Volume 129, Issue 7 p. 1497-1505
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Beta-3 adrenergic stimulation of L-Type Ca2+ channels in rat portal vein myocytes

Patricia Viard

Patricia Viard

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

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Nathalie Macrez

Nathalie Macrez

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

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Frédéric Coussin

Frédéric Coussin

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

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Jean-Luc Morel

Jean-Luc Morel

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

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Jean Mironneau

Corresponding Author

Jean Mironneau

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS UMR 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail: [email protected]Search for more papers by this author
First published: 02 February 2009
Citations: 23

Abstract

  • The effects of β3-adrenergic stimulation were studied on the L-type Ca2+ channel in single myocytes from rat portal vein using the whole-cell mode of the patch-clamp technique.

  • Reverse transcription-polymerase chain reaction showed that β1-, β2- and β3-adrenoceptor subtypes were expressed in rat portal vein myocytes. Application of both propranolol (a non-selective β1- and β2-adrenoceptor antagonist) and SR59230A (a β3-adrenoceptor antagonist) were needed to inhibit the isoprenaline-induced increase in L-type Ca2+ channel current.

  • L-type Ca2+ channels were stimulated by CGP12177A (a β3-adrenoceptor agonist with potent β1- and β2-adrenoceptor antagonist property) in a manner similar to that of isoprenaline. The CGP12177A-induced stimulation of Ca2+ channel current was blocked by SR59230A, cyclic AMP-dependent protein kinase inhibitors, H-89 and Rp 8-Br-cyclic AMPs, but was unaffected by protein kinase C inhibitors, GF109203X and 19-31 peptide. This stimulation was mimicked by forskolin and 8-Br-cyclic AMP. In the presence of okadaic acid (a phosphatase inhibitor), the β3-adrenoceptor-induced stimulation was maintained after withdrawal of the agonist.

  • The β3-adrenoceptor stimulation of L-type Ca2+ channels was blocked by a pretreatment with cholera toxin and by the intracellular application of an anti-Gαs antibody. This stimulation was unaffected by intracellular infusion of an anti-Gβcom antibody and a βARK1 peptide.

  • These results show that activation of β3-adrenoceptors stimulates L-type Ca2+ channels in vascular myocytes through a Gαs-induced stimulation of the cyclic AMP/protein kinase A pathway and the subsequent phosphorylation of the channels.

British Journal of Pharmacology (2000) 129, 1497–1505; doi:10.1038/sj.bjp.0703187

Abbreviations:

  • βARK
  • β-adrenergic receptor kinase
  • cyclic AMP
  • cyclic adenosine monophosphate
  • CTX
  • cholera toxin
  • ERK
  • extracellular signal-regulated kinase
  • MAPK
  • mitogen-activated protein kinase
  • PCR
  • polymerase chain reaction
  • PI3K
  • phosphatidylinositol-3-kinase
  • PKA
  • protein kinase A
  • PKC
  • protein kinase C
  • Introduction

    Previous data have reported that β-adrenergic activation stimulates L-type Ca2+ channels in vascular and visceral smooth muscles (Fukumitsu et al., 1990; Loirand et al., 1992, Muraki et al., 1993). Several reports support a stimulatory effect of the cyclic AMP/protein kinase A (PKA) pathway on L-type Ca2+ channels in vascular myocytes (Shi & Cox, 1995; Tewari & Simard, 1994; Ruiz-Velasco et al., 1998). These results are in contrast with other data indicating that isoprenaline and cyclic AMP exert a transient increase followed by a decrease in L-type Ca2+ channel activity (Xiong et al., 1994; Xiong & Sperelakis, 1995). The stimulatory effect of β-adrenergic receptor activation has been proposed to depend on a direct modulation of L-type Ca2+ channels by the activated Gs protein whereas the inhibitory effect has been attributed to Gs activation of adenylyl cyclase and the subsequent phosphorylation of the Ca2+ channel by PKA (Xiong & Sperelakis, 1995). However, there are increasing evidence that three or four β-adrenoceptor subtypes may exist in different cell types including heart and visceral smooth muscle (Kaumann, 1997), so that β-adrenergic stimulation cannot be considered as a single mechanism as long as β-adrenoceptors have not been identified in the studied cells. β3-adrenoceptors have been identified in adipocytes in which they are predominantly expressed (Emorine et al., 1989) but also in cardiac and smooth muscles (Kaumann, 1997; Anthony et al., 1998). Activation of β3-adrenoceptors has been shown to induce negative inotropic effect in human heart (Gauthier et al., 1996) and relaxation in visceral smooth muscle (Fujimura et al., 1999), but the cellular mechanisms activated by these receptors in vascular myocytes have not been elucidated.

    In the present study, we identified by RT–PCR the mRNAs corresponding to β1-, β2- and β3-adrenoceptor subtypes and demonstrated the existence of a β3-adrenoceptor activated-stimulation of L-type Ca2+ channels in rat portal vein myocytes. We show that the transduction pathway activated by β3-adrenoceptors is mediated through a Gαs-induced stimulation of the cyclic AMP/PKA pathway and the subsequent phosphorylation of L-type Ca2+ channels.

    Methods

    The investigation conforms with the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J noL358, 18 December 1986) and the French decree no87/748 of October 19, 1987 (J Off République Française, 20 October 1987, pp. 12245–12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture et de la Pêche.

    Cell preparation

    Isolated myocytes from rat portal vein were obtained by enzymatic dispersion, as described previously (Leprêtre et al., 1995). Cells were seeded at density of ∼103 cells mm−2 on glass slides and maintained in short-term primary culture (2–36 h) in M199 containing 5% foetal calf serum, 2 mM glutamine, 1 mM pyruvate, 20 u ml−1 penicillin and 20 μg ml−1 streptomycin.

    Membrane current measurement

    Voltage-clamp and membrane current recordings were performed with a standard patch-clamp technique using an EPC-7 amplifier (List, Darmstadt-Eberstadt, Germany). Whole-cell recordings were performed with patch pipettes having resistances of 2–4 MΩ. Membrane potential and membrane currents were stored and analysed using a PC computer (P-clamp system, Axon Instruments, Foster City, CA, U.S.A.). Ba2+ current density is expressed as peak current amplitude per capacitance unit (in pA/pF). All experiments were performed at 30±1°C. Results are expressed as means±s.e.mean. Significance was tested by Student's t-test. P values >0.05 were considered as significant.

    Solutions

    The physiological solution used to record Ba2+ currents contained (in mM): NaCl 130, KCl 5.6, MgCl2 1, BaCl2 5, glucose 11, HEPES 10, pH 7.4 with NaOH. The basic pipette solution contained (in mM): CsCl 130, EGTA 10, ATPNa2 5, GTP 0.1, MgCl2 2, HEPES 10, pH 7.3, with CsOH. Isoprenaline and CGP12177A were extracellularly applied to the recorded cell by pressure ejection from a glass pipette.

    RNA purification and reverse transcription-polymerase chain reaction (PCR)

    Total RNA was extracted from about 500 cells dissociated from rat portal vein and detrusor muscles by using RNeasy mini kit (Qiagen, Hilden, Germany) and following the instructions of the supplier. The reverse transcription reaction was performed using Sensiscript RT kit (Qiagen, Hilden, Germany). Briefly, total RNA was first incubated with random primers (Promega, Charbonnières, France) at 65°C for 5 min and cooled down 60 min at 37°C. The resulting cDNA was stored at −20°C. PCR was performed with 1 μl of cDNA, 1.25 units of HotStartTaq DNA polymerase (Qiagen), 0.5 μM of each primer and 200 μM of each deoxynucleotide triphosphate, in a final volume of 50 μl. The PCR conditions were 95°C for 15 min for HotStartTaq activation, then 35 cycles were performed as follows: 94°C for 1 min, 55°C (β1- and β2-adrenoceptors) or 62°C (β3-adrenoceptor) for 1.5 min and 72°C for 1 min. At the end of PCR, samples were kept at 72°C for 10 min for final extension before being stored at 4°C. Reverse transcription and PCR were performed with a thermal cycler (Techne, Cambridge, U.K.). Amplification products were separated by electrophoresis (2% agarose gel) and visualized by ethidium bromide staining. Gels were photographed with EDAS 120 and analysed with KDS1D 2.0 software (Kodak Digital Science, Paris, France). Sense (s) and antisense (as) primer pairs specific for β1-, β2 and β3-adrenoceptors were designed on the known cloned rat receptor sequences deposited in GenBank (accession numbers D00634, X17607 and S73473 for β1-, β2- and β3-adrenoceptors, respectively) with Lasergene software (DNASTAR, Madison, WI, U.S.A.). The nucleotide sequences and the length of the expected PCR products (in parentheses) for each primer pair were respectively: β1-adrenoceptor (s) TC  GT  G T  GC  A C  CG  T G  TG  G G  CC , (as) AG  GA AA CG GC GC TC GC AG CT (264 bp); β2-adrenoceptor (s) GC CT GC TG AC CA AG AA TA AG, (as) CC CA TC CT GC TC CA CC TG G (328 bp); β3-adrenoceptor (s) AC CT TG GC GC TG AC TG G, (as) AT GG GC GC AA AC GA CA C (229 bp).

    Chemicals and drugs

    Isoprenaline, propranolol, prazosin and rauwolscine were from Sigma (Saint Quentin Fallavier, France). Forskolin, 8-Br-cyclic AMP, Rp-8-Br-cyclic AMPs, H-89, 19-31 peptide and cholera toxin (CTX) were from Calbiochem (Meudon, France). Phorbol ester 12,13-dibutyrate and 4α-phorbol 12,13-dibutyrate were from LC Laboratories (Woburn, MA, U.S.A.). The protein kinase C (PKC) inhibitor, GF109203X, was a gift from Glaxo (Les Ulis, France). CGP12177A was from RBI (Natick, MA, U.S.A.). SR59230A (3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronapth-1-ylaminol] - (2S)-propanolol-oxalate) was from Sanofi (Milano, Italy). M199 medium was from Flow Laboratories (Puteaux, France). Streptomycin, penicillin, glutamine and pyruvate were from Gibco (Paisley, U.K.). Rabbit anti-Gαs subunit antibody (371732) raised to the carboxyl-terminal animo acids, RMHLRQYELL, of Gαs was from Calbiochem. Rabbit anti-Gβcom antibody (SC 378) raised to the carboxyl-terminal amino acids, TDDGMAVATGSWDSFLKIWN, of Gβ1 subunit was from Santa-Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Peptides corresponding to the Gβγ binding domain of β-adrenergic receptor kinase-1 or to a region outside the Gβγ binding site (Viard et al., 1999) were synthetized by Genosys (Cambridge, U.K.). PCR primers were synthetized by Eurogentec (Seraing, Belgium). Molecular-weight marker, HinfI, was from Promega (Charbonnières, France).

    Results

    Effects of isoprenaline on voltage-dependent Ba2+ current

    Inward currents were elicited every 20 s in single myocytes bathed in 5 mM BaCl2 solution by 200 ms depolarization to +10 mV from a holding potential of −40 mV and they gradually increased in size over 2–3 min in almost all cells, as previously reported (Viard et al., 1999). After the amplitude of inward current became steady application of 10 μM isoprenaline (in the continuous presence of 10 nM prazosin and 10 nM rauwolscine to inhibit α1- and α2-adrenoceptors, Leprêtre et al., 1994) resulted in an increase in Ba2+ current reaching 39±8% (n=5) within 3–4 min (Figure 1). Under both conditions (with or without isoprenaline), the recorded Ba2+ currents were completely blocked by 1 μM oxodipine or isradipine (data not shown). After a 10 min pre-treatment with either 1 μM propranolol (a non-selective β1- and β2-adrenoceptor antagonist) or 0.1 μM SR59230A alone (a β3-adrenoceptor antagonist, Manara et al., 1996), the isoprenaline-induced stimulation of Ba2+ current was not significantly affected (Figure 1B). In contrast, application of both compounds inhibited the stimulation of the Ba2+ current evoked by 10 μM isoprenaline (Figure 1A,B). Figure 1C illustrates the inhibitory effect of increasing concentrations of SR59230A in the absence or in the presence of 1 μM propranolol. It appears that the inhibitory concentration-response curve for SR59230A was shifted to lower concentrations in the presence of propranolol, as the SR59230A concentration corresponding to half-maximal inhibition decreased from 1.2 μM to 14 nM.

    Details are in the caption following the image

    Effects of isoprenaline and β-adrenoceptor antagonists on L-type Ca2+ channels in rat portal vein myocytes. (A) Ba2+ currents evoked by a depolarization to +10 mV from a holding potential of −40 mV before (a) and during the application of 10 μM isoprenaline for 3 min (b) in control conditions (1), in the presence of 1 μM propranolol for 10 min (2) or in the presence of both 1 μM propranolol and 0.1 μM SR59230A for 10 min (3). (B) Compiled data showing the effects of β-adrenoceptor antagonists on the isoprenaline-induced increase in L-type Ba2+ current. Currents are expressed as a fraction of their control values (I/Ic). Results are means±s.e.mean, with the number of cells tested indicated in parentheses. ★, Values significantly different from control values (P<0.05). (C) Concentration-response curves showing the inhibition of isoprenaline-induced increase in L-type Ba2+ current by SR59230A in the absence or in the presence of 1 μM propranolol. External solution contained 5 mM Ba2+, 10 mM prazosin and 10 nM rauwolscine.

    In order to identify the β-adrenoceptors potentially involved in the effects of isoprenaline, mRNA purified from rat portal vein myocytes was reversibly transcribed into cyclic DNA, and a fragment of cyclic DNA of each receptor subtype was amplified using subtype-specific primers for the PCR. As illustrated in Figure 2, products of expected sizes corresponding to β1-, β2- and β3-adrenoceptor mRNAs were detected in rat portal vein and detrusor muscle (used as a positive control, Seguchi et al., 1998). These results suggest that the three β-adrenoceptor subtypes are involved in the stimulation of Ba2+ current through independent transduction pathways which, however, present a common step since their effects are not additive. Furthermore, it appears that SR59230A is a specific antagonist of β3-adrenoceptors at concentrations lower than 100 nM.

    Details are in the caption following the image

    Expression of β-adrenoceptor mRNA in rat portal vein and detrusor smooth muscles. Amplified cDNA fragments corresponding to β1-, β2- and β3-adrenoceptors (AR) were separated on a 2% agarose gel and visualized by staining with ethidium bromide. HinfI: molecular size standards in base pairs (bp). For RNA purification and PCR conditions, see Methods.

    Stimulation of Ba2+ current by the selective β3 agonist CGP12177A

    In the following experiments, CGP12177A was used as a β3-adrenergic agonist and 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine were continuously added to the perfusion solution. Stimulation of Ba2+ current was detectable with 10 nM CGP12177A and increased with the concentration of CGP12177A to reach a maximum with 10 μM CGP12177A (Figure 3A,B). The maximal stimulation of Ba2+ current in the presence of 10 μM CGP12177A was 43±4% (n=25), not significantly different from that obtained with 10 μM isoprenaline (Figure 3B). The concentration of CGP12177A and isoprenaline producing a half-maximal stimulation of Ba2+ current were 0.14 and 1.35 μM, respectively (Figure 3B). CGP12177A-induced stimulation of Ba2+ current was concentration-dependently inhibited by SR59230A with a half-maximal inhibition obtained at 18 nM (Figure 3C). As illustrated by the current-voltage relationship (Figure 4A), the maximal Ba2+ current was increased by 10 μM CGP12177A without any change in the voltage threshold, the potential for the maximal current, and the extrapolated reversal potential. Steady-state inactivation of the Ba2+ current was examined with a two-pulse protocol (Figure 4B). A test pulse to +10 mV (V2) from a holding potential of −40 mV was preceded by a prepulse (V1) of 20 s duration and of variable amplitude (−60 to −20 mV). For each prepulse, the amplitude of the test current was taken as an index of the remaining activatable channels. Relative availability was expressed by plotting the test current against the prepulse potential value. The amplitude of the test current was expressed as a fraction of the current obtained at the most negative prepulse. As shown in Figure 4B, the curves obtained in the absence and presence of 10 μM CGP12177A were superimposed. Since the Ba2+ currents in cells stimulated by CGP12177A appeared to be slightly more enhanced at negative potentials than at positive potentials, we calculated the conductance (G) by dividing the peak Ba2+ current at each negative potential tested by the driving force (V-Vrev). The normalized conductance (G/Gmax) is plotted as a function of membrane potential in Figure 4C. In control conditions we found a half-activation potential of −14.0±2.0 mV (n=4). In the presence of 10 μM CGP12177A, the half-activation potential was not significantly modified (−16.5±2.5 mV, n=4, P>0.05). Taken together, these results indicate that CGP12177A stimulates L-type Ca2+ channels without affecting the gating properties of these channels.

    Details are in the caption following the image

    Effects of CGP12177A, a β3-adrenoceptor selective agonist, on L-type Ca2+ channels. (A) Ba2+ currents evoked by a depolarization to +10 mV from a holding potential of −40 mV before (a) and during the application of 10 μM CGP12177A for 3 min (b) in control conditions (1) or in the presence of 0.1 μM SR59230A for 10 min (2). (B) Concentration-response curves showing the stimulatory effect of CGP12177A and isoprenaline. Ba2+ currents are expressed as a percentage of the maximal response obtained with 10 μM CGP12177A, on each cell tested. Results are means±s.e.mean for 5–10 cells. (C) Inhibition curve for CGP12177A-induced increase in L-type Ba2+ current by SR59230A. Values are expressed as a percentage of the maximal response obtained with 10 μM CGP12177A. Results are means±s.e.mean for 4–8 cells. External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    Details are in the caption following the image

    Effect of CGP12177A on the current-voltage relationship and the activation and inactivation curves of L-type Ca2+ channels. (A) Current-voltage relationships obtained from a holding potential of −40 mV in control conditions and during the application of 10 μM CGP12177A for 3 min. Currents are expressed as a fraction of the maximal current obtained in control conditions (I/Ic). Data are means±s.e.mean for 4–7 cells. (B) Steady-state inactivation curve obtained with the two-pulse protocol (inset). Currents are expressed as a fraction of maximal current (I/Imax) obtained in control conditions or during application of 10 μM CGP12177A. (C) Activation curve expressed as a fraction of G/Gmax in control conditions or during application of 10 μM CGP12177A. Data are fitted by curves of form 1/1 [1+exp (Vm–Vh)/k], in which Vh is the potential at which half of the current is inactivated, Vm is the membrane potential, and k is the slope factor. Data are given as means±s.e.mean for 5–7 cells. External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    Transduction pathway activated by β3-adrenoceptors

    Modulation of L-type Ca2+ channels in vascular myocytes by β-adrenoceptor activation is believed to involve the cyclic AMP/PKA pathway through phosphorylation of channel subunits by PKA (Ishikawa et al., 1993) and/or a possible direct modulation of the channel activity by α subunits of Gs protein (Xiong et al., 1995).

    To elucidate the mechanism of stimulation of Ba2+ current by CGP12177A, we applied forskolin, a direct activator of adenylyl cyclase. Forskolin (5 μM) increased the Ba2+ current by 40±6% (n=10) within 4–5 min (Figure 5A,B). Similarly, 8-Br-AMPc (0.3 mM), applied extracellularly, stimulated the Ba2+ current by 35±6% (n=5; Figure 5A) indicating that activation of PKA may stimulate L-type Ca2+ channels in vascular myocytes. To evaluate whether PKA is involved in the intracellular pathway activated by β3-adrenoceptors, we tested the effects of PKA inhibitors (H-89 and Rp 8-Br-cyclic AMPs) on the stimulation of Ba2+ current evoked by CGP12177A. Superfusion of cells with H-89 (0.1 μM) or Rp 8-Br-cyclic AMPs (10 μM) for 10–15 min did not change the peak Ba2+ current in control cells (data not shown) nor affect the stimulatory response of 1 μM phorbol dibutyrate, an activator of PKC (control: 37±4%, n=4; in the presence of H-89: 42±4%, n=4; Figure 5B). In contrast, the stimulation of Ba2+ current evoked by forskolin, 8-Br-AMPc (not shown) or CGP12177A was inhibited by H-89 (Figure 5B) and Rp 8-Br-cyclic AMPs (not shown), suggesting that PKA is involved in the stimulation of Ca2+ channel activity by β3-adrenergic agonist. To ensure that the stimulation of Ba2+ current was mediated by a phosphorylation of L-type Ca2+ channels, we applied a protein phosphatase inhibitor, okadaic acid, before stimulation with CGP12177A or forskolin. Okadaic acid (5 μM) by itself had no significant effect on the Ba2+ current density when applied for 10–15 min (control: 8.1±0.6 pA/pF; in the presence of okadaic acid: 8.9±0.7 pA/pF, n=5) in agreement with previous data on L-type Ca2+ channels (Chahine et al., 1996). In control conditions, stimulation of the Ba2+ current by 10 μM CGP12177A or 5 μM forskolin was maximal within 3–4 min. After withdrawal of the stimulating substances, the Ba2+ current returned to its control value within 4–5 min (Figure 6A–C). In the presence of 5 μM okadaic acid, the CGP12177A-induced increase in Ba2+ current was similar to that obtained in control conditions but persisted after wash-out of CGP12177A (Figure 6B,C). Similar results were obtained with forskolin (not shown). These results support the idea that a phosphorylation mechanism is involved in the effects of CGP12177A and forskolin on L-type Ca2+ channels.

    Details are in the caption following the image

    Effects of activators and inhibitors of PKA on L-type Ca2+ channels. (A) Ba2+ currents evoked by a depolarization to +10 mV from a holding potential of −40 mV before (a) and during the external application of 0.3 mM 8-Br-AMPc (b), 5 μM forskolin (c) or 10 μM CGP12177A (d), in control conditions (1). In the presence of 0.1 μM H-89 for 10 min (2), currents before (a) and during application of 5 μM forskolin (e) or 10 μM CGP12177A (f). (B) Compiled data showing the effect of 0.1 μM H-89 on the increase in L-type Ba2+ current evoked by 5 μM forskolin, 0.1 μM phorbol dibutyrate (PDBu) and 10 μM CGP12177A. Currents are expressed as a fraction of their control values (I/Ic). Data are means±s.e.mean with the number of cells tested indicated in parentheses. ★, Values significantly different from control values (P<0.05). External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    Details are in the caption following the image

    Effects of okadaic acid on CGP12177A-induced stimulation of L-type Ca2+ channels. (A) Ba2+ currents on control conditions (a), in the presence of 10 μM CGP12177A for 4 min (b), and after withdrawal of the agonist for 5 min (c). (B) Ba2+ currents in the presence of 5 μM okadaic acid before (a) and during the applications of 10 μM CGP12177A (b), and after withdrawal of the agonist for 5 min (c). (C) Compiled data showing the stimulatory effects of 10 μM CGP12177A and the recovery after withdrawal of the agonist for 5 min in control conditions and in the presence of 5 μM okadaic acid. Currents are expressed as a fraction of their control values (I/Ic). Data are means±s.e.mean with the number of cells tested indicated in parentheses. External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    To identify the G protein coupling the β3-adrenoceptors to adenylyl cyclase we used cholera toxin (CTX) which ADP ribosylates Gs proteins, leading to their permanent activity. Cells were incubated in a culture medium containing 160 ng ml−1 CTX for 20 h. This pretreatment suppressed the CGP12177A-induced stimulation of Ba2+ current (Figure 7A) whereas it did not affect the clonidine-induced stimulation of Ba2+ current (control: 41±5%, n=5; CTX-pre-treated cells: 40±6%, n=5) which has been demonstrated to depend on activation of a Gi protein (Leprêtre et al., 1995). Antibodies directed against the carboxyl terminus of the α subunits of G proteins have been shown to be useful tools for identifying transduction couplings. When a carboxyl-terminal anti-Gαs antibody was added to the basic pipette solution for 4 min, the CGP12177A-induced stimulation of Ba2+ current was concentration-dependently inhibited, with maximal inhibition obtained at 10 μg ml−1 anti-Gαs antibody (Figure 7B,C). Intracellular application of the anti-Gαs antibody inactivated by heating at 95°C for 30 min did not significantly affect the CGP12177A-induced stimulation of Ba2+ current (Figure 7B). These results indicate that the Gs protein transduces the signal for stimulation of Ca2+ channels in response to activation of β3-adrenoceptors.

    Details are in the caption following the image

    Effects of cholera toxin and anti-Gαs antibody on the CGP12177A-induced stimulation of L-type Ca2+ channels. (A) Ba2+ currents evoked by a membrane depolarization to +10 mV from a holding potential of −40 mV before (a) and during application of 10 μM CGP12177A (b) or 10 μM clonidine (c) in control conditions (1) or after a pretreatment with 160 μg ml−1 CTX for 20 h (2). (B) Ba2+ currents before (a) and during application of 10 μM CGP12177A in control conditions (1), in cells dialyzed with 10 μg ml−1 anti-Gαs antibody for 4 min (2) or boiled anti-Gαs antibody (3). (C) Compiled data showing the inhibition of CGP12177A-induced increase in L-type Ba2+ current by increasing concentrations of anti-Gαs antibody. Currents are expressed as a fraction of their control values (I/Ic). Data are means±s.e.mean, with the number of cells tested indicated in parentheses. ★, Values significantly different from control values (P<0.05). External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    The anti-Gαs antibody, however, cannot distinguish whether α or βγ subunits interact with adenylyl cyclase. Therefore, intracellular infusion of either an anti-Gβcom antibody or a peptide corresponding to a fragment of βARK1 was used to bind Gβγ subunits and to block activation of effectors. Applications of βARK1 peptide and anti-Gβcom antibody had no significant effects on the Ba2+ current density in non-stimulated myocytes. The mean Ba2+ current density was 8.5±0.5 pA/pF in control conditions (n=21), 7.8±0.7 pA/pF in the presence of 10 μM βARK1 (n=11) and 7.6±0.6 pA/pF in the presence of 10 μg ml−1 anti-βcom antibody for 5–7 min (n=9). As shown in Figure 8A, 10 μg ml−1 anti-Gβcom antibody or 10 μM βARK1 peptide (corresponding to the Gβγ binding region of βARK1, Nair et al., 1995; Stehno-Bittel et al., 1995) had no significant effects on CGP12177A-induced stimulation of Ba2+ current (control: 40±3%, n=8; in the presence of βARK1: 43±4%, n=5, or anti-Gβcom antibody: 41±4%, n=5). In contrast, in the same cell batches, the angiotensin II-induced stimulation of Ba2+ current was selectively inhibited by both the anti-Gβcom antibody and the βARK1 peptide (Figure 8B; control: 44±4%, n=10; in the presence of βARK1: 2±1%, n=5, or anti-Gβcom antibody: 3±2%, n=5, P<0.05). Taken together, these results suggest that Gβγ subunits are not involved in the β3-adrenergic stimulation of adenylyl cyclase in vascular myocytes.

    Details are in the caption following the image

    Effects of anti-Gβcom antibody and βARK1 peptide on CGP12177A- and angiotensin II-induced stimulation of L-type Ca2+ channels. (A) Typical Ba2+ currents evoked by a depolarization to +10 mV from a holding potential of −40 mV before (a) and during application of 10 μM CGP12177A (b) in control conditions (1), in cells dialyzed with 10 μM βARK1 peptide (2) or 10 μg ml−1 anti-Gβcom antibody for 4 min (3). (B) Typical Ba2+ currents before (a) and during applications of 10 nM angiotensin II (b) in control conditions (1), in cells dialyzed with 10 μM βARK1 peptide (2) or 10 μg ml−1 anti-Gβcom antibody for 4 min (3). External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    We have previously shown that the vascular L-type Ca2+ channels may be stimulated by PKC-dependent mechanisms (Leprêtre et al., 1994; Viard et al., 1999). Stimulation of Ba2+ current evoked by 0.1 μM PDBu was completely blocked by PKC inhibitors, i.e. external application of 1 μM GF109203X (Figure 9A,B) or infusion of 1 μM 19–31 peptide. Both GF109203X and 19–31 peptide had no direct effects on the control Ba2+ current activated by membrane depolarization (data not shown) as well as on the stimulation of Ba2+ current evoked by forskolin and CGP12177A (Figure 9A,B). In addition, simultaneous applications of both 0.1 μM PDBu and 5 μM forskolin resulted in a further increase in Ba2+ current. This stimulatory effect is higher than that obtained with PDBu or CGP12177A alone (Figure 9B) and suggests an additive effect of PKC and PKA on L-type Ca2+ channel activity. These results confirm that a PKC-dependent pathway does not mediate the β3-adrenergic-induced stimulation of Ca2+ channels.

    Details are in the caption following the image

    Effects of PKC inhibitor on the PDBu-, forskolin- and CGP12177A-induced stimulation of L-type Ca2+ channels. (A) Ba2+ currents evoked by a depolarization to +10 mV from a holding potential of −40 mV before (a) and during application of 10 μM PDBu (b) or 5 μM forskolin (c) in control conditions (1) and in the presence of 0.1 μM GF109203X for 10 min (2). (B) Compiled data showing the effects of 0.1 μM GF109203X on PDBu-, forskolin- and CGP12177A-induced increase of L-type Ba2+ current. Currents are expressed as a fraction of their control values (I/Ic). Data are means±s.e.mean with the number of cells tested indicated in parentheses. ★, Values significantly different of those obtained in the absence of GF109203X. External solution contained 5 mM Ba2+, 1 μM propranolol, 10 nM prazosin and 10 nM rauwolscine.

    Discussion

    The results of the present study indicate that in vascular myocytes: (1) β1-, β2- and β3-adrenoceptor mRNAs are expressed, as identified by RT–PCR; (2) the stimulatory effect of the β3-adrenoceptor agonist CGP12177A on L-type Ca2+ channels is selectively prevented by SR59230A (the β3-adrenoceptor antagonist); (3) the CGP12177A-induced stimulation of Ca2+ channels is blocked by cyclic AMP dependent protein kinase inhibitors, H-89 and Rp 8-Br-cyclic AMPs, but not by PKC inhibitors, GF109203X and 19–31 peptide. This stimulation was mimicked by forskolin and 8-Br-AMPc. In the presence of okadaic acid, inhibition of recovery after withdrawal of β3-adrenoceptor agonist and forskolin suggests that phosphorylation plays a major role on L-type Ca2+ channel modulation; (4) the β3-adrenoceptor stimulation of L-type Ca2+ channels was removed by a pretreatment with CTX and by the intracellular application of an anti-Gαs antibody but was unaffected by intracellular infusion of an anti-Gβcom antibody and a βARK1 peptide. These observations indicate that the transduction coupling activated by β3-adrenoceptors involves a Gαs-induced stimulation of the cyclic AMP/PKA pathway leading to phosphorylation and subsequent activation of L-type Ca2+ channels.

    The β3-adrenoceptors have been cloned and sequenced in several species including rat and human (Strosberg, 1997). Recent studies have suggested the existence of putative β4-adrenoceptors which have not been cloned and sequenced up to now (Kaumann et al., 1998). However, since these putative β4-adrenoceptors have been shown to be insensitive to SR59230A (Galitzky et al., 1997), our results showing a complete inhibition with SR59230A support the idea that only β3-adrenoceptors mediate the CGP12177A-induced stimulation of L-type Ca2+ channels in vascular myocytes. Activation of the Gs/PKA pathway by β3-adrenoceptors has been previously reported in rodent adipocytes (Strosberg, 1997). Although it is generally accepted that α subunits of Gs protein play an important role in regulation of L-type Ca2+ channels in cardiac and smooth muscles during β-adrenergic stimulation, the signalling pathways underlying the modulation of L-type Ca2+ channels by activated Gs proteins remain controversial. In cardiac myocytes, the β-adrenergic-induced stimulation of Ca2+ channels is essentially via activation of adenylyl cyclase and subsequent phosphorylation of the channel (McDonald et al., 1994). In addition, a direct G protein activation of Ca2+ channels has been also proposed in the heart in response to β-adrenergic stimulation (Yatani et al., 1987). In rabbit portal vein myocytes, intracellular application of activated Gαs subunits mimics the stimulatory effect of isoprenaline, and this effect has been interpreted as the result of a direct action of Gs protein on L-type Ca2+ channels (Xiong & Sperelakis, 1995). However, recent reports have shown that 8-Br-cyclic AMP and the catalytic subunit of PKA significantly increased peak Ba2+ currents, and their effects could be blocked by PKA inhibitors (Ruiz-Velasco et al., 1998). In the present study, the β3-adrenoceptor-induced stimulation of Ba2+ current was entirely blocked by H-89 and Rp 8-Br-cyclic AMPs, inhibitors of PKA, and by intracellular infusion of the anti-Gαs antibody. In addition, application of okadaic acid, a protein phosphatase inhibitor, suppressed the CGP12177A- and forskolin-induced recovery of Ba2+ current after withdrawal of the stimulating substances. These results support the idea that the activated αs subunits of Gs proteins elicit their stimulatory effect through the cyclic AMP/PKA pathway and the subsequent phosphorylation of L-type Ca2+ channels.

    In addition to Gs protein, β3-adrenoceptors have been shown to interact with Gi/o proteins in adipocytes (Chaudhry et al., 1994) and human cardiac cells (Gauthier et al., 1996). Recently, it has been demonstrated that the human β3-adrenoceptor may activate the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) 1 and 2 through Gi/o protein and phosphatidylinositol-3 kinase (PI3K) (Gerhardt et al., 1999). Moreover, the isoprenaline-induced activation of MAPK has been reported to involve the Gβγ dimers derived from Gi/o protein (Crespo et al., 1995). Using an anti-Gβcom antibody and a βARK1-derived Gβγ binding peptide, we have reported that the βγ dimer from G13 is responsible for the transduction pathway during angiotensin II-induced stimulation of L-type Ca2+ channels (Macrez et al., 1997). Intracellular infusion of purified Gβγ proteins has been shown to stimulate the activity of L-type Ca2+ channels; this effect is largely inhibited by PKC inhibitors but remains insensitive to PKA inhibitors (Viard et al., 1999). In contrast, we show here that the β3-adrenoceptor-induced stimulation of Ba2+ current is removed by H-89 but not affected by PKC inhibitors and after intracellular infusion of an anti-Gβcom antibody or a βARK1 peptide, which act as Gβγ scavengers. Thus, our results suggest that in vascular myocytes only the α subunits of Gs may play a role in the regulation of L-type Ca2+ channels during β3-adrenoceptor activation. Although different combinations of β and γ subunits of G proteins may have similar actions on Ca2+ channels (Dolphin, 1998), recent data have shown that activation of MAPK/ERK and inhibition of adenylyl cyclases V and VI appear to be Gβ isoform specific (Gβ1 being more efficient than Gβ5) (Zhang et al., 1996; Bayewitch et al., 1998). As the subunit composition of the Gs protein that interacts with the β3-adrenoceptor of vascular myocytes has not been identified, it can be postulated that this βγ dimer may be unable to stimulate the Gβγ-sensitive pathway leading to stimulation of L-type Ca2+ channels.

    Activation of β-adrenoceptors is known to induce a relaxation in smooth muscle cells and several hypotheses have been proposed including Ca2+-dependent and Ca2+-independent mechanism. Although global increases in [Ca2+]i regulate contraction, local Ca2+ transients (Ca2+ sparks) caused by opening of ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum may activate KCa channels in the surface membrane (Mironneau et al., 1996; Perez et al., 1999) leading to hyperpolarization and closing of voltage-dependent Ca2+ channels (Knot & Nelson, 1998). We have shown that Ca2+ sparks can be triggered by activation of L-type Ca2+ currents, particularly in the voltage range between −20 and 0 mV (Arnaudeau et al., 1997). In contrast, propagated Ca2+ waves are obtained only when large (+10 mV) and durable (>200 ms) depolarizations are applied (Arnaudeau et al., 1997). Preliminary results indicate that 1 μM CGP12177A and 5 μM forskolin increase the frequency of Ca2+ sparks in portal vein myocytes held at −50 mV (F. Coussin and J. Mironneau, unpublished data), then promoting increased activation of KCa channels, hyperpolarization and, ultimately, vasodilation. Therefore, it is proposed that the β3-adrenoceptor-mediated modulation of L-type Ca2+ channels may be important in the regulation of smooth muscle tension.

    In conclusion, the present study shows that in vascular myocytes activation of β3-adrenoceptors stimulates the L-type Ca2+ channels though a Gαs-induced activation of the cyclic AMP/PKA pathway and the subsequent phosphorylation of the channels.

    Acknowledgments

    This work was supported by grants from Centre National de la Recherche Scientifique and Fondation pour la Recherche Médicale (to P. Viard), France. We thank N. Biendon for secretarial assistance and C. Le Sénéchal for participating to PCR experiments.