pm-Epac2-camps (MOI 600 pfu/cell) in DMEM containing 10% FBS. FRET experiments were performed 42 hours after transfection with Epac1-camps or 24 hours after infection with pm-Epac2-camps. We previously determined these incubation times to get an optimal subcellular localization of the sensors.

Cells were maintained in a K + -Ringer solution containing (in mM): NaCl 121.6, KCl 5.4, MgCl2 1.8, CaCl2 1.8, NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10 (pH 7.4), at room temperature. To study changes in FRET signals in response to pharmacological agents, the cell of interest was continuously and locally perfused with K + -Ringer solutions containing or not these agents, using a microperfusion system allowing rapid applications of these solutions. Iso was applied either at steady state to perform cumulative concentration-response curves or as a pulse of 15 s (at a submaximal concentration) to evaluate the effect of pharmacological agents on the dynamics of its response. PDE inhibitors and -AR antagonists were applied 3 min before the brief application of Iso and maintained throughout the experiment.

Images were captured every 5 s using the 40X oil immersion objective of an inverted microscope (Nikon TE 300) connected to a software-controlled (Metafluor, Molecular Devices) cooled charge-coupled device camera (Sensicam PE; PCO, Kelheim, Germany). CFP was excited during 150-300 ms by a Xenon lamp (100 W, Nikon, Champigny-sur-Marne, France) using a 440/20BP filter and a 455LP dichroic mirror. Dual emission imaging of CFP and YFP was performed using an Optosplit ІІ emission splitter (Cairn Research, Faversham, UK) equipped with a 495LP dichroic mirror and BP filters 470/30 and 530/30, respectively. Average fluorescence intensity was measured in a region of interest comprising the entire cell. For analysis, background was subtracted and YFP intensity was corrected for CFP spillover into the 535 nm channel before calculating the ratio of CFP/YFP emitted fluorescence intensities. Data were expressed as a percentage of the CFP/YFP ratio measured before application of the drug. Ratio images were obtained with ImageJ software (National Institutes of Health). Kinetic parameters of the Iso-induced FRET signal (tmax: time to peak, t1/2on: time to half-peak, t1/2off: time from the peak to obtain half recovery) were determined using Microsoft Excel software. EC50 value (concentration that produces 50% of the maximum response) was estimated in each individual concentration-response curves using the Boltzman equation fit using Origin 6 software.

Data analysis and statistics

Data are represented as mean ± SEM of N experiments in RT-PCR studies and cAMP-PDE activity assay or n cells in FRET imaging experiments. The different parameters were compared using Student's t-test for paired or unpaired data, where appropriate. A difference was considered statistically significant when P<0.05.

Results

Expression pattern of PDE isoforms in cultured RASMCs

mRNA species encoding PDEs 1A, 1C, 3A, 3B, 4A, 4B, 4D, 7A, 7B and 8A were detected in cultured RASMCs (Figure 1). RT-PCR products for PDE 1B, 2A, 4C and 8B isoforms were absent.

cAMP-PDE activity in cultured RASMCs

Total cAMP-PDE activity measured in lysates from cultured RASMCs was 37.47.1 pmol/min/mg protein (n=6). To determine which PDE families contribute to this total hydrolyzing activity, the assay was also conducted in the presence of different PDE inhibitors (Figure 2). Consistent with the absence of PDE2 mRNA expression, BAY (100 nM), a selective PDE2 inhibitor [START_REF] Boess | Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance[END_REF], had no effect on cAMP-PDE activity. Cil (1 µM, a selective PDE3 inhibitor [START_REF] Sudo | Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity[END_REF]) and Ro (10 µM, a selective PDE4 inhibitor [START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF]) reduced cAMP-PDE activity by 20% and 40%, respectively. As no perfect PDE1 inhibitor is commercially available, we used MIMX which blocks PDE1 activity by interfering with its catalytic site with a low micromolar affinity and exhibits a selectivity over other PDEs of 30-to 50-fold [START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF][START_REF] Wells | Methylxanthine inhibitors of phosphodiesterases[END_REF][START_REF] Goncalves | The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue[END_REF]. MIMX decreased cAMP-PDE activity by 22% and 37%, when used at 10 µM and 50 µM, respectively. In the simultaneous presence of MIMX (10 µM), Cil and Ro, total cAMP-PDE activity was reduced by 75%. Increasing the concentration of MIMX to 50 µM had no further effect under this condition. The PDE7 inhibitor BRL (50 µM) [START_REF] Smith | Discovery of BRL 50481[END_REF] lowered the total cAMP-PDE activity by 16%. However, BRL had no additive inhibitory effect on the cAMP-PDE activity measured in the presence of MIMX (10 µM), Cil and Ro which questions its specificity on PDE7. Finally, the broad-spectrum PDE inhibitor IBMX (1 mM) [START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF] inhibited the total cAMP-PDE activity by 96%. In summary, the rank order of PDE families contributing to global cAMP-PDE activity in cultured RASMCs was PDE4 > PDE3=PDE1. PDE2 activity is absent and PDE7 activity uncertain.

FRET measurements of cytosolic cAMP signals in cultured RASMCs

We then evaluated the functional contribution of the different PDE isoforms in controlling intracellular cAMP concentration ([cAMP]i) in RASMCs. [cAMP]i changes in response to Iso were monitored in real-time by fluorescence imaging using the FRET-based cAMP sensor Epac1-camps. As illustrated on the images in Figure 3A and Figure 4A, the CFP/YFP ratio fluorescence was distributed throughout the cytosol in cells expressing Epac1-camps, indicating a cytosolic localization of the probe. Cumulative increasing concentrations of Iso (0.1 nM to 1 µM) induced a significant increase in CFP/YFP ratio in a concentration-dependent manner, with a maximum response of 27.92.0% and an EC50 value of 18.12.2 nM (n=7) (Figure 3B). These alterations of FRET signal reflect the production of cytosolic cAMP upon β-AR stimulation. To analyze the dynamics of this cAMP signal, Iso was then applied transiently at the concentration of 0.1 µM. As shown in Figure 4, a 15 s-pulse of Iso induced a rapid increase in the CFP/YFP ratio to reach a maximum of 14.10.7% at 48.71.4 s (n=124) before returning to baseline with a t1/2off of 51.53.0 s. Thus, a short β-AR stimulation induced a transient increase in cytosolic [cAMP]i, suggesting that cAMP is rapidly metabolized in RASMCs, most likely through cAMP hydrolysis by PDEs.

Role of PDE families in the control of cytosolic cAMP concentrations in cultured

RASMCs

To evaluate the functional contribution of the different PDE families in regulating the cAMP response to β-AR stimulation, RASMCs were incubated in the presence of PDE inhibitors during 3 min before Iso pulse. Application of PDE inhibitors alone, with the exception of BAY and BRL, induced a slight but significant increase in basal FRET ratio, which was maximal with 50 µM MIMX (the PDE1 inhibitor) and 100 µM IBMX (the non-selective PDE inhibitor) (Table 2). 50 µM MIMX (Figure 5A), 100 nM BAY (the PDE2 inhibitor, Figure 5B) and 1 µM Cil (the PDE3 inhibitor, Figure 5C) had no effect on the dynamics of the Iso-induced FRET signal. 50 µM BRL (the PDE7 inhibitor, Figure 5E) had only minor effects on its kinetics (with a slight increase in the time to peak of 17%, n=9, P<0.05), whereas 10 µM Ro (the PDE4 inhibitor, Figure 5D) markedly increased its amplitude (by 52%, n=13, P<0.01) and significantly delayed its onset and recovery phases. IBMX (Figure 5I) also increased the amplitude of Iso response (by 76%, n=9, P<0.05) and dramatically prolonged its duration. The IBMX effect on the decay phase was significantly higher than that induced by Ro (t1/2off increased by 99% and 770% with Ro and IBMX, respectively, P<0.001; Figure 6B). This indicates that, in RASMCs, PDE4 is an essential regulator of β-AR-elicited cytosolic cAMP signals, but that other PDEs might also contribute to this regulation.

We then evaluated the effect of the simultaneous inhibition of PDE4 and one of the 3 other PDEs detected in these cells (as shown by PDE activity assay: PDE1, PDE3 and also PDE7) on the Iso response. Ro+MIMX significantly increased the amplitude of Iso response (by 109%, n=12) and delayed its onset and recovery phases (Figure 5F). Only the effect of Ro+MIMX on the decay phase was significantly higher than that induced by Ro alone [t1/2off increasd by 99% (n=13) and 280% (n=12) with Ro and Ro+MIMX, respectively, P<0.001; Figure 6]. However, this effect was still lower than that induced by IBMX. It should be noticed that a similar potentiating effect of MIMX in the presence of Ro was also observed when MIMX was used at a smaller concentration, 10 µM (Figure S2). Ro+Cil significantly increased the amplitude of Iso response (by 80%, n=9) and drastically prolonged its duration (Figure 5G), with an effect on the decay phase which was significantly higher than that induced by Ro alone [t1/2off increased by 99% (n=13) and 377% (n=9) with Ro and Ro+Cil, respectively, P<0.001; Figure 6B] and by Ro+MIMX [t1/2off increased by 280% (n=12) and 377% (n=9) with Ro+MIMX and Ro+Cil, respectively, P<0.001;

Figure 6B]. In the presence of Ro+BRL (Figure 5H), the Iso response was significantly increased in amplitude (by 90%, n=9) and duration (t1/2off increased by 160%), but to a similar extent to that obtained in the presence of Ro alone (Figure 6).

Altogether, these results suggest that PDE4 inhibition unmasks the effect of PDE3 and PDE1 on cytosolic cAMP generated by β-AR stimulation. .

Role of β1-and β2-AR subtypes in cytosolic cAMP production in cultured

RASMCs

To evaluate the contribution of β1-and β2-AR subtypes in cytosolic cAMP production elicited by Iso, RASMCs were incubated in the presence of β1-or β2-AR antagonists during 3 min before the Iso pulse. The selective β1-AR antagonist CGP-20712A was used at a concentration of 100 nM, which is about 100-fold higher than its binding affinity [START_REF] Leblais | β-Adrenergic relaxation in pulmonary arteries: preservation of the endothelial nitric oxide-dependent β2 component in pulmonary hypertension[END_REF][START_REF] Hoffmann | Comparative pharmacology of human β-adrenergic receptor subtypes characterization of stably transfected receptors in CHO cells[END_REF]. At this concentration, CGP-20712A did not modify the FRET signal induced by a selective β2-AR agonist (Figure S3), confirming its selectivity towards β1-ARs in our experimental conditions. By contrast, CGP-20712A significantly decreased the amplitude of Iso response by 39% without affecting its kinetics (Figure 7A), indicating that β1-ARs are involved in this response. To antagonize β2-ARs, we used ICI 118,551, which exhibits a binding affinity for β2-ARs of around 1 nM and a selectivity ratio for β2-ARs over β1-ARs of about 70 [START_REF] Hoffmann | Comparative pharmacology of human β-adrenergic receptor subtypes characterization of stably transfected receptors in CHO cells[END_REF]. We evaluated the effect of different concentrations of ICI 118,551 (1, 5, 10 and 100 nM) on FRET signal induced by Iso in RASMCs. ICI 118,551 significantly reduced the amplitude of Iso response in a concentration-dependent manner [by 14% (n=6), 48% (n=7), 55% (n=5) and 90% (n=9) at 1, 5, 10 and 100 nM, respectively], without affecting its kinetics (Figure S4 and Figure 7B for ICI 118,551 at 5 nM), indicating that β2-ARs are involved in this response. In the following experiments, we decided to use ICI 118,551 at 5 nM, a concentration which should preserve its selectivity towards β2-ARs according to its binding affinities.

As PDE4 is the main PDE family controlling Iso-induced cytosolic cAMP signals, we then evaluated the role of PDE4 in hydrolyzing cAMP produced by either β1or β2-AR stimulation. The β1-AR response was elicited through an Iso pulse in the presence of the β2-AR antagonist ICI 118,551, whereas the β2-AR response was elicited through an Iso pulse in the presence of the β1-AR antagonist CGP-20712A.

The PDE4 inhibitor (10 µM Ro) slightly but not significantly increased the amplitude of the Iso+CGP-20712A response, and significantly prolonged its duration with a strong effect on its recovery phase (t1/2off increase of 155%, n=9, P<0.01; Figure 7C).

Similarly, the Iso+ICI 118,551 response (Figure 7D) was markedly increased by Ro in amplitude (by 120%, n=7-8, P<0.01) and duration (t1/2off increase of 178%, n=9, P<0.01). This indicates that PDE4 hydrolyses cytosolic cAMP pool generated by both β1-and β2-AR stimulations.

Role of PDE families in the control of submembrane cAMP concentrations in cultured RASMCs

To directly monitor cAMP dynamics in the submembrane compartment, we used the modified FRET-based cAMP sensor, pm-Epac2-camps, which was effectively targeted to the plasma membrane (Figure 8A). As shown in Figure 8B, pm-Epac2-camps generated a FRET response upon transient stimulation with Iso at the concentration of 10 nM. The FRET signal reached a maximum of 11.9±1.3% at 99.8±4.4 s (n=23 cells) before returning to baseline with a t1/2off of 78.48.8 s (Figure 8C). We then investigated the role of PDE3 and PDE4 families in regulating submembrane cAMP produced by -AR stimulation. Application of 1 µM Cil or 10 µM Ro slightly increased the basal FRET ratio (Table 3). Both PDE3 and PDE4 inhibitors significantly affected the Iso-induced FRET signal by increasing its amplitude (by 51% and 38%, respectively) and delaying its recovery phase (Figure 9A andB). When Cil and Ro were applied concomitantly, the basal FRET ratio was markedly enhanced (Table 3) and the Iso response was significantly increased in amplitude by 59% (P<0.05, n=8) and dramatically prolonged in duration (t1/2off increased by 548%, compared to 150% and 85% with Cil and Ro alone, respectively, P<0.001; Figure 9C). It should be noted that the effect of PDE inhibitors on the amplitude of Iso response was underestimated here given their effect on the basal FRET ratio. Altogether, these results suggest that, unlike in the cytosol, both PDE3 and PDE4 control submembrane cAMP concentration in RASMCs. Cell culture is known to induce a phenotypic switch of vascular smooth cells associated with modifications of PDE expression [START_REF] Dunkerley | Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues[END_REF][START_REF] Rybalkin | Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype[END_REF][START_REF] Liu | Expression of phosphodiesterase 4D (PDE4D) is regulated by both the cyclic AMP-dependent protein kinase and mitogen-activated protein kinase signaling pathways. A potential mechanism allowing for the coordinated regulation of PDE4D activity and expression in cells[END_REF]. In this study, we performed a large PDE activity assay to characterize our cultured RASMC model in terms of biochemical cAMP-PDE activity. By using selective pharmacological inhibitors of PDE3 and PDE4 (cilostamide and Ro-20-1724, respectively) [START_REF] Sudo | Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity[END_REF][START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF], we observed that the most important cAMP-PDE was PDE4, accounting for around 40% of total cAMP hydrolyzing activity, whereas PDE3 accounted only for 20%. In this regard, mRNAs encoding 2 PDE3 isoforms (3A and 3B) and 3 PDE4 isoforms (4A, 4B and 4D) were detected in these cells. This is in accordance with Dunkerley et al who described a reduced PDE3/PDE4 activity ratio in cultured rat aorta cells compared to fresh cells; this modification appeared from the passage 1 of culture and was correlated with a reduced PDE3 activity [START_REF] Dunkerley | Reduced phosphodiesterase 3 activity and phosphodiesterase 3A level in synthetic vascular smooth muscle cells: implications for use of phosphodiesterase 3 inhibitors in cardiovascular tissues[END_REF]. Other classical cAMP-PDEs include PDE1 and PDE2. Here, we show that mRNAs encoding PDE1A and 1C but not 1B were expressed and that PDE1 accounted for at least 20% of total PDE activity according to the inhibitory effect of 10 µM MIMX. PDE1 protein expression was previously described in cultured aorta SMCs isolated from several species including rat [START_REF] Rybalkin | Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype[END_REF], but PDE1 hydrolyzing cAMP activity was essentially shown in cells from other species [START_REF] Rybalkin | Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype[END_REF][START_REF] Palmer | Dual expression and differential regulation of phosphodiesterase 3A and phosphodiesterase 3B in human vascular smooth muscle: implications for phosphodiesterase 3 inhibition in human cardiovascular tissues[END_REF][START_REF] Orallo | Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin[END_REF]. Increasing MIMX concentration to 50 µM further increased the inhibitory effect of MIMX when used alone, but had no additive effect in the presence of PDE3 and PDE4 inhibitors suggesting that increasing MIMX concentration induces non-selective inhibition, probably towards PDE3 or PDE4, according to its affinity values [START_REF] Rich | In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors[END_REF][START_REF] Goncalves | The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue[END_REF]. Finally, we confirmed the absence of PDE2 expression and activity in our conditions, as shown in previous works [START_REF] Rose | Cyclic AMP-mediated regulation of vascular smooth muscle cell cyclic AMP phosphodiesterase activity[END_REF]. Thus, cultured RASMCs exhibited PDE1+PDE3+PDE4 activities, which contributed to 80% of total cAMP-PDE activity. However, IBMX-sensitive PDE activity represented about 96% of total activity. This suggests either that the concentrations of PDE inhibitors are not sufficient to fully block the related PDE activities, or that PDEs other than PDE1, 3 and 4 participate in the cAMP hydrolyzing PDE activity. Other IBMX-sensitive cAMP-PDEs include PDE7, PDE10 and PDE11. Phillips et al have shown that PDE7 mRNA is expressed in cultured RASMCs (at passage 2), and proposed that PDE7-PDE11 account for less than 10% of total cAMP-PDE activity of these cells [START_REF] Phillips | cAMP phosphodiesterase inhibitors potentiate effects of prostacyclin analogs in hypoxic pulmonary vascular remodeling[END_REF]. Here, we observed that cultured RASMCs (at passages 2-6) express both PDE7A and PDE7B mRNAs, PDE7A being dominant. We further investigated the potential role of PDE7 in total cAMP-hydrolyzing activity by using a recently described PDE7 inhibitor (BRL) [START_REF] Smith | Discovery of BRL 50481[END_REF]. BRL decreased total cAMP-PDE activity by 16%, but had no additive effect to that induced by the association of PDE1+PDE3+PDE4 inhibitors. Furthermore, BRL did not alter the cAMP concentration measured by FRET-based imaging in intact living cells. Thus, the effect of BRL observed in vitro in cell lysates might be explained by a non-selective action on other PDEs than PDE7, particularly PDE4, for which BRL exhibits some affinity [START_REF] Smith | Discovery of BRL 50481[END_REF]. Therefore, the function of PDE7 isoforms in RASMCs remains to be elucidated by using more specific tools. In the absence of commercialized selective PDE10 and PDE11 inhibitors, we were unable to evaluate the contribution of these two isoforms in our PDE activity assay. To conclude, despite some limits of the pharmacological approach used in the PDE activity assay regarding the selectivity of PDE inhibitors, our data clearly show that the rank order of PDE activity in cultured RASMCs is PDE4 > PDE1 = PDE3. The PDE2 activity is absent, and the PDE7 activity uncertain.

Discussion

In the second part of the work, we analyzed the functional contribution of these PDE isoforms in controlling intracellular cAMP concentration. For this purpose, we took advantage of the FRET technique using an Epac1-based sensor which allows a real-time monitoring of cAMP concentrations in living cells [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF][START_REF] Nikolaev | Monitoring of cAMP synthesis and degradation in living cells[END_REF]. This approach has been applied extensively in several cell types, including cell lines [START_REF] Nikolaev | Monitoring of cAMP synthesis and degradation in living cells[END_REF] or freshly isolated cells such as cardiomyocytes [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF], but rarely in vascular smooth muscle cells [START_REF] Sassi | Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation[END_REF][START_REF] Von Hayn | Gq-mediated Ca 2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells[END_REF]. In mice aorta SMCs maintained one week in culture after dissociation, Iso increased intracellular cAMP with an EC50 of about 40 nM [START_REF] Von Hayn | Gq-mediated Ca 2+ signals inhibit adenylyl cyclases 5/6 in vascular smooth muscle cells[END_REF]. In our study, we also used the physiological -AR stimulation as cAMP-elevating agent. Cumulative application of Iso produced a significant rise in [cAMP]i, with an EC50 value of about 18 nM which is in the same range of concentration as that related to its functional relaxant effect in intact rat aorta [START_REF] Trochu | β3-Adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium-derived nitric oxide in rat thoracic aorta[END_REF]. Furthermore, both 1and 2-AR subtypes contributed to this response, the 2 subtype being predominant according to the efficiency of the selective 1and 2-AR antagonists to inhibit this response. This is consistent with the fact that both 1and 2-AR are localized at the SMC membrane and contribute to the -adrenergic relaxation in rat aorta [START_REF] Brawley | β1-, β2and atypical β-adrenoceptor-mediated relaxation in rat isolated aorta[END_REF][START_REF] Guimaraes | Vascular adrenoceptors: an update[END_REF]. This also indicates that our culture protocole maintains the expression of these receptors [START_REF] Ostrom | Localization of adenylyl cyclase isoforms and G protein-coupled receptors in vascular smooth muscle cells: expression in caveolin-rich and noncaveolin domains[END_REF]. We did not explore the role of the 3-AR as this subtype has been shown to be expressed and functional in endothelial cells of rat aorta [START_REF] Trochu | β3-Adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium-derived nitric oxide in rat thoracic aorta[END_REF][START_REF] Rautureau | β3-adrenoceptor in rat aorta: molecular and biochemical characterization and signalling pathway[END_REF].

A short β-AR stimulation of RASMCs during 15 s induced a fast but transient increase in the cytosolic cAMP concentration with a return to baseline in about 200 s. This is similar to what was observed in other cell types, including rat ventricular cardiomyocytes [START_REF] Leroy | Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca 2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases[END_REF]. The broad spectrum PDE inhibitor, IBMX, markedly prolonged the Iso-triggered cAMP signal, by increasing the t1/2off by almost 800%, demonstrating that PDEs play a key role in modulating cytosolic cAMP concentrations in RASMCs. However, even in the presence of IBMX, the cAMP signal was not stable in time and slowly decreased. Several hypotheses might be proposed to explain this observation: (1) the failure of IBMX to totally block PDEs;

(2) the role of an IBMX-insensitive PDE isoform in cAMP degradation, such as PDE8; accordingly, we detected PDE8A mRNA in RASMCs. However, we were unable to characterize PDE8 function in our model as no selective PDE8 inhibitor is commercially available; (3) the decrease in cAMP concentration due to its extrusion out of the cell through a multidrug resistance-associated protein, the MRP-4, which inhibition has been shown to slightly increase cAMP concentration in response to forskolin in RASMCs [START_REF] Sassi | Multidrug resistance-associated protein 4 regulates cAMP-dependent signaling pathways and controls human and rat SMC proliferation[END_REF]; and (4) the biophysical properties of the Epac1-camps sensor [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF], as the decrease of the signal in the presence of IBMX was slowed down when a different Epac-based cAMP sensor was used, the Epac2-camps sensor, characterized by a two times higher affinity than Epac1-camps [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF]. Nevertheless, the strong effect of IBMX demonstrates that IBMX-sensitive PDEs play a major role in regulating the dynamic of the β-adrenergic cytosolic cAMP signal.

The PDE subtypes involved in this effect were further characterized using the PDE inhibitors at the concentrations established in the PDE activity assay. We observed that: (1) PDE4 was the main isoform hydrolyzing the cytosolic cAMP produced after β-AR stimulation in RASMCs; (2) PDE1 and PDE3 also contributed to this activity but only when PDE4 was inhibited; (3) PDE2 and PDE7 were absent or inactive. This functional PDE activity pattern is in perfect adequacy with the biochemical PDE activity pattern described above in the same cells, PDE4 being the main active and functional isoform. Furthermore, we observed that PDE4 similarly controlled the cytosolic cAMP pool produced by both 1and 2-AR stimulations in RASMCs. This is in agreement with studies showing that PDE4 regulates both 1and 2-AR signaling in heterologous expression systems as well as native cardiomyocytes [START_REF] Nikolaev | Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined β2-adrenergic receptor-mediated signaling[END_REF][START_REF] Richter | Signaling from β1and β2-adrenergic receptors is defined by differential interactions with PDE4[END_REF]. However, the nature of the PDE4 isoform and the mode of interaction between PDE4 and -AR subtypes remain to be identified. Interestingly, we showed that PDE4 masked the functional contribution of both PDE1 and PDE3 which was revealed only upon PDE4 inhibition. One explanation might be that under PDE4 inhibition, PDE1 and PDE3 activities are efficiently enhanced to a level allowing them to hydrolyze cAMP in living cells. In fact, PDE3 activity is known to be increased by PKA phosphorylation [START_REF] Degerman | Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3)[END_REF] that might occur following the cAMP increase induced by PDE4 inhibition. However, an opposite regulation pathway is described for PDE1, as its phosphorylation by PKA is known to decrease its activity [START_REF] Omori | Overview of PDEs and their regulation[END_REF], suggesting that this hypothesis is unlikely, at least for PDE1. A more relevant explanation is that PDE4 confines the cytosolic cAMP generated under β-AR stimulation into a subcellular compartment devoid of PDE1 and PDE3, and that PDE4 inhibition allows cAMP to diffuse in other subcellular compartments enriched in PDE1 and PDE3. The role of PDEs in shaping enzymatic barriers to limit cAMP diffusion has been extensively studied, particularly in cardiomyocytes [START_REF] Fischmeister | Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases[END_REF].

To get some insight on the subcellular cAMP compartmentation in our cellular model, we measured cAMP underneath the plasma membrane. To do this, we used a FRET-based sensor which was modified by adding to its N-terminal the SH4 motif of Lyn kinase. We confirmed that this modification targeted the sensor to the plasma membrane of RASMCs, as previously published in HEK cells [START_REF] Wachten | Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells[END_REF]. We observed that PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced by -AR stimulation in RASMCs. This is clearly different from what happened in the cytosolic compartment, suggesting that PDEs delineate distinct subcellular cAMP pools. However, these results should to be interpreted with caution because of some differences between the cytosolic and the plasma membrane-targeted sensors. First, the cytosolic and plasma membrane-targeted sensors operate under 2 different vectors of expression, namely a plasmid and an adenovirus, respectively. This implies appropriate culture conditions for each vector, that might be associated with distinct phenotypes [START_REF] Rovner | Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells[END_REF]. Second, the two sensors possess distinct Epac-derived cAMP-binding domains, Epac1 and Epac2, which are known to exhibit different affinity for cAMP [START_REF] Nikolaev | Novel single chain cAMP sensors for receptor-induced signal propagation[END_REF][START_REF] Wachten | Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells[END_REF].

In conclusion, our study underlines the importance of cAMP-PDEs for the spatiotemporal control of intracellular cAMP in synthetic RASMCs, and demonstrates the prominent role of PDE4 in the control of both 1and 2-AR responses. The synthetic phenotype of SMCs is representative of the phenotype of proliferative SMCs observed in vascular diseases, like atherosclerosis and restenosis [START_REF] Owens | Molecular regulation of vascular smooth muscle cell differentiation in development and disease[END_REF], and cAMP exerts anti-proliferative and anti-migratory properties in vascular smooth muscle cells. Thus, a therapeutic strategy to counter the pathological vascular proliferative state is to increase cAMP concentrations in these cells. Our data suggest that in this context, a combination of a PDE4 inhibitor with a PDE1 or PDE3 inhibitor would be more potent than a single inhibitor. Furthermore, the effectors regulated by cAMP differ regarding the SMC phenotype (synthetic versus contractile). It is thus tempting to speculate that PDE isoforms contribute to the functional specificity of cAMP by delineating distinct subcellular cAMP pools involved in distinct physiological processes. Further work is needed to explore this hypothesis. The expression of PDE1A (1A), PDE1B (1B), PDE1C (1C), PDE2A (2A), PDE3A (3A), PDE3B (3B), PDE4A (4A), PDE4B (4B), PDE4C (4C), PDE4D (4D), PDE7A (7A), PDE7B (7B), PDE8A (8A) and PDE8B (8B) was analyzed. A: PCR products were resolved by electrophoresis on a 3% agarose gel. Shown is the picture of a representative gel stained with GelRed  . Position of molecular weight markers is indicated in base pairs (Bp). B: mRNA expression was expressed using the 2 Ct method as described in Materials and methods. Data are meanSEM of 9 independent experiments. n.d. not detectable.

A B C

Iso [START_REF] Piggott | Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments[END_REF] Iso+Cil [START_REF] Castro | Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes[END_REF] Iso (8) Iso [START_REF] Delpy | Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3[END_REF] Iso+Ro [START_REF] Nikolaev | Monitoring of cAMP synthesis and degradation in living cells[END_REF] Iso+Ro+Cil

  experiments performed conform to the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, December 18, 1986), the local ethics committee (CREEA Ile-de-France Sud) guidelines, and the French decree no. 97-848 of October 19, 1987 (J Off République Française, October 20, 1987, pp 12245-12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministry of Agriculture, Fisheries and Food (no. 92-283, June 27, 2007). Pharmacological agents CGP-20712A methanesulfonate salt (CGP), ICI 118,551 hydroclhoride (ICI), 3-isobutyl-1-methylxanthine (IBMX) and (-)-isoproterenol hydrochloride (Iso) were purchased from Sigma Aldrich (St Quentin, Fallavier, France). Cilostamide (Cil) and BRL 50481 (BRL) were from Tocris Bioscience (Bristol, UK), 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIMX) and Ro-20-1724 (Ro) from Calbiochem (Merck Chemicals Ltd, Nottingham, UK), and BAY-60-7550 (BAY) from Cayman Chemical (Bertin Pharma, Montigny-le-Bretonneux, France). As all PDE inhibitors stock solutions were prepared in dimethylsulfoxide (DMSO; Sigma), control experiments were performed in the presence of equivalent concentrations of DMSO.

  In this study, we evaluated the role of individual PDEs in regulating the spatiotemporal dynamics of cAMP signals in cultured RASMCs. The main findings are as follows: (1) mRNAs encoding several cAMP-PDE isoforms (including PDE7A, 7B, 8A) are present in cultured RASMCs; (2) the rank order of PDE activity in RASMCs is PDE4 > PDE1 = PDE3; (3) Iso strongly increases cytosolic cAMP concentration, through both 1and 2-AR activation; (4) PDE4 is the main PDE controlling the cytosolic cAMP signal elicited by -AR stimulation. However, PDE4 inhibition unmasks an effect of PDE3 and PDE1 in hydrolyzing cytosolic cAMP; (5) both PDE3 and PDE4 contribute to regulate basal and β-AR-stimulated cAMP levels in the submembrane compartment.

Figure 1 .

 1 Figure 1. Expression analysis of mRNA encoding cAMP-PDE isoforms inRASMCs. RT-qPCR reactions were carried out on mRNAs isolated from RASMCs.

Figure 2 .

 2 Figure 2. cAMP-PDE activity in RASMCs. cAMP-PDE activity was determined in lysates of cultured RAMSCs in the absence (vehicle) or presence of selective PDE inhibitors (PDE1: 10 or 50 µM MIMX; PDE2: 100 nM BAY; PDE3: 1 µM Cil; PDE4: 10 µM Ro; PDE7: 50 µM BRL) or a non-selective PDE inhibitor (1 mM IBMX) or a combination of several inhibitors as indicated. Results are expressed in % of cAMP-PDE activity measured in the absence of inhibitors (vehicle). Data are meanSEM of 3-6 independent experiments. *** P<0.001 versus vehicle; ## P<0.01 and ### P<0.001 versus Ro; $$$ P<0.001 as indicated.

Figure 3 .

 3 Figure 3. Effect of steady-state activation of -AR on cytosolic [cAMP] in RASMCs. Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in cultured RAMSCs cells incubated with cumulative increasing concentrations of isoproterenol (0.1 nM to 1 µM). A: Representative cAMP signals monitored in one cell. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph. B: Concentration-response curve of isoproterenol effect as shown in A. Data are meanSEM of 7 independent cells.

Figure 4 .

 4 Figure 4. Effect of transient activation of -AR on cytosolic [cAMP] in RASMCs. Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s). A: Representative cAMP signals monitored in one cell. Pseudocolor images reflecting the CFP/YFP ratio were recorded at the times indicated by the letters on the graph. B: Dynamic parameters (peak amplitude, tmax, t1/2on, and t1/2off) of isoproterenol-induced FRET signal as shown in Data are meanSEM of 124 independent cells.

Figure 5 .

 5 Figure 5. Effect of PDE inhibition on -AR-induced cytosolic cAMP signal in RASMCs. Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence () or presence () of one selective PDE family inhibitor (A: 50 µM MIMX for PDE1; B: 100 nM BAY for PDE2; C: 1 µM Cil for PDE3; D: 10 µM Ro for PDE4; E: 50 µM BRL for PDE7) or a combination of several inhibitors (F: 10 µM Ro + 50 µM MIMX +; G: 10 µM Ro + 1 µM Cil; H: 10 µM Ro + 50 µM BRL) or the non-selective PDE inhibitor (I: 100 µM IBMX). Top and lower panels represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are meanSEM of 8-13 independent cells as indicated. * P<0.05, ** P<0.01, *** P<0.001 versus Iso.

Figure 6 .

 6 Figure 6. Effect of PDE inhibitors on the peak amplitude and t1/2off of -AR-induced cytosolic cAMP signal in RASMCs. Dynamic parameters (peak amplitude, A and t1/2off, B) obtained in Figure 5 are expressed in % of isoproterenol-induced response (Control). Data are meanSEM of 8-13 independent cells as indicated. * P<0.05, ** P <0.01, *** P<0.001 versus Control; $ P<0.05, $$ P<0.01 and $$$ P<0.001 versus IBMX; # P<0.05, ### P<0.001 as indicated.

Figure 7 .

 7 Figure 7. Effect of 1-AR and 2-AR antagonists on -AR-induced cytosolic cAMP signals in RASMCs. Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence or presence of -AR antagonists and PDE4 inhibitor. A, B: Effect of 1-AR (100 nM CGP-20712A, CGP; A) and 2-AR (5 nM ICI 118,551, ICI; B) antagonists on Iso response. C, D: Effect of PDE4 inhibitor (10 µM Ro) on Iso response obtained in the presence of (Iso+CGP; C) or 2-AR (Iso+ICI; D) antagonists. Top and lower panels represent the mean variation of CFP/YFP ratio and the corresponding kinetic parameters, respectively. Data are meanSEM of 7-13 independent cells as indicated. * P<0.05, ** P<0.01 versus Iso (A, B), Iso+CGP (C) or Iso+ICI (D).

Figure 8 .

 8 Figure 8. Effect of transient activation of -AR on submembrane [cAMP] in RASMCs. Submembrane cAMP measurements were conducted in cultured RASMCs cells using the plasma membrane-targeted FRET-based cAMP sensor pm-Epac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s). A: Submembrane localization of pm-Epac2-camps was ascertained by recording the CFP emission. B: Variation of the CFP/YFP ratio monitored in one cell. B: Dynamic parameters (peak amplitude, tmax, t1/2on, and t1/2off) of isoproterenol-induced FRET signal as shown in B. Data are meanSEM of 23 independent cells.

Figure 9 .Table 1 .

 91 Figure 9. Effect of PDE inhibition on -AR-induced submembrane cAMP signal in RASMCs. Submembrane cAMP measurements were conducted in cultured RASMCs cells using the FRET-based cAMP sensor pmEpac2-camps in response to a short application of isoproterenol (Iso, 0.01 µM, 15 s) after a pre-treatment in the absence () or presence () of one selective PDE family inhibitor (A: 1 µM Cil for PDE3; B: 10 µM Ro for PDE4) or a combination of 10 µM Ro + 1 µM Cil (C). Top and lower panels represent the mean variation of CFP/YFP ratio and the

  Figure1

  Figure5

  Figure9

Figure S2 Effect

 S2 Figure S2

  Figure S3Effect of the 1-AR antagonist on 2-AR-induced cytosolic cAMP signals in cultured RASMCs. Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in response to a maintained application of the 2-AR agonist formoterol (0.1 µM, 3 min) after a pre-treatment in the absence or presence of the 1-AR antagonist (100 nM CGP-20712A). Data are meanSEM of 4 independent cells.

Figure S4 Effect of different

  Figure S4 Effect of different concentrations of the 2-AR antagonist on -AR-induced cytosolic cAMP signals in cultured RASMCs. Cytosolic cAMP measurements were conducted using the FRET-based cAMP sensor Epac1-camps in response to a short application of isoproterenol (Iso, 0.1 µM, 15 s) after a pre-treatment in the absence or presence of increasing concentrations of the 2-AR antagonist (1, 5, 10 and 100 nM ICI 118,551, ICI). The peak amplitude of the FRET-signal obtained in the different conditions is expressed in % of the isoproterenol-induced response (Control).Data are meanSEM of 4-9 independent cells. * P<0.05 versus Iso.

Table 2 . Effect of PDE inhibition on basal cytosolic cAMP signal in RASMCs.

 2

	PDE inhibitor	% increase in CFP/YFP
	Vehicle	0.8 ± 0.1 (n = 9)
	MIMX (50 µM)	3.1 ± 0.3 (n = 8) ***
	BAY (100 nM)	0.3 ± 0.1 (n = 13)
	Cil (1 µM)	1.7 ± 0.4 (n = 12) *
	Ro (10 µM)	1.8 ± 0.4 (n = 13) *
	BRL (50 µM)	1.4 ± 0.3 (n = 9)
	Ro (10 µM) + MIMX (50 µM)	2.8 ± 0.3 (n = 12) *** , #
	Ro (10 µM) + Cil (1 µM)	2.4 ± 0.7 (n = 9) *
	Ro (10 µM) + BRL (50 µM)	1.8 ± 0.2 (n = 9) **
	IBMX (100 µM)	3.4 ± 1.1 (n = 8) *
	Cytosolic cAMP measurements were conducted in cultured RAMSCs cells using the
	FRET-based cAMP sensor Epac1-camps. The % increase in CFP/YFP ratio induced
	by the different PDE inhibitors or the vehicle was calculated after a 3 min-incubation.
	Data are meanSEM of 8-13 independent cells as indicated. * P<0.05, ** P<0.01, ***
	P<0.001 versus vehicle;

# P<0.05 versus Ro.

Table 3 . Effect of PDE inhibition on basal submembrane cAMP signal in RASMCs.

 3 Submembrane cAMP measurements were conducted in cultured RASMCs cells using the FRET-based cAMP sensor pm-Epac2-camps. The % increase in CFP/YFP ratio induced by the different PDE inhibitors or the vehicle was calculated after a 3 min-incubation. Data are meanSEM of 8-14 independent cells as indicated. *** P<0.001 versus vehicle; ## P<0.01 versus Cil and versus Ro. antagonist (1, 5, 10 and 100 nM ICI 118,551, ICI). The peak amplitude of the FRET-signal obtained in the different conditions is expressed in % of the isoproterenol-induced response (Control). Data are meanSEM of 4-9 independent cells. * P<0.05 versus Iso.

	PDE inhibitor	% increase in CFP/YFP
	Vehicle	0.8 ± 0.1 (n = 10)
	Cil (1 µM)	1.8 ± 0.5 (n = 9) ***
	Ro (10 µM)	2.6 ± 0.3 (n = 14) ***
	Ro (10 µM) + Cil (1 µM)	5.2 ± 0.9 (n = 8) *** , ##

Acknowledgements:

We thank Valérie Domergue-Dupont (IPSIT, Faculté de Pharmacie, Châtenay-Malabry, France) and the animal core facility for efficient animal care, Patrick Lechêne (Inserm UMR-S 769) for technical assistance in FRET experiments, Claudine Deloménie (Transcriptomic platform of IPSIT IFR141, Faculté de Pharmacie, Châtenay-Malabry, France) for technical assistance in RT-PCR experiments and Dr Grégoire Vandecasteele (Inserm UMR-S 769) for helpful discussions. We are grateful to Dr. V.O. Nikolaev (Universitätsmedizin Göttingen, Germany) for providing us the FRET-based cAMP sensors, Epac1-camps and pm-Epac2-camps.

Author Contributions