{beta}-Adrenergic receptor stimulation of L-type Ca2+ channels in rabbit portal vein myocytes involves both {alpha}s and {beta}{gamma} G protein subunits

HOME

HELP

FEEDBACK

SUBSCRIPTIONS

$beta$ -Adrenergic receptor stimulation of L-type Ca²⁺ channels in rabbit portal vein myocytes involves both $alpha$ s and $beta$ $gamma$ G protein subunits

Juming Zhong, Joseph R. Hume and Kathleen D. Keef

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA

MS 11079 Received 5 May 2000; accepted after revision 18 October 2000

ABSTRACT

Top
Abstract
Introduction
Methods
Results
Discussion
References

Previous studies have shown that purified G protein $alpha$ s and $beta$ $gamma$ subunits stimulate vascular L-type Ca²⁺ channels through protein kinase A and C (PKA and PKC), respectively. The present study tested whether activation of endogenous G proteins via $beta$ -adrenergic receptor binding also stimulates vascular Ca²⁺ channels through both G $alpha$ s and G $beta$ $gamma$ and the subsequent activation of PKA and PKC.

Peak Ba²⁺ current (I_Ba) in freshly isolated rabbit portal vein smooth muscle cells was significantly increased by bath application of 0.5 $mu$ M isoproterenol (isoprenaline; ISO) when measured using the whole-cell patch clamp method (53 ± 3 % increase, n = 15). Stimulation of I_Ba by ISO was partially reversed by a PKA inhibitor, KT 5720, or a PKC inhibitor, calphostin C, and completely blocked when cells were pretreated with both KT 5720 and calphostin C.

Dialysis of cells with polyclonal antibody to G $alpha$ s significantly reduced but did not completely eliminate ISO-induced stimulation of I_Ba. The remaining stimulation was abolished by calphostin C. Dialysis of cells with a polyclonal antibody to G $beta$ also significantly reduced ISO-induced stimulation and the remaining stimulation was abolished by KT 5720. Dialysis of cells with both antibodies completely prevented the stimulation of I_Ba by ISO.

ISO-induced stimulation of I_Ba was reversed by ICI-118,551, a specific $beta$ ₂-adrenoceptor antagonist, but not by CGP 20712A, a specific $beta$ ₁-adrenoceptor antagonist. In addition, the $beta$ ₂-adrenoceptor agonist zinterol significantly increased peak I_Ba while the $beta$ ₁-adrenoceptor agonist dobutamine and $beta$ ₃-adrenoceptor agonist BRL 37344A had little effect on peak I_Ba.

These data suggest that $beta$ -adrenergic receptor stimulation of vascular L-type Ca²⁺ channels involves both $alpha$ s and $beta$ $gamma$ G-protein subunits, which exert their effects through PKA and PKC, respectively.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Ca²⁺ influx through L-type Ca²⁺ channels plays an important role in the regulation of contraction in vascular smooth muscle. In recent years a number of studies have suggested that $beta$ -adrenergic receptor stimulation leads to enhancement of Ca²⁺ channel activity via G protein-dependent activation of cAMP-dependent protein kinase (PKA). Upon binding with agonist, the $beta$ -adrenergic receptor promotes replacement of GDP with GTP on the G protein $alpha$ s subunit (G $alpha$ s). This in turn leads to dissociation of the GTP-bound G $alpha$ s subunit from the $beta$ $gamma$ dimer (G $beta$ $gamma$ ). At present, the role of these G protein subunits in the excitatory effects of $beta$ -adrenergic receptors on L-type Ca²⁺ channels in smooth muscle cells is still unclear.

Dissociated G protein subunits may interact with a number of different effectors such as adenylyl cyclases and ion channels (Hepler & Gilman, 1992; Clapham, 1994). Previous studies from our laboratory demonstrated that both purified G $alpha$ s and G $beta$ $gamma$ subunits, when dialysed into cells, lead to the stimulation of L-type Ca²⁺ channel activity in rabbit portal vein myocytes (Zhong et al. 1999). G $alpha$ s was associated with activation of the adenylyl cyclase-PKA pathway whereas G $beta$ $gamma$ was associated with activation of PKC. However, in the light of these observations the possible role of both endogenous G $alpha$ s and G $beta$ $gamma$ in the $beta$ -adrenergic receptor stimulation of L-type Ca²⁺ channels is still unknown. Whereas G $alpha$ s has long been considered to play a central role in the actions of $beta$ -adrenergic receptor stimulation, the role of G $beta$ $gamma$ is much less clear. Indeed, at present there is no information available concerning the potential role of G $beta$ $gamma$ as a possible contributor to the actions of $beta$ -adrenergic receptors on L-type Ca²⁺ channels.

In the present study, we investigated the role of endogenous G $alpha$ s and G $beta$ $gamma$ in the modulation of L-type Ca²⁺ channels by $beta$ -adrenergic receptor stimulation in rabbit portal vein smooth muscle myocytes. We used polyclonal antibodies directed against either the $alpha$ s or the $beta$ G protein subunit. In addition, inhibitors of PKA and PKC were tested to determine whether one or both of these kinases contribute to the response, and specific $beta$ -adrenoceptor agonists and antagonists were used to characterize the receptor subtype involved. Our results suggest that both G $alpha$ s and G $beta$ $gamma$ participate in $beta$ -adrenergic receptor stimulation of L-type Ca²⁺ channels, mediated by the PKA and PKC pathways, respectively.

METHODS

Top
Abstract
Introduction
Methods
Results
Discussion
References

Isolation of rabbit portal vein myocytes

Myocytes were isolated using previously described methods (Zhong et al. 1999). Male albino rabbits (1.5-2.0 kg) were killed with an intravenous overdose of sodium pentobarbital (50 mg kg^-1). The portal vein was rapidly removed and cleaned of connective tissue in ice-cold Krebs solution (mM): 125 NaCl, 4.2 KCl, 1.2 MgCl₂, 1.8 CaCl₂, 11 glucose, 1.2 K₂HPO₄, 23.8 NaHCO₃ and 11 Hepes, pH 7.4 with NaOH and bubbled with 95 % O₂ and 5 % CO₂. The portal vein was then cut into small segments (~4 mm X 4 mm) and pre-incubated for 30 min in a shaking water bath at 35 °C in a dispersion solution (enzyme-free, mM): 90 NaCl, 1.2 MgCl₂, 1.2 K₂HPO₄, 20 glucose, 50 taurine and 5 Hepes, pH 7.1 with NaOH. Following pre-incubation, the segments were incubated in the dispersion solution containing 2 mg ml^-1 collagenase type I (Sigma), 0.5 mg ml^-1 protease type XXVII (Sigma) and 2 mg ml^-1 bovine serum albumin (Sigma) for 10-14 min at 35 °C, and then rinsed 4 times with enzyme-free dispersion solution. Smooth muscle cells were dispersed by gentle trituration of the segments with a wide-tipped fire-polished Pasteur pipette. The cell suspension was stored in enzyme-free dispersion solution containing BSA (1 mg ml^-1) and Ca²⁺ (0.1 mM) at 4 °C and used within 10 h. The animal use protocol was reviewed and approved by the Animal Care and Use Committee of the University of Nevada.

Electrophysiology

Ba²⁺ currents (I_Ba) in portal vein smooth muscle cells were measured using the whole-cell patch clamp technique. A drop of cell suspension was added to a small recording chamber mounted on the stage of an inverted microscope (Nikon, Japan). The cells were allowed to settle down to the bottom of the chamber and superfused at a constant rate (~1-2 ml min^-1). Inward currents were measured using an Axopatch-1D patch clamp amplifier (Axon Instruments). Patch electrodes were made from borosilicate glass pulled with a Sutter P-87 Flaming/Brown micropipette horizontal puller and fire polished with a MF-83 Narishige microforge (Japan). Pipette resistance was 3-5 M $Omega$ when filled with the pipette solution. After establishing the whole-cell configuration, cell membrane capacitance and series resistance were determined using a 20 mV hyperpolarizing pulse and were partially compensated. Inward current was elicited by stepping the voltage to 0 mV from a holding potential of -70 mV at 30 s intervals. Voltage clamp protocols were applied to the cells using the data acquisition package pCLAMP 7 (Axon Instruments) and filtered at 2 kHz. Data analysis was performed using the pCLAMP 7 software package. All the experiments were performed at room temperature (20-23 °C).

The bath solution used to record I_Ba in portal vein cells was composed of (mM): 117.5 NaCl, 10 TEACl, 5 BaCl₂, 0.5 MgCl₂, 5.5 glucose, 5 CsCl and 10 Hepes, pH 7.40 with NaOH. Both TEACl and CsCl were used to block K⁺ currents. The pipette solution contained (mM): 120 CsCl, 20 TEACl, 1 K₂HPO₄, 5.5 glucose, 5.7 MgCl₂, 5 ATP, 0.5 GTP, 10 EGTA and 10 Hepes, pH 7.2 with CsOH.

Drugs

Isoproterenol (ISO) and chemicals were purchased from Sigma. Dobutamine, ICI 118,551 and CGP 20712A were from RBI (Natick, MA, USA). KT 5720 and calphostin C were from CalBiochem (La Jolla, CA, USA). Rp-8-Br-PET-cGMPS was from Biolog (La Jolla, CA, USA). Zinterol was a generous gift from Dr Iain Buxton, Department of Pharmacology, University of Nevada School of Medicine. Antibodies to G protein subunits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-G $alpha$ s antibody is a rabbit polyclonal IgG raised against an epitope mapping within the N-terminal domain of human G $alpha$ s. The anti-G $beta$ _com antibody is a rabbit polyclonal antibody raised against a peptide mapping to the C-terminal domain of mouse G $beta$ and is reactive with all four subtypes of G $beta$ . Drugs insoluble in water were first dissolved in dimethylsulphoxide (DMSO) and were then further diluted in the solution with the final concentration of DMSO less than 0.2 %. DMSO alone at a concentration of 0.2 % had no effect on I_Ba.

Data analysis

All experimental values are presented as means ± S.E.M., and n refers to the number of cells tested. Differences between the values from different groups were compared using Student's paired and unpaired t tests, and two-way analysis of variance, where appropriate. P values of less than 0.05 were considered significantly different.

RESULTS

Top
Abstract
Introduction
Methods
Results
Discussion
References

ISO-induced stimulation of I_Ba is completely abolished by inhibitors of both PKA and PKC

Ba²⁺ currents through L-type Ca²⁺ channels were recorded in freshly isolated rabbit portal vein myocytes using the whole-cell voltage clamp technique. The $beta$ -adrenergic receptor agonist isoproterenol (ISO) was used to stimulate $beta$ -adrenoceptors. Previous studies from our laboratory have shown that the inward current recorded from these cells under similar experimental conditions was blocked by nicardipine. In addition, low concentrations of ISO (< 1 $mu$ M) produced constant stimulation of Ca²⁺ channel currents in rabbit portal vein cells while higher concentrations of ISO were associated with a transient increase followed by a reduction in current due to 'cross-activation' of protein kinase G (PKG; Ishikawa et al. 1993). Thus low concentrations (0.5-1 $mu$ M) of ISO were used in this study. Once steady-state current amplitudes were obtained in the whole-cell configuration, ISO (0.5 $mu$ M) was added to the superfusate, which caused a significant increase in peak I_Ba (mean peak I_Ba with ISO was 155 ± 5 % of basal current, n = 10; Fig. 1). Application of KT 5720 (0.2 $mu$ M), a specific PKA inhibitor, significantly reduced, but did not abolish, the ISO-induced stimulation of peak I_Ba. The peak I_Ba that remained in the presence of KT 5720 was 21 ± 4 % higher than the peak I_Ba recorded under basal conditions (Fig. 1). The current stimulation that remains is unlikely to be due to incomplete blockade of PKA since we have previously shown that this concentration of KT 5720 completely blocks the stimulation that occurs when cells are dialysed with the catalytic subunit of PKA or activated G protein $alpha$ s subunits (Ruiz-Velasco et al. 1998; Zhong et al. 1999). These results suggest that PKA significantly contributes to $beta$ -adrenergic receptor stimulation of Ca²⁺ channels in these cells but that another pathway(s) independent of PKA is also likely to be involved.

View larger version
[in this window]
[in a new window]

Figure 1 ISO-enhanced Ca²⁺ channel current in rabbit portal vein myocytes is not entirely reversed by PKA inhibitor

Currents were elicited by stepping the membrane potential to 0 mV from a holding potential of -70 mV. A, superimposed recordings from a cell in the absence (1) or presence (2) of ISO (0.5 $mu$ M) or presence (3) of ISO and KT 5720 (PKA inhibitor, 0.2 $mu$ M). B, time course of peak I_Ba from a cell before and after addition of ISO and KT 5720. ISO and KT 5720 were added to the superfusate when peak current reached a steady state. C, percentage change of peak I_Ba in the presence of ISO or ISO plus KT 5720 compared with the values in the absence of any drugs. Values represent means ± S.E.M. and asterisks indicate a significant difference from values before ISO application (P < 0.05).

To further investigate the nature of the PKA-independent response to ISO, cells were superfused with KT 5720 (0.2 $mu$ M), calphostin C (a PKC inhibitor, 0.2 $mu$ M), or KT 5720 plus calphostin C (0.2 $mu$ M each), before and during application of ISO. When ISO (0.5 $mu$ M) was added in the presence of KT 5720 it still resulted in a 17 ± 1 % increase in peak I_Ba (n = 13, Fig. 2C). This is very similar to the magnitude of current stimulation that remained when KT 5720 was added after ISO (Fig. 1C). Similarly, when ISO was added in the presence of calphostin C peak I_Ba increased by 31 ± 3 % (Fig. 2C), which is very similar to the magnitude of current stimulation that remained when calphostin C was added after ISO (i.e. 30 ± 4 %, n = 3, data not shown). On the other hand, the combined application of KT 5720 plus calphostin C produced complete blockade of ISO-induced stimulation of I_Ba (n = 11, Fig. 2). These data suggest that the PKA-independent response to ISO is likely to be due to PKC.

Since high levels of cAMP can lead to cross-activation of PKG (Lincoln et al. 1990), some experiments with ISO were repeated when cells were first superfused with the PKG inhibitor Rp-8-Br-cGMPS (Rp-PET). The increase in peak current produced with ISO (1 $mu$ M) plus Rp-PET was not significantly greater than the response measured in the absence of Rp-PET (i.e. 67 ± 7 vs. 55 ± 5 %). To further investigate the possible role of PKG in the ISO response, calphostin C and KT 5720 were added to cells pretreated with Rp-PET and stimulated with ISO. Combined calphostin C plus KT 5720 entirely abolished ISO-induced stimulation of I_Ba in the presence of Rp-PET (n = 7, data not shown) as it did in the absence of Rp-PET (Fig. 2C), suggesting that PKG effects do not significantly contaminate the results obtained with 1 $mu$ M ISO.

View larger version
[in this window]
[in a new window]

Figure 2 ISO-enhanced Ca²⁺ channel current in rabbit portal vein myocytes is entirely abolished by combined PKA and PKC inhibitors

A, time course of peak current recordings from a cell before and after application of ISO (0.5 $mu$ M) in the presence of KT 5720 (0.2 $mu$ M). B, time course of peak current recordings from a cell before and after application of ISO in the presence of KT 5720 and calphostin C (0.2 $mu$ M). C, percentage change of peak I_Ba after application of ISO in the presence of KT 5720, calphostin C, or KT 5720 plus calphostin C. Numbers in parentheses indicate the number of cells tested. Values represent means ± S.E.M. and asterisks indicate a significant difference from values before ISO application (P < 0.05).

ISO-induced stimulation of I_Ba is completely attenuated by antibodies to G $alpha$ s and G $beta$

A previous study from our laboratory showed that either purified $alpha$ s or $beta$ $gamma$ G protein subunits stimulate Ca²⁺ channel currents in rabbit portal vein myocytes (Zhong et al. 1999). However, it is unclear whether both of these G protein subunits are involved in the response to $beta$ -adrenergic receptor stimulation. To examine the role of G $beta$ $gamma$ in the actions of ISO, cells were first dialysed with anti-G $alpha$ s antibody (10 $mu$ g ml^-1) and Ba²⁺ currents were measured before and after addition of ISO. In the presence of anti-G $alpha$ s antibody, ISO still gave rise to an increase in peak I_Ba, but this increase was significantly smaller than the stimulatory effect of ISO in control cells without antibody (18 ± 3 vs. 55 ± 5 % increase, respectively; see Figs 1 and 3). The stimulation of peak I_Ba by ISO in the presence of anti-G $alpha$ s antibody was reversed by calphostin C, but not by KT 5720 (Fig. 3). When the amount of anti-G $alpha$ s antibody in the patch pipette was doubled (20 $mu$ g ml^-1) there was no further reduction in the stimulatory effect of ISO on I_Ba (i.e. 19 ± 2 % increase, n = 5, data not shown) suggesting that 10 $mu$ g ml^-1 fully blocks G $alpha$ s. To isolate the G $alpha$ s component of the response to ISO, cells were first dialysed with anti-G $beta$ antibody (10 $mu$ g ml^-1) and Ba²⁺ currents were measured before and after addition of ISO. In the presence of anti-G $beta$ antibody, ISO still elicited a 19 ± 3 % increase in peak I_Ba. This increase of peak I_Ba was entirely reversed by KT 5720, but not by calphostin C (Fig. 4). When the amount of anti-G $beta$ antibody in the patch pipette was doubled (20 $mu$ g ml^-1) there was no further reduction in the stimulatory effect of ISO (16 ± 1 % increase, n = 3, data not shown) suggesting that the G $beta$ subunit was fully blocked with 10 $mu$ g ml^-1 of antibody. These data suggest that neither antibody, when used alone, completely prevents the stimulatory effect of ISO on Ca²⁺ channel activity. In contrast, 10 $mu$ g ml^-1 of the same anti-G $beta$ antibody was sufficient to abolish the angiotensin II-induced stimulation of Ca²⁺ channels in rat portal vein myocytes (Macrez et al. 1997).

View larger version
[in this window]
[in a new window]

Figure 3 The G $alpha$ s-independent effect of ISO is abolished by PKC inhibitor

Cells were dialysed with anti-G $alpha$ s antibody (10 $mu$ g ml^-1) and currents recorded every 30 s. ISO (1 $mu$ M) was added to the bath solution when current reached a steady state. A, time course of peak current recordings from a cell dialysed with anti-G $alpha$ s antibody in the absence and presence of ISO and calphostin C (0.2 $mu$ M). B, percentage change of peak I_Ba after application of ISO and kinase inhibitors. Numbers in parentheses indicate the number of cells tested. Values represent means ± S.E.M. and asterisks indicate a significant difference from values before ISO application (P < 0.05).

View larger version
[in this window]
[in a new window]

Figure 4 The G $beta$ $gamma$ -independent effect of ISO is abolished by PKA inhibitor

Cells were dialysed with anti-G $beta$ antibody (10 $mu$ g ml^-1) and currents recorded every 30 s. ISO (1 $mu$ M) was added into the bath solution when current reached a steady state. A, time course of peak current recordings from a cell dialysed with anti-G $beta$ antibody in the absence and presence of ISO and KT 5720 (0.2 $mu$ M). B, percentage change of peak I_Ba after application of ISO and kinase inhibitors. Numbers in parentheses indicate the number of cells tested. Values represent means ± S.E.M. and asterisks indicate a significant difference from values before ISO application (P < 0.05).

Our results suggest that the ISO-induced response consists of two components, one initiated by G $alpha$ s, which leads to the stimulation of PKA, and one initiated by G $beta$ $gamma$ , which leads to the stimulation of PKC. To further explore this hypothesis, additional experiments were undertaken in which cells were dialysed with combined anti-G $alpha$ s antibody and anti-G $beta$ $gamma$ antibody (10 $mu$ g ml^-1 each). Peak I_Ba measured following addition of ISO was not different from that recorded before addition of ISO when cells were dialysed with both anti-G $alpha$ s and anti-G $beta$ antibodies (Fig. 5). This complete block was not due to the greater amount of antibody used since neither 20 $mu$ g ml^-1 of anti-G $alpha$ s antibody alone nor 20 $mu$ g ml^-1 of anti-G $beta$ antibody alone produced complete block of ISO-induced stimulation (see above).

View larger version
[in this window]
[in a new window]

Figure 5 Combined antibodies to G $alpha$ s and G $beta$ subunits prevent ISO-induced stimulation of peak I_Ba in rabbit portal vein myocytes

A, time course of peak current recordings from a cell dialysed with anti-G $alpha$ s and anti-G $beta$ (10 $mu$ g ml^-1 each) in the absence and presence of ISO. Cells were dialysed with anti-G $alpha$ s and anti-G $beta$ antibodies and currents recorded every 30 s. ISO (1 $mu$ M) was added to the bath solution when current reached a steady state. B, peak I_Ba in cells dialysed with anti-G $alpha$ s plus anti-G $beta$ (10 $mu$ g ml^-1) before and after application of ISO (n = 11). Values represent means ± S.E.M. There was no significant difference between the values before and after application of ISO.

$beta$ ₂-Adrenoceptor agonist is a potent stimulator of I_Ba in rabbit portal vein myocytes

Results from the above experiments as well as other reports indicate that $beta$ -adrenergic receptor activation leads to the stimulation of L-type Ca²⁺ channels in vascular smooth muscle cells. Although it is well established that $beta$ ₂-adrenoceptors mediate relaxation of vascular smooth muscle, it is not clear which subtype of $beta$ -adrenergic receptor is responsible for the $beta$ -adrenoceptor-induced stimulation of Ca²⁺ channels. This question was examined for rabbit portal vein cells in two ways. First, we tested subtype-specific $beta$ -adrenoceptor antagonists. A previous study from our laboratory showed that the ISO-induced stimulation of I_Ba was completely abolished by propranolol, a non-selective $beta$ ₁- and $beta$ ₂-adrenoceptor antagonist (Ishikawa et al. 1993). In the present study we tested the effects of the more specific $beta$ ₁- and $beta$ ₂-adrenoceptor antagonists CGP 20712A and ICI 118,551, respectively (Bilski et al. 1983; Laflamme & Becker, 1998; Schroder & Herzig, 1999). Figure 6 shows the effects of either ICI 181,551 or CGP 20712A on ISO-induced stimulation of Ca²⁺ channel current. In the presence of 5 $mu$ M ICI 181,551, stimulation of peak I_Ba by ISO was entirely abolished (Fig. 6A). On the other hand, 5 $mu$ M CGP 20712A failed to block the ISO-induced stimulation (Fig. 6B). The average increase of peak I_Ba by ISO in the presence of CGP 20712A was not significantly different from control, while there was no increase of peak I_Ba by ISO in the presence of ICI 181,551 (Fig. 6C).

View larger version
[in this window]
[in a new window]

Figure 6 ISO-induced stimulation of I_Ba is abolished by $beta$ ₂-adrenoceptor antagonist

Cells were superfused with ICI 181,551 ( $beta$ ₂-adrenoceptor antagonist) or CGP 20712A ( $beta$ ₁-adrenoceptor antagonist) before application of ISO (1 $mu$ M). ISO was added into the bath solution when peak current reached a steady state. A, time course of peak current recordings from a cell superfused with ICI 181,551 (5 $mu$ M) before and after addition of ISO. B, time course of peak current recordings from a cell superfused with CGP 20712A (5 $mu$ M) before and after addition of ISO. C, percentage change of peak current after application of ISO in the absence or presence of ICI 181,551 or CGP 20712A. Numbers in parentheses indicate the number of cells tested. Values represent means ± S.E.M. and asterisks indicate a significant difference from values before application of ISO (P < 0.05).

The role of $beta$ -adrenoceptor subtypes was also examined by testing the effects of more specific $beta$ -adrenoceptor subtype agonists on Ca²⁺ channel currents. Surprisingly, zinterol, a potent $beta$ ₂-adrenoceptor agonist (Laflamme & Becker, 1998; Schroder & Herzig, 1999), at concentrations from 1 up to 10 $mu$ M did not stimulate Ba²⁺ current in most cells and even reduced peak I_Ba in some cells tested, and dobutamine, a potent $beta$ ₁-adrenoceptor agonist (Doggrell, 1990), at 10 $mu$ M concentration did not have any detectable effect on Ca²⁺ channels (data not shown). We then repeated the experiments in the presence of Rp-8-Br-PET-cGMPS (Rp-PET, 1 $mu$ M), a specific PKG inhibitor, to determine whether the effects of the $beta$ -adrenoceptor agonists might be masked by the well-established ability of high cAMP concentrations to cross-activate PKG (Lincoln et al. 1990; Ruiz-Velasco et al. 1998). Application of Rp-PET itself increased peak I_Ba by about 10 %. In the presence of Rp-PET, zinterol (1 $mu$ M) led to a large increase in peak I_Ba of approximately 80 %. On the other hand, neither the $beta$ ₁-adrenoceptor agonist dobutamine (1 $mu$ M) nor the $beta$ ₃-adrenoceptor agonist BRL 37344A (1 $mu$ M) (Oriowo et al. 1996) had a detectable effect on I_Ba in the presence of Rp-PET (Fig. 7). These experiments suggest that $beta$ ₂-adrenoceptors are the predominant receptor subtype underlying $beta$ -adrenoceptor-induced stimulation of L-type Ca²⁺ channels in rabbit portal vein myocytes.

View larger version
[in this window]
[in a new window]

Figure 7 The $beta$ ₂-adrenoceptor agonist zinterol is a potent stimulator of Ca²⁺ channels in rabbit portal vein myocytes

Cells were first superfused with normal bath solution and current recorded. When peak current reached a steady state, cells were then superfused with bath solution containing Rp-8-Br-PET-cGMPS (Rp-PET; PKG inhibitor, 1 $mu$ M), followed by addition of Rp-PET plus dobutamine (dob, 1 $mu$ M), BRL 37344A (BRL, 1 $mu$ M) or zinterol (zin, 1 $mu$ M). The inset shows superimposed representative recordings from a cell before (1) and after application of Rp-PET (2), and after application of zinterol in the presence of Rp-PET (3). The bar graph shows the averaged percentage increase of peak current after application of different drugs in the presence of Rp-PET. Numbers in parentheses indicate the number of cells tested. Values represent means ± S.E.M. and asterisks indicate a significant difference from the value with Rp-PET alone (control, P < 0.05).

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

The $beta$ -adrenergic receptor is coupled to the G protein Gs, which is composed of a G $alpha$ s and a G $beta$ $gamma$ subunit. G $alpha$ s has long been known to play a central role in responses evoked with $beta$ -adrenergic receptor stimulation because of its ability to activate adenylyl cyclase, leading to the stimulation of PKA and phosphorylation of various proteins. On the other hand, there is increasing evidence to suggest that G $beta$ $gamma$ also plays an important regulatory role in modulating a variety of downstream effectors (Hepler & Gilman, 1992; Clapham, 1994). Our results suggest that stimulation of vascular Ca²⁺ channels via activation of $beta$ -adrenergic receptors involves both the G $alpha$ s and the G $beta$ $gamma$ subunit and that different downstream mediators are involved, namely PKA and PKC, respectively.

In cardiac muscle, $beta$ -adrenergic receptor stimulation has long been known to lead to the activation of L-type Ca²⁺ channels and the primary mediator of this effect has generally been assumed to be the G $alpha$ s-adenylyl cyclase-PKA pathway (McDonald et al. 1994; Xiong & Sperelakis, 1995). In addition, PKA phosphorylation sites on both the $alpha$ and $beta$ subunits of the cardiac L-type Ca²⁺ channel have also been identified (Gao et al. 1997; Gerhardstein et al. 1999). The vascular L-type Ca²⁺ channel $alpha$ 1C subunit shares 93 % homology with the cardiac Ca²⁺ channel $alpha$ 1C subunit and contains the same phosphorylation site on the carboxy terminus (Stea et al. 1995). In addition, vascular L-type Ca²⁺ channels have been shown to be stimulated by exogenous G $alpha$ s, by 8-Br-cAMP and forskolin and by application of the catalytic subunit of PKA (Fukumitsu et al. 1990; Loirand et al. 1992; Ishikawa et al. 1993; Tewari & Simard, 1994; Shi & Cox, 1995; Farrugia, 1997; Liu et al. 1997; Ruiz-Velasco et al. 1998). The present study provides additional evidence suggesting an important role for the G $alpha$ s-PKA pathway in the regulation of L-type Ca²⁺ channels by $beta$ -adrenergic receptors. The ISO-induced stimulation of Ca²⁺ channels was reduced to less than half by dialysis of cells with anti-G $alpha$ s antibody or by superfusion of cells with the PKA inhibitor KT 5720. Furthermore, when the G $beta$ $gamma$ pathway was eliminated by dialysis of cells with anti-G $beta$ antibody, the remaining ISO-induced stimulation of Ca²⁺ channel current was entirely abolished by KT 5720.

Modulation of L-type Ca²⁺ channels by G protein $beta$ $gamma$ subunits has received much less attention although $beta$ $gamma$ subunits of G proteins have been shown to modulate many effectors in different pathways (Clapham & Neer, 1993, 1997; De et al. 1997; Dolphin, 1998). Using an anti-G protein $beta$ subunit antibody, Macrez et al. (1997) provided evidence that the $beta$ $gamma$ dimer of G₁₃ was responsible for the angiotensin II-induced stimulation of L-type Ca²⁺ channels in rat portal vein myocytes. In addition, direct dialysis of cells with purified G $beta$ $gamma$ subunits has been shown to increase Ca²⁺ channel currents in both rabbit and rat portal vein myocytes (Zhong et al. 1999; Viard et al. 1999). The present study provides evidence that the G $beta$ $gamma$ pathway also contributes to $beta$ -adrenergic receptor stimulation in rabbit portal vein. ISO-induced stimulation of Ca²⁺ channel current was reduced when cells were dialysed with anti-G $beta$ antibody and complete blockade only occurred when anti-G $alpha$ s antibody was combined with anti-G $beta$ antibody. Thus in rabbit portal vein myocytes the G $beta$ $gamma$ subunit, in addition to G $alpha$ s, appears to be involved in the actions of ISO.

The G $alpha$ s antibody used in the present study was a rabbit polyclonal IgG raised against an epitope mapping within the N-terminal domain of human G $alpha$ s. The N-terminus of G $alpha$ subunits interacts strongly with G $beta$ $gamma$ in the GDP-binding state and is located near the plasma membrane surface (Conklin & Bourne, 1993). Thus, anti-G $alpha$ s antibody may not have access to the binding region when G $alpha$ s is associated with G $beta$ $gamma$ . Blockade with anti-G $alpha$ s antibody is therefore predicted to occur following dissociation of G $alpha$ s from G $beta$ $gamma$ . Under these conditions a specific block of G $alpha$ s can be achieved as opposed to the general G protein block attained when antibody directed towards the C-terminus of the G $alpha$ subunit is used. The C-terminal region of the G $alpha$ subunit is the site at which receptor activation occurs. Without receptor activation the G $alpha$ subunit no longer dissociates from G $beta$ $gamma$ and both G $alpha$ and G $beta$ $gamma$ effects are removed (Macrez et al. 1997).

In a previous study we concluded that the action of exogenous G $beta$ $gamma$ on L-type Ca²⁺ channels was due to activation of PKC since the enhancement of current by G $beta$ $gamma$ was blocked by calphostin C, a specific PKC inhibitor, but not by PKA inhibitors (Zhong et al. 1999). No evidence for a direct membrane-delimited action of G $beta$ $gamma$ on the Ca²⁺ channel was obtained. This conclusion is in agreement with studies by others reporting that G $beta$ $gamma$ subunits have no direct effect on $alpha$ 1C L-type Ca²⁺ channel subunits (Dolphin, 1998). Furthermore, G $beta$ $gamma$ does not appear to have a direct effect on either type V or type VI adenylyl cyclases (cardiac and smooth muscle types) (Tang & Gilman, 1992; Ishikawa & Homcy, 1997). In the present study, complete blockade of ISO-induced stimulation of Ca²⁺ channels required a combination of PKA and PKC inhibitors, suggesting that PKC contributes to the actions of ISO on Ca²⁺ channels. In addition, the ISO-induced stimulation of Ca²⁺ channel currents obtained in the presence of anti-G $alpha$ s antibody was completely reversed by calphostin C, suggesting that PKC is activated by the G $beta$ $gamma$ subunit. There are a number of different possible pathways by which G $beta$ $gamma$ may lead to stimulation of PKC, such as G $beta$ $gamma$ -activated phospholipase C, D and A₂ (Cockcroft, 1992; Clapham & Neer, 1997). In cultured vascular smooth muscle cells it was suggested that angiotensin II stimulates phospholipase D (PLD) via G $beta$ $gamma$ since PLD activation was inhibited when cells were electroporated with anti-G $beta$ antibody as well as in cells over-expressing the G $beta$ $gamma$ -binding region of $beta$ ark-1 (Ushio-Fukai et al. 1999). In a recent study of L-type Ca²⁺ channels in rat portal vein cells (Viard et al. 1999) it was suggested that G $beta$ $gamma$ may activate PKC via phosphoinositide 3-kinase (PI3K) since infusion of cells with purified PI3K $gamma$ mimicked G $beta$ $gamma$ -induced stimulation of Ca²⁺ channels, and since both G $beta$ $gamma$ - and PI3K $gamma$ -induced stimulation of Ca²⁺ channel currents were reduced by PKC inhibitors. Although it is not clear what specific $beta$ $gamma$ combination of G protein is coupled with $alpha$ s in the present study, it appears that many different combinations of $beta$ and $gamma$ subunits (except $beta$ 1 $gamma$ 1) have similar actions (Ueda et al. 1994; Dolphin, 1998). In fact, recent studies from our laboratory (Zhong et al. 1999) and the Mironneau laboratory (Viard et al. 1999) demonstrated that both the recombinant G protein $beta$ 1 $gamma$ 2 from SF9 cells and a purified $beta$ $gamma$ subunit from bovine brain Gi/Go protein could stimulate L-type Ca²⁺ channels via PKC activation in rabbit and rat portal vein smooth muscle cells.

Although there is a diversity of functional $beta$ -adrenoceptor subtypes, these different subtypes (especially $beta$ ₁ and $beta$ ₂) may mobilize similar signal transduction pathways. Specifically, available evidence suggests that both $beta$ ₁- and $beta$ ₂-adrenoceptor stimulation of cardiac L-type Ca²⁺ channels involves activation of the adenylyl cyclase- cAMP-PKA pathway. On the other hand, $beta$ ₂-adrenoceptors may also exert an effect through other signalling pathways, such as coupling to Gi protein as well as some G protein-independent signalling pathway (Steinberg, 1999). In the present study, the stimulatory effect of ISO on L-type Ca²⁺ channels was antagonized by dialysing cells with antibodies to G protein $alpha$ s and $beta$ $gamma$ subunits, suggesting a requirement for the Gs protein in the signalling pathway. The results expand upon earlier studies in suggesting that PKC as well as PKA contribute to the actions of Gs on I_Ba.

In conclusion, our results suggest that $beta$ -adrenergic receptor stimulation of vascular L-type Ca²⁺ channels involves both $alpha$ s and $beta$ $gamma$ G protein subunits and that these subunits exert their effects through the PKA and PKC pathways, respectively. This conclusion is in agreement with our previous studies in which activated G protein subunits were applied exogenously to cells via dialysis (Zhong et al. 1999). These data, along with results obtained for angiotensin II receptor stimulation by others (Macrez et al. 1997), suggest that the G $beta$ $gamma$ subunit and PKC may make an important contribution to the regulation of vascular L-type calcium channels following stimulation of a diverse range of G protein-coupled receptors. It is possible that this novel signalling pathway involving G $beta$ $gamma$ and PKC may in fact be responsible for the PKA-independent regulation of vascular L-type Ca²⁺ channels by ISO described in earlier studies (Xiong & Sperelakis, 1995) and attributed to a membrane-delimited action of G protein $alpha$ s subunits.

Acknowledgements

This study was supported by NIH grants HL-40399 to (K.D.K.) and HL-49254 (to J.R.H.). J.Z. is a recipient of a NRSA postdoctoral fellowship from NIH (HL-10119).

Corresponding author

K. D. Keef: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.

Email: kathy{at}physio.unr.edu

This article has been cited by other articles:

G. C. Amberg, M. F. Navedo, M. Nieves-Cintron, J. D. Molkentin, and L. F. Santana
Calcium sparklets regulate local and global calcium in murine arterial smooth muscle
J. Physiol., February 15, 2007; 579(1): 187 - 201.
[Abstract] [Full Text] [PDF]

X. Gao, R. Sadana, C. W. Dessauer, and T. B. Patel
Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein beta{gamma} Subunits
J. Biol. Chem., January 5, 2007; 282(1): 294 - 302.
[Abstract] [Full Text] [PDF]

B. Callaghan, J. Zhong, and K. D. Keef
Signaling pathway underlying stimulation of L-type Ca2+ channels in rabbit portal vein myocytes by recombinant Gbeta{gamma} subunits
Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2541 - H2546.
[Abstract] [Full Text] [PDF]

Y. Ding, D. Schwartz, P. Posner, and J. Zhong
Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC
Am J Physiol Cell Physiol, August 1, 2004; 287(2): C413 - C421.
[Abstract] [Full Text] [PDF]

B. Callaghan, S.D. Koh, and K.D. Keef
Muscarinic M2 Receptor Stimulation of Cav1.2b Requires Phosphatidylinositol 3-Kinase, Protein Kinase C, and c-Src
Circ. Res., March 19, 2004; 94(5): 626 - 633.
[Abstract] [Full Text] [PDF]

H. M. ROSENFELDT, Y. AMRANI, K. R. WATTERSON, K. S. MURTHY, R. A. PANETTIERI JR, and S. SPIEGEL
Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells
FASEB J, October 1, 2003; 17(13): 1789 - 1799.
[Abstract] [Full Text] [PDF]

This Article

Abstract

Full Text (PDF)

Services

Email this article to a friend

Similar articles in this journal

Similar articles in PubMed

Alert me to new issues of the journal

Download to citation manager

Citing Articles

Citing Articles via HighWire

Citing Articles via Google Scholar

Google Scholar

Articles by Zhong, J.

Articles by Keef, K. D.

Search for Related Content

PubMed

PubMed Citation

Articles by Zhong, J.

Articles by Keef, K. D.