Protein kinase regulation of muscarinic receptor signalling in colonic smooth muscle

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1 Br. J. Pharmacol. (1993), 18, t"i Macmillan Press Ltd, 1993 Protein kinase regulation of muscarinic receptor signalling in colonic smooth muscle Lubo Zhang & 'lain L.O. Buxton Department of Pharmacology/318, University of Nevada School of Medicine, Reno, NV 89557, U.S.A. Keywords: 1 We have previously demonstrated that M2 and M3 muscarinic receptors coexist in the circular smooth muscle of canine promixal colon. Activation of receptors of the M2 subtype leads to inhibition of adenylyl cyclase activity through the GTP-binding protein, Gi, while M3 receptors are coupled to a pertussis toxin-insensitive GTP-binding protein and mediate phosphoinositide hydrolysis. 2 In the present study, the interactions between these second messenger systems were examined. Activation of either protein kinase C or adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase attenuated carbachol-stimulated phosphoinositide hydrolysis without affecting basal activity. Activation of both protein kinases produced greater attenuation of inositol 1,4,5-trisphosphate formation than activation of either kinase alone. 3 In contrast to its inhibitory effect on phosphoinositide hydrolysis, activation of protein kinase C had no effect on adenylyl cyclase activity. 4 Activation of protein kinase C by phorbol ester treatment resulted in the sequestration of M3 muscarinic receptors from the cell surface without effecting the M2 muscarinic receptor population. Sequestered M3 muscarinic receptors were not rapidly degraded. 5 In contrast, elevation of cellular cyclic AMP decreased the affinity of cell surface muscarinic receptors for an antagonist radioligland without affecting their density. 6 Muscarinic agonist binding was not affected by either activation of protein kinase C or elevation of cellular cyclic AMP. 7 These data support the notion of negative feedback by protein kinase C and cyclic AMP-dependent protein kinase on phosphoinositide hydrolysis. In canine colonic circular smooth muscle this negative feedback regulation of inositol phosphate generation by muscarinic receptor stimulation does not appear to involve the guanine nucleotide binding protein:receptor interaction. Muscarinic receptors; protein kinase C, cyclic AMP-dependent protein kinase; phosphoinositide hydrolysis; smooth muscle, colon Introduction Parasympathetic innervation of the colon resulting in the action of acetylcholine at postsynaptic muscarinic receptors on the smooth muscle provides the principal stimulus for normal colonic motility. We have demonstrated the coexistence of muscarinic receptors of the M2 and M3 subtype in canine colonic circular. smooth muscle (Zhang et al., 1991). Activation of M2 receptors leads to inhibition of adenylyl cyclase activity through a pertussis toxin-sensitive guanine nucleotide binding protein (G-protein), GI, while M3 receptors are coupled to a pertussis toxin-insensitive G-protein and mediate phosphatidylinositol (PI) hydrolysis (Zhang & Buxton, 1991). While a precise role for the M2 receptor in the contraction of gastrointenstinal smooth muscle has not been conclusively established (McDaniel et al., 1991), removing the relaxation produced by adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent phosphorylations is likely to be necessary, if not sufficient, for contraction (Candell et al., 199; Fernandes et al., 1992). On the other hand, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and diacyglycerol (DAG) following activation of the M3 receptor is likely to play a direct role in initiation of contraction of the muscle (Candell et al., 199). Ins-1,4,5-P3 is known to release Ca2+ from sarcoplasmic stores in smooth muscle (Somlyo & Himpens, 1989), while DAG together with Ca2, would be expected to activate protein kinase C (PKC) (Nishizuka, 1986). Author for correspondence. It is generally agreed that protein phosphorylations mediated by specific cellular kinases, such as PKC and cyclic AMP-dependent protein kinase (cyclic AMP-PK), play a critical role in cellular physiology, including regulation of receptor responsiveness. Activation of PKC, an expected consequence of PI hydrolysis, blocks muscarinic agonist-induced stimulation of phospholipase C (PLC) in the brain as well as other cell types (Orellana et al., 1985; Blackmore & Exton, 1986; Lai & El-Fakahany, 1988); while the action of cyclic AMP-PK leads to inhibition of inositol phosphate formation mediated by al-adrenoceptors in rat aorta (Neylon & Summers, 1988; Manolopoulos et al., 1991). Such protein kinase regulation of PI turnover could occur at any of a number of possible steps. The most probable of these are: (i) phosphorylation of receptors leading to their sequestrian (Scherer & Nathanson, 199; Abdallah et al., 199; Hu et al., 1991); (ii) phosphorylation of the G-protein coupling receptors to PLC activation (Brostom et al., 1989; Scherer & Nathanson, 199; Imaizumi et al., 1991); or (iii) phosphorlylation of the PLC enzyme itself (Kim et al., 1989; Rhee et al., 1989; Ryu et al., 199). The functions of many members of the G- protein-linked receptor family, including muscarinic receptors, are regulated by receptor phosphorylation. It has been shown that purified Ml muscarinic receptors can be phosphorylated in vitro by both PKC and cyclic AMP-PK and that purified M2 receptors can be phosphorylated by cyclic AMP-PK (Rosenbaum et al., 1987; Haga et al., 1988). In this regard, it is intriguing that phosphorylation of muscarinic receptors shows subtype specificity with Ml and M3 receptor

2 614 L. ZHANG & I.L.O. BUXTON subtypes being more sensitive to the effects of PKC (Scherer & Nathanson, 199; Hu et al., 1991). Since M2 and M3 muscarinic receptors coexist on the same smooth muscle cell in colonic circular muscle and mediate their effects through distinct signal transduction pathways, it is of interest to determine the potential interactions between these second messenger systems. In the present study, we employ the phorbol ester PKC agonist, phorbol 12,13- dibutyrate (PDBu) and the adenylyl cyclase agonist, forskolin (FSK), to examine the effects of activation of cyclic AMP-PK and PKC on PI hydrolysis stimulated by occupation of muscarinic receptors in canine colonic circular smooth muscle. In addition, we extend our observations of muscarinic receptor heterogeneity on individual smooth muscle cells to include the fraction of receptors present on the cell surface. Methods Tissue and cell preparation Mongrel dogs of either sex were anaesthetized with pentobarbitone sodium (3 mg kg-'). A segment of proximal colon (6-14 cm from the ileocecal spincter) was removed and cut into 2 x 3 cm pieces in a dissecting dish bathed with Krebs- Ringer bicarbonate solution of the following composition (in mm): Na' 142.3, K+ 5.9, Ca2+ 2.5, Mg2+ 1.2, Cl , HCO3-23.8, H2P4-1.2, dextrose 11.. This solution had a ph of 7.4 at 37C when bubbled to equilibrium with 95% 2/5% CO2. The longitudinal smooth muscle was carefully removed and the circular smooth muscle separated from the submucosa by the method of Smith et al. (1987). Isolated circular smooth muscle cells were prepared as described previously (Zhang et al., 1991). Briefly, isolated circular muscle strips were minced and digested in calcium-free Hank's solution containing (mgml-'): collagenase type II (1.3); bovine serum albumin (2.); trypsin inhibitor (2.); and adenosine triphosphate (.56). Digestion was carried out with gentle agitation at 37 C for 3 min. The supernatant was collected and replaced alternatively with calcium-free Hank's solution and the buffer containing enzyme, every 2min until the tissue was totally digested. Intact cells were found in the supernatant to which a final concentration of 1% foetal calf serum was added. Final purification of cells with an anisodiametric appearance (>9%) excluding trypan blue (.3%) was achieved by unit gravity sedimentation. Cells not excluding dye were stubby or rounded and represented dead or dying cells. The final preparation was devoid of small debris present in the original digestion. The use of unit gravity sedimentation in the preparation of cells is believed to purify in favour of the relatively more dense smooth muscle cells over other cells types present in the original digestion. Radioligand binding in membrane homogenates Tissues prepared as described above were washed three times with ice-cold hypotonic buffer A (5 mm Tris base, O mm MgCl2, 1 mm EGTA, ph 7.4) and homogenized in 1 vol. of the same buffer. The homogenate was centrifuged as 2,5 x g for 2 min, the supernatant collected and the pellet again homogenized and centrifuged under the same conditions. This second pellet, containing nuclei and mitochondria, was discarded and the supernatants combined and centrifuged at 5, g for 6 min at 4 C. The resulting supernatant was discarded and the pellet quickly frozen in liquid nitrogen and stored as a frozen powder at - 8 C for 1-2 weeks until employed in radioligand binding studies. Competition between the non selective muscarinic receptor antagonist radioligand [3H]-QNB and the muscarinic agonist, carbachol (CCh) was measured as previously described (Zhang et al., 1991). Protein was determined by the method of Bradford (1976). Radioligand binding to intact cells Binding of ligands to freshly dispersed intact colonic circular smooth muscle cells was measured by a rapid filtration method similar to that described previously (Zhang et al., 1991). Cells were washed three times with oxygenated Krebs solution (composition mm: NaCl 118, KCI 4.7, KH2PO4.6, Na2HPO4.6, MgCI2 1.2, destrose 2, CaC12 1, HEPES 25, ph 7.4). Following incubation with the permeant phorbol ester, PDBu, FSK or diluent at 32 C for the time period indicated, equilibrium binding of [3H]-NMS and [3H]-QNB to intact cells was carried out in Krebs solution at 32 C for 6 min. For saturation binding experiments, cells were incubated with [3H]-NMS (25 to 32 pm) in the presence and absence of 1 gm atropine to define nonspecific binding. Specific binding of [3H]-NMS averaged approximately 9% of total binding at its KD. Bound and free radioligand were separated by rapid filtration over Whatman GF/C filters washed with two 5 ml aliquots of ice-cold.9% NaCl and counted at 45% efficiency in a Beckman LS 6IC liquid scintillation counter (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). All determinations were performed in triplicate, and specific binding of [H]-NMS and [H]-QNB at.2 nm was examined in competition studies employing increasing concentrations of unlabelled competitor. Measurement ofprotein kinase activity Circular muscles were homogenized in ice-cold 2 mm Tris/ containing 2 mm EDTA,.5 mm EGTA, and HCI, ph 7.4, 2 mm phenyl methyl-sulphonylfluoride and centrifuged at 1, g for 3 min at 4 C. Assay of the supernatant was defined as cytoplasmic PKC activity. Membrane-bound PKC activity was extracted from the pellet by resuspension in 2 mm Tris/HCl, ph 7.4, containing 1% Triton X-1 (4 C for 6 min) and assayed in the supernatant following centrifugation at 1, g for 3 min. Both cytosolic and membrane-bound fractions were purified over DEAE cellulose columns washed with 5 ml of 2 mm Tris/HCl, ph 7.4, and eluted with 3 ml of the same buffer containing 12 mm NaCl. Fractions were assayed for PKC activity by the method of Secrest et al. (1991) using phosphorylation of histone (Type VII-S). DEAE fractions (35 fil) were incubated at 3 C for O min in 2 mm Tris HCI buffer (ph 7.4) containing: 75 pg mlh' histone, 5 mm magnesium acetate, 2 mg ml-' phosphatidylserine, 3 mm CaCl2 and 2 mm ATP (-4-6 x 16d.p.m. [32P]-ATP) in a total volume of 15 gll. The incubation was stopped on ice for 3min, spotted onto 2.1 cm circles of Whatman P-81 phosphocellulose paper and immediately placed into hot (6 C) 1% trichloroacetic acid (TCA)/2% sodium phosphate for 5min. Filters were removed and washed in two changes of fresh TCA/sodium phosphate solution at 21C. Incorporation of [2P]-ATP into histone was determined by counting radioactivity remaining on the filter. Protein kinase activity other than PKC activity was determined in the absence of phosphatidylserine and CaCl2 and the presence of 1 mm EGTA. Specific PKC activity was calculated by sub-tracting activity in the absence of lipid and Ca2+. Data are expressed as pmol-p4 incorporated min' mg' protein. Phosphoinositide hydrolysis Circular smooth muscle was dissected into 5 mg (wet wt.) pieces and placed into oxygenated labeling buffer of the following composition (mm): NaCl 118., KCl 4.7, KH2PO4.6, Na2HPO4.6, MgCl2 1.2, dextrose 5., CaCl2.5, HEPES 1., NaHCO3 5. (ph 7.4). Tissues were incubated with [3H]-myo-inositol (2 gci ml-') for 3 h at 35 C and washed with six changes of fresh buffer over 3 min to remove unincorporated radioactivity. At this concentration of the radiolabel, incorporation of [3H]-myo-inositol into the inositol containing phospholipids, phosphatidylinositol (PI),

3 MUSCARINIC RECEPTOR SIGNALLING IN SMOOTH MUSCLE 615 phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) reached apparent steady state by 3 h (Zhang & Buxton, 1991). Following various treatments determined by experimental design, tissues were challenged with the muscarinic agonist carbachol (CCh) in the presence of Li' (1 mm) and the reactions terminated by quick freezing in liquid N2. Tissues were then homogenized in 2ml ice-cold CHCl3:CH3H:HCl (66:33:1), centrifuged at 4 g for 1 min and the aqueous phase collected and lyophilized to dryness. [3H]-inositol phosphates (InsPs) were separated by high performance liquid chromatography (h.p.l.c.) over Whatman Partisil 1 SAX eluted with an ammonium phosphate gradient as described previously (Zhang & Buxton, 1991). The identities of the [3H]-InsPs were verified by comparison with authentic standards. Quantification was achieved by determining peak area as recorded in counts per min by the radioactive flow scintillation counter (3H Eff. = 26%). Cyclic AMP assay Cyclic AMP content of circular smooth muscle was determined by the method of Gilman (197). Freshly dissected tissues (3 mg wet wt.) were incubated with the cyclic AMPphosphodiesterase inhibitor, isobutylmethyl xanthine (1 mm) for 3 min prior to treatment with agonists for the determination of cyclic AMP. experiments (data not shown) revealed that without the presence of this submaximal block of cyclic AMP metabolism, changes in cyclic AMP content following either a-receptor or FSK stimulated adenylyl cyclase activity could not be reproducibly measured. After treatment, tissues were homogenized in 5% TCA and the deproteinized supernatant purified over Dowex AG 5W- X4 (2-4 mesh). The recovery of cyclic AMP after purification was determined by the addition of.25 pmol [3H]-cyclic AMP to the sample before column purification. Values are expressed as pmol cyclic AMP mg' protein. Materials Drugs and chemicals were purchased from the following sources: carbachol, pirenzepine, atropine, isoprenaline, PDBu, FSK, ATP, cyclic AMP, GTP, creatine phosphate, creatine phosphokinase, cyclic AMP-PK, phosphatidylserine, HEPES, EGTA, EDTA, histone VII-S, bovine serum albumin, soybean trypsin inhibitor, and isobutylmethyl xanthine were from Sigma Chemical Co. (St. Louis, MO, U.S.A.); 4a-phorbol-12,13-dideconoate was from Calbiochem (San Diego, CA, U.S.A.); collagenase type II was from Worthington (Freehold, NJ, U.S.A.); anion exchange resin from BioRad Laboratories (Richmond, CA, U.S.A.); DEAE- Sephacel was obtained from Pharmacia LKB (Piscataway, NJ, U.S.A.); P-81 phosphocellulose paper from Whatman (Hillsboro, OR, U.S.A.); [3H]-quinuclidinyl benzilate ([3H]- QNB; 45.7 Ci mmol- ), Vlf-N-methyl scopolamine ([3H]- NMS, 7 Ci mmol '), [3H]-cyclic AMP (44.5 Ci mmol -), V2P]- ATP (3 Ci mmol '), [3H]-myo-inositol (58.1 Ci mmol ') and [3H]-inositol phosphate standards were obtained from Dupont-NEN (Boston, MA, U.S.A.). Data analysis Saturation binding data were analyzed by Scatchard plot. For competition binding data, the computer assisted (Inplot, GraphPad Software, San Diego, CA, U.S.A.) non-linear least-squares approach fits the data to either one or two classes of binding sites and assists in determining if the two-site model is significantly better than the one-site fit of the data (F-test). Data are expressed as the mean ± s.e.mean. Statistal significance was determined by Students t test where a P value less than.5 was considered significant. Results Phorbol 12,13-dibutyrate and carbachol treatment increases protein kinase C activity Since activation of PKC has been shown to be associated with the translocation of the enzyme from the cell cytosol to the plasma membrane (Kraft & Anderson, 1983), we evaluated the effects of PDBu and CCh on PKC subcellular distribution and activity in colonic smooth muscle. Basal, membrane-bound PKC activity was 7.3 pmol min-' mg-' protein which represented 29% of total cellular PKC activity, while the remaining 71% of activity (17.6 pmol min' mg' ) was found in the cytosolic fraction. In the presence of PDBu (.1 and 1 pm), the activity of membrane-bound PKC was increased to 191% and 224% of the control, respectively (Figure 1). As expected, these increases in membrane-bound PKC activity were accompanied by stimultaneous decreases in the amount of PKC present in the cytosol. Concentrations of the muscarinic receptor agonist CCh (.1 to 1 tim) known to stimulate PI turnover (Zhang & Buxton, 1991) produced dose-dependent increases in PKC activity associated with the plasma membrane (Figure 1). Phorbol 12,13-dibutyrate andforskolin treatment decreases phosphatidylinositol-turnover stimulated by carbachol Treatment of colonic circular smooth muscle with FSK (1 nm+ 1 gm) for O min produced a dose-dependent increase in cyclic AMP (25 ± 18+67± 33 pmol mg-' protein) with an EC5 of 1.9 pm. This change in cyclic AMP accumulation is associated with an inhibition of acetycholineinduced contractions (Smith et al., 1992). Pretreatment of colonic circular smooth muscle with either PDBu (1 tim; 3 min) or FSK (1I M; Omin) did not modify the basal concentration of inositol phosphates (measured 3 min later in the presence of 1mM Li'), suggesting that neither PKC nor cyclic AMP-PK exert a tonic control over PI turnover in unstimulated tissues. As shown in Figure 2, after 3 min in i- ŪW cd ~ Carbachol (t±m) PDBu (gmm) Figure 1 Effects of carbachol (CCh) and phorbol 12,13-dibutyrate (PDBu) on the subcellular distribution of protein kinase C (PKC) activity in canine colon circular smooth muscle. PKC activity in cytosolic and membrane fractions of colonic smooth muscle were assayed as described in Methods. Open column; cytosolic PKC; solid column: membrane-bound PKC. Enzyme activity is expressed as percentage increase over basal values determined in the absence of agonist or phorbol ester pretreatment (cytosolic control = ± 8.3 pmol min-' mg-'; membrane bound activity = 7.3 ± 2. pmol min' I mg-'). Activation of the enzyme is associated with translocation of activity from soluble to particulate fractions. Data points are mean values ± range of two experiments performed in duplicate.

4 616 L. ZHANG & I.L.O. BUXTON : a ( Total IP Figure 2 Effects of phorbol 12,13-dibutyrate (PDBu) on carbachol (CCh)-stimulated [3H]-inositol phosphate accumulation. Circular smooth muscle tissues, pre-labelled with [3H]-myo-inositol, were incubated with 1 AM CCh for 3 min in the presence of 1 mm LiCl (control). Open column: control; solid column: PDBu I AM for 3 min prior to addition of CCh; cross-hatched column: forskolin (FSK) I AM 1O min pretreatment; stippled column: PDBu- + FSK; hatched column: 4ac-phorbol 12,13-dideconoate (4o-PDD) I AM, 3 min pretreatment. Individual [3H]-inositol phosphates were separated by SAX-h.p.l.c. as described under Methods. Data, determined as the area under the h.p.l.c. peak in c.p.m. for inositol 1,4,5-trisphosphate (ins-1,4,5-p3) and each of the inositol phosphate isomers, are added together to generate total inositol phosphates. Values shown (mean ± s.e.mean, n = 7) are expressed as 1 AM CCh-induced fold increase of total InsP or Ins-1,4,5-P3 over basal (total InsP and Ins-l,4,5-P3: 55 ± 22 and 48 ± 4 c.p.m. mg-' tissue wet weight). *Significant difference from the control; P<.5. tsignificant difference from PDBu or FSK alone; P<.5. the presence of 1 mm Li', CCh (1 JiM) produced a 2 fold increase in Ins-1,4,5-P3 accumulation, a result mirrored by a 3.4 fold increase in total InsP accumulation. This CChinduced InsP accumulation was significantly attenuated when tissues were preincubated for 3 min with 1 JlM PDBu, a treatment which significantly increased membrane-bound PKC activity (see Figure 1). The effect of PDBu on CCh stimulated InsP accumulation is probably the result of PKC activation since the biologically inactive phorbol ester, 4mphorbol 12,13-dideconoate (1 JM), was without effect (Figure 2). Pretreatment of tissues with 1 JAM FSK for O min produced similar attenuation of InsP accumulation in response to CCh (Figure 2). The combined treatment of tissues with concentrations of both PDBu and FSK known to be maximal at the incubation times selected (data not shown) produced an even further reduction on CCh stimulated InsP accumulation, suggesting that activation of PKC and cyclic AMP-PK may act in disparate ways to produce inhibition of PLC activity. Effects ofphorbol 12,13-dibutyrate on cyclic AMP responses In order to evaluate the specific effects of PDBu on PI turnover stimulated by CCh in colonic circular smooth muscle, we examined the effects of the phorbol ester on CChinduced inhibition of FSK-stimulated cyclic AMP accumulation. The majority (8%) of muscarinic receptors present in colonic circular smooth muscle are of the M2 subtype which mediate inhibition of agonist stimulated cyclic AMP accumulation via Gi (Zhang & Buxton, 1991). As shown in Figure 3, CCh (IO M) reduced FSK-stimulated cyclic AMP accumulation by 5%. In contrast to its inhibitory effects on CCh-stimulated InsP accumulation, preincubation of the tissues with 1 JAM PDBu for 3 min had no effect on either basal, on P adrenoceptor-mediated stimulation or on CCh- IP3 Figure 3._ 8 Q E 6 E - 4 = 2 1 T,- T I- I- Effects of phorbol 12,13-dibutyrate (PDBu) on cyclic AMP accumulation in canine colon circular smooth muscle. Cyclic AMP was measured in colonic circular smooth muscle tissue before and after treatment of tissues with PDBu as described in Methods. Open column: basal; solid column: forskolin (FSK) I JAM for 1 min; crosshatched column: FSK 1 JAM for 1 min in the presence of carbachol 1 JAM; stippled column: isoprenaline 1O JiM for 1 min. No effect of PDBu treatment on basal, on FSK stimulated, or on receptormediated stimulation or inhibition of adenylyl cyclase could be detected. Data are mean ± s.e.mean (vertical bars) of four experiments determined in duplicate. *Significant difference from FSK alone; P <.5. tsignificant difference from basal; P <.5. mediated inhibition of cyclic AMP accumulation, suggesting no effect of PKC on either the M2 muscarinic receptor or the P-adrenoceptor. Moreover, these results demonstrate that in colonic smooth muscle, PDBu has no functional effect on either the G-proteins responsible for the dual regulation of adenylyl cyclase or adenylyl cyclase itself. Regulation of muscarinic receptor antagonist binding by phorbol 12,13-dibutyrate and forskolin Activation of PKC causes internalization of muscarinic receptors in neuroblastoma cells that requires the presence of functional cyclic AMP-PK (Scherer & Nathanson, 199). To determine whether activation of PKC and/or cyclic AMP-PK could regulate cell surface muscarinic receptors in smooth muscle, freshly dispersed colonic smooth muscle cells were employed in muscarinic receptor antagonist radiogland binding. Cell surface, rather than total, muscarinic receptor number was determined by specific [3H]-NMS binding. Under our assay conditions, the quaternary muscarinic receptor antagonist radioligand [3H]-NMS labelled 22% fewer sites than [3H]-QNB (Table 1). This suggests that muscarinic receptors are present at both cell surface and sequestered sites in colonic smooth muscle cells. Preincubation of colonic smooth muscle cells with 1 JM PDBu for 3 min at 32 C reduced subsequent maximal specific binding of [3H]-NMS by 25% (Table 2) without altering the affinity of remaining cell surface receptors for [3H]-NMS. In contrast, FSK (1 JAM, 1 min) treatment significantly decreased the affinity of receptors for [3H]-NMS (KD:.2 nm-*.47 nm) without affecting maximal specific binding (Table 2). In order to evaluate the effects of PDBu on M2 and M3 subtypes of the muscarinic receptor, we examined the ability of pirenzepine to compete for [3H]-NMS binding to intact colonic myocytes. While muscarinic receptors of the Ml subtype are thought to have a higher affinity for pirenzepine than M3 receptors (Hulme et al., 199) our previous finding suggests that pirenzepine is also useful in studies of M3 receptors. As we have described previously using [3H]-QNB as the radiogland, (Zhang et al., 1991), pirenzepine can discriminate the presence of both M2 (low affinity for

5 MUSCARINIC RECEPTOR SIGNALLING IN SMOOTH MUSCLE 617 Table 1 Comparison of [3H]-N-methyl scopolamine ([3H]-NMS) and [3H]-quinuclidinyl benzilate ([3H]-QNB) saturation binding in canine colonic circular smooth muscle cells [3H]-NMS [3H]-QNB B.X (sites/cell) 93, * 12,396 ± 5721 KD (nm).38 ±.11.8 ±.2 Increasing concentrations (.6-6 nm) of [3H]-QNB and [3H]-NMS were incubated with freshly isolated colonic circular smooth muscle cells at 32 C for 6 min. Specific binding was defined as the arithmetic difference between total binding and nonspecific binding in the presence of 1 M atropine. Analysis of the specific binding data by nonlinear computer-based methods (fit to a rectangular hyperbola) confirmed that both [3H]-QNB and [3H]-NMS bound to a single class of binding sites in the cells. Data are means ± s.d. of two experiments determined in triplicate. *Statistically significant decrease compared to [3H]-QNB binding (P <.5). 1 c. C.1 c co no Table 2 Effects of phorbol 12,13-dibutyrate (PDBu) and forskolin (FSK) on [3H]-N-methyl scopolamine ([3H]-NMS) binding in canine colonic circular smooth muscle cells KD (nm).2 ±.4 BmXa 74,61 ± 6223 (sites/cell) PDBu FSK.21.7 *56,336 ± 582 * ,27 ± 458 Saturation binding of [3H]-NMS to intact colon circular smooth cells was measured in the absence () or presence of PDBu (1 fim, 3 min) and FSK (1 fim, 1O min). KD and B. values were determined by Scatchard analysis. Data are means ± s.e.mean of three experiments determined in triplicate. Receptor density on control cells = 12 muscarinic receptors per #tm2 and is 6% of that determined using [H]-QNB (see discussion and data of Zhang et al., 1991). *Statistically significant changes compared to control (P<.5). pirenzepine; -3.5 gsm) and M3 (high affinity for pirenzepine; -25 nm) muscarinic receptors in the membrane homogenate of colonic circular smooth muscle. Pirenzepine competed completely for specific [3H]-NMS binding in intact cells (Figure 4a). Under control conditions, competition data were best-fit when analyzed according to a two-site model (F<.1). Thus, in control cells, pirenzepine interacted with an almost equal proportion of high- (M3) and low-affinity (M2) sites in a ratio of high to low affinities of 2:3 (Table 3). When the cells were pretreated with PDBu (1 JAM, 3 min), however, there was a marked rightward shift and steepening in the pirenzepine competition curve (Figure 4a) associated with a 15% decrease in bound radioactivity. Analysis of these competition curves (Table 3) confirmed that pirenzepine bound preferentially to a single low-affinity site (K1 = 27 ± 3 nm). The loss of high-affinity binding sites for pirenzepine on the surface of PDBu-pretreated cells suggests that activation of PKC may cause preferential sequestration of M3 muscarinic receptors. These sequestered or internalized receptor sites may not be accompanied by a significant change in the number of total receptor sites (Galper et al., 1982; Feigenbaum & El-Fakahany, 1985), or such sites could be degraded or unavailable to radioligand and thus, total cellular receptor number decreased (Siman & Klein, 1979; Shifrin & Klein, 198). To determine whether M3 muscarinic receptors sequestered from the cell-surface following a short-term exposure to PDBu (3 min) were degraded intracellularly, we examined total cellular receptor number using the lipophilic muscarinic antagonist radioligand [3H]-QNB. In untreated cells, pirenzepine competition curves were again best-fit log [Pirenzepinel (M) Figure 4 Effects of phorbol 12,13-dibutyrate (PDBu) on pirenzepine competition of [HJ-N-methylscopolamine ([3H]NMS) (a) and [3H]- quinuclidinyl benzilate ((H]-QNB) (b) binding in intact colonic circular smooth muscle cells. Competition of [3H]-NMS (.2 nm) and [H]-QNB (.1 nm) binding to intact smooth muscle cells by the M3-more selective muscarinic antagonist pirenzepine was analyzed by nonlinear computer-based methods. (e) ; (e) cells pretreated with I $M PDBu for 3 min prior to start of equilibrium binding. Data, normalized to binding in the absence of competitor (1%) and that occuring in the presence of 1iM atropine (%), are expressed as the percentage of maximal radioligand binding for each radioligand ([3H]-NMS = 965 d.p.m. per 15 cells; [H]-QNB = 128 d.p.m. per 11 cells). In the absence of PDBu pretreatment, competition curves are best fit, assuming the presence of two receptor binding sites. Competition of V3H]-NMS binding in (a) following PDBu pretreatment of cells is significantly reduced (15%) and now best-fit assuming the presence of a single receptor site. Competition of [H]-QNB binding to cells (b) is unaltered by PDBu pretreatment. The binding parameters determined in several such experiments are summarized in Table 3. assuming the presence of two receptor sites (Figure 4b; Table 3). The equilibrium dissociation constant determined for the high-affinity pirenzepine binding site was similar to that seen when [3H]-NMS was employed. However, the binding was significantly greater (Table 3) and the relative contribution of high-affinity binding sites (M3) to total binding sites (M3 + M2) was lower for pirenzepine competition with [3H]- QNB (26%) compared to pirenzepine competition with [3H]- NMS (42%; Table 3). Furthermore, pirenzepine competition was not altered in cells exposed first to PDBu, suggesting that sequestered receptors are not quickly degraded. These results show that the relative distribution of M2 and M3 muscarinic receptors on the cell surface is different from that inside the cell with the proportion of surface M3 receptors being about equal to that of surface M2 receptors. Effects ofphorbol 12,13-dibutyrate andforskolin treatment on agonist binding One of the steps involved in protein kinase-regulated receptor response is the uncoupling of receptors from G-protein,

6 618 L. ZHANG & I.L.O. BUXTON Table 3 Effects of phorbol 12,13-dibutyrate (PDBu) on pirenzepine binding to intact canine colonic circular smooth muscle cells [3H]-NMS PDBu [3H]-QNB PDBu Kjh(nM) Kl(,uM) 1.3 ± ±.5.27 ±.3 Rh (%) 42± ± ± ± ± ±.39 3 ± 3.5 RI (%) 58 ± ± ± 3.5 Pirenzepine affinities (Kjh, K11), and proportions of high- (Rh%) and low-affinity sites (RI%) in antagonist competition binding for [3H]-N-methylscopolamine ([3H]-NMS,.18 nm) and [3H]-quinuclidinyl benzilate (ph]-qnb,.1 nm) binding in intact colonic circular smooth muscle cells (16 cells per tube) were determined by computer fit as described in the text. Cells were preincubated with PDBu (I JLM) at 32 C for 3 min. In the presence of PDBu, pirenzepine competition data were significantly (F-test) better-fit assuming the presence of a single low affinity site for PH]-NMS. Total specific binding (1%) in cells incubated with [3H]-NMS at.2 nm was 3.7 ±.12 fmol per 15 cells, while [3H]-QNB binding was 9.9 ±.47 fmol per 1i cells under similar conditions. Data are the mean ± s.e.mean of four experiments performed in triplicate. Table 4 Effects of phorbol 12,13-dibutyrate (PDBu) and forskolin (FSK) on agonist binding to colonic circular smooth muscle muscarinic receptors FSK PDBu FSK + PDBu -GTPyS Klh(nM) K11( 11M) 24.1 ± ± ± ± ± ± ± ± 4.9 Rl(%) 43.2± ± ± ± 5.7 +GTP-tyS 1 1M Klh(nM) K11(nM) Rl(%) 137 ± ± ± 1 59 ± ± ± ± ± ± ± ± ± 8. Carbachol affinities (Kjh, K11) and proportion of high- (Rh%) and low-affinity sites (RI%) in agonist competition for [3H]-QNB (.2 nm) binding in colonic membranes prepared as described in the text, were determined in the absence or presence of I IM PDBu and/or I plm FSK. Data are mean values ± s.e.mean of two to seven experiments determined in triplicate. evidenced by the loss of high-affinity guanine nucleotidesensitive agonist binding (Lefkowitz & Caron, 1988). Therefore, the effects of PDBu and FSK treatment on the ability of the muscarinic receptor agonist CCh to compete for [3H]-QNB binding in the absence and presence of the stable GTP analogue GTP'yS was tested in colonic circular smooth muscle. As might be expected in the untreated tissue, competition curves were shallow and best-fit by a two-site model (Figure 5). In the absence of GTPyS, the relative contribution of the high-and low-affinity agonist binding sites was about equal (Table 4). In the presence of a maximally effective concentration of GTP'yS (1 pm), the CCh competition curve was significantly shifted to the right and the population of high-affinity agonist binding sites decreased from 57% to 15% of sites (Figure 5a; Table 4). Pretreatment of tissues with FSK (1 gtm, O min) had no effect on either high- or low-affinity CCh binding sites or the effect of GTPyS on the agonist competition (Figure Sb; Table 4). Similar results were obtained with PDBu treatment or the combination of FSK and PDBu (Table 4) CO 6 a ~4- )R log [Carbachol] (M) Figure 5 Effects of forskolin (FSK) on agonist competition of [3H]- quinuclidinyl benzilate ([3H1-QNB) binding in membranes of canine colon circular smooth muscle. Competition of [3H-QNB (.1 nm) binding by the muscarinic agonist carbachol was determined in control (a) and FSK (b)-treated membranes of colonic smooth muscle as described in Methods. Competition curves were generated in the absence () and presence () of a saturating concentration of GTPyS (1pM). Data were analyzed by nonlinear computer-based methods which yielded best-fits assuming two components of agonist binding in all cases. Values, normalized to binding in the absence of competitor (1%) and that occurring in the presence of 1 #M atropine (%), are expressed as the percentage of maximal PH]-QNB binding. Binding parameters determined in several such experiments and in the presence of PDBu and the combination of PDBu plus FSK are summarized in Table 4. b Discussion We have examined the relationship between two second messengers, cyclic AMP and Ins-1,4,5-P3, and the two protein kinases, PKC and cyclic AMP-PK, that are the consequence of elevation of these second messengers in canine colonic circular smooth muscle. Results presented have demonstrated that the activation of either PKC or cyclic AMP-PK partially blocks muscarinic receptor mediated PI hydrolysis. Although the combined effect of activation of PKC and cyclic AMP-PK was greater than activation of either kinase alone, the combined result is not strictly additive. Since we employ conditions for the activation of each kinase that we believe to be maximal, the data may suggest the involvement of distinct mechanisms underlying the inhibition of PLC activity. Stimulation of PI turnover is a critical intracellular signalling pathway linked to activation of muscarinic receptors in many tissues and is thought to play an important role in the contraction of smooth muscle. We have recently demonstrated the presence of both M2 and M3 muscarinic receptor subtypes in the canine colonic circular smooth muscle (Zhang et al., 1991). Activation of the M3 muscarinic receptor mediates PI hydrolysis (Zhang & Buxton, 1991) and activates PKC. Activation of PKC results in phosphorylation of numerous protein substrates, and such action has been linked to a wide variety of biological responses, including negative feedback on PI turnover (El-Fakahany et al., 1988). In the

7 MUSCARINIC RECEPTOR SIGNALLING IN SMOOTH MUSCLE 619 present study, preincubation of colonic circular smooth muscle with PDBu resulted in inhibition of the PI response to CCh. The concentration of PDBu used for these effects elicted a significant increase of membrane-bound PKC activity. In addition, 4oc-phorbol dideconoate, which does not activate PKC, was ineffective in blocking Ins-1,4,5,-P3 accumulation. These results suggest that the observed effects of PDBu treatment are due to activation of PKC. Although hormonal regulation of adenylyl cyclase is not affected by PKC, activation of adenylyl cyclase by FSK does affect hormonal regulation of PLC. It has been well documented that PLC-mediated hydrolysis of PIP2 is affected by cyclic AMP-dependent phosphorylation. Elevations in cyclic AMP have been shown to inhibit agonist-dependent PI turnover in many cell types (Watson et al., 1984; Neylon & Summers, 1988; Manolopoulos et al., 1991) and to augment it in others (Blackmore & Exton, 1986; Olashaw & Pledger, 1988). While we measure no effect of cyclic AMP-PK activation on basal PLC activity in colon, such an effect has been measured in C6Bul cultured cells (Ehlert & Tran, 199). Decreased Ins-1,4,5-P3 formation following cyclic AMP elevation suggests that activation of cyclic AMP-PK may regulate PKC activity by decreasing the availability of DAG which would serve to assist translocation of the enzyme to the membrane. Indeed, McAtee & Dawson (1989) demonstrated that DAG was lowered substantially in NCB-2 cells treated with FSK and that this was associated with translocation of PKC into the cytosol. Interestingly, the combined effects of forskolin and PDBu treatment on CCh stimulated Ins-1,4,5-P3 formation in colonic smooth muscle were greater than their individual effects. This result may suggest the involvement of the distinct sites by which forskolin and PDBu inhibit PLC activity. Recently, it has been demonstrated that PKC and cyclic AMP-PK phosphorylate disparate PLC isozymes (Kim et al., 1989; Ryu et al., 199). Treatment of, C6Bul cells with FSK increased serine phosphate in PLCI, while the phosphate content of PLCP and PLC6 were unchanged. In contrast, treatment of the cells with phorbol ester stimulated the phosphorylation of serine residues in PLCP, while the phosphorylation state of PLCy and PLC6 were unchanged. Reasoning that the absence of obvious membrane spanning domains in the known sequences of PLCP, I and 6 isoforms of PLCs means that the enzyme must bind to the membrane prior to hydrolysis of its substrate, PIP2; it is possible that this event is the target of PKC and cyclic AMP-PK phosphorylation. It is clear that PLC is not the only target protein likely to be phosphorylated by PKC and cyclic AMP-PK. Phosphorylation has also been implicated in the regulation of muscarinic receptors. The purified Ml receptor can be phosphorylated in vitro by both PKC and cyclic AMP-PK and the purified M2 receptor can be phosphorylated by cyclic AMP- PK (Rosebaum et al., 1987; Haga et al., 1988). Phosphorylation of muscarinic receptors can be correlated with either a decrease in affinity or with sequestration of the receptor. In neuroblastoma cells, muscarinic receptors are rapidly sequestered following activation of PKC (Liles et al., 1986) while increasing cellular cyclic AMP leads to no such effect (Scherer & Nathanson, 199). The present study demonstrates that activation of PKC results in a partial loss of muscarinic receptors from the surface of colonic circular smooth muscle cells within 3 min. [H]-NMS labelled 22% fewer sites than (3H]-QNB in freshly isolated colonic smooth muscle cells (see Table 1). A similar result was obtained in freshly isolated intact chick heart cells (Brown & Goldstein, 1986). This suggests that muscarinic receptors are present at both cell surface and sequestered sites in colonic smooth muscle cells. Although it has been reported that in cultured human neuroblastoma cells all muscarinic receptors are accessible to [3H]-NMS binding at 37 C (Fisher, 1988), this is not the case in freshly isolated smooth muscle cells. Pretreatment of colonic circular smooth muscle with PDBu decreased [3H1-NMS maximal binding to cell surface receptors but did not affect their affinity for radioligand. We have determined that the relative distribution of M2 and M3 receptors between cell surface and cell interior is different. Our previous results with a lipophilic radioligand in tissue membranes (Zhang et al., 1991) demonstrated that 2% of muscarinic receptors were of the M3 subtype, but could not assign their location. The [3HJ-NMS binding in the intact smooth muscle cells shows that the relative density of M3 muscarinic receptors is higher on the cell surface than the cell interior suggesting the relative importance of M3 receptor signalling in the contractile response to acetylcholine in vivo. In fact, when the extent of [H]-QNB binding to cells determined to be M3 sites is reconciled with the extent of [H]- NMS bound and predicted to be M3 sites (Table 3), it is apparent that the absolute number of such sites is similar and thus, argues that most of the M3 binding sites are on the cell surface. In accordance with published reports (Lai & El- Fakahany, 1986; Abdallah et al., 199; Lai et al., 199), our results demonstrate that activation of PKC selectively decreases high-affinity pirenzepine binding sites from the smooth muscle cell surface. Our reasoning that M2 muscarinic receptors on colonic cells are not affected by PKC is supported by the observation that the purified M2 muscarinic receptor from porcine atria is not phosphorylated by purified PKC (Rosenbaum et al., 1987; Haga et al., 1988). The M3 receptors sequestered from the surface of the colonic smooth muscle cell surface following PDBu treatment are not rapidly degraded since [H]-QNB binding is not affected by PDBu. Such a result is consistent with the notion that cells modulate their responsiveness to activation of protein kinase C, acutely, by removing receptors from the extracellular environment. In contrast to our findings in cells treated with PDBu, increasing cellular cyclic AMP content following FSK treatment decreased the affinity of cell surface receptors for [H]- NMS without affecting maximal binding (see Table 2). The effects of FSK treatment were most likely due to cyclic AMP-PK activation since incubation of FSK with cell membranes in the radioligand binding assay failed to affect binding. Non-specific affects of isobutyl methylxanthine (IBMX), the cyclic AMP-phosphodiesterase inhibitor used to ensure sustained elevation of cyclic AMP, were ruled out since it had no effect in the absence of FSK. Although we do not know the identity, M2 vs. M3, of the receptors whose affinity is altered by FSK treatment, it is known that M2 receptors can be phosphorylated by cyclic AMP-PK in vitro and exist as a phosphoprotein in intact tissue (Rosenbaum et al., 1987; Kwatra et al., 1989). Pretreatment of human neuroblastoma cells with phorbol ester converted multiple agonist binding sites to a single site with low affinity for agonist (Serra et al., 1986). In colonic circular smooth muscle, despite effects of PDBu and FSK on antagonist radioligand binding and PLC activity, such treatment does not eliminate high-affinity agonist binding. Indeed, the lack of effects. of PDBu and FSK added together on GTP-dependent agonist binding suggests that the receptor effects of activation of PKC and cyclic AMP-PK in this tissue do not occur at the level of guanine nucleotide binding protein:receptor interactions. These data are in agreement with findings in brain in which PDBu does not alter agonist binding (Abdallah et al., 199). In addition, Rosenbaum et al. (1987) demonstrated that phosphorylation of atrial M2 receptors did not alter either agonist binding or the GTPase activity of Gi. These results, taken together with our own, suggest the notion that receptor phosphorylation serves to target receptors for events such as sequestration or internalization rather than regulating agonist binding. This notion is further supported by data showing that receptor phosphorylation is required for receptor internalization (Scherer & Nathanson, 199). Our results also provide for the possibility that the effects of activation of PKC and cyclic AMP- PK contribute to altered interaction between the activated G-protein and PLC.

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9 MUSCARINIC RECEPTOR SIGNALLING IN SMOOTH MUSCLE 621 SMITH, T.K., WARD, S.M., ZHANG, L., BUXTON, I.L.O., GERTHOF- FER, W.T., SANDERS, K.M. & KEEF, K.D. (1992). P-Adrenergic inhibition of electrical and mechanical activity in canine colon: Role of cyclic AMP. Am. J. Physiol. (in press). SOMLYO, A.P. & HIMPENS, B. (1989). Cell calcium and its regulation in smooth muscle. FASEB J., 3, WATSON, S.P., MCCONNELL, R.T. & LAPETINA, E.G. (1984). The rapid formation of inositol phosphates in human platelets by thrombin is inhibited by prostacyclin. J. Biol. Chem., 259, ZHANG, L. & BUXTON, I.L.O. (1991). Muscarinic receptors in canine colonic circular smooth muscle II. Signal transduction pathways coupled to the muscarinic receptors. Mol. Pharmacol., 4, ZHANG, L., HOROWITZ, B. & BUXTON, I.L.O. (1991). Muscarinic receptors in canine colonic circular smooth muscle I. Coexistence of M2 and M3 subtypes. Mol. Pharmacol., 4, (Received July 21, 1992 Revised October 6, 1992 Accepted October 16, 1992)