Inhibition of Muscarinic-Stimulated Polyphosphoinositide Hydrolysis and Ca2+ Mobilization in Cat Iris Sphincter Smooth Muscle Cells By cAMP-Elevating Agents

Cell. Signal. Vol. 9, No. 6, pp. 411–421, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0898-6568/97 $17.00 PII S0898-6568(97)00018-1

Inhibition of Muscarinic-Stimulated Polyphosphoinositide Hydrolysis and Ca21 Mobilization in Cat Iris Sphincter Smooth Muscle Cells By cAMP-Elevating Agents Ke-Hong Ding,† Shahid Husain,† Rashid A. Akhtar,† Carlos M. Isales‡ and Ata A. Abdel-Latif†* †Department of Biochemistry and Molecular Biology and ‡Institute for Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-2100, USA

ABSTRACT. The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca21]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 mM, respectively. The muscarinic agonist provoked a transient increase in [Ca21 ]i which reached maximum within 77 s, and increased [Ca21]i mobilization in a concentration-dependent manner with an EC50 of 1.4 mM. Thapsigargin, a Ca21-pump inhibitor, caused a rapid rise in [Ca21 ]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CChinduced [Ca21]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca21]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca21]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca21 ]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 5 80 nM) and [Ca21]i mobilization (IC50 5 0.17 lM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 mM) and GTPgS (0.1 mM). Pretreatment of the cells with ISO or forskolin, 5 mM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTPgS-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca21]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca21 ]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes. cell signal 9;6:411–421, 1997  1997 Elsevier Science Inc. KEY WORDS. Iris sphincter smooth muscle cells, Carbachol, Phosphoinositide metabolism, Intracellular calcium, Isoproterenol, Cyclic AMP

INTRODUCTION It is well established now that in smooth muscle, cAMPelevating agents, such as b-adrenergic agonists, cAMP phosphodiesterase inhibitors, and E-type prostaglandins, inhibit agonist-stimulated hydrolysis of the polyphosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), Ca21 mobilization and contraction. A large body of evidence has recently accumulated, in both vascular and *Author to whom all correspondence should be addressed. Received 25 July 1996; and accepted 30 September 1996.

nonvascular smooth muscle, which indicates that cross talk between the cAMP and the polyphosphoinositide signalling cascade plays an important role in the functional antagonism between the sympathetic and parasympathetic nervous systems [1–5]. Activation of muscarinic M3 receptors in smooth muscle results in phospholipase C (PLC)-mediated hydrolysis of PIP2 into IP3 and DAG and muscle contraction. IP3 releases Ca21 from the sarcoplasmic reticulum (SR) which produces a transient rise in [Ca21]i and initiates the rapid phase of the contractile response. Muscarinic receptor-mediated Ca21 influx causes a rise in [Ca21]i which is involved in the sustained phase of muscle contraction [6].

412

The receptor-stimulated influx of Ca21 is thought to cross the plasma membrane in part through a family of poorly characterized voltage-insensitive Ca21 channels [7]. Elevation of intracellular cAMP concentration leads to activation of protein kinase A (PKA) which is involved in smooth muscle relaxation, and may involve phosphorylation of a number of effector proteins that cause either reduction of [Ca21]i and/or reduction of myosin light chain kinase (MLCK) sensitivity to Ca21-calmodulin [2, 4, 8, 9]. Cyclic AMP-elevating agents, such as isoproterenol (ISO) and forskolin, have been reported to cause a marked attenuation of histamine-induced inositol phosphates formation in tracheal smooth muscle [9–12], of carbachol (CCh)-induced IP3 production in bovine iris sphincter smooth muscle [13, 14], of CCh-induced IP3 production and Ca21 mobilization in cultured canine tracheal smooth muscle cells [5], and of CCh-induced Ca21 mobilization in airway smooth muscle cells [9]. The site and mechanism of cAMP inhibition of agonist-induced IP3 production, Ca21 mobilization and contraction remain unclear [1–5, 9]. Possible target sites for PKA modulation of the CCh-induced responses include phosphorylation of the muscarinic receptor, G protein, PLC, IP3 metabolizing enzymes and/or IP3 receptor [4]. In the iris sphincter smooth muscle elevation of intracellular cAMP concentration results in attenuation of IP3 production and muscle contraction [13, 14]. Whether the inhibitory effect of cAMP on CCh-induced muscle contraction occurs at the level of IP3 production or at the level of Ca 21 mobilization is unclear. The effects of cAMP elevating agents on agonist-induced Ca21 mobilization in the iris sphincter has not yet been investigated. In the present study, we addressed these questions by first characterizing the effects of CCh on IP3 production and [Ca21]i mobilization in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and then investigated the effects of cAMP-elevating agents on these responses. These cells retain morphological characteristics of primary cultured cat iris sphincter smooth muscle cells and are capable of responding to CCh stimulation by eliciting increased PIP2 hydrolysis, arachidonic acid release and MLC phosphorylation [15]. We found that the kinetics of Ca 21 mobilization correlate well with those of IP3 production and that cAMP attenuates muscarinic receptor-mediated PIP2 hydrolysis and Ca 21 mobilization.

MATERIAL AND METHODS Materials L-a-phosphatidylserine (PS) and L-a-phosphatidylethanolamine (PE) were purchased from Avanti Polar Lipids, Alabaster, AL. L-a-phosphatidylinositol 4,5-bisphosphate (PIP2), leupeptin, aprotinin, phenylmethanesulphonyl fluoride (PMSF), carbachol, atropine, thapsigargin, catalytic subunit of PKA (bovine heart), isoproterenol, forskolin, and ryanodine were obtained from Sigma Chemical Co., St. Louis, MO. Pirenzepine was from Boehringer Ingelheim, Ridgefield, CT, and 4-diphenylacetoxy N-methyl-piperi-

K. -H. Ding et al.

dine (4-DAMP) and U73122 (PLC inhibitor) were obtained from Research Biochemical International, Natick, MA. Fura-2 acetoxymethyl ester (fura-2/AM) was obtained from Molecular Probes, Eugene, OR. Growth media and other reagents needed for cell culture were obtained from Gibco/Life Technologies Ltd., Grand Island NY, Myo[3H]inositol (specific radioactivity 15.5 Ci/mmol) was purchased from Amersham, Arlington Heights, IL, and [3H]PIP2 (specific radioactivity 5.4 Ci/mmol) were obtained from DuPont New England Nuclear, Boston, MA. Methods The SV-CISM-2 cells were maintained in DMEM supplemented with 10% foetal calf serum and 50 mg/ml gentamicin (culture medium) as described previously [15]. To initiate subculture, the confluent SV-CISM-2 cells were washed with Ca21-Mg21-free Dulbecco phosphate buffer and treated with 0.05% trypsin in 0.5 mM EDTA for 3 min at 378C. This was followed by addition of Dulbeccomodified Eagle‘s medium (DMEM) and the cell suspension centrifuged at 200 g for 5 min. The pelleted cells were suspended in DMEM supplemented with 10% foetal calf serum (FCS) and 50 mg/ml gentamicin. The cells were seeded in 6-well plates for PLC assay and in 35 mm dishes for [Ca21 ]i mobilization at a split ratio of 1:4. The cultures were maintained by changing the medium every other day until the cells became confluent. CELL CULTURE.

RADIOLABELLING OF SV-CISM-2 CELLS WITH MYO-[3H] INOSITOL AND ANALYSIS OF INOSITOL PHOSPHATES. To label SV-CISM-2 cells with myo-[3H]inositol, the cells were incubated in 2 ml DMEM, without unlabelled inositol, that contained 5 mCi myo-[3H]inositol for 24 h. At the end of incubation, the cells were washed 33 with non-radioactive medium and then incubated for 5 min in 2 ml medium containing 10 mM LiCl. At this time, CCh or other agents were added and incubation continued for various time intervals as indicated. When the effects of ISO or muscarinic antagonists were to be examined, the drugs were added to the cells 5 min prior to the addition of CCh. The reactions were terminated by aspirating the medium and adding 1 ml ice-cold 10% (w/v) TCA. The cells were scraped off the wells and centrifuged at 1,000 g for 10 min. The supernatant containing inositol phosphates was washed 43 with an equal volume of diethyl ether and analysed by means of anionexchange chromatography as described previously [16]. MEMBRANE PREPARATION AND ASSAY OF PLC. To prepare the membrane fraction, the confluent SV-CISM-2 cells were washed with ice-cold PBS, scraped and then pelleted by low-speed centrifugation. The procedure used to prepare the microsomal fraction was the same as described previously [17]. Briefly, the cell pellet was resuspended in 20 mM Tris-HCl (pH 7.4) containing 1 mM EGTA, 1 mM EDTA, 5 mM DTT, 0.3 M Sucrose, 2 mM PMSF, 10 mg/ ml leupeptin and 10 mg/ml aprotinin. The cells were homogenized and centrifuged at 600 g for 15 min. The cell de-

cAMP and Muscarinic Stimulation of IP3 and Ca21 Mobilization

bris was discarded and the supernatant centrifuged at 100,000 g for 90 min. The pelleted membranes were resuspended in the homogenization buffer and protein content determined by the method of Lowry et al. [18]. Suitable aliquots of the membrane fraction were frozen at 2808C until used in the PLC assay. PLC was assayed as described previously [19]. The assay was conducted at 378C for 10 min. The reaction mixture contained 20 mM Hepes buffer (pH 7.0), 0.1 M NaCl, 1.5 mM MgCl2, 1 mM EGTA, 0.2 mM free Ca21 and 30 mM [3H] PIP2 (specific radioactivity 1000 dpm/nmol) in a total volume of 100 ml. The PIP2 was added to the assay mixture in the form of lipid vesicles prepared as described previously [19]. The low concentration of free Ca21 in the assay mixture was maintained by using Ca 21/ EGTA buffer. The free Ca21 concentration was calculated using the Bathe Constituent program [20]. The reaction was started by adding approximately 15 mg membrane protein and incubation conducted for 10 min at 378C. The reaction was terminated by adding 0.5 ml chloroform/ methanol/ HCl (100:100:0.6, by vol) followed by addition of 0.15 ml 1 M HCl containing 5 mM EGTA. The mixture was vortexed and the phases separated by centrifugation. Suitable aliquots of the upper aqueous phase containing inositol phosphates were taken for determination of radioactivity by liquid scintillation counting.

413

FIGURE 1. Time-course of CCh-induced IP3 production in SV-

CISM-2 cells. Confluent SV-CISM-2 cells were prelabelled with [3H]inositol as described under Materials and Methods. CCh (10 mM) was then added and incubations continued for various time intervals as indicated. The incubations were terminated by adding 10% TCA and the water-soluble inositol phosphates analysed by anion-exchange chromatography. The results are expressed as percentage of control (without CCh), and each data point represents the mean 6 SEM of triplicate determinations of two separate experiments.

PHOSPHORYLATION OF MEMBRANE FRACTION BY CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE A.

Phosphorylation of membrane fractions prepared from SVCISM-2 cells was performed as described previously [21]. Briefly, the membrane fraction (100-200 mg protein) was incubated at 308C in a total volume of 100 ml buffer that contained 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 80 mM EDTA, 3 mM MgCl2, 3 mM DTT and 10-50 units of PKA (catalytic subunit). The reaction was started by the addition of 50 mM ATP (final concentration). After 30 min of incubation, 10 ml of the reaction mixture was immediately used to assay for the PLC activity. MEASUREMENT OF CYTOSOLIC CA21 CONCENTRATION.

SV-CISM-2 cells were subcultured on glass coverslips until the cells became confluent. [Ca21 ]i was measured in confluent monolayers with the calcium-sensitive dye fura-2/AM as described previously [22]. Briefly, the cells were washed once with 2 ml Krebs-Ringer bicarbonate buffer (KRB; pH 7.4) containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 10 mM dextrose and 1.25 mM CaCl2. Following this, the cells were loaded with 5 mM fura-2/AM in KRB buffer at room temperature for 45 min. At the end of the loading period, the coverslips were washed twice with KRB buffer and then incubated in KRB buffer without fura-2/AM for another 30 min. The coverslip was inserted into a quartz cuvette in a dual-wavelength spectrofluorimeter (Photon Technologies International, South Brunswick, New Jersey). Fluorescence of Ca21-bound and unbound fura-2 was measured at room temperature by rapidly alternating the dual excitation

wavelengths between 340 and 380 nm and electronically separating the resultant fluorescence signals at an emission wavelength of 510 nm. Autofluorescence was measured in unloaded cells and this value was subtracted from all the measurements. Minimum emission (Rmin) was measured upon addition of EGTA (8 mM) containing Tris buffer (pH 8.1) followed by addition of ionomycin (10 mM). Maximum emission (Rmax) was measured by adding 20 mM CaCl2 to the cuvette. Free [Ca21]i concentration was calculated using the equation: [Ca21] 5 Kd 3 (F380 free/F380 sat) 3 (R-Rmin)/ (Rmax-R). The Kd of fura-2 for Ca 21 was assumed to be 224 nM [23]. When [Ca21]i mobilization was to be measured in Ca21-free medium, 1.25 mM CaCl2 was replaced by 0.1 mM EGTA in the Ca21 mobilization buffer. DATA ANALYSIS. Each well was considered an n of 1. Comparisons between two treatment groups were performed by the unpaired t-test and a p , 0.05 was considered significant. Unless otherwise stated, the data are mean 6 SEM of two experiments with triplicate incubations for each data point.

RESULTS CCh-induced IP3 production in SV-CISM-2 cells TIME-COURSE STUDIES. To investigate the effects of CCh on IP3 production, the cells were labelled with myo[3H]inositol and changes in water soluble inositol phosphates analysed by anion-exchange chromatography. Fig. 1 shows the time course of CCh-induced IP3 accumulation in the cells. After 1 min of incubation with the muscarinic

414

K. -H. Ding et al.

FIGURE 2. Concentration-response effect of CCh on IP3 pro-

duction in SV-CISM-2 cells. SV-CISM- 2 cells prelabelled with [3H]inositol were incubated with different concentrations of CCh for 10 min. The water-soluble inositol phosphates were extracted and analysed for radioactivity. The data are means 6 SEM of two separate experiments each run in triplicate.

agonist, IP3 formation was increased by 18%, and after 5 min it increased by a maximal value of 35%. There was no change in IP3 formation after 10 min of incubation. The t1/2 (effective time for half-maximal response) value for IP3 accumulation was 68 s. The results of studies on the effects of different concentrations of CCh on IP3 accumulation are shown in Fig. 2. A significant increase in IP3 production was observed at 0.1 mM CCh, and increasing the agonist concentration provoked a corresponding increase in IP3 accumulation, reaching maximum at 10 mM. At 25 mM CCh, IP3 accumulation was somewhat decreased, this could be due to desensitization of the muscarinic receptors. The EC50 (effective concentration for half-maximal response) value for IP3 accumulation was 0.5 mM. All subsequent experiments were performed using 10 mM CCh with an incubation time of 10 min. CONCENTRATION-RESPONSE STUDIES.

EFFECTS OF MUSCARINIC ANTAGONISTS. To examine the involvement of muscarinic receptor subtypes, the effects

FIGURE 3. Effect of CCh on [Ca21]i mobilization in SV-CISM-2

cells. The SV-CISM-2 cells, grown as monolayer on glass cover slip, were loaded with 5 mM fura-2/AM for 45 min at room temperature, washed, and then incubated in KRB buffer that contained 1.25 mM Ca21. The fluorescence of the Ca21 bound and unbound fura-2 was measured using a dual-wavelength spectrofluorimeter as described in the Materials and Methods section. Spectrofluorimeter tracing shows the time course of the increase in [Ca21]i concentration following the addition of CCh. The inset shows a trace from an experiment in which SV-CISM-2 cells were stimulated with CCh in a Ca21-free KRB buffer.

of muscarinic receptor antagonists were investigated. As shown in Table 1, 1 mM atropine, a non-selective muscarinic receptor antagonist, abolished the CCh-stimulated increase in IP3 formation, thus suggesting the involvement of cholinergic muscarinic receptors. To investigate further the muscarinic receptor subtype(s) mediating CCh-induced IP3 accumulation, the effects of muscarinic receptor subtypeselective antagonists were employed. As shown in Table 1, at equimolar concentrations, 4-DAMP (M3-selective) was more potent than pirenzepine (M1-selective) in inhibiting IP3 accumulation. The data presented demonstrate that CCh produces a time- and concentration-dependent rise in IP3 production in SV-CISM-2 cells, and that it stimulates PLC activity in these cells through activation of the M3 muscarinic receptor subtype.

TABLE 1. Effects of atropine, pirenzepine and 4-DAMP on CCh-induced IP3 produc-

tion in SV-CISM-2 cells Addition None CCh (10 CCh (10 CCh (10 CCh (10

mM) mM) 1 atropine (1 mM) mM) 1 pirenzepine (0.1 mM) mM) 1 4-DAMP (0.1 mM)

Radioactivity in [3H]IP3 (dpm/mg protein)

% of control

6 6 6 6 6

100 131 113 121 111

448 586 506 542 497

11 15 6 9 4

SV-CISM-2 cells prelabelled with [3 H]inositol were incubated with 10 mM CCh for 10 min as described in the Methods section. When used, the muscarinic antagonists were added 5 min before CCh. The inositol phosphates were extracted and analysed for radioactivity by anion-exchange chromatography. The data are means 6 SEM of two separate experiments each run in triplicate.

cAMP and Muscarinic Stimulation of IP3 and Ca21 Mobilization

FIGURE 4. Concentration-response effect of CCh on [Ca21]i in

SV-CISM-2 cells. The SV-CISM-2 cells were loaded with fura2/AM and stimulated with different concentrations of CCh. (A) Curves obtained following stimulation with different concentrations of CCh have been superimposed. The CCh concentrations used were: (a) 0.1 mM; (b) 0.5 mM; (c) 1 mM; (d) 10 mM; (e) 25 mM. (B) Peak [Ca21]i concentration at different CCh concentrations have been plotted. Each data point represents net increase in [Ca21]i and is an average of three determinations.

CCh-induced [Ca21]i mobilization in SV-CISM-2 cells TIME-COURSE STUDIES. The elevation of [Ca2 1]i after receptor stimulation is biphasic, consisting of an initial rapid spike of [Ca21]i release, the result of Ca21 mobilization from intracellular stores mediated mainly by IP3 generation [24], which is followed by a sustained extracellular Ca21 influx. To demonstrate a relationship between CCh-induced IP3 generation and CCh-induced [Ca21 ]i mobilization in SVCISM-2 cells, we have investigated the latter in these cells. Fig. 3 shows the response of SV-CISM-2 cells, loaded with the fluorescent [Ca21 ]i indicator fura-2/AM, to the addition of 1 mM CCh in a complete incubation medium (containing 1.25 mM Ca 21). 10-20 s after the CCh addition, [Ca21]i increased from an average basal value of 50

415

FIGURE 5. Effect of thapsigargin and ryanodine on CChinduced [Ca21]i mobilization in SV-CISM-2 cells. The fura2/AM loaded cells were treated with (A) 2 mM thapsigargin and (B) 100 mM ryanodine and then stimulated with 10 mM CCh as shown in the figure.

nM to an average peak value of 350 nM (seven-fold increase). Following the initial rise in [Ca21]i, which reached maximum within 70-77 s, a delayed and sustained increase was observed. Thus, even after 20 min from the CCh addition, the level of [Ca21]i remained significantly higher (z80 nM) than that of the basal level. However, when the experiment was carried out in Ca21 -free medium, the CCh-induced initial rise in [Ca21]i was still present (z70%), but the sustained increase in [Ca21]i was abolished (Fig. 3, inset). The [Ca21]i level returned to the resting basal value within 2 min. These results demonstrate that the CCh-induced [Ca21]i increase in SV-CISM-2 cells has a dual origin, intracellular (mobilization from the SR) and extracellular (increased influx). CONCENTRATION-RESPONSE STUDIES. The results of studies on the effects of different concentrations of CCh on [Ca21]i mobilization are given in Fig. 4. As low as 0.5 mM

416

K. -H. Ding et al.

CCh was effective in mobilizing [Ca21]i, and increasing the CCh concentration to 10 mM markedly enhanced the peak [Ca21]i. The EC50 value for CCh-induced Ca21 mobilization was 1.4 mM. Increasing CCh concentration to 25 mM resulted in a slight attenuation of [Ca21]i mobilization; again this could be due to desensitization of the muscarinic receptors. EFFECTS OF THAPSIGARGIN AND RYANODINE. Since agonist-stimulated increase in [Ca21]i could also result from inhibition of Ca21 reuptake into SR and/or stimulation of Ca 21-induced Ca21 release from intracellular Ca21 stores, we used thapsigargin and ryanodine to investigate whether the CCh-stimulated increase in [Ca21]i in SV-CISM-2 cells was due to mobilization from IP3 sensitive Ca 21 stores. Thapsigargin has been shown to prevent the reuptake of Ca 21 into SR by inhibiting the Ca 21-pump of the SR in smooth muscle [25]. On the other hand, ryanodine depletes the intracellular Ca21 stores by maintaining the Ca21 release channel in a partially open state [26]. At high concentrations (z100 mM), ryanodine inhibits [Ca21]i release from the SR by binding to its low affinity receptors. As shown in Fig. 5, addition of thapsigargin to fura-2/AMloaded cells resulted in a rapid rise in [Ca21]i which was followed by a sustained increase in Ca21. When the cells were subsequently stimulated with CCh, there was no increase in [Ca21]i normally observed in untreated cells. The data demonstrate that once the intracellular Ca21 store is depleted by thapsigargin, the IP3 generated by CCh fails to elicit any increase in [Ca21]i . Treatment of the cells with 100 mM ryanodine had no effect on basal level of [Ca21]i, and CCh was fully effective in mobilizing [Ca21]i, suggesting that the CCh-stimulated increase in [Ca21]i was indeed due to its release from an IP3-sensitive Ca21 store. EFFECTS OF MUSCARINIC ANTAGONISTS. To investigate the muscarinic receptor subtype which mediates the CCh-induced [Ca21]i mobilization, the effects of atropine, pirenzepine and 4-DAMP were investigated. As shown in Table 2, 1 mM atropine inhibited CCh-induced peak [Ca21]i by 74%. Under similar experimental conditions, 0.1 mM 4-DAMP was more effective in inhibiting CChinduced [Ca21]i mobilization than 0.1 mM pirenzepine. These data suggest that, as with IP3 production, CChinduced Ca21 mobilization is mediated by the M3 muscarinic receptor subtype.

FIGURE 6. Concentration-response effect of U73122 on CCh-

induced IP3 production in SV-CISM-2 cells. The [3H]inositollabelled SV-CISM-2 cells were incubated with different concentrations of U73122 for 5 min prior to stimulation with 10 mM CCh for 10 min. The water-soluble inositol phosphates were isolated and analysed for radioactivity. The data shown are means 6 SEM of two separate experiments with triplicate incubations for each data point.

Effects of U73122 on CCh-induced IP3 production and [Ca21]i mobilization U73122 is a new PLC inhibitor often used to study the involvement of PLC-mediated PIP2 hydrolysis in cellular processes [27]. We used this inhibitor to determine whether the generation of IP3 in SV-CISM-2 cells causes mobilization of [Ca21]i. As shown in Fig. 6, addition of U73122 to cells pre-labelled with myo-[3H]inositol dose-dependently inhibited CCh stimulation of IP3 production. Respectively, 10 nM and 1 mM U73122 inhibited CCh- induced IP3 production by 50 and 100%. Under similar experimental conditions, U73122 inhibited CCh-induced [Ca21 ]i mobilization in a concentration-dependent manner (Fig. 7). Again, 10 nM and 1 mM U73122 inhibited CCh-induced [Ca21 ]i by 50 and 100%, respectively. U73122 had no effect on the basal levels of IP3 and [Ca21 ]i in the smooth muscle cells.

TABLE 2. Effects of atropine, pirenzepine and 4-DAMP on CCh-induced [Ca21]i

mobilization in SV-CISM-2 cells Addition CCh CCh CCh CCh

(10 (10 (10 (10

mM) mM) 1 atropine (1 mM) mM) 1 pirenzepine (0.1 mM) mM) 1 4-DAMP (0.1 mM)

Intracellular Ca21 (nM)

% Inhibition

6 6 6 6

0 74 41 52

749 195 439 363

17 8 15 19

The fura-2/AM loaded cells were stimulated with 10 mM CCh and intracellular Ca21 measured as described in the Methods section. When used, the muscarinic antagonists were added 3 min before the addition of CCh. The results are means 6 SEM of peak [Ca2 1]i concentrations reached following the addition of CCh from two different experiments each run in triplicate.

cAMP and Muscarinic Stimulation of IP3 and Ca21 Mobilization

417

Furthermore, U73343, a structural analogue of U73122, had no effect on CCh-induced IP3 production and [Ca21]i mobilization (data not shown). These data suggest that enhanced IP3 generation could be involved in the increased [Ca21]i mobilization in SV-CISM-2 cells. Effects of ISO on CCh-induced IP3 production and [Ca21]i mobilization ISO, a b-adrenergic agonist, increases cAMP level in SVCISM-2 cells [15]. To investigate whether ISO exerts an inhibitory effect on CCh-induced IP3 production and [Ca21 ]i mobilization, the cells were treated with ISO for 5 min prior to the addition of CCh. As shown in Fig. 8, when the cells were incubated with 0.1 mM ISO, the CCh-induced IP3 production was inhibited by about 60% and complete inhibition was observed at 1 mM of the adrenergic agonist. The IC50 value for ISO inhibition of CCh-induced IP3 production was 80 nM. Under similar experimental conditions, pretreatment of the cells with 0.5 mM ISO resulted in complete inhibition of the CCh-stimulated transient increase in [Ca21]i (Fig. 9). However, the late sustained increase in [Ca21]i was only partially decreased by ISO treatment. When the cells were incubated with different concentrations of ISO, a dose-dependent inhibition of CCh-induced [Ca21]i mobilization was observed with an IC50 value of 0.17 mM. As with ISO, treatment of the cells with forskolin, which directly stimulates adenylate cyclase, resulted in inhibition of CCh-induced IP3 production and [Ca21]i mobilization (data not shown). To rule out the possibilty that ISO may interfere with the storage and/or release of Ca 21, experiments were carried out to determine if ISO could inhibit the elevation of [Ca21 ]i induced by thapsigargin. As shown in Fig. 10, when the cells were preincubated with ISO and then stimulated with thapsigargin, the thapsigargininduced [Ca21]i increase was not affected. Similarly, the thapsigargin-induced [Ca21]i increase was not inhibited by U73122. These data suggest that in SV-CISM-2 cells, ISO exerts its inhibitory effects at a site proximal to CCh-induced IP3 generation and [Ca21]i mobilization. Effects of CCh on PLC activity in membrane fractions isolated from ISO-treated SV-CISM-2 cells To confirm that in SV-CISM-2 cells ISO exerts its inhibitory effects at a site proximal to CCh-induced IP3 generation and Ca 21 mobilization, PLC was assayed in membrane fractions prepared from cells that were treated with ISO. As shown in Table 3, addition of 10 mM CCh to microsomal fractions prepared from untreated cells increased IP3 production by 53%. However, when CCh was added to membranes isolated from ISO-treated cells, IP3 production was inhibited by 55%. Similar results were obtained in membrane fractions prepared from forskolin-treated cells. These data suggest that ISO, presumably via cAMP and PKA phosphorylation, inhibits CCh-induced IP3 production by inhibiting PLC activity.

FIGURE 7. Effect of U73122 on CCh-induced mobilization of

[Ca21]i in SV-CISM-2 cells. (A) Spectrofluorimeter traces showing CCh-induced changes in [Ca21]i with or without U73122 treatment. CCh (10 mM) and U73122 were added at the indicated times. The U73122 concentrations used were: (a) none; (b) 0.01 mM; (c) 0.1 mM; (d) 1 mM; (e) 5 mM. (B) Peak [Ca21]i levels obtained with various U73122 concentrations were plotted. Each data point represents an average of three determinations.

Effect of protein phosphorylation by PKA on CCh- and GTPgS-induced IP3 production in membrane fractions isolated from SV-CISM-2 cells To show whether PKA phosphorylation is involved in cAMP inhibition of CCh-induced IP3 production, we investigated the effects of CCh on PIP2 hydrolysis in membrane fractions phosphorylated with PKA in vitro. As shown in Table 4, in control membranes CCh and GTPgS increased IP3 production by 33 and 30%, respectively, and these effects were significantly inhibited in membranes which were phosphorylated with PKA. In addition, the effects of CCh plus GTPgS on IP3 production in control and PKA phos-

418

K. -H. Ding et al.

FIGURE 8. Concentration-response effect of ISO on CCh-in-

duced formation of IP3 in SV-CISM-2 cells. SV-CISM-2 cells prelabeled with [3H]inositol were exposed to ISO for 5 min before incubation with 10 mM CCh for 10 min. The data represent means 6 SEM of two different experiments each run in triplicate.

phorylated membranes were 52 and 31%, respectively. These data suggest that in SV-CISM-2 cells PKA phosphorylation inhibits CCh-induced IP3 production. DISCUSSION In the present work we first characterized the effects of CCh on IP3 production and [Ca21]i mobilization in SV-CISM-2 cells and showed that [Ca21]i mobilization correlates well with IP3 production, and then showed that in these cells cAMP attenuates muscarinic receptor-mediated PIP2 hydrolysis and Ca21 mobilization. Previously, we have reported that when iris sphincter smooth muscle is treated with CCh there is rapid increase in IP3 that leads to muscle contraction [16]. Here, we have demonstrated that in Fura2/AM loaded cells, CCh increases [Ca21 ]i concentration in two phases, an initial major transient peak followed by a smaller sustained phase (Fig. 3). The transient phase is essentially complete by 77 s after the addition of the agonist, but the sustained phase continues for about 20 min. Our data further show that the transient peak is mainly due to release of Ca21 from intracellular stores and that the sustained phase is due to influx of Ca21 from extracellular source. This conclusion is based on the finding that when the cells were stimulated with CCh in a Ca21-free medium, the transient peak could still be observed, whereas the sustained phase was completely abolished. Additionally, CChinduced [Ca21 ]i mobilization was completely abolished by pretreatment of the cells with thapsigargin, suggesting that [Ca21]i mobilization was dependent upon intracellular IP3sensitive Ca 21 store. These data are consistent with several other reports in which CCh was shown to elicit a similar Ca 21 response in other cell types [5, 28, 29]. An important

FIGURE 9. Effect of ISO on CCh-induced mobilization of

[Ca21]i in SV-CISM-2 cells. (A) Spectrofluorimeter traces showing CCh-induced changes in [Ca21]i concentrations with or without treatment of the cells with 0.5 mM ISO. The concentration of CCh used was1 mM. (B) Concentration-dependent effects of ISO on CCh-induced [Ca21]i. Each data point represents peak [Ca21]i level attained with 1 mM CCh and is an average of three determinations.

observation here is that in these cells the transient Ca21 peak due to CCh could be due to the concomitant generation of IP3. This conclusion is supported by the good correlation between the temporal effects of CCh on both IP3 production (T1/2 value 5 68 s) and the maximal increase in [Ca21]i concentration (77 s) (Figs. 1 and 3). In addition, the EC50 values for CCh-induced IP3 production (0.5 mM) and Ca21 mobilization (1.4 mM) are also comparable. Additional support for the suggestion that the CCh-stimulated increase in [Ca21 ]i is due to enhanced production of IP3 comes from the data obtained from the experiments on the effects of the PLC inhibitor, U73122. This agent has been shown to inhibit agonist-induced PIP2 hydrolysis and [Ca21]i mobilization in several cell systems [27, 30, 31].

cAMP and Muscarinic Stimulation of IP3 and Ca21 Mobilization

FIGURE 10. Effects of U73122 and ISO on thapsigargin-induced

increase in [Ca21]i in SV-CISM-2 cells. Fura-2/AM loaded SVCISM-2 cells were exposed to thapsigargin (2 mM) and increase in [Ca21]i measured as described in the Materials and Methods section (A). The cells were treated with 5 mM U73122 (B) or 5 mM ISO (C) prior to the addition of thapsigargin.

419

Thus, when SV-CISM-2 cells were treated with U73122, both CCh-induced IP3 production (Fig. 6) and [Ca21 ]i mobilization [Fig. 7] were significantly inhibited. The inhibitory effects of U73122 on the two responses were dosedependent with identical IC50 values (10 nM). Taken together, these data provide reasonable experimental evidence for a causal relationship between CCh-induced IP3 generation and [Ca21]i mobilization in the SV-CISM-2 cells. For continuous and repetitive agonist-induced Ca21 release it is necessary that the intracellular Ca21 stores are refilled. Under normal physiological conditions, the refilling of the intracellular Ca21 stores occurs by activation of the Ca21 pump. Thapsigargin, a plant alkaloid, has been shown to inhibit selectively the SR Ca21-ATPase, thus resulting in elevation of [Ca21 ]i [25]. It has been reported that thapsigargin inhibited the repletion of the phenylephrine-sensitive store in both rat aorta and dog mesenteric artery [31]. When thapsigargin was added to the Fura-2/AM loaded SVCISM-2 cells, there was a transient gradual rise in [Ca21]i concentration which was followed by a smaller sustained increase in [Ca21]i concentration (Fig. 5). Subsequent addition of CCh to these cells did not result in additional [Ca21]i release typically observed in cells treated with CCh alone. These data suggest that once the intracellular stores are depleted of Ca21, IP3, generated by CCh-stimulated PIP2 hydrolysis, is ineffective in elevating [Ca21]i. Additionally, unlike the CCh effect on [Ca21]i mobilization, the effect of thapsigargin was not inhibited by pretreatment of the cells with U73122 or ISO (Fig. 10), thus suggesting that U73122 and ISO specifically inhibit the CCh-induced IP3-mediated Ca21 release. Whereas, ryanodine, at low concentrations, increases [Ca21]i, at high concentrations this alkaloid inhibits [Ca21]i mobilization [26]. Treatment of SV-CISM-2 cells with 100 mM ryanodine had no effect on basal or the CChstimulated [Ca21]i levels, indicating that the ryanodine-sensitive Ca21 store does not contribute to the CCh-induced Ca21 mobilization. Lack of ryanodine effect in SV-CISM-2 cells could also suggest that, like myometrium and other smooth muscles [32, 33], the ryanodine receptors are poorly expressed in these cells. Both CCh-induced IP3 production and CCh-induced [Ca21]i mobilization are more potently antagonized by 4-DAMP than by pirenzepine (Tables 1 and 2), suggesting that both responses are mediated by activation of M3 muscarinic receptors. These data on SV-CISM-2 cells are consistent with our previous work on the type of muscarinic receptors involved in CCh-induced PIP2 hydrolysis and contraction in the iris sphincter smooth muscle [16, 34]. There is substantial evidence that Ca 21 and cAMP signal transduction systems in most tissues are coupled, and that interaction between the two messenger systems probably plays an important role in various physiological processes, including smooth muscle contraction/relaxation. In bovine iris sphincter smooth muscle, CCh increases IP3 production which results in contraction of the muscle. Subsequent treatment with ISO or forskolin increases cAMP and de-

420

K. -H. Ding et al. TABLE 3. Effects of ISO and forskolin treatment on CCh-induced

production of IP3 in microsomal fraction prepared from SVCISM-2 cells Radioactivity in [3H] Inositol Phosphates (dpm) Additions None CCh (10 mM)

Control

ISO-treated

FSK-treated

2262 6 101 (100) 3473 6 86 (153%)†

2290 6 29 (100) 2954 6 65 (124%)

2081 6 43 (100) 2601 6 155 (125%)

The SV-CISM-2 cells were incubated with or without ISO (5 mM) or forskolin (5 mM) for 30 min in the presence of 0.1 mM IBMX. Following this, the cells were homogenized and microsomal fractions prepared as described in the Methods section. Ten micrograms of the microsomal fraction were then assayed for PLC using [ 3H]PIP2 as substrate. The data are means 6 SEM of two experiments each run in triplicate. † % of control.

creases IP3 which leads to relaxation of the muscle [13, 14]. Similar functional antagonism between cAMP and Ca21 responses has been reported in other smooth muscles [for reviews, 3, 4, 8]. The exact locus of cAMP inhibition of the IP3 /Ca21 system is not well established yet. Thus, cAMP has been shown to decrease [Ca21]i as a result of hyperpolarization due to stimulation of Ca21 -activated K1 efflux, inhibit Ca21 influx into cells, increase Ca21 efflux from cells through stimulation of plasma membrane Ca21 pump, and increase Ca 21 uptake into intracellular stores [35]. The data presented herein indicate that in SV-CISM-2 cells cAMP exerts its inhibitory effect at the level of IP3 production. This conclusion is based on the finding that ISO dosedependently decreased CCh-stimulated IP3 production with a corresponding decrease in CCh-induced [Ca21]i mobilization. The ISO IC50 values for CCh-stimulated IP3 production and CCh-stimulated Ca21 release were 80 and 170 nM, respectively (Figs. 8 and 9). The inhibitory effect of ISO was predominantly exerted on the transient phase of [Ca21]i with slight attenuation of the sustained phase. These data

provide further evidence that ISO inhibits [Ca21]i mobilization by attenuating the effect of CCh on IP3 production. To investigate further whether cAMP acts at a site proximal to the IP3 receptor, the effect of CCh on PIP2 hydrolysis was examined in membranes prepared from ISO or forskolin treated and untreated SV-CISM-2 cells. The data obtained showed that, as compared to the control, the CCh-stimulated PIP2 hydrolysis was significantly inhibited in membranes prepared from the treated cells (Table 3). Furthermore, when the membranes were phosphorylated in vitro with the catalytic subunit of PKA, the CCh- and GTPgSstimulated PIP2 hydrolysis was significantly inhibited, suggesting that cAMP decreases IP3 production by a mechanism involving phosphorylation of PLC and/or G-protein. The contribution of inhibition of PIP2 metabolism to a reduced [Ca21]i mobilization and subsequent relaxation by cAMP-elevating agents remains to be established. Thus, Hointing et al. [9], working on histamine-induced IP3 production and Ca21 mobilization in bovine isolated tracheal smooth muscle cells, concluded that the cAMP-dependent

TABLE 4. Effect of phosphorylation by PKA on CCh- and GTPgSinduced IP3 production by membrane fractions prepared from SVCISM-2 cells

Radioactivity in [3H] Inositol Phosphates (dpm) Addition None CCh (10 mM) GTPgS (0.1 mM) CCh (10 mM) 1 GTPgS( 0.1 mM)

Control

PKA-treated

4336 6 85 (100%) 5766 6 75 (133%)† 5637 6 37 (130%) 6591 6 120 (152%)

5375 6 150 (100%) 6344 6 96 (118%) 6397 6 64 (119%) 7042 6 125 (131%)

The membrane fraction was phosphorylated in the presence of 50 units of PKA (catalytic subunit) as described in the Methods section. Following the phosphorylation the membranes (20 micrograms) were used for the PLC assay. The data are mean 6 SEM of 6 determinations from two separate experiments. † % of control.

cAMP and Muscarinic Stimulation of IP3 and Ca21 Mobilization

421

inhibition of Ca21 mobilization and subsequent relaxation of these cells are not primarily caused by attenuation of IP3 production. Yang et al. [5], working with cultured canine tracheal smooth muscle cells, concluded that since generation of IPs and increase in [Ca]i are very early events in the activation of muscarinic receptors, attenuation of these events by cAMP-elevating agents might contribute to the inhibitory effect of cAMP on tracheal smooth muscle function. The inhibitory effect of cAMP on muscarinic receptors mediating both the contraction and the Ca21-activated K1 current is partly due to the inhibition of IP3-induced Ca21 release from intracellular stores in rat gastric muscle cells [28]. Murthy et al. [36 ], working with isolated gastric muscle cells, concluded that when the stimulus is maximal, inhibition of initial contraction by cyclic nucleotides is mediated by inhibition of IP3-dependent Ca21 release. Schramm et al. [37] reported that cAMP generation inhibits IP3 binding in rabbit tracheal smooth muscle. In bovine iris sphincter smooth muscle, PKA phosphorylates and activates IP3 3-kinase; this could attenuate IP3 accumulation and subsequently decrease Ca21 release from the SR [21]. Our studies here demonstrate that SV- CISM-2 cells are a good system for investigating mechanisms involved in the regulation of muscarinic-stimulation of PIP2 hydrolysis by cAMP-elevating agents. We can conclude that in these cells activation of M3 receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca21 ]i, and that cAMP, via PKA, may inhibit IP3 production and [Ca21]i mobilization through mechanisms involving phosphorylation of PLC, G-proteins, or the proteins involved in IP3 metabolism and IP3-induced Ca21 release.

8. Abdel-Latif A. A. (1991) Cell. Signal. 3, 371–385. 9. Hoiting B. H., Meurs H., Schuiling M., Kuipers R., Elzinga C. R. S. and Zaagsma J. (1996) Br. J. Pharmacol. 117, 419– 426. 10. Madison J. M. and Brown J. K. (1988) J. Clin. Invest. 82, 1462–1465. 11. Hall I. P. and Hill S. J. (1988) Br. J. Pharmacol. 95, 1204– 1212. 12. Offer G. J., Chilvers E. R. and Nahorski S. R. (1991) Eur. J. Pharmacol. 207, 243–248. 13. Tachado S. D., Akhtar R. A. and Abdel-Latif A. A. (1989) Invest. Ophthalmol. Vis. Sci. 30, 2332–2239. 14. Tachado S. D., Akhtar R. A., Zhou C-J. and Abdel-Latif A. A. (1992) Cell. Signal. 4, 61–75. 15. Ocklind A., Yousufzai S. Y. K., Ghosh S., Coca-Prados M., Stjernschantz J. and Abdel- Latif A. A. (1995) Exp. Eye Res. 61, 535–545. 16. Howe P. H., Akhtar R. A., Naderi S. and Abdel-Latif A. A. (1986) J. Pharmacol. Exp. Ther. 239, 574–583. 17. Husain S. and Abdel-Latif A. A. (1996) Curr. Eye Res. 15, 329–334. 18. Lowry O. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) J. Biol. Chem. 193, 265–275. 19. Zhou C-J., Akhtar R. A. and Abdel-Latif A. A. (1994) Exp. Eye Res. 59, 377–384. 20. Fabiato A. and Fabiato F. (1979) J. Physiol. Paris 75, 463–505. 21. Wang X.-L., Akhtar R. A. and Abdel-Latif A. A. (1995) Biochem. J. 308, 1009–1016. 22. Gasalla-Herraiz J., Rhee S. and Isales C. M. (1995) Biochem. Biophys. Res. Commun. 214, 373–388. 23. Grynkiewicz G., Poenie M. and Tsein R. Y. (1985) J. Biol. Chem. 260, 3440–3450. 24. Berridge M. J. (1993) Nature 361, 315–325. 25. Shima H. and Blaustein M. P. (1992) Circ. Res. 70, 968–977. 26. Fleischer S. and Inui M. (1989) Ann. Rev. Biophys. Chem. 18, 333–364. 27. Bleasdale J. E., Thakur N. R., Gremban R. S., Bundy G. L., Fitzpatrick F. A., Smith R. J. and Bunting S. (1990) J. Pharmacol. Exp. Ther. 255, 756–768. 28. Ohta T., Ito S., Noto T., Tachibana R., Nakazato Y. and Ohga A. (1992) J. Physiol. 453, 367–384. 29. Denning G. M., Clark R. A. and Welsh M. J. (1994) Am. J. Physiol. 267, C776–C784. 30. Mecrez-Lepretre N., Morel J-L. and Mironneau J. (1996) Biochem. Biophys. Res. Commun. 218, 30–34. 31. Low A. M., Gaspar V., Kwan C. Y., Darby P. J., Bourreau J. P. and Daniel E. E. (1991) J. Pharmacol. Exp. Ther. 258, 1105– 1113. 32. Sorrentino V. and Volpe P. (1993) Trends Pharmacol. Sci. 14, 98–103. 33. Missiaen L., De Smedt H., Droogmans G., Himpens B. and Casteels R. (1992) Pharmac. Ther. 56, 191–231. 34. Honkanen R. E., Howard E. F. and Abdel-Latif A. A. (1990) Invest. Ophthalmol. Vis. Sci. 31, 590–593. 35. Prestwich S. A. and Bolton T. B. (1995) Brit. J. Pharmacol. 114, 602–611. 36. Murthy K. S., Severi C., Grider J. R. and Makhlouf M. (1993) Am. J. Physiol. 264, G967–G974. 37. Schramm C. M., Chuang S. T. and Grunstein M. M. (1995) Am. J. Physiol. 269, (Lung Cell. Mol. Physiol. 13) L715– L719.

This work was supported by NIH grants R37 EY-04171, EY-04387 and EY-05738. The authors wish to thank Ms. Ashley Skinner for typing the manuscript.

References 1. Rasmussen H., Kelly G. and Douglas J. S. (1990) Am. J. Physiol. 258, L279–L288. 2. De Lanerolle P. and Paul R. J. (1991) Am. J. Physiol. 261, L1– L14. 3. Challis R. A. J. and Boyle J. P. (1994) Airways Smooth Muscle: Biochemical Control of Contraction and Relaxation (Raeburn D. and Giembycz M. A., Eds) pp. 309–327. Birkhauser Verlag, Basel, Switzerland. 4. Abdel-Latif A. A. (1996) Proc. Soc. Exp. Biol. Med. 211, 163–177. 5. Yang C-M., Hsu M-C., Tsao H-L., Chiu C-T., Ong R., Hsieh J. T. and Fan L-W. (1996) Cell Calcium 19, 243–254. 6. Murray R. K. and Kotlikoff M. I. (1991) J. Physiol. 435, 123– 144. 7. Felder C. C., Singer-Lahat D. and Mathes C. (1994) Biochem. Pharmacol. 48, 1997–2004.