Differential Coupling of Muscarinic m2 and m3 Receptors to Adenylyl Cyclases V/VI in Smooth Muscle

Muscarinic m2 and m4 receptors couple preferentially to inhibition of adenylyl cyclase, whereas m1, m3, and m5 receptors couple preferentially to activation of phospholipase C-β and in some cells to stimulation of cAMP. Smooth muscle cells were shown to express adenylyl cyclases types V and/or VI. Acetylcholine (ACh) stimulated the binding of [35S]GTPγS·Gα complexes in smooth muscle membranes to Gαq/11 and Gαi3 antibody. Binding to Gαq/11 antibody was inhibited by the m3receptor antagonist, 4-DAMP, and binding to Gαi3 antibody was inhibited by the m2 receptor antagonist,N,N′-bis[6[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride (methoctramine). The decrease in basal cAMP (35 ± 5%) induced by ACh in dispersed muscle cells was accentuated by 4-DAMP or Gβ antibody (55 ± 8 to 63 ± 6%). In contrast, methoctramine, pertussis toxin (PTx), or Gαi3 antibody converted the decrease in cAMP to increase above basal level (+28 ± 5 to +32 ± 6%); the increase in cAMP was abolished by 4-DAMP or Gβ antibody. In muscle cells where only m3 receptors were preserved by selective receptor protection, ACh caused only an increase in cAMP that was abolished by 4-DAMP. Conversely, in muscle cells where only m2 receptors were preserved, ACh caused an accentuated decrease in cAMP that was abolished by methoctramine or PTx. In conclusion, m2 receptors in smooth muscle couple to inhibition of adenylyl cyclases V/VI via Gαi3, and m3 receptors couple to activation of the enzymes via Gβγq/11.

Complementary DNA clones encoding the full sequences of eight isoforms of mammalian adenylyl cyclase (types I-VIII) and the partial sequences of two additional isoforms (types IX and X) have been isolated (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The amino acid sequences (range 1064 -1248 residues) are arranged in two cassettes of six transmembrane-spanning domains (9 -11). Overall homology is 60% with some cytoplasmic regions exhibiting up to 93% amino acid identity. The presumed catalytic domains (C 1a and/or C 2a ) are homologous to the corresponding domains of membranebound homodimeric and soluble heterodimeric guanylyl cyclases (11,12). Structural homology among the various isoforms is most evident between types II and IV and types V and VI. All the isoforms are expressed in the brain, and types V and VI are the predominant isoforms in the periphery (8 -11).
Although functional regulation of adenylyl cyclases is diverse, three broad categories can be distinguished comprising types I, III, and VIII, types II, IV, and probably VII, and types V and VI (10,11). All adenylyl cyclases are activated by the diterpene, forskolin, and the ␣ subunit of G s . Types I and VIII, which are expressed exclusively in neurons, are stimulated by submicromolar concentrations of Ca 2ϩ and calmodulin, whereas type III, which is more widely expressed, is stimulated by low micromolar concentrations of Ca 2ϩ (13)(14)(15)(16); type I is effectively inhibited by G␤␥ but only moderately inhibited by G i and G o (17)(18)(19)(20)(21). Types II and IV are not stimulated by Ca 2ϩ / calmodulin or inhibited by G i but are stimulated by G␤␥ (19,(21)(22)(23)(24). Stimulation by G␤␥, initially thought to be conditional on concurrent stimulation by G␣ s , is now viewed as highly synergistic, with only modest stimulation by G␤␥ alone (21). Types V and VI are inhibited by G i and by submicromolar concentrations of cytosolic Ca 2ϩ elicited by capacitative Ca 2ϩ influx but not by Ca 2ϩ release from sarcoplasmic stores (24,(25)(26)(27)(28); inhibition by G o or stimulation by G␤␥ remains uncertain. Both types V and VI contain consensus sequences for phosphorylation and exhibit feedback inhibition, by cAMP-dependent protein kinase (29,30).
The expression of adenylyl cyclase isoforms in smooth muscle has not been determined. The regulatory pattern suggested by our previous studies in gastrointestinal smooth muscle is consistent with the presence of types V and/or VI. Agonist-induced cAMP formation is mediated by G␣ s (31,32), and forskolinstimulated cAMP formation is inhibited, depending on the agonist, by G i1 , G i2 , G i3 , and G o (33)(34)(35). Thus, inhibition induced by somatostatin (acting via sstr3) is mediated by G i1 and G o (33), and inhibition induced by opioid agonists (acting via , ␦, and receptors) is mediated by G i2 and G o (34); inhibition induced by adenosine (acting via A 1 receptors) is mediated by G i3 but not by G o (35). Forskolin-stimulated cAMP formation is inhibited in feedback fashion by cAMP-dependent protein kinase (36). Phorbol esters have no effect on basal or forskolinstimulated cAMP formation (37). However, inhibition of cAMP formation induced by agonists acting via G i1 or G i2 (but not G i3 ) is partly reversed by concomitant activation of PKC 1 ; the effect reflects selective PKC-dependent phosphorylation of G␣ i1 and G␣ i2 but not G␣ i3 or G␣ o (37).
It is well established that the muscarinic receptors, m 2 and m 4 , are preferentially coupled to inhibition of adenylyl cyclase; the odd-numbered receptors, m 1 , m 3 , and m 5 , are preferentially coupled to phosphoinositide hydrolysis but, in some cells, can also increase the levels of cAMP (38 -44). The mechanisms responsible for the increase in cAMP are likely to reflect the type of adenylyl cyclase expressed in various cells. In cells expressing predominantly Ca 2ϩ /calmodulin-sensitive adenylyl cyclases types I, III, and VIII, activation could result from Ca 2ϩ mobilization and activation of PKC (43). This, however, is unlikely in cells expressing predominantly Ca 2ϩ /calmodulin-insensitive types II and IV or in cells expressing types V and VI that are inhibited by physiological levels of Ca 2ϩ (10,28). In the present study, we show that types V and/or VI, the isoforms of adenylyl cyclase expressed in smooth muscle cells, are inhibited by m 2 receptors via the ␣ subunit of G i3 and concurrently activated by m 3 receptors via the ␤␥ subunits of G q/11 .

EXPERIMENTAL PROCEDURES
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 (31,33). Briefly, muscle strips were incubated for 30 min at 31°C 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 KH 2 PO 4 , 2 mM CaCl 2 , 0.6 mM MgCl 2 , 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's essential amino acid mixture. After washing, the tissues were re-incubated in the same medium for 30 min. The digested tissue was washed with enzyme-free medium, and the cells were allowed to disperse spontaneously for 30 min. Suspensions of single muscle cells were harvested by filtration through 500-m Nitex mesh. The suspensions were centrifuged twice for 10 min at 350 ϫ g.
In experiments with G protein antibodies, the cells were permeabilized as described previously (31,33) by incubation for 10 min with 35 g/ml saponin in a medium containing 20 mM NaCl, 100 mM KCl, 5 mM MgSO 4 , 1 mM NaH 2 PO 4 , 25 mM NaHCO 3 , 0.34 mM CaCl 2 , 1 mM EGTA, and 1% bovine serum albumin. The cells were centrifuged at 350 ϫ g for 5 min, washed free of saponin, and resuspended in the same medium.
Radioligand Binding to Muscarinic Receptors in Dispersed Muscle Cells-Radioligand binding to dispersed muscle cells was done as described previously (33). Muscle cells were suspended in HEPES medium containing 1% bovine serum albumin. Triplicate aliquots (0.5 ml) of cell suspension (10 6 cells/ml) were incubated for 15 min with 1 nM [ 3 H]scopolamine alone or in the presence of acetylcholine, methoctramine, or 4-DAMP. Bound and free radioligand were separated by rapid filtration under reduced pressure through 5-m polycarbonate Nucleopore filters followed by repeated washing (4 times) with 3 ml of ice-cold HEPES medium containing 0.2% bovine serum albumin. Nonspecific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of 10 M unlabeled ligand. Specific binding was calculated as the difference between total and nonspecific binding (mean Ϯ S.E. 33 Ϯ 6%). IC 50 values were calculated from competition curves using the P.fit program (Biosoft; Elsevier Publishing, Cambridge, UK).
Measurement of cAMP in Dispersed Muscle Cells by Radioimmunoassay-cAMP was measured in dispersed cells by radioimmunoassay as described previously (33,34). Aliquots (0.5 ml) containing 10 6 cells/ml were incubated with 0.1 M acetylcholine, and the reaction was terminated after 60 s with 6% cold trichloroacetic acid (v/v). The mixture was centrifuged at 2,000 ϫ g for 15 min at 4°C. The supernatant was extracted three times with 2 ml of diethyl ether and lyophilized. The samples were reconstituted for radioimmunoassay in 500 l 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-l aliquots and expressed as pmol/10 6 cells.
Selective Protection of Muscarinic Receptors-A technique of selective receptor protection was used to determine the presence and function of m 2 and m 3 receptors. The technique was previously used to determine the co-existence and function of opioid , ␦, and receptors (45), 5-hydroxytryptamine 5-HT 2 and 5-HT 4 receptors (32), histamine H 1 and H 2 receptors (46), and tachykinin NK 1 , NK 2 , and NK 3 receptors (47). The technique involves protection of one receptor type with a selective agonist or antagonist followed by inactivation of all unprotected receptors by brief treatment with a low concentration of Nethylmaleimide. In the present study, the selective m 2 receptor antagonist, methoctramine (10 nM), and m 3 receptor antagonist, 4-DAMP (10 nM), were used in separate experiments to protect m 2 and m 3 receptors, respectively. Freshly dispersed muscle cells were incubated with one antagonist at 31°C for 2 min followed by addition of 5 M N-ethylma-leimide for 20 min. The cells were centrifuged twice at 150 ϫ g for 10 min to eliminate the protective antagonist and N-ethylmaleimide and resuspended in fresh HEPES medium. The cAMP response of cells treated in this fashion was compared with the response of untreated (naive) cells. Previous studies have shown that the coupling of protected receptors to signaling pathways remains intact (32,46,47). Smooth muscle cells incubated with N-ethylmaleimide without protective ligand lost their ability to respond to receptor-linked agonists but retained their ability to respond to agents that bypass receptors (e.g. ionomycin, KCl, forskolin), implying that post-receptor mechanisms were intact (46,47).

Identification of Adenylyl Cyclase Isoforms in Smooth
Muscle by Western Blot-Cell homogenates were prepared from dispersed gastric muscle cells and solubilized on ice for 1 h in 20 mM Tris (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 0.5% sodium cholate. For control studies, homogenates were prepared from rat brain. The suspension was centrifuged at 13,000 ϫ g for 5 min. Solubilized membrane proteins (60 -70 g) were resolved by 7.5% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. After incubation in 5% non-fat dry milk to block nonspecific antibody binding, the blots were incubated for 12 h at 4°C with antibodies to types II, III, and IV, and a common antibody to types V and VI, and then for 1 h with anti-rabbit IgG conjugated with horseradish peroxidase. The bands were identified by enhanced chemiluminescence.
Identification of Muscarinic Receptor-activated G Proteins-G proteins selectively activated by acetylcholine were identified by the method of Okamoto et al. (48). Ten ml of muscle cell suspension (2 ϫ 10 6 cells/ml) were homogenized in 20 mM HEPES medium (pH 7.4) containing 2 mM MgCl 2 , 1 mM EDTA, and 2 mM dithiothreitol. After centrifugation at 27,000 ϫ g for 15 min, the crude membranes were solubilized for 60 min at 4°C in 20 mM HEPES medium (pH 7.4) containing 2 mM EDTA, 240 mM NaCl, and 1% CHAPS. The membranes were incubated for various periods at 37°C with 60 nM [ 35 S]GTP␥S in a solution containing 10 mM HEPES (pH 7.4), 100 M EDTA, and 10 mM MgCl 2 . The reaction was stopped with 10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM MgCl 2 , 100 mM NaCl, and 20 M GTP, and the mixture was placed in wells pre-coated with specific G protein antibodies. After incubation for 2 h on ice, the wells were washed three times with phosphate buffer solution containing 0.05% Tween 20, and the radioactivity from each well was counted. Coating with G protein antibodies (1:1000) was done after the wells were first coated with anti-rabbit IgG (1:1000) for 2 h on ice. The selective m 2 receptor antagonist, methoctramine, and m 3 receptor antagonist, 4-DAMP, were used to identify the receptor subtype coupled to a given G protein.
Data Analysis-Results were expressed as means Ϯ S.E. of n separate experiments and evaluated statistically using Student's t test for paired or unpaired values.
Materials-125 I-cAMP, [ 3 H]scopolamine, and [ 35 S]GTP␥S were obtained from NEN Life Science Products; HEPES was from Research Organics, Cleveland, OH; soybean trypsin inhibitor and collagenase (type II) were from Worthington; 4-DAMP, methoctramine, p-fluorohexahydro-siladifenidol (p-F-HHSiD) were from Research Biochemicals International, Natick, MA; N-ethylmaleimide and all other chemicals were from Sigma. Antibodies to adenylyl cyclase types II, III, and IV, a common antibody to types V and VI, and antibody to G ␤ were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Pertussis toxin and antibodies to G␣ q/11 , G␣ i1-2 , G␣ i3 , G␣ o , and G␣ s and peptide fragments against which antibodies to G␣ q/11 (QLNLKEYNLV) and G␣ i3 (KNN-KECGLY) were raised were obtained from Calbiochem. The ability of these antibodies to block activation or inhibition of specific effector enzymes (phospholipase-␤1, phospholipase-␤3, nitric oxide synthase, adenylyl cyclase) has been demonstrated in recent studies (31)(32)(33)(34)(35), and a concentration of 10 g/ml was found to be maximally effective. Schild analysis of the relative potencies of the two antagonists in inhibiting acetylcholine-induced contraction confirmed that 4-DAMP was about 6 ϫ 10 3 -fold more potent than methoctramine as an antagonist of m 3 receptors that mediate contraction. Contraction in dispersed muscle cells was measured by scanning micrometry as described previously (33,34). Muscle cells in which only m 2 receptors were preserved lost the ability to contract in response to acetylcholine, whereas muscle cells in which only m 3 receptors were preserved retained fully their ability to contract (EC 50

Expression of Adenylyl Cyclase Isoforms in Gastric Smooth
Muscle-Western blot analysis of homogenates derived from dispersed gastric smooth muscle cells using antibodies to adenylyl cyclase type II, type III, type IV, and a common antibody to types V and VI disclosed the presence of type V and/or type VI only (Fig. 2). In contrast, homogenates from rat brain disclosed the presence of types II, III, IV, and V/VI (Fig. 2). The selective expression of types V and/or type VI in smooth muscle conforms to the predominant expression of these two types in peripheral tissues (8 -11). The properties of adenylyl cyclase in gastric and intestinal smooth muscle are consistent with the properties of types V and VI; both isozymes are inhibited by G␣ i and G␣ o (unlike types II and IV) (33,34) and are not stimulated by Ca 2ϩ or calmodulin (unlike types I, III, and VIII) (10,28).
Identification of G Proteins Coupled to Muscarinic m 2 and m 3 Receptors-Incubation of solubilized muscle cell membranes with acetylcholine (0.1 M) and [ 35 S]GTP␥S (60 nM) for 20 min caused a significant, time-dependent increase in the binding of [ 35 S]GTP␥S⅐G␣ complexes to wells pre-coated with specific antibody to G␣ q/11 and G␣ i3 but not to wells pre-coated with antibodies to G␣ s , G␣ i1-2 , or G␣ o (Fig. 3 and Table I). The increase in bound radioactivity reflected acetylcholine-dependent activation of the dissociated ␣ subunits of G q/11 and G i3 by  2. Expression of adenylyl cyclase types V and/or VI in gastric smooth muscle. Western blot analysis was performed on homogenates prepared from dispersed gastric smooth muscle cells and rat brain. The homogenates were solubilized with sodium cholate in Tris buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and probed with specific antibodies to the adenylyl cyclase (AC) types II, III, IV, and a common antibody to types V and VI. Immunoreactive bands for types II, III, IV, and V/VI were detected in rat brain; a band corresponding to type V/VI only was detected in smooth muscle.
tors were coupled to G i3 .
Peptides I and II, comprising the G protein sequences against which the G␣ i3 and G␣ q/11 antibodies, respectively, were raised, were used to block the binding of GTP␥S⅐G␣ complexes to the corresponding antibody. Peptide I inhibited the binding of GTP␥S⅐G␣ complexes to G␣ i3 antibody in a concentration-dependent fashion, whereas peptide II had no effect ( Fig. 3 and Table I). Conversely, peptide II inhibited the binding of GTP␥S⅐G␣ complexes to G␣ q/11 antibody in a concentrationdependent fashion, whereas peptide I had no effect (Fig. 3 and Table I). It is noteworthy that the peptides inhibited the control binding of GTP␥S⅐G␣ complexes to the corresponding antibody as well as the increase in binding induced by acetylcholine.
Dual cAMP Response of Dispersed Smooth Muscle Cells to Acetylcholine-Acetylcholine (0.1 M) caused a significant 35 Ϯ 5% decrease in basal cAMP levels of dispersed gastric muscle cells (basal level, 3.9 Ϯ 0.6 pmol/10 6 cells) (Fig. 4). Neither 4-DAMP nor methoctramine alone had any effect on basal cAMP (4.5 Ϯ 0.4 pmol/10 6 cells). Pretreatment of the cells with 0.1 M methoctramine converted the decrease induced by acetylcholine to a significant 32 Ϯ 6% increase above basal level (Fig. 4); the increase in the presence of methoctramine was inhibited in a concentration-dependent fashion by 4-DAMP (EC 50 0.5 nM) and abolished by 0.1 M 4-DAMP (Figs. 4 and 5). Pretreatment of the cells for 1 h with 200 ng/ml PTx so as to uncouple m 2 receptors from G i3 also converted the acetylcholine-induced decrease in cAMP to a significant 36 Ϯ 5% increase above basal level; the increase in cAMP was abolished by 0.1 M 4-DAMP (Fig. 4).
Conversely, pretreatment of the cells with 0.1 M 4-DAMP accentuated the decrease in cAMP induced by acetylcholine (Ϫ63 Ϯ 6% versus control response Ϫ35 Ϯ 5% with acetylcholine alone; p Ͻ 0.01); the decrease was reversed in a concentration-dependent fashion by methoctramine (EC 50 (Fig. 6). The pattern of response to acetylcholine reflected concurrent inhibition of cAMP mediated by m 2 receptors and stimulation mediated by m 3 receptors.
The protein kinase C inhibitor, calphostin C (1 M), had no effect on the increase in cAMP induced by acetylcholine in the presence of methoctramine or PTx (ϩ37 Ϯ 4 and ϩ39 Ϯ 5%, respectively, versus ϩ35 Ϯ 5%) or the accentuated decrease in cAMP induced by acetylcholine in the presence of 4-DAMP (Ϫ59 Ϯ 5% versus Ϫ63 Ϯ 6%).

Identification of G Proteins Coupled to m 2 and m 3 Receptors by Functional
Blockade with Antibodies-Permeabilized muscle cells were used to identify the G proteins coupled to m 2 and m 3 receptors by functional blockade with G protein antibodies. Basal cAMP and the decrease in cAMP induced by acetylcholine were not affected by permeabilization (basal level, 4.2 Ϯ 0.5 pmol/10 6 cells; acetylcholine-induced decrease of cAMP, Ϫ33 Ϯ 3%). Pretreatment of permeabilized muscle cells with methoctramine, PTx, or G␣ i3 antibody (10 g/ml) converted the decrease in cAMP to an increase above basal level (ϩ27 Ϯ 5, ϩ35 Ϯ 5, and ϩ28 Ϯ 5%, respectively) (Fig. 7). Preincubation of the cells for 1 h with G ␤ antibody (10 g/ml) abolished the increase in cAMP induced by all three agents (Fig. 7), and preincubation with antibodies to G␣ q/11 or G␣ i1-2 (each 10 g/ml) had no effect (range of response ϩ27 Ϯ 7 to ϩ30 Ϯ 6%). The increase in cAMP induced by G␣ i3 antibody was also abolished by 4-DAMP (2 Ϯ 5%).
Binding of complexes to G␣ q/11 antibody was blocked by 4-DAMP (0. 1  M) (B, open circles), and binding of complexes to G␣ i3 antibody was blocked by methoctramine (0.1 M) (A, open circles). Peptide I against which G␣ i3 antibody was raised abolished binding to G␣ i3 antibody but not G␣ q/11 antibody (see Table I). Peptide II against which G␣ q/11 antibody was raised abolished binding to G␣ q/11 antibody but not G␣ i3 antibody (see Table I). Values are means Ϯ S.E. of four experiments.
The pattern of response elicited by treatment of permeabilized muscle cells with specific G protein antibodies implied that the acetylcholine-induced decrease in cAMP was mediated by m 2 receptors via G␣ i3 , whereas the increase in cAMP was mediated by m 3 receptors via G␤␥ q/11 . cAMP Response in Muscle Cells with One Muscarinic Receptor Type-The results obtained in naive muscle cells expressing both m 2 and m 3 receptors were corroborated in cells where only one receptor type was preserved. In cells where only m 3 receptors were preserved, acetylcholine caused only an increase in cAMP (ϩ27 Ϯ 3%), similar to that elicited in naive cells when m 2 receptors were blocked with methoctramine or uncoupled with PTx, or when G␣ i3 was blocked with G␣ i3 antibody (Fig. 9). The increase in cAMP induced by acetylcholine was abolished by 4-DAMP but was not affected by methoctramine or PTx.
In cells where only m 2 receptors were preserved, acetylcholine elicited an accentuated decrease in cAMP (Ϫ52 Ϯ 4%) (Fig.  9), similar to that elicited in naive cells in the presence of 4-DAMP or after treatment with G ␤ antibody (Figs. 6 and 9). The accentuated decrease in cAMP was abolished by methoctramine or PTx but was not affected by 4-DAMP (Fig. 9). DISCUSSION The present study confirmed the co-existence of muscarinic m 2 and m 3 receptors on gastric smooth muscle cells and demonstrated the differential coupling of the two receptor types to adenylyl cyclase. Muscarinic m 2 receptors, the predominant receptor type expressed in gastrointestinal smooth muscle (40,53), are known to be coupled to inhibition of adenylyl cyclase via a PTx-sensitive G protein (40, 54 -56). The present study identified this G protein as G i3 . In addition, the present study demonstrated a direct coupling of m 3 receptors to activation of adenylyl cyclase via the ␤␥ subunits of G q/11 . Although coupling of m 3 receptors to activation of adenylyl cyclase was known to occur in some cell types (38, 41), the mechanism(s) underlying this effect had not been determined.
The evidence for the differential coupling of m 2 and m 3 receptors was based on a combination of experimental strategies. (a) GTP␥S⅐G␣ complexes activated by m 3 receptors bound selectively to G␣ q/11 antibodies, whereas GTP␥S⅐G␣ complexes activated by m 2 receptors bound selectively to G␣ i3 antibodies. No acetylcholine-induced increase in binding to G␣ i1-2 or G␣ o antibodies could be detected. The binding of GTP␥S⅐G␣ complexes to G␣ i3 or G␣ q/11 antibodies was selectively blocked by peptide fragments against which these antibodies were raised. (b) Blockade of m 2 receptors or their uncoupling from G proteins by PTx converted the decrease in cAMP induced by acetylcholine to increase above basal level; the increase was blocked by a selective m 3 receptor antagonist and by a common antibody to G ␤ implying that activation of adenylyl cyclase was mediated by G␤␥ derived from m 3 -dependent activation of G q/ 11. (c) Concurrent activation of adenylyl cyclase mediated by m 3 receptors attenuated the predominant inhibition mediated by m 2 receptors; blockade of the stimulatory effect with an m 3 receptor antagonist or G ␤ antibody accentuated the decrease in cAMP; the accentuated decrease was abolished by methoctramine, PTx, and G␣ i3 antibody. (d) The results obtained in naive muscle cells expressing both receptor types were corroborated using muscle cells in which only one receptor type was preserved. Thus, cells where only m 2 receptors were preserved responded to acetylcholine by an accentuated decrease in cAMP which was abolished by an m 2 receptor antagonist or PTx; conversely, cells where only m 3 receptors were preserved responded to acetylcholine with only an increase in cAMP that was abolished by an m 3 receptor antagonist.
The selectivity of the antagonists used for receptor protection was demonstrated by radioligand binding, and the measured IC 50 values closely matched those derived from measurements in cells expressing cloned m 2 or m 3 receptors (39, 49 -52). Pharmacological analysis confirmed the validity of the receptor protection technique that had previously been used to characterize a variety of receptors co-expressed on smooth muscle cells and coupled to the same or distinct signaling pathways (e.g. histamine H 1 and H 2 receptors (46), 5-HT 2 and 5-HT 4 receptors (32), adenosine A 2b and A 1 receptors (35), tachykinin NK 1 , NK 2 , and NK 3 receptors (47), and opioid , ␦, and receptors (34)). Only receptors were inactivated while postreceptor mechanisms were spared; in particular, neither basal nor forskolin-stimulated cAMP formation was affected (32,35).
Increasing awareness of the diverse regulation of various isoforms of adenylyl cyclases requires that the proposed mechanisms for activation or inhibition of the enzymes be consistent with the properties of the adenylyl cyclase(s) expressed in a given cell type (8 -11). In the present study, adenylyl cyclase types II, III, and IV could not be detected in dispersed gastric muscle cells. Type V and/or type VI was detected by Western blot analysis since the common antibody could not distinguish between the two types. The tissue expression and regulatory features of adenylyl cyclase in smooth muscle are consistent with the absence of types I and VIII which are confined to neurons (13)(14)(15)(16), and with the absence of types II, IV, and possibly VII which are not susceptible to inhibition by G i (11,(21)(22)(23).
The types of adenylyl cyclase (V and/or VI) expressed in smooth muscle are regulated in similar fashion and known to be inhibited by various isoforms of G i and by feedback phosphorylation by cAMP-dependent protein kinase (25,26,29,30). Both these properties are evident in smooth muscle. Forskolinstimulated cAMP formation in gastric smooth muscle, for example, is augmented by selective inhibition of cAMP-dependent protein kinase activity with myristoylated protein kinase A inhibitor (36). Inhibition of adenylyl cyclase in smooth muscle by various isoforms of G i and G o appears to be receptor-specific. Inhibition by somatostatin sstr 3 is mediated additively by G i1 and G o (33), whereas inhibition by opioid , ␦, and receptors is mediated additively by G i2 and G o (34). G o is not involved in inhibition by adenosine A 1 or muscarinic m 2 receptors that are  . 7. Effect of G protein antibodies on the cAMP response to ACh in permeabilized smooth muscle cells. cAMP was measured in permeabilized smooth muscle cells and expressed as percent change from basal levels. Permeabilization had no effect on basal levels (4.2 Ϯ 0.5 pmol/10 6 cells). Cells were treated for 10 min with methoctramine (0.1 M), 60 min with PTx (200 ng/ml), or 60 min with G␣ i3 antibody (10 g/ml). Treatment with methoctramine, PTx, or G␣ i3 antibody converted the decrease in cAMP induced by ACh (0.1 M) to increase above basal level; the increase was abolished by preincubation of the cells for 60 min with G ␤ antibody (10 g/ml). Values are means Ϯ S.E. of four experiments. **, p Ͻ 0.01. coupled to G i3 (35,37).
Inhibition by submicromolar concentrations of Ca 2ϩ resulting from capacitative entry of Ca 2ϩ is a distinctive regulatory feature of adenylyl cyclases types V and VI (10,21); this feature, however, could not be demonstrated under our experimental conditions. The measurement of cAMP in the present study was made during the first 60 s when the concomitant rise in cytosolic Ca 2ϩ induced by acetylcholine is determined by inositol 1,4,5-trisphosphate-dependent release of Ca 2ϩ from sarcoplasmic stores and thus precedes store depletion and capacitative Ca 2ϩ entry (57,58). Furthermore, suppression of phosphoinositide hydrolysis and inositol 1,4,5-trisphosphatedependent Ca 2ϩ release by G␣ q/11 antibody had no effect on the increase in cAMP mediated by m 3 receptors. The concurrent activation of PKC also had no effect on adenylyl cyclase activity in smooth muscle since inhibition of PKC activity by calphostin C had no effect on cAMP levels. As previously shown (37), however, PKC can influence adenylyl cyclase activity in gastrointestinal smooth muscle indirectly by selective phosphorylation of G␣ i1 and G␣ i2 ; concurrent activation of PKC attenuated the inhibition of forskolin-stimulated cAMP mediated by somatostatin sstr 3 receptors coupled to G i1 and by opioid , ␦, and receptors coupled to G i2 , but not by adenosine A 1 or muscarinic m 2 receptors coupled to G i3 (33)(34)(35).
A novel aspect of this study was the ability of G␤␥ derived from the dissociation of G q/11 to activate smooth muscle adenylyl cyclases type V and/or VI. Activation was not conditional on concurrent activation of G s , as is the case for activation of types II and IV (11,21). The low abundance of G␤␥ in peripheral tissues, particularly when derived from the dissociation of G q/ 11, raises the possibility that, when expressed in smooth muscle, types V and VI may be unusually sensitive to activation by G␤␥ or by specific combinations of ␤␥ subunits.
In summary, muscarinic m 2 receptors are coupled to inhibition of adenylyl cyclases V and/or VI in smooth muscle via the ␣ subunit of G i3 , whereas m 3 receptors are coupled to activation of the enzymes via the ␤␥ subunits of G q/11 . The cAMP response to muscarinic agonists reflects the predominant inhibitory influence of m 2 receptors.