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(Hypertension. 1996;27:774-780.)
© 1996 American Heart Association, Inc.

Articles

cAMP Signaling Inhibits Dihydropyridine-Sensitive Ca²⁺ Influx in Vascular Smooth Muscle Cells

Sergei N. Orlov; Johanne Tremblay; Pavel Hamet

From the Centre de Recherche Hôtel-Dieu de Montréal, Université de Montréal (Québec), Canada.

Correspondence to Pavel Hamet, MD, PhD, Laboratory of Molecular Pathophysiology, Centre de Recherche Hôtel-Dieu de Montréal, 3850 St. Urbain St, Montréal, Québec H2W 1T8, Canada.

	Abstract

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Abstract This study examines the role of the cAMP signaling pathway in the regulation of ⁴⁵Ca influx in cultured vascular smooth muscle cells from the rat aorta. K⁺_o-induced depolarization of smooth muscle cells increased the rate of ⁴⁵Ca uptake by twofold to threefold. This effect was completely abolished by the dihydropyridine derivatives nifedipine and nicardipine, with a K_i of 3 and 10 nmol/L, respectively. Activators of cAMP signaling (isoproterenol, forskolin, cholera toxin) increased cAMP content by 50- to 100-fold and decreased voltage-dependent ⁴⁵Ca uptake by 50% to 70%. Neither the dihydropyridines nor the cAMP activators affected basal ⁴⁵Ca influx. Direct addition of the protein kinase inhibitor H-89 to the incubation medium in the 1- to 10-µmol/L range did not alter basal ⁴⁵Ca uptake but completely abolished voltage-dependent Ca²⁺ transport. Preincubation of cells for 1 hour with 10 µmol/L H-89 did not modify basal and depolarization-induced ⁴⁵Ca uptake in H-89–free medium but prevented forskolin-induced inhibition of voltage-dependent Ca²⁺ influx. The addition of cytoskeleton-active compounds reduced voltage-dependent Ca²⁺ transport and completely abolished its regulation by cAMP. Activation of cAMP signaling decreased the volume of smooth muscle cells by 12% to 15%. The same cell volume diminution in hyperosmotic medium did not alter voltage-dependent ⁴⁵Ca uptake. Thus, data obtained in this study show that in contrast to cardiac and skeletal myocytes, in vascular smooth muscle cells, ⁴⁵Ca influx, putatively due to L-type channels, is inhibited by cAMP. This regulatory pathway appears to be mediated via protein kinase A activation and cytoskeleton reorganization.

Key Words: muscle, smooth, vascular • calcium channels • cyclic AMP • cytoskeleton

	Introduction

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Several years ago, we presented evidence that isoproterenol infusion induced a stronger increment of plasma cAMP levels in young subjects with labile hypertension than in age-matched control subjects. We compared this exaggerated responsiveness to ß-adrenergic stimulation with the diminished reaction observed in older hypertensive patients with low plasma renin activity.^{1 2} Viewing these results together with data on abnormalities of the cAMP signaling system obtained in cells and isolated membrane fractions from patients with essential hypertension and from spontaneously hypertensive rats, we proposed that augmented responsiveness to ß-adrenergic stimulation plays a key role in enhanced cardiac output revealed in labile hypertension, whereas decreased responsiveness of this system in chronic hypertension is involved in the progressive increase of total peripheral resistance via cAMP-mediated modulation of VSMC relaxation and/or growth and proliferation (for more details, see Reference 3).

Despite numerous data on cAMP-induced vasorelaxation, the mechanism of this phenomenon is poorly understood. The initial hypothesis on cAMP-induced decrease of Ca²⁺ sensitivity of myosin light-chain kinase was based on reversible phosphorylation of this enzyme from turkey gizzard smooth muscle by protein kinase A in vitro.⁴ This observation was subsequently confirmed in tracheal smooth muscle cells.⁵ However, to the best of our knowledge, there are no data on cAMP-induced phosphorylation of myosin light-chain kinase in VSMCs or on the correlation of smooth muscle relaxation and cAMP-induced phosphorylation of this enzyme in vivo. On the contrary, it has been shown that forskolin-induced relaxation of carotid arteries is not associated with altered Ca²⁺ sensitivity of myosin light-chain kinase.⁶

It may be assumed that apart from the modulation of activity of Ca²⁺-sensitive enzymes involved in excitation-contraction coupling, cAMP affects intracellular Ca²⁺ concentration. Indeed, in cardiac muscle, ß-adrenergic activation of cAMP signaling is accompanied by phosphorylation of the 22-kD protein phospholamban, which leads to severalfold activation of the sarcoplasmic reticulum Ca²⁺ pump.⁷ In VSMCs, phospholamban phosphorylation occurs at the same site as in cardiac cells but is mediated predominantly by cGMP-dependent protein kinase (for more details, see Reference 8). Several types of protein kinases, in particular protein kinase A, inhibit pharmacomechanical coupling triggered by the activation of ion channel and phospholipase C–coupled receptors.⁹ However, this mechanism cannot explain the inhibition of electromechanical coupling in VSMCs by cAMP.

Several years ago, Ousterhout and Sperelakis¹⁰ reported that both isoproterenol and forskolin depressed Ca²⁺-dependent action potential in depolarized cultured VSMCs. These results suggest that cAMP inhibits electromechanical coupling by membrane hyperpolarization and/or inactivation of VDCCs. Pharmacological studies indicate that cAMP-induced membrane hyperpolarization of VSMCs is mediated by activation of K⁺ channels.¹¹ Indeed, in vascular and tracheal smooth muscles, direct evidence has been obtained by the patch-clamp technique of membrane-delimited G_s protein–mediated and cytoplasmic protein kinase A–mediated pathways of ß-adrenergic receptor coupling with Ca²⁺-activated K⁺ channels.^{12 13 14}

In contrast to K⁺ channels, data on the modulation of VDCCs in VSMCs by cAMP are contradictory. Thus, dialysis with cAMP solutions had no effect on Ca²⁺ current in VSMCs from the rabbit saphenous artery¹⁵ and rat portal vein.¹⁶ Benham and Tsien¹⁷ reported a threefold increase of DHP-sensitive long-lasting (L-type) Ca²⁺ current in smooth muscle cells from the rabbit ear artery treated with norepinephrine. However, this effect was not mediated via known subtypes of ß- or -adrenergic receptors.¹⁷ Ishikawa and coworkers¹⁸ reported that low concentrations of intracellular cAMP produce modest increases in whole-cell L-type Ca²⁺ channel current, whereas higher cAMP concentrations result in strong inhibition of L-type channel activity in VSMCs from the rabbit portal vein. Membrane-permeable cAMP analogues slightly inhibit slow, inward, long-lasting (L-type) Ca²⁺ channels in VSMCs isolated from the rat tail artery¹⁹ and in cultured cells from the rabbit aorta.²⁰ Sustained inhibition of L-type Ca²⁺ was shown in freshly isolated smooth muscle cells from the rabbit portal vein treated with forskolin and 8-bromo-cAMP.²¹ In the A7r5 cell line derived from rat embryonic smooth muscle²² and in VSMCs from the porcine coronary artery,²³ forskolin increases L-type VDCC-mediated Ca²⁺ current. In contrast, Lorenz and coworkers²⁴ demonstrated that both cAMP analogues and forskolin inhibit L-type VDCCs in A7r5 cells.

These contradictory results on the regulation of VDCCs by cAMP signaling are probably caused by difficulties in obtaining satisfactory whole-cell voltage-clamp recordings from small myocytes and by rapid rundown of VDCC activity. Furthermore, enzymatic digestion in Ca²⁺-free medium during VSMC preparation can leave dispersed or primary cultured cells with drastically altered calcium homeostasis and membrane-bound protein function.²⁴ To overcome these problems, we studied the regulation of Ca²⁺ inward fluxes in long-term cultured VSMCs by radioisotope methods. We report here that DHP-sensitive VD ⁴⁵Ca influx in VSMCs from the rat aorta is inhibited by activators of cAMP signaling. Our results suggest that this regulatory pathway is mediated by activation of protein kinase A and cytoskeleton rearrangement.

	Methods

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VSMCs were obtained by the explant method from the aortas of 10- to 13-week-old Brown Norway (BN.lx) male rats (Department of Biology, Charles University, Prague, Czech Republic) as described previously in detail.²⁵ The cells reached confluency at 7 to 10 days and exhibited a hill-and-valley pattern typical of smooth muscle cells in culture. They were then passaged by treatment with 0.05% trypsin (Gibco) in Ca²⁺- and Mg²⁺-free Dulbecco's phosphate-buffered saline and incubated in 80-cm² tissue culture flasks at a density of 10⁵ cells/mL. This study was performed on VSMCs between passages 14 and 18. Under these conditions, the VSMCs reacted positively to specific smooth muscle myosin antibodies, as examined by fluorescence microscopy.²⁶ Before experimentation, the cells were plated and allowed to grow for 20 to 24 hours in DMEM containing 10% calf serum. To establish quiescence, the growing medium was aspirated and the cells were supplied with DMEM containing 0.2% calf serum. After 48 to 72 hours, this medium was aspirated and replaced by a physiologically balanced salt solution immediately before the experiments began (see below). Cellular protein content was determined by a modification of the Lowry method.²⁷ Intracellular cAMP content in VSMCs was assessed by radioimmunoassay.²⁸

⁴⁵Ca influx was measured essentially as described earlier,²⁹ with changes in composition of the incubation media. Confluent, quiescent cultures of VSMCs (in 24-well plates) were washed twice at room temperature with 2-mL aliquots of medium A containing 150 mmol/L NaCl and 10 mmol/L HEPES-Tris (pH 7.4). One milliliter of medium B (in mmol/L: NaCl 140, KCl 5, MgCl₂ 1, CaCl₂ 1, glucose 5, HEPES-Tris 20, pH 7.4) was added to each well. After 30 minutes of preincubation at 37°C, the medium was replaced by 0.25 mL of medium B containing 0.2 mmol/L CaCl₂, and the cells were incubated for a further 10 to 20 minutes at 37°C. Where appropriate, the preincubation medium contained different compounds, as indicated in the figure and table legends. Thereafter, each well was supplemented with 0.25 mL of prewarmed (37°C) low-potassium or high-potassium medium containing 5 mmol/L KCl and 140 mmol/L NaCl, respectively (final concentration of K⁺ and Na⁺, 5 and 140 mmol/L, respectively) or 125 mmol/L KCl and 20 mmol/L NaCl (final concentration of K⁺ and Na⁺, 65 and 80 mmol/L, respectively) with 2 to 4 µCi/mL ⁴⁵CaCl₂. The osmolality of these media was decreased by reduction of the NaCl concentration or increased by the addition of sucrose. Previously, it was shown that under these conditions, the kinetics of ⁴⁵Ca uptake by VSMCs is linear up to 10 minutes.²⁹ To estimate the rate of unidirectional inward Ca²⁺ fluxes, the time of incubation with the isotope was limited to 5 minutes. Incubation was terminated by the addition of 2.5 mL ice-cold solution (medium C) containing 100 mmol/L MgCl₂ and 10 mmol/L HEPES-Tris (pH 7.4). The dishes were transferred onto ice, and the cells were washed 5 times with 2.5 mL of ice-cold medium C. The cells were lysed with 1 mL of 4 mmol/L EDTA/1% sodium dodecyl sulfate, and radioactivity was quantified by liquid scintillation counting. ⁴⁵Ca influx was calculated as A/an, where A is radioactivity in the cell lysate (cpm), a is specific radioactivity of the incubation medium (cpm/pmol), and n is the protein content per well (in micrograms). VD (depolarization-induced) Ca²⁺ influx was determined as the difference between the rate of ⁴⁵Ca uptake in high- and low-potassium media. The volume of intracellular water in VSMCs was determined as [¹⁴C]urea available space.³⁰

Chemicals
⁴⁵CaCl₂ was obtained from Amersham Inc; isoproterenol bitartrate, cytochalasin B, vinblastine, nifedipine, and nicardipine were purchased from Sigma; forskolin, cholera toxin, pertussis toxin, and H-89 (N-[2-([3-(40 bromophenyl)-2-propenyl]-lamino)ethyl]-5-isoquinolinesulfonamide) were procured from Calbiochem Novabiochem Corp; D-glucose, salts, and buffers were from Sigma, Gibco, and Anachemia, respectively.

	Results

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Effect of Extracellular Potassium
The increase of extracellular K⁺ concentration in incubation medium from 5 to 10 or 15 mmol/L slightly decreased ⁴⁵Ca influx in VSMCs (Fig 1). This effect was probably caused by VSMC hyperpolarization due to [K⁺]_o-dependent activation of the electrogenic Na⁺,K⁺ pump.⁸ A further increase of [K⁺]_o up to 40 mmol/L was accompanied by a sharp twofold to threefold elevation of ⁴⁵Ca uptake by VSMCs.

View larger version (15K): [in this window] [in a new window]   Figure 1. Dependence of 45Ca uptake by VSMCs on extracellular potassium concentration. Medium B was aspirated 30 minutes after preincubation of cells, and 45Ca uptake was initiated by the addition of medium B containing 0.1 to 60 mmol/L KCl, 145 to 85 mmol/L NaCl (total concentration of monovalent cations, 145 mmol/L), 0.1 mmol/L CaCl2, and 2 µCi/mL 45CaCl2. Means±SEM were obtained from three experiments performed in quadruplicate.

Dose Dependence of ⁴⁵Ca Uptake on Nicardipine and Nifedipine in Low- and High-Potassium Media
Increased nicardipine and nifedipine concentrations from 10^-9 to 10^-6 mol/L did not significantly modify ⁴⁵Ca uptake by VSMCs under basal (low-potassium medium) conditions (data not shown). Both DHP derivatives completely blocked depolarization-induced ⁴⁵Ca uptake in a concentration-dependent manner with a K_i of 3 and 10 nmol/L for nifedipine and nicardipine, respectively (Fig 2).

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of nicardipine and nifedipine on depolarization-induced 45Ca uptake by VSMCs. After 30 minutes of preincubation of cells, medium B was changed to 0.25 mL medium B containing 0.2 mmol/L CaCl2 without or with 2x10-9 to 2x10-6 mol/L nicardipine or nifedipine. Calcium uptake was initiated after 10 minutes by the addition of 0.25 mL low-potassium or high-potassium medium containing 4 µCi/mL 45CaCl2. Depolarization-induced 45Ca uptake values in the absence of the DHPs were taken as 100%. Means±SEM were obtained in three experiments performed in duplicate.

Effect of cAMP Signaling System Modulators
Activators of ß-adrenergic receptors, adenylate cyclase, and G_s proteins (isoproterenol, forskolin, and cholera toxin, respectively) increased cAMP content in VSMCs by 50- to 100-fold (Table 1A). These compounds did not affect basal ⁴⁵Ca influx and decreased depolarization-induced ⁴⁵Ca uptake by 50% to 70%. Neither basal nor depolarization-induced ⁴⁵Ca was sensitive to pertussis toxin, an inhibitor of G_i proteins. Nicardipine did not significantly modify the effect of isoproterenol on cAMP content but completely abolished cAMP-induced inhibition of ⁴⁵Ca uptake in high-potassium medium (Table 1A).

Effect of Protein Kinase A Inhibitor
H-89 is known to be a powerful and selective inhibitor of cAMP-dependent protein kinase. Thus, in cell-free systems and at micromolar ATP concentrations, half-maximal inhibition of protein kinase A, protein kinase G, and protein kinase C activity by H-89 was observed at 0.05, 0.5, and 30 µmol/L, respectively.³¹ Due to the high intracellular ATP concentration and limited permeability across plasma membrane, this compound inhibits protein kinase A activity in intact cells with an ED₅₀ of 5 to 10 µmol/L.³¹ As seen in Fig 3, simultaneous addition of 10 µmol/L H-89 with ⁴⁵Ca did not modify basal ⁴⁵Ca uptake but completely abolished the increment of ⁴⁵Ca uptake in high-potassium medium. These results demonstrate that at the same concentration range as used for inhibition of protein kinase A activity in vivo, H-89 completely suppressed VD Ca²⁺ transport in VSMCs, probably because of the direct interaction with L-type Ca²⁺ channels. To overcome this problem, we treated VSMCs with 10 µmol/L H-89, washed the cells with BSA-containing medium to remove extracellular H-89, and then measured ⁴⁵Ca uptake in H-89–free medium. As seen in Fig 4b, H-89 did not modify depolarization-induced ⁴⁵Ca uptake under this protocol but abolished its regulation by forskolin. It is important to mention that neither basal nor forskolin-induced cAMP production was altered in H-89–treated VSMCs (Fig 4a).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of H-89 on 45Ca uptake by VSMCs in low- and high-potassium media. Cells were incubated for 30 minutes in medium B and then for 10 minutes in medium B containing 0.2 mmol/L CaCl2. 45Ca uptake was initiated by the addition of 0.25 mL of low-potassium or high-potassium medium containing 4 µCi/mL 45CaCl2. H-89, 2x10-7 to 2x10-5 mol/L, was added simultaneously with 45Ca. The data from two experiments performed in quadruplicate are presented as percentages compared with 45Ca uptake in low-potassium medium without H-89.

View larger version (17K): [in this window] [in a new window]   Figure 4. Effect of forskolin and H-89 on cAMP content (a) and depolarization-induced 45Ca uptake (b) by VSMCs. Cells were preincubated for 1 hour in 1 mL medium B (control VSMCs) or in medium B containing 10 µmol/L H-89 (H-89–treated VSMCs). This medium was aspirated, and cells were washed with 3x2-mL aliquots of medium A and incubated for 5 minutes with 1 mL of medium B containing 0.1% BSA. The latter medium was aspirated, and cells were incubated in medium B without or with 10 µmol/L forskolin. In 15 minutes, VSMCs were used for measurement of cAMP content or 45Ca uptake. 45Ca uptake was initiated by the addition of 0.25 mL low-potassium or high-potassium medium containing 4 µCi/mL 45CaCl2. Means±SEM were obtained from two (cAMP content) and four (45Ca uptake) experiments performed in triplicate (cAMP content) or quadruplicate (45Ca uptake). *P<.01.

Effect of Cytoskeleton Assembly Modulators
It is known that activation of cAMP signaling in VSMCs is accompanied by cell shape transition^{32 33} and cytoskeleton reorganization.³⁴ We therefore compared the effects of modulators of cytoskeleton assembly and cell volume on ⁴⁵Ca uptake by VSMCs. As seen in Table 1B, cytochalasin B and vinblastine decreased depolarization-induced ⁴⁵Ca uptake in BN.lx VSMCs by 70% and 45%, respectively. It should be underscored, however, that the effects of these cytoskeleton modulators on Ca²⁺ transport in VSMCs were probably mediated by distinct mechanisms. Indeed, cytochalasin B, an inhibitor of actin polymerization in microfilament bundles, did not modify basal ⁴⁵Ca uptake and decreased it by 35% in high-potassium medium. In contrast to cytochalasin B, vinblastine, a microtubule-disrupting compound, increased basal ⁴⁵Ca uptake by 60% but did not affect it in high-potassium medium. Neither cytochalasin B nor vinblastine modified forskolin-induced cAMP production. However, in VSMCs treated with these compounds, forskolin did not lead to further decreases of depolarization-induced Ca²⁺ influx (Table 1B).

Effect of Cell Volume
Table 2 shows that treatment of VSMCs with forskolin reduced VSMC volume by 12% to 15%, which is in accordance with data obtained on VSMCs treated with isoproterenol.³³ The same cell volume decrease (from 3.1 to 2.4 µL/mg protein) induced by hyperosmotic shrinkage did not alter depolarization-induced ⁴⁵Ca uptake by VSMCs (Fig 5, curve 3). Under the same conditions of cell shrinkage, we did not observe any changes of forskolin-induced cAMP production and its regulation of VD ⁴⁵Ca uptake (Table 2). A further decrease of VSMC volume from 2.4 to 1.6 µL/mg protein completely blocked depolarization-induced ⁴⁵Ca uptake (Fig 5).

View this table: [in this window] [in a new window]   Table 2. Cell Volume, cAMP Content and Depolarization-Induced 45Ca Uptake in VSMCs

View larger version (21K): [in this window] [in a new window]   Figure 5. Dependence of 45Ca uptake by VSMCs in low-potassium (curve 1) and high-potassium (curve 2) media on intracellular water volume. Curve 3 represents a depolarization-induced component of 45Ca uptake. After 30 minutes in medium B, the cells were aspirated, and 45Ca uptake was initiated by the addition of 0.5 mL low- or high-potassium medium B containing 0.1 mmol/L CaCl2 and 4 µCi/mL 45CaCl2. The osmolality of these media was increased or decreased by the addition of sucrose or by reduction of NaCl concentration, respectively. Means±SEM were obtained from two experiments performed in quadruplicate.

	Discussion

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The data obtained in the present study show that an increase of cAMP content in long-term cultured VSMCs from the rat aorta via activation of ß-adrenergic receptors (isoproterenol), G_s proteins (cholera toxin), or adenylate cyclase (forskolin) inhibits depolarization-induced ⁴⁵Ca influx (Table 1). This parameter was completely blocked by DHPs with a K_i of 3 to 10 nmol/L (Fig 2), which is in accordance with the sensitivity of VSMC L-type Ca²⁺ channels to these compounds under depolarization conditions obtained with the patch-clamp technique.³⁵ In the presence of DHPs, we did not observe any effect of activation of cAMP signaling on ⁴⁵Ca uptake by VSMCs (Table 1). On the basis of these results, it may be concluded that the cAMP-induced decrease of VD ⁴⁵Ca uptake in VSMCs is caused by inhibition of L-type Ca²⁺ channels. This conclusion is concordant with data on the effect of cAMP analogues^{19 20} and forskolin²⁴ obtained by patch clamp in primary cultured VSMCs from the rabbit aorta²⁰ and rat tail artery¹⁹ and in the A7r5 cell line derived from vascular smooth muscles²⁴ but contradicts results obtained in other laboratories by the same methods for dispersed or primary cultured VSMCs from the rabbit saphenous artery,¹⁵ rat portal vein,¹⁶ rabbit ear artery,¹⁷ porcine coronary artery,²³ and A7r5 cells.²² This discrepancy may be due to (1) different properties of signaling pathway in smooth muscle from different vessels and species, (2) modification of the properties of L-type Ca²⁺ channels during preparation of dispersed and primary cultured VSMCs, and (3) difficulties in recording L-type VDCCs by the electrophysiological approach because of rapid rundown of their activity under cell dialysis.

View this table: [in this window] [in a new window]   Table 1. Effect of Modulators of cAMP Signaling (A) and Cytoskeleton Assembly (B) on cAMP Content and 45Ca Uptake by VSMCs

Recently, it was reported that cAMP analogues inhibit L-type Ca²⁺ current in rat osteoblastic cells,³⁶ indicating that this mechanism of VDCC regulation is not limited to VSMCs. On the other hand, it is well documented that in most electrically excitable tissues (cardiomyocytes, skeletal fibers, neurons), cAMP enhances L-type VDCCs (see References 37 through 39 for review). ß-Adrenergic regulation of ion channels can be mediated by cAMP-independent and cAMP-dependent pathways. The cAMP-independent membrane-delimited pathway is triggered by direct interaction of ion channels with the activated -subunit of G_s proteins,⁴⁰ whereas the cAMP-dependent cytoplasmic pathway probably involves the activation of protein kinase A.⁴¹ In our study, we did not find significant differences between isoproterenol, forskolin, and cholera toxin in the inhibition of VD Ca²⁺ influx in VSMCs (Table 1). In the presence of forskolin, isoproterenol did not lead to a further reduction of VD ⁴⁵Ca uptake (data not shown). Inhibition of VD Ca²⁺ influx by forskolin (Fig 4) and isoproterenol (data not shown) was completely abolished by pretreatment of VSMCs with H-89, an inhibitor of protein kinase A. These results are in accordance with data on partial inhibition of L-type channels in VSMCs from rabbit portal vein by a catalytic subunit of protein kinase A²¹ and indicate that ß-adrenergic–induced inhibition of L-type VDCCs in VSMCs is mediated by the cytoplasmic protein kinase A pathway.

Protein kinase A–induced inhibition of VD Ca²⁺ influx in VSMCs may be caused by phosphorylation of (1) the L-type channel itself and, in particular, its ₁-subunit possessing several potential phosphorylation sites or (2) a contiguous regulatory type of protein(s). The first hypothesis seems unlikely. Indeed, despite opposite effects of cAMP on L-type VDCCs in VSMCs and cardiomyocytes, cDNA sequencing has predicted high homology of their ₁-subunits.⁴² Moreover, it has been shown that cAMP does not affect Ca²⁺ channel currents in Xenopus oocytes resulting from expression of the ₁-subunit of VSMC VDCCs alone.⁴³ Apart from the ₁-subunit, a potential phosphorylation site for protein kinase A has also been demonstrated in the ß-subunit of skeletal muscle L-type VDCCs (see Reference 38 for review). Coexpression of the VSMC ₁-subunit with the skeletal muscle ß-subunit led to twofold activation of VD Ca²⁺ current in Xenopus oocytes. It should be underscored, however, that the same results were obtained with coexpressed ₁-subunit from cardiac myocytes.⁴³

Searching for candidate proteins that could be involved in the inhibition of VD Ca²⁺ influx in VSMCs, we focused on cAMP-induced VSMC shape transition and cytoskeleton reorganization first reported by Smith³² and later confirmed in other laboratories.^{33 34} Data obtained in the present study show that reorganization of the cytoskeleton network with cytochalasin B and vinblastine inhibits VD ⁴⁵Ca uptake in VSMCs and abolishes its regulation by cAMP signaling (Table 1B). These results demonstrate that intact cytoskeleton is necessary for proper Ca²⁺ channel functions and suggest that cAMP-induced cytoskeleton reorganization is involved in the inhibition of L-type channel activity. Here, it is important to underscore that cytoskeleton-mediated regulation of plasma membrane ion currents is not limited by L-type Ca²⁺ channels. Thus, it was recently shown that cytochalasin D reduces whole-cell peak Na⁺ current in rat and rabbit ventricular cardiac myocytes⁴⁴ and prevents activation of cystic fibrosis transmembrane conductance regulator-mediated Cl^- conductance in mouse mammary adrenocarcinoma by cAMP analogues.⁴⁵

Apart from shape transition, cAMP induces a decrease of VSMC volume by 12% to 15% (Table 2). Keeping in mind numerous data on the volume-dependent regulation of ion transport,⁴⁶ we assumed that cAMP-induced inhibition of VD Ca²⁺ influx could be mediated by VSMC shrinkage. However, results obtained with hyperosmotically shrunken VSMCs contradict this assumption. Indeed, as seen in Fig 5, moderate hyperosmotic shrinkage did not modify VD ⁴⁵Ca uptake by VSMCs.

In conclusion, the present study demonstrates that in contrast to skeletal and cardiac myocytes, cAMP inhibits VD DHP-sensitive Ca²⁺ influx in cultured smooth muscle cells from the rat aorta. It may be assumed that this signaling pathway plays a key role in vasorelaxation triggered by adenylate cyclase–coupled receptors. The inhibitory effect of cAMP on L-type Ca²⁺ channels deserves further investigation to determine the mechanism of its interrelation with protein phosphorylation and cytoskeleton network rearrangement.

	Selected Abbreviations and Acronyms

 

DHP = dihydropyridine DMEM = Dulbecco's modified Eagle's medium VD = voltage-dependent VDCC = voltage-dependent Ca2+ channels VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by grants from the Medical Research Council of Canada (MT-10803 and MT-11463), the Heart and Stroke Foundation of Canada, and Bayer Canada. Dr Tremblay is the recipient of a senior scholarship from Fonds de la Recherche en Santé du Québec, and Dr Orlov received a visiting professorship from the International Society of Hypertension, Pfizer Pharmaceutical Co. The technical assistance of Monique Poirier, the secretarial skills of Josée Bédard-Baker, and the editorial help of Ovid Da Silva are appreciated.

	References

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