Introduction

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Functional regulation of the 10 cloned isoforms of adenylyl cyclase is diverse, with no two isoforms displaying identical regulation (Cooper et al., 1995; Sunahara et al., 1996). Nevertheless, three broad categories can be distinguished comprising (1) types I and VIII, predominantly expressed in neurons and stimulated by submicromolar concentrations of Ca²⁺ and calmodulin and a more widely expressed type III, stimulated by low micromolar concentrations of Ca²⁺ (Choi et al., 1992a, 1992b; Xia et al., 1992; Cali et al., 1994), (2) types II, IV, and IX, which are not affected by Ca²⁺ or inhibited (in the case of types II and IV) by the GTP-binding proteins G_i and G_o (Tang and Gilman, 1992; Taussig et al., 1993, 1994; Premont et al., 1996), and (3) types V and VI, which are inhibited by G_i and G_o and by submicromolar concentrations of Ca²⁺ elicited by Ca²⁺ influx but not by Ca²⁺ release from intracellular stores (Boyajian et al., 1991; Yoshimura and Cooper, 1992; Chiono et al., 1995; Taussig and Gilman, 1995).

We have shown recently that adenylyl cyclase types V and VI, but not types II, III, or IV, are expressed in gastrointestinal smooth muscle (Murthy and Makhlouf, 1997). The cyclases are inhibited, depending on the agonist, by G_i1, G_i2, G_i3, and G_o. Inhibition via somatostatin sst3 receptors is mediated by G_i1 and G_o (Murthy et al., 1996), whereas inhibition via opioid µ, , or  receptors is mediated by G_i2 and G_o (Murthy and Makhlouf, 1996). Inhibition via adenosine A₁ receptors, muscarinic m₂ receptors, and P_2Y2 receptors is mediated by G_i3 (Murthy and Makhlouf, 1995a, 1997, 1998). Stimulation of adenylyl cyclase via muscarinic m₃ receptors is mediated by the subunit of G_q/11; the stimulation is masked by the predominant inhibition mediated via m₂ receptors by G_i3 (Murthy and Makhlouf, 1997).

The coexistence of receptor subtypes coupled to distinct signaling pathways is likely to elicit various patterns of regulation of adenylyl cyclase V/VI in smooth muscle. For example, stimulation of adenylyl cyclase activity by adenosine A₂ receptors coupled to G_s is attenuated by A₁ receptors coupled to inhibition of adenylyl cyclase via the  subunit of G_i3 and to activation of PLC-3 and IP₃-dependent Ca²⁺ release via the  and subunits of G_i3 (Murthy and Makhlouf, 1995a): inhibitory regulation could be mediated by Ca²⁺ influx, inhibitory G protein, or both. Whether these inhibitory mechanisms operate in concert or are mutually exclusive has not been determined.

In the current study, we examined the characteristics of inhibitory regulation of adenylyl cyclase V/VI by Ca²⁺ and G proteins in smooth muscle. Experiments were designed in which the mechanisms were evoked separately, sequentially, or concurrently using a variety of agonists and probes. G protein-independent Ca²⁺ influx was elicited by the P_2X receptor agonist ,-methylene-ATP (Fredholm et al., 1997; Surprenant et al., 1995), and G protein-dependent and -independent capacitative Ca²⁺ influx was elicited by agonists (cholecystokinin-octapeptide) and thapsigargin, respectively. Both mechanisms could be elicited concurrently or sequentially with the P_2Y2 agonist UTP, which is coupled to both G_q/11 and G_i3 (Harden et al., 1995; Nicholas et al., 1996; Murthy and Makhlouf, 1998), or the mixed P_2X and P_2Y2 agonist ATP. PTX was used to uncouple P_2Y2 receptors from G_i3. The results indicate that inhibition can be independently mediated by Ca²⁺ influx and inhibitory G proteins, but when both mechanisms are triggered, inhibition is exclusively mediated by inhibitory G proteins.

	Experimental Procedures

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Dispersion of smooth muscle cells. Muscle cells were isolated from the circular muscle layer of the rabbit stomach by successive enzymatic digestion, filtration, and centrifugation as described previously (Murthy and Makhlouf, 1997). Briefly, slices of gastric muscle were obtained from the body of circular muscle; the initial slices at the boundaries presumed to contain the majority of interstitial cells of Cajal were discarded. The muscle slices were incubated for 30 min at 31° in 15 ml of HEPES medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor. The composition of the medium was 120 mM NaCl, 4 mM KCl, 2.6 mM KH₂PO₄, 0.6 mM MgCl₂, 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's essential amino acid mixture; no Ca²⁺ was added to the medium. The partly digested tissue was washed with 100 ml of enzyme-free medium and reincubated for 30 min, during which the cells were allowed to disperse spontaneously without tissue trituration. Suspensions of single muscle cells (~20 × 10⁶ cells) were harvested by filtration through 500-µm Nitex mesh. The suspensions were centrifuged twice for 10 min at 350 × g to eliminate cell debris and organelles, in particular, neural membranes as shown previously using [³H]saxitoxin binding (Murthy and Makhlouf, 1994). Muscle cells prepared in this fashion exclude trypan blue (95-98%) and were studied no later than 1-2 hr after dispersion. Muscle cell length ranged in length from 70 to 150 µm.

Measurement of Ca²⁺ release and uptake in dispersed smooth muscle cells. Ca²⁺ release and uptake were measured in dispersed muscle cells as described previously (Poggioli and Putney, 1982; Bitar et al., 1986). The muscle cells (10⁷ cells in 10 ml) were incubated in a medium containing ⁴⁵Ca²⁺ (10 µCi/ml) and antimycin (10 µM), and Ca²⁺ uptake into nonmitochondrial Ca²⁺ stores was measured at intervals for 60 min when a steady state was attained (steady state ⁴⁵Ca²⁺ cell content, 2.46 ± 0.12 nmol/10⁶ cells). UTP (10 µM) was added, and ⁴⁵Ca²⁺ cell content was measured at intervals for 10 min and expressed in nanomoles or percent change from steady state ⁴⁵Ca²⁺ cell content. Decrease in ⁴⁵Ca²⁺ cell content during the initial 15-30 sec reflected net Ca²⁺ release.

Measurement of cAMP in dispersed smooth muscle cells by radioimmunoassay. cAMP was measured in dispersed cells by radioimmunoassay as described previously (Murthy and Makhlouf, 1996 and 1997). Aliquots (0.5 ml) containing 10⁶ cells/ml were incubated with 10 µM forskolin and the test agent in the presence of 10 µM isobutyl methylxanthine, and the reaction was terminated after 60 sec with 6% cold trichloroacetic acid (v/v). The mixture was centrifuged at 2000 × g for 15 min at 4°. The supernatant was extracted three times with 2 ml of diethyl ether and lyophilized. The samples were reconstituted for radioimmunoassay in 500 µl of 50 mM sodium acetate, pH 6.2, and acetylated with triethylamine/acetic anhydride (3:1, v/v) for 30 min. cAMP was measured in duplicate using 100-µl aliquots and expressed as pmol/10⁶ cells.

Experimental design. Several experimental approaches were devised to distinguish between the effects of Ca²⁺ and inhibitory G proteins. (1) In one set of experiments, forskolin (10 µM) was added to dispersed smooth muscle cells either alone or together with a Ca²⁺-mobilizing agonist for 60 sec, and cAMP formation during this period was measured. Measurements were made in Ca²⁺-containing and Ca²⁺-free medium (0 Ca²⁺ plus 1 mM EGTA) and in muscle cells preincubated for 60 min with 400 ng/ml PTX. (2) In another set of experiments, the muscle cells were treated for 5 min with a maximally effective concentration of a Ca²⁺-mobilizing agonist so as to elicit capacitative Ca²⁺ influx and then were treated with forskolin for 60 sec. Measurements were made in Ca²⁺-containing and Ca²⁺-free medium and in muscle cells treated for 10 min with the PLC- inhibitor U-73122 or for 60 min with PTX. (3) In control experiments, agonist-independent capacitative Ca²⁺ influx was elicited by treating the muscle cells with thapsigargin (2 µM) for 30 min in Ca²⁺-free medium followed by restitution of normal Ca²⁺; alternatively, the muscle cells were treated with ionomycin (10 µM), which induces both Ca²⁺ release and Ca²⁺ influx. Measurements of forskolin-stimulated cAMP formation were made in the presence or absence of Ca²⁺.

Data analysis. Results were expressed as mean ± standard error and were evaluated statistically using Student's t test for paired or unpaired values.

Materials. [¹²⁵I]cAMP and ⁴⁵Ca²⁺ were obtained from DuPont-New England Nuclear (Boston, MA). HEPES was from Research Organics (Cleveland, OH). Soybean trypsin inhibitor and collagenase (type II) were from Worthington Biochemicals (Freehold, NJ). ,-Methylene-ATP and ,-methylene-ATP were from Research Biochemicals (Natick, MA). PTX, vinpocetine, U-73122, and thapsigargin were from Calbiochem (San Diego, CA). All other chemicals were from Sigma Chemical (St. Louis, MO).

	Results

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The effect of forskolin on cAMP formation and agonist-induced Ca²⁺ release in dispersed smooth muscle cells. Forskolin caused a prompt increase in cAMP formation above basal level that attained a peak within 1 min declining slowly over a period of 10 min (Fig. 1). CCK-8 had no effect on basal cAMP (basal cAMP, 4.8 ± 0.4 pmol/10⁶ cells; CCK-8, 4.8 ± 0.5 pmol/10⁶ cells) or on forskolin-stimulated cAMP (peak forskolin response, 21.9 ± 2.5 pmol/10⁶ cells above basal level; forskolin plus CCK-8, 21.7 ± 3.8 pmol/10⁶ cells). In contrast, the P_2Y2 receptor agonist UTP inhibited forskolin-stimulated cAMP (forskolin plus UTP, 5.5 ± 0.8 pmol/10⁶ cells above basal level); the inhibition by UTP was completely blocked by preincubation of the muscle cells for 60 min with 400 ng/ml PTX.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Time course of forskolin-stimulated cAMP formation in dispersed smooth muscle cells. cAMP formation attained a peak 1 min after addition of forskolin (10 µM) and declined slightly over a 10-min period (). UTP (10 µM; ) but not CCK-8 (1 nM; ) inhibited cAMP formation; inhibition by UTP was reversed by preincubation of the muscle cells for 60 min with 400 ng/ml PTX (). The results are expressed as pmol of cAMP/106 cells above basal levels (basal level, 4.81 ± 0.42 pmol/106 cells). Values are mean ± standard error of three or four experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Time course of Ca2+ release and reuptake induced by UTP and CCK-8 in smooth muscle cells. Muscle cells were incubated in a medium containing 45Ca2+ (10 µCi/ml) and antimycin (10 µM), and Ca2+ uptake into nonmitochondrial Ca2+ stores was measured at intervals for 60 min when a steady state 45Ca2+ cell content (2.5-2.8 nmol Ca2+/106 cells) was attained. UTP (10 µM) or CCK-8 (1 nM) was added with or without nifedipine (1 µM) or forskolin (10 µM), and 45Ca2+ cell content was measured at intervals for 10 min. Nifedipine and forskolin had no effect on Ca2+ release (first minute) but inhibited Ca2+ reuptake. Data are mean ± standard error of four experiments. *, p < 0.05, **, p < 0.01 from control levels.

Differential inhibition of adenylyl cyclase activity by G protein-coupled P_2Y receptor and ligand-gated P_2X receptor agonists. We have shown recently that the initial increase in [Ca²⁺]_i induced by UTP, a P_2Y2 receptor agonist in gastric smooth muscle cells, was mediated by IP₃-dependent Ca²⁺ release, whereas the increase in [Ca²⁺]_i induced by ,-methylene-ATP, a P_2X1 receptor agonist in these cells, was mediated by Ca²⁺ influx via voltage-sensitive Ca²⁺ channels (Murthy and Makhlouf, 1998). The increase in [Ca²⁺]_i induced by ATP, a mixed P_2Y2/P_2X1 receptor agonist in these cells, was mediated by both Ca²⁺ release and Ca²⁺ influx. The P_2Y2 receptors were coupled to both G_q/11 and G_i3, and the stimulation of IP₃ formation and Ca²⁺ release resulted from concurrent activation of PLC-1 by G_q/11 and PLC-3 by G_i3. The distinctive properties of these agonists were used to evaluate the regulation of adenylyl cyclase V/VI in dispersed gastric smooth muscle cells. Selective adenosine A₁ and A₂ receptor antagonists [1 µM DPCPX cyclopentyl-1,3-dipropylxanthine and 0.1 µM CGS-15943 (9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine] were added to the medium to prevent effects that could result from degradation of purine agonists (Murthy and Makhlouf, 1995a).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves for ,-methylene-ATP-induced inhibition of forskolin-stimulated cAMP formation in smooth muscle cells. The effects of ,-methylene-ATP on forskolin-stimulated cAMP formation in dispersed gastric smooth muscle cells were measured during the first 60 sec of agonist stimulation in the presence or absence of extracellular Ca2+ (0 Ca2+/1 mM EGTA) and after treatment with PTX (400 ng/ml) for 1 hr. Forskolin was added either alone or together with the agonist for 1 min. The results were expressed as pmol of cAMP/106 cells above basal levels (basal level, 4.59 ± 0.42 pmol/106 cells; basal level after PTX, 4.51 ± 0.34 pmol/106 cells; basal level in 0 Ca2+, 4.57 ± 0.34 pmol/106 cells). Values are mean ± standard error of four experiments. *, p < 0.02; **, p < 0.01.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves for UTP-induced inhibition of forskolin-stimulated cAMP formation in smooth muscle cells. The effects of UTP on forskolin-stimulated cAMP formation in dispersed gastric smooth muscle cells were measured during the first 60 sec of agonist stimulation in the presence or absence of extracellular Ca2+ and after treatment with PTX for 1 hr. Forskolin (10 µM) was added either alone or together with the agonist for 1 min. The results were expressed as pmol of cAMP/106 cells above basal levels (basal level, 4.62 ± 0.36 pmol/106 cells; basal level after PTX, 4.50 ± 0.31 pmol/106 cells; basal level in 0 Ca2+, 4.70 ± 0.42 pmol/106 cells). Values are mean ± standard error of four experiments. **, p < 0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Concentration-response curves for ATP-induced inhibition of forskolin-stimulated cAMP formation in smooth muscle cells. The effects of ATP on forskolin-stimulated cAMP formation in dispersed gastric smooth muscle cells were measured during the first 60 sec of agonist stimulation in the presence or absence of extracellular Ca2+ (0 Ca2+/EGTA) and after treatment with 400 ng/ml PTX for 1 hr. Forskolin (10 µM) was added alone or together with the agonist for 1 min. The results were expressed as pmol of cAMP/106 cells above basal levels (basal level, 4.59 ± 0.36 pmol/106 cells; basal level after PTX, 4.74 ± 0.34 pmol/106 cells; basal level in 0 Ca2+, 4.57 ± 0.38 pmol/106 cells; basal levels in 0 Ca2+ after treatment with PTX, 4.80 ± 0.52 pmol/106 cells). Values are mean ± standard error of four experiments. *, p < 0.05; **, p < 0.01.

Inhibition of adenylyl cyclase activity in smooth muscle cells by agonist-dependent capacitative Ca²⁺ influx. To examine the effects of capacitative Ca²⁺ influx triggered by depletion of intracellular Ca²⁺ stores on adenylyl cyclase activity, the design of the experiments was altered as follows. The muscle cells were first treated for 5 min with the agonist so as to evoke capacitative Ca²⁺ influx, after which 10 µM forskolin was added for 1 min. The two agonists used in these experiments were CCK-8, which activates G_q/11 (Murthy and Makhlouf, 1995b), and UTP, which activates both G_q/11 and G_i3 (Murthy and Makhlouf, 1998) in these cells.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Inhibition of forskolin-stimulated cAMP formation in smooth muscle cells by CCK-induced capacitative Ca2+ influx. The effects of capacitative Ca2+ influx on adenylyl cyclase activity were determined after the smooth muscle cells were treated for 5 min with CCK-8 (1 nM). The cells were treated for 1 min with 10 µM forskolin. U-73122 (10 µM) was used to inhibit PLC- activity and IP3-dependent Ca2+ release. The results were expressed as pmol of cAMP/106 cells above basal levels (basal level, 4.51 ± 0.3 pmol/106 cells). Values are mean ± standard error of four experiments. **, p < 0.01.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Inhibition of forskolin-stimulated cAMP formation in smooth muscle cells by UTP-induced capacitative Ca2+ influx. To examine the effects of capacitative Ca2+ influx triggered by depletion of intracellular Ca2+ stores on adenylyl cyclase activity, the muscle cells first were treated for 5 min with the Ca2+ mobilizing agonist UTP (10 µM), after which 10 µM forskolin was added for 1 min. U-73122 (10 µM) was used to inhibit PLC- activity and IP3-dependent Ca2+ release. PTX was used to block the inhibition mediated by Gi. The results are expressed as pmol of cAMP/106 cells above basal level (basal level, 4.30 ± 0.53 pmol/106 cells). Values are mean ± standard error of four experiments. **, p < 0.01.

Inhibition of adenylyl cyclase activity in smooth muscle cells by agonist-independent capacitative Ca²⁺ influx. The ability of capacitative Ca²⁺ influx to inhibit adenylyl cyclase activity was examined further using the sarcoplasmic Ca²⁺-ATPase inhibitor thapsigargin, which depletes intracellular Ca²⁺ stores independently of receptor activation (Thastrup, 1990). The muscle cells were incubated for 30 min with 2 µM thapsigargin in 0 Ca²⁺ plus 1 mM EGTA, followed by restitution of control Ca²⁺ levels (2 mM), and then treated with 10 µM forskolin for 1 min. In muscle cells treated with thapsigargin followed by restitution of extracellular Ca²⁺, forskolin-stimulated cAMP formation was inhibited by 41 ± 3% (forskolin alone, 18.4 ± 2.1 pmol/10⁶ cells; forskolin plus thapsigargin, 11.4 ± 1.7 pmol/10⁶ cells; p < 0.01, n = 4) (Fig. 8). Inhibition was not observed when the muscle cells were maintained in Ca²⁺-free medium after treatment with thapsigargin (18.8 ± 1.6 pmol/10⁶ cells).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Inhibition of forskolin-stimulated cAMP formation in smooth muscle cells by thapsigargin- and ionomycin-induced Ca2+ influx. Top, cAMP was measured before and after treatment with the sarcoplasmic Ca2+/ATPase inhibitor thapsigargin. Muscle cells were incubated for 30 min with 2 µM thapsigargin in 0 Ca2+/1 mM EGTA and then treated with 10 µM forskolin for 1 min in either the presence of 2 mM Ca2+ or absence of Ca2+. Bottom, The muscle cells were treated for 1 min with 10 µM ionomycin and 10 µM forskolin in the presence or absence of Ca2+. The results were expressed as pmol of cAMP/106 cells above basal levels (basal level, 4.62 ± 0.49 pmol/106 cells; basal level after thapsigargin treatment, 4.76 ± 0.31 pmol/106 cells; basal level in 0 Ca2+/1 mM EGTA, 4.70 ± 0.35 pmol/106 cells). Values are mean ± standard error of four experiments. **, p < 0.01.

	Discussion

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The results of the current study show the operation of two distinct mechanisms for the inhibitory regulation of adenylyl cyclase types V/VI in smooth muscle cells: a G protein-dependent mechanism and a Ca²⁺-dependent mechanism that seems to operate only in the absence of inhibitory G protein regulation. The mechanisms could be activated separately by agonists acting on G protein-coupled receptors (UTP, ATP, CCK-8) and ligand-gated receptors (,-methylene-ATP and ATP) and by agents that bypass receptors, such as the sarcoplasmic Ca²⁺/ATPase inhibitor, thapsigargin, and ionomycin. The agonists acting on G protein-coupled receptors provided distinctive patterns of G protein activation that facilitated analysis of the role of each inhibitory mechanism. The Ca²⁺-dependent mechanisms had in common the ability to induce Ca²⁺ influx via voltage-sensitive Ca²⁺ channels and did not involve activation of a Ca²⁺-stimulated PDE1.

During the initial 1-min period of agonist stimulation that coincided with Ca²⁺ release from intracellular stores, UTP, which activates P_2Y2 receptors coupled to G_q/11 and G_i3 in visceral and vascular smooth muscle (Pacaud et al., 1995; Murthy and Makhlouf, 1998), inhibited forskolin-stimulated cAMP formation in a concentration-dependent fashion; the inhibition was blocked by PTX but not by 0 Ca²⁺, implying that it was mediated exclusively by G_i3. CCK-8, which activates receptors coupled to G_q/11 only, had no effect on cAMP formation. ,-Methylene-ATP, which selectively activates ligand-gated P_2X receptors in smooth muscle cells, causing membrane depolarization and dihydropyridine-sensitive Ca²⁺ influx (Murthy and Makhlouf, 1998), also inhibited cAMP formation, but in contrast to UTP, the inhibition was blocked by 0 Ca²⁺ but not by PTX, implying that it was mediated exclusively by Ca²⁺ influx via voltage-sensitive Ca²⁺ channels.

The effect of ATP, which activates both G protein-coupled P_2Y2 and ligand-gated P_2X receptors, demonstrated the preferential operation of the inhibitory mechanism mediated by G_i3. Withdrawal of Ca²⁺ from the medium had no effect on ATP-induced inhibition of cAMP formation. However, when the inhibitory effect mediated by G_i3 was blocked with PTX, the inhibitory effect of Ca²⁺ influx mediated by P_2X receptors was unmasked and could be blocked by withdrawal of Ca²⁺ from the medium. The pattern implied that when both mechanisms were elicited by different receptors, adenylyl cyclase activity was preferentially inhibited by the G protein.

Preferential inhibition by G protein also was observed with UTP during the period of capacitative Ca²⁺ influx, that is, 5 min after exposure to the agonist. Inhibition of forskolin-stimulated cAMP formation during this period was mediated by G_i3 and could be blocked by PTX but not by withdrawal of Ca²⁺. However, after G_i3-mediated inhibition was blocked with PTX, inhibition by capacitative Ca²⁺ influx was unmasked and could be blocked by withdrawal of Ca²⁺ from the medium. This pattern also implied that inhibition of adenylyl cyclase activity was preferentially mediated by the G protein that masked or suppressed the inhibitory effect of capacitative Ca²⁺ influx. In cell lines (e.g., NCB-20) in which UTP activates P_2Y receptors coupled to a PTX-insensitive G protein, inhibition of cAMP formation was mediated by capacitative Ca²⁺ influx (Garritsen et al., 1992).

The independent inhibitory effect of capacitative Ca²⁺ influx was seen to best advantage after 5-min treatment with CCK-8 or 30-min treatment with thapsigargin to deplete the Ca²⁺ stores where inhibition of forskolin-stimulated cAMP formation was abolished by withdrawal of Ca²⁺ from the medium. Similar inhibition was obtained after 1-min treatment with ionomycin: the effect of the ionophore that induces both Ca²⁺ influx and Ca²⁺ release was mediated by Ca²⁺ influx because it was blocked on withdrawal of Ca²⁺ from the medium.

It is worth noting that inhibition of adenylyl cyclase V/VI activity by Ca²⁺ influx in smooth muscle could be elicited whether Ca²⁺ influx was triggered by (1) activation of ligand-gated P_2X receptors/channels (,-methylene-ATP and ATP), (2) capacitative Ca²⁺ influx resulting from depletion of Ca²⁺ stores by agonists (CCK, UTP) or thapsigargin, or (3) Ca²⁺ influx via ionophore (ionomycin). Earlier studies (Bitar et al., 1986) have shown that repletion of Ca²⁺ stores after agonist (CCK-8) stimulation in smooth muscle cells is mediated by Ca²⁺ influx via voltage-sensitive Ca²⁺ channels; this notion was confirmed in the current study with both CCK-8 and UTP as agonists (Fig. 2). More recent studies have shown that activation of P_2X receptors results in membrane depolarization and Ca²⁺ influx via dihydropyridine-sensitive Ca²⁺ channels (Murthy and Makhlouf, 1998). These channels seem to be the preferred route for inhibition of adenylyl cyclase V/VI in smooth muscle as they are in cardiac muscle, which expresses the same adenylyl cyclase isoforms (Yu et al., 1993; Cooper et al., 1995; Gao et al., 1997). The Ca²⁺ channels are colocalized with adenylyl cyclase in the plasma membrane of cardiac myocytes, providing a structural basis for the ability of Ca²⁺ influx to regulate adenylyl cyclase (Gao et al., 1997). The strict requirement for regulation by Ca²⁺ influx seems to prevail for other isoforms of adenylyl cyclase (e.g., types I, III, VIII) that are stimulated by Ca²⁺ (Fagan et al., 1996). Membrane colocalization and functional interplay of adenylyl cyclases and Ca²⁺ channels seem to be maintained even when the cyclases are expressed heterologously. However, neither the mechanism of inhibition of adenylyl cyclase by Ca²⁺ nor the mechanism by which concurrent inhibition by G protein precludes inhibition by Ca²⁺ influx have been defined. The absence of binding sites for Ca²⁺ or calmodulin on adenylyl cyclase seems to preclude competitive interplay between the  subunit of inhibitory G proteins and Ca²⁺.

In summary, inhibition of adenylyl cyclase V/VI activity in smooth muscle can be mediated by inhibitory G proteins or Ca²⁺ influx independently of whether the latter is elicited by activation of ligand-gated or G protein-coupled receptors. When both mechanisms are triggered concurrently, inhibition is exclusively mediated by inhibitory G proteins.