Introduction

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The interaction of agonists with plasma membrane receptors triggers the activation of an intracellular cascade of biochemical events that leads to the final biological response. A single cell that possesses multiple membrane receptors for such agents (Makhlouf, 1987; Gardner and Jensen, 1993) could be exposed simultaneously to several of them. Inside the cell are various feedback mechanisms that integrate and regulate overall receptor activation. The most widespread regulatory mechanism is desensitization, which consists of a loss of responsiveness to an agonist that develops after prior or persistent exposure to the same, or to another, agonist. Desensitization is involved in the regulation of cellular responses to different agonists and in the prevention of cellular overstimulation, thus protecting the cells or tissues from abnormal stimuli. All biological systems in which desensitization occurs exhibit membrane receptors that mediate the effects induced by a specific agonist (Sibley and Leftkowitz, 1985).

Depending on its time of appearance, desensitization can be acute or chronic. Furthermore, according to the agonist that triggers the phenomenon, it can be either homologous or heterologous. These classifications are complementary. Acute desensitization appears rapidly (seconds/minutes) and disappears quickly after the removal of the stimuli, which seems to be due to an uncoupling between the receptors and the intracellular cascade with a consequent loss of membrane receptor function. Chronic desensitization is observed after hours or days of exposure to the agonist and intracellular proteolytic degradation of the receptors occurs, requiring a resynthesis to restore normal cellular response. Desensitization can also be classified as agonist-specific (homologous) and agonist-nonspecific (heterologous), the former being restricted to the ligand used as a desensitizer and stimulus. In heterologous desensitization cells become refractory to several classes of agonists (Lohse, 1993).

As for acute desensitization, the most extensively studied receptor is the beta-2 adrenoreceptor (Hausdorff et al., 1990), a G-coupled receptor that stimulates cellular responses by activating adenylate cyclase with a subsequent increase in intracellular cAMP. Much less is known regarding the desensitization of G protein-coupled receptors that stimulate cellular responses by activating the phospholipase C (PLC)-dependent intracellular pathway. Desensitization of this class of G protein receptors appears to be a multistep phenomenon (Chuang et al., 1996). There is considerable variation in the susceptibility of this class of receptors to desensitization as well as in the intracellular mechanisms involved in the phenomenon (Wojcikiewicz et al., 1993; Logston, 1994). It appears from recent studies that these receptors can be acutely desensitized by multiple mechanisms, causing either homologous or heterologous desensitization, and that different desensitization mechanisms are used by a given receptor in different cells (Hishinuma and Uchida, 1989; Battistini et al., 1990; Bolger et al. 1992; Lods et al., 1995).

A group of agonists that interacts with G protein-coupled receptors linked to phosphatidylinositol hydrolysis and to Ca⁺⁺-dependent intracellular mechanisms is represented by the peptidergic excitatory neurotransmitters present in the enteric nervous system. These neurotransmitters, some of which are coexpressed by the same neurons, may activate smooth muscle cells, causing contraction, by a combined action (plurichemical transmission) (Furness et al., 1989; Torsoli and Severi, 1993). After neural stimulation, various neurotransmitters may be released by enteric nerves, and, for this reason, smooth muscle cells can be exposed simultaneously to multiple agonists. Several mechanisms of regulation then must occur in these cells to prevent overstimulation, but few studies have been carried out in this area. Muscarinic receptors have been the most extensively studied (Mita and Uchida, 1987; Hishinuma et al., 1990).

The aim of the present study was to investigate acute desensitization of contraction caused by excitatory enteric neuropeptides in smooth muscle cells isolated from guinea pig stomach. The mechanisms involved in receptor desensitization were studied by evaluating contractions and intracellular inositol phosphate (InsP) and Ca⁺⁺ levels, together with selective activation of the different steps in the intracellular cascade. Our data show that smooth muscle cells are desensitized by enteric excitatory neurotransmitters and, depending on the duration of pre-exposure of smooth muscle cells to an agonist, that desensitization was either homologous or heterologous and occurred at receptor and postreceptor levels. The dual pattern of desensitization was associated with a different relative involvement of the various intracellular mechanisms responsible for the contractile response.

	Materials and Methods

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Materials

Chemicals. Cholecystokinin (CCK), gastrin-releasing peptide (GRP), and Substance P (SP) were obtained from Bachem (Bubendorf, Switzerland); Fura-2-free acid, 1-[2-(carboxyoxazol-2yl)-6-aminobenzofuran-5-oxyl]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'tetraacetic acid, sodium salt (Fura-2-AM), inositol trisphosphate (InsP₃), 2-[1-(3-dimethylaminopropyl)-1H-indol-3yl]-3-(1H-indol3-yl) maleimide (GF109203X), and saponin were purchased from Calbiochem (La Jolla, CA); collagenase CLS Type II and soybean trypsin inhibitor were obtained from Worthington (Freehold, NJ); Eagle's minimum essential amino acid mixture was obtained from GIBCO (Paisley, UK); D-myo[³H]inositol (specific activity, 814 GBq/mmol) was obtained from DuPont-NEN (Bad Hamburg, Germany); and phorbol 12-O-tetradecanoylphorbol-13-acetate, HEPES, Dowex 1X8-200 (100-200 mesh), and all other reagents were obtained from Sigma (St. Louis, MO).

Animals. Male guinea pigs (200-300 g, body weight) were purchased from Charles River Italia (Milan, Italy).

Methods

Preparation of Smooth Muscle Cells. Smooth muscle cells were isolated from guinea pig stomach as described previously (Bitar and Makhlouf, 1982). In brief, muscle strips, free of mucosa, were incubated at 31°C for two successive 45-min periods in 15 ml of HEPES medium, pH 7.4, containing 0.1% collagenase (150 U/ml). The composition of the medium was as follows: 115 mM NaCl, 5.8 mM KCl, 2.1 mM KH₂PO₄, 2.0 mM CaCl₂, 0.6 mM MgCl₂, 25 mM HEPES, and 14 mM glucose. The medium also contained 0.01% soybean trypsin inhibitor and 2.1% essential amino acid mixture. At the end of the second incubation period, the muscle strips were washed with enzyme-free medium on 500-µm Nitex mesh and resuspended in the same medium for 30 min to allow spontaneous dispersion of cells. In some experiments, cell dispersion was accelerated by gentle suction of the suspension using an inverted 10-ml pipette. The cells then were harvested by filtration through 500-µm Nitex mesh and then centrifuged for 5 min at 350g to adjust the selected cellular concentration required for desensitization protocols (i.e., 10⁴ cells/ml in contraction experiments or 10⁶ cells/ml in InsP and Ca⁺⁺ measurements).

Measurement of Contraction. Contraction was measured in suspensions of smooth muscle cells as described previously (Bitar and Makhlouf, 1982). Cell suspension (0.5 ml) was added to 0.2 ml of incubation medium containing the agent to be tested, and the reaction was stopped at fixed time intervals by adding acrolein (final concentration, 1%). The interval was timed to elicit a peak contraction with each agent tested (30 s with receptor agonists; 5 s with InsP₃). For measurement of control cell length, the agent was omitted and an equivalent volume of medium was added. In control samples and upon addition of test agents, the length of 50 cells in sequential microscopic fields was measured by image-scanning micrometry (Lasico; Los Angeles, CA). Contraction was expressed as the mean percentage decrease in cell length with control length taken as 100%.

Measurements of Cytosolic-Free Ca⁺⁺ ([Ca⁺⁺]_i). [Ca⁺⁺]_i was measured in intact and permeabilized smooth muscle cells, as already described (Murthy et al., 1992, 1993), using the fluorescent Ca⁺⁺ dyes Fura-2-AM and Fura-2-free acid, respectively. For [Ca⁺⁺]_i measurements, dispersed intact cells were resuspended in HEPES medium consisting of: 125 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM MgSO₄, 1 mM CaCl₂, 5 mM glucose, 20 mM taurine, 5 mM sodium pyruvate, and 5 mM creatine, pH 7.4. Two milliliters of cell suspension (10⁶ cells/ml) was incubated with Fura-2-AM (2 µM) for 30 min at 31°C, centrifuged at 350g for 20 min, and resuspended in 2 ml of HEPES medium. Fluorescence was measured within 5 min. Maximal fluorescence was determined after addition of 100 µg/ml digitonin (81 µM) and minimal fluorescence was determined after addition of 7.5 mM EGTA plus 60 mM Tris.

Measurement of InsPs. InsPs were determined in circular smooth muscle cells by ion-exchange chromatography as described (Berridge et al., 1983; Murthy and Makhlouf, 1991). Five milliliters of cell suspension were labeled with myo[³H]inositol (15 µCi/ml) for 90 min at 31°C. At the end of the incubation, nonincorporated myo[³H]inositol was removed by centrifugation and the cells were resuspended in 3 ml of fresh medium. The cells were then desensitized, washed twice, and resuspended for an additional 10 min in fresh medium to which LiCl was added to a final concentration of 10 mM. CCK-8 (1 nM) was then added to 0.5 ml of cell suspension at 31°C for 10 min, and the reaction was stopped by addition of 940 µl of chloroform/methanol/HCl (50:100:1). After standing 2 min in ice, chloroform and distilled water (310 µl each) were added; the mixture was vortexed vigorously and then centrifuged at 1000g for 15 min. The upper aqueous phase was then poured on a column loaded with 1 ml of Dowex 1X8 (formate form) in distilled water. The column was washed with 10 ml of water followed by 10 ml of 5 mM sodium tetraborate/60 mM ammonium formate to remove [³H]glycerophosphoinositols. Total InsPs were eluted with 6 ml of 1.0 M ammonium formate/0.1 M formic acid. The results were expressed as cpm per 10⁶ cells.

Experimental Protocols for Evaluating Desensitization. Desensitization was evaluated as the difference in the ability of an agonist to stimulate a contractile response or to increase InsPs or [Ca⁺⁺]_i in cells pre-exposed to one of the peptides and the responses observed in cells preincubated without agonist. For this purpose, isolated smooth muscle cells were preincubated at 31°C for up to 15 min (first incubation) without (control) or with one of the agonists, then washed twice to remove the agonist and re-exposed (second incubation) to the agent to be assayed. Washings were performed using HEPES buffer containing 1% bovine serum albumin in which CaCl₂ was omitted. Contraction was measured at the end of the second incubation, and desensitization was evaluated as described previously. In those experiments where the effect of InsP₃ was evaluated, dispersed isolated cells were permeabilized before starting the desensitization protocols. In the experiments carried out to explore protein kinase C (PKC) involvement, dispersed smooth cells were preincubated for 60 min with 10 µM GF109203X before starting desensitization protocols in normal or permeabilized smooth muscle cells.

Statistical Analysis. Results were expressed as mean ± S.E. of n experiments, and the data were calculated with Student's t test (p < 0.05 was considered statistically significant). Cells for each experiment were obtained from different animals. The agonist concentration causing half-maximal response, indicated as ED₅₀, was obtained by linear regression analysis.

	Results

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Smooth muscle cells isolated from guinea pig stomach had a mean cellular length of 101.0 ± 2.2 µm and a maximal contraction of 24.9 ± 1.5% in response to 1 nM CCK.

Control experiments were performed initially to verify whether preincubation of smooth muscle cells, followed by the two wash-out steps, could cause modifications of the basal experimental conditions. For this purpose, resting cell length before and after preincubation with or without a given peptide was measured. In the absence of the agonist, only a small reduction of cell length (3.0 ± 1.1%) from the initial value was observed after preincubation and subsequent washings. Agonist preincubation also did not induce any significant modification of cell length. In fact, no difference was observed between resting cell length of cells preincubated with the peptide after removal of the agonist (95.3 ± 3.0 µm) and cells preincubated without the agonist (98.3 ± 3.0 µm), showing that no residual contraction was present after agonist preincubation and the washings that followed.

When smooth muscle cells were desensitized with increasing doses of CCK (10 pM-1 µM), a progressive shift to the right of the dose-response curves to CCK was observed in these cells. As shown in Fig. 1, a decrease in both maximal response and in potency of the peptide was observed in cells preincubated for 15 min with submaximal (10 pM; ED₅₀, 4.5 pM) or maximal dose of CCK (1 nM; ED₅₀ 9 pM). In cells preincubated with supramaximal doses of CCK (10 nM-1 µM) there was a further decrease in potency of the peptide (ED₅₀: from 45 pM to 2.2 nM) associated with a rightward shift in the maximal dose. In control cells, namely, in cells preincubated in the absence of any peptide, CCK had a potency (ED₅₀ 1.2 pM) similar to that normally observed in isolated guinea pig gastric smooth muscle cells (Bitar and Makhlouf, 1982). CCK-induced desensitization therefore was strongly dose-dependent.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Dose dependence of CCK-induced desensitization of guinea pig gastric smooth muscle cell contraction. Muscle cells were preincubated during the first incubation with no addition or with increasing doses of CCK for 15 min (data are the mean ± S.E. of three to six experiments).

To investigate whether this decreased response of desensitized cells might be due to a difference in time required to reach peak contraction, response kinetics were studied in both control cells (no addition) and cells preincubated with 1 nM CCK (Fig. 2). No differences were observed in the time course of response. In both preparations, peak contraction was observed at 30 s and was followed by a decrease of the response to approximately 40% of maximal contraction, which remained constant for up to 2 min.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Kinetics of contractile response of guinea pig gastric smooth muscle cells preincubated during the first incubation with no addition () or with 1 nM CCK () (data are the mean ± S.E. of four to seven experiments).

The time course of desensitization then was evaluated. Smooth muscle cells preincubated with 1 nM CCK for up to 15 min displayed a progressive time-dependent decrease in subsequent CCK-induced maximal contraction (Fig. 3). The decrease in contraction was detectable after 30 s of preincubation, increased during the following 5 min, and then remained constant. In cells preincubated without agonist, no inhibition of maximal contraction was observed.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Time dependence of CCK-induced desensitization of guinea pig gastric smooth muscle cell contraction (data are the mean ± S.E. of five to seven experiments).

To elucidate whether CCK caused homologous or heterologous desensitization, smooth muscle cells were preincubated with either different doses of CCK or a maximal dose of CCK for different times to be re-exposed subsequently to CCK, GRP, or SP. A short, 30-s pre-exposure to 1 nM CCK caused only a decrease of subsequent response to CCK without affecting those maximal contractile responses caused by the other peptides, thus indicating homologous desensitization (Table 1). A longer pre-exposure (5 min) inhibited instead to the same extent either CCK-, GRP-, and SP-induced contractions (Table 1), thus indicating heterologous desensitization. A similar trend was observed with submaximal doses of CCK (data not shown). To exclude that a heterologous desensitization could have been missed after short CCK pre-exposure by the use of maximal agonists concentrations, GRP and SP dose-response curves were constructed before and after a 30-s preincubation with 1 nM CCK. Similar potency for either peptide was observed before (ED₅₀: GRP = 10 pM and SP = 20 pM) or after short-term desensitization to CCK (ED₅₀: GRP = 11 pM and SP = 35 pM), thus confirming that the homologous pattern occurred on maximal responses. The observed time dependence of CCK-induced desensitization then was associated with different patterns of desensitization.

To elucidate the cellular mechanisms responsible for this dual pattern of desensitization, the different intracellular steps involved in contractile responses were investigated using direct activators and inhibitors of intracellular responses and measuring intracellular mediators. For this purpose, InsP accumulation and Ca⁺⁺ mobilization induced by CCK were evaluated in control and desensitized cells. In control cells preincubated without agonist, CCK induced an intracellular InsP production almost two times greater than basal level, while in desensitized cells this effect was limited to a 20% increase (Table 2). The degree of inhibition in InsPs response was similar after a 30-s or a 5-min preincubation, therefore resulting independently from the time of pre-exposure of the cells to the agonist.

Intracellular Ca⁺⁺ mobilization induced by CCK and InsP₃ then was evaluated in control and desensitized permeabilized cells (Fig. 4). In permeabilized control cells preincubated without agonist, CCK induced an intracellular increase of [Ca⁺⁺] five times greater than basal values, an increase similar to that observed in intact gastric smooth muscle cells (5.2 ± 1.5 times). InsP₃ induced an increase of intracellular [Ca⁺⁺] similar to that observed in response to CCK. In permeabilized cells pre-exposed for 30 s to 1 nM CCK, a decrease in intracellular [Ca⁺⁺] mobilization was observed in response to CCK and InsP₃. The decrease in InsP₃-induced mobilization was significantly lower than the decrease in CCK-induced response. On the contrary, in permeabilized cells pre-exposed for 5 min to the same dose of CCK, CCK- and InsP₃-induced responses were almost equally inhibited. In desensitized cells the inhibition of CCK-induced intracellular [Ca⁺⁺] mobilization therefore was as already observed for CCK-induced InsP production, independent from the time of pre-exposure of the cells to the agonist. The inhibition of InsP₃-induced response instead was time-dependent, being somewhat lower after a shorter pre-exposure time as suggested by the statistically different degree of [Ca⁺⁺] mobilization induced by CCK and InsP₃ after a 30-s pre-exposure.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Basal () and stimulated [Ca++]i levels in response to 1 nM CCK () and 1 µM InsP3 () in permeabilized cells previously pre-exposed to no agonist or to CCK for 30 s or 5 min (data are the mean ± S.E. of 10-11 experiments; *p < .05 versus CCK-induced release for each treatment).

To further investigate the time dependence observed in InsP₃-induced Ca⁺⁺ release in desensitized cells, the effect of exogenous InsP₃ on smooth muscle cell contraction in control and desensitized cells was evaluated. For this purpose, isolated smooth muscle cells were previously permeabilized, then desensitized for 30 s or 5 min with 1 nM CCK, and, after two washings to remove the agonist, re-exposed to 1 nM CCK or to 1 µM InsP₃. Control experiments were carried out to evaluate the effect of cell permeabilization on the contractile response and on the time dependence of desensitization. In control cells (no addition), CCK-induced contraction in permeabilized cells (24.7 ± 1.4%) was similar to the response observed in intact cells (22.5 ± 1.3%). Furthermore, permeabilized cells pre-exposed for different times to a maximal dose of CCK showed a time-dependent decrease in contraction (14.6 ± 3.9% after 30 s; 9.0 ± 2.0% after 5 min) similar to that observed in intact desensitized cells (13.8 ± 1.8% after 30 s; 9.4 ± 0.9% after 5 min). Smooth muscle cell membrane permeabilization therefore did not modify the time dependence of CCK-induced desensitization. Moreover, in permeabilized cells, the time dependence of CCK-induced desensitization was associated with different patterns of desensitization. A 30-s pre-exposure to CCK did not modify GRP- nor SP-induced contraction (20.3 ± 2.4% and 25.9 ± 1.7%, respectively), while longer pre-exposure inhibited both these responses (13.5 ± 2.4% and 13.6 ± 1.4%, respectively).

In permeabilized cells, contraction induced by maximal doses of CCK and InsP₃ then was evaluated (Fig. 5). The two agonists, which activate contraction at different sequential steps, caused a similar maximal response in control permeabilized cells, but this response was inhibited in desensitized cells. In desensitized cells, the pattern of response to the two agonists depended on the time of pre-exposure to CCK. Comparing the contraction induced by the two agonists in permeabilized cells pre-exposed for 30 s to 1 nM CCK, the response induced by InsP₃ was reduced to a lesser extent than the contraction induced by CCK. A 5-min preincubation with 1 nM CCK instead caused an equal reduction in CCK- and InsP₃-induced contractions.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   CCK, 1 nM (), and InsP3, 1 µM (), induced contraction of permeabilized guinea pig gastric smooth muscle cells preincubated with no addition or with 1 nM CCK for 30 s or 5 min (data are the mean ± S.E. of three to five experiments; *p < .05 versus CCK-induced contraction for each treatment).

To investigate the role played by PKC in desensitization, smooth muscle desensitization experiments were carried out before or after a 60-min preincubation with 10 µM GF109203X, a PKC inhibitor (Toullec et al., 1991). For this purpose CCK- and SP-induced maximal contractions were evaluated in desensitized cells that had been preincubated with or without the PKC inhibitor. This pre-exposure to GF109203X was effective in inhibiting PKC in gastric smooth muscle cells because the contraction induced by 1 nM PKC-stimulating phorbol ester phorbol 12-O-tetradecanoylphorbol-13-acetate (14.1 ± 1.1%) was inhibited when the cells were preincubated with GF109203X (8.6 ± 0.7%). Pretreatment of smooth muscle cells with the PKC inhibitor did not modify CCK-induced homologous desensitization as no differences were observed in the selective inhibition of CCK-induced contraction in smooth muscle cells pre-exposed for 30 s or 5 min to 1 nM CCK (Fig. 6). The time dependence of CCK-induced homologous desensitization was not PKC-dependent. On the contrary, CCK-induced heterologous desensitization was PKC-dependent. In fact, the inhibition of the SP-induced maximal contraction observed after 5-min pre-exposure to CCK in cells not treated with the specific PKC inhibitor was overcome in 5-min desensitized cells pretreated with GF109203X (Fig. 6). The effect of GF109203X pretreatment on InsP₃-induced contraction was evaluated in normal and desensitized cells. For this purpose, smooth muscle cells were pre-incubated for 60 min with or without 10 µM GF109203X, then permeabilized and finally incubated for 5 min in the absence or presence of 1 nM CCK. Pretreatment with the PKC inhibitor reduced the inhibition of InsP₃-induced contraction observed after a 5-min pre-exposure to CCK (Table 3). These data parallel the previous observation on restoration of SP-induced contraction in desensitized cells pretreated with GF109203X and desensitized by 5-min preincubation with 1 nM CCK.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   CCK-induced, 1 nM (), and SP-induced, 100 nM (), contraction of guinea pig gastric smooth muscle cells preincubated in the absence or presence of 1 nM CCK for 30 s or 5 min before or after preincubation for 60 min with 10 µM GF109203X.

Finally, to examine whether CCK-induced desensitization of contraction was a reversible phenomenon, smooth muscle cells were pre-exposed for 15 min to 1 nM CCK and, from the end of the second washing, CCK-induced contraction was measured until restoration of the maximal response at 15-min intervals. As shown in Fig. 7, 15 min after the end of the washings, contraction in response to a maximal dose of CCK was already higher than that observed in the same cells at time 0 (i.e., at the end of preincubation). After 30 min, a complete recovery of contractile response in desensitized smooth muscle cells was detected. Thirty minutes after the first incubation, maximal CCK-induced contraction in previously desensitized cells was similar to that observed in cells preincubated without agonist.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Reversibility of CCK-induced desensitization of guinea pig gastric smooth muscle cell contraction (data are the mean ± S.E. of four to five experiments).

	Discussion

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In this study we have shown that pre-exposure of smooth muscle cells to CCK, GRP, and SP, whose receptors couple to phosphatidylinositol hydrolysis and to intracellular Ca⁺⁺-dependent mechanisms, caused desensitization of contractile responses that, depending on the time of preincubation, was either homologous or heterologous. A 30-s preincubation with CCK caused an inhibition of subsequent responses to CCK only, without affecting GRP- and SP-induced responses. A 5-min or longer pre-exposure to CCK inhibited GRP- and SP-induced contraction as well, resulting in a heterologous pattern of desensitization. The progression from homologous to heterologous desensitization was not related to the dose of agonist present in the preincubation medium. A similar behavior has been observed in endothelial (Wilkinson et al., 1994) but not in other cells (Rahman and Neuman, 1993; Sjödin and Gylfe, 1994). Thus, in gastric smooth muscle cells, the interaction of CCK with its specific receptor first activates contraction and almost simultaneously inactivates the response to CCK itself. Subsequently, desensitization to other agonists acting through the same intracellular pathway occurs. Such a rapid onset of homologous desensitization may be a characteristic of smooth muscle tissue because a similar latency of appearance has been observed in guinea pig taenia coli (Hishinuma et al., 1993). Desensitization of contractile response also occurs when cells are exposed continuously to the same peptide. In fact, after reaching peak contraction a decrease in response was observed in kinetic studies that were performed in the continuous presence of the agonist.

In smooth muscle cells, homologous and heterologous desensitization occurs at receptor and postreceptor levels. Excitatory enteric neuropeptides induce smooth muscle contraction by interacting with specific membrane receptors, which, in turn, activate PLC via a regulatory G protein. This enzyme is responsible for hydrolysis of the plasma-membrane phospholipid phosphatidylinositol 4,5-biphosphate, thus generating the second messengers InsP₃ and 1,2-diacylglycerol (DAG). InsP₃ triggers the release of Ca⁺⁺ from intracellular stores producing a rise in [Ca⁺⁺]_i, whereas DAG activates cytosolic PKC. An additional aim of this study was to clarify the relative role of different intracellular mechanisms involved in the observed dual pattern of desensitization.

Homologous desensitization, which was studied in more detail after pre-exposure to CCK, appeared after a pre-exposure of 30 s and was reversible and dose-dependent. Both membrane receptors and intracellular mechanisms were involved in this pattern. The decrease in potency and efficacy observed in CCK dose-response curves in desensitized cells suggests an involvement of membrane receptors in the inhibition of contractile responses. Desensitization caused by increasing concentrations of CCK caused variations in dose-response curves that were anomalous with respect to predictions of classical receptor theory (Kenakin, 1985). First, there was a decrease both in potency and in efficacy of the contractile agent, while more intense desensitization was limited to a further rightward shift of the curves without a further reduction in the maximal amplitude of the response. It should be noted from previous work (Makhlouf, 1987) and as shown in Fig. 1 that dose-response curves of contractile agents in smooth muscle cells are biphasic, with the maximal contractile response being strongly affected by inhibition caused by supramaximal stimuli. Upon removal of the agonist, there was a rapid recovery of the contractile response, indicating that receptor modification was temporary. Because the response recovered fully within 30 min from the end of preincubation, degradation of membrane receptors probably can be excluded. It has been postulated that homologous desensitization, as opposed to heterologous desensitization, is a result of receptor modifications not involving alterations of intracellular mechanisms triggered by receptor activation (Triggle, 1980). The broad range of CCK concentrations (10 pM-1 µM) that induce desensitization of smooth muscle cell response, however, might reflect an involvement of more than one mechanism, as observed for bombesin on pancreatic acinar cells (Sjödin and Gylfe, 1994). In fact, in our cell system, intracellular mechanisms modulate homologous desensitization. One mechanism might be a decrease in InsPs formation, because the InsPs increase in response to CCK was inhibited after pre-exposure of the cells for 30 s to 1 nM CCK. Reduced PLC-catalyzed hydrolysis with a subsequent decrease in InsP₃ and DAG production has been observed in other cellular systems (Larsson and Simonsson, 1993; Shinohara and Kawasaki, 1994; Servant et al., 1995). The reduced InsPs production is reinforced further by the observation that in smooth muscle cells pre-exposed to CCK for 30 s, the InsP₃-induced contractile response as well as intracellular Ca⁺⁺ mobilization were inhibited to a lesser extent than were CCK-induced responses. The higher degree of inhibition observed in InsP₃-induced Ca⁺⁺ mobilization than in InsP₃-induced contraction might be due to desensitization of the former with respect to the latter (Masters et al., 1985). The inhibition observed in InsP₃-induced responses, however, suggests that some additional intracellular mechanism is involved in homologous desensitization of smooth muscle cells, acting most likely at the level of InsP₃ metabolism and/or of InsP₃ receptor desensitization (Ferris et al. 1992; Alblas et al. 1995). PKC, instead, seems not to play an active role in the induction of acute homologous desensitization in gastric smooth muscle cells because the PKC inhibitor GF109203X did not alter the pattern of desensitization of the contractile agent used as desensitizer (i.e., CCK). A similar result has been obtained in other cell types (Walsh et al. 1993), although it is well known that specific agonist-induced desensitization can differ in the various cellular systems (Shih and Malbon, 1994; Lods et al., 1995) and that agonists can activate different regulatory mechanisms in the same cells (Kurose and Lefkowitz, 1994).

A longer pre-exposure of the cells to the agonist caused an increase in the extent of homologous desensitization and the appearance of heterologous desensitization. In fact, after a 5-min pre-exposure to CCK, inhibition of subsequent CCK-induced contraction was higher than that observed after a 30-s preincubation, and GRP- and SP-induced contractions were inhibited as well. Other regulatory mechanisms appeared to be involved in the inhibition of responses caused by a longer pre-exposure of smooth muscle cells to CCK. Most of the effects observed, after a 5-min pre-exposure, appeared to be PKC-dependent. Preincubation with the PKC inhibitor GF109203X, while failing to remove homologous inhibition to CCK, abolished heterologous desensitization to SP and reversed the inhibition of InsP₃-induced response. Inhibition of PKC therefore is able to restore the contractile machinery but not the response induced by the desensitizing agonist itself. Homologous desensitization therefore appears to involve some PKC-independent mechanisms as suggested (Inglese et al., 1993). This hypothesis is supported by the observation that the inhibition of CCK-induced release of InsPs and Ca⁺⁺ was practically completed within the first 30 s. Neither inhibition of Ca⁺⁺ release in desensitized cells, whose extent was similar to that previously found in guinea pig pancreatic acini (Servant et al., 1995), nor the inhibition of the InsP release was critical for the greater homologous desensitization, because a similar inhibition was detected in smooth muscle cells pre-exposed for 30 s or 5 min to CCK. In smooth muscle cells, as in other cellular systems, homologous and heterologous desensitization appear to be completely independent processes (Chuang et al. 1996).

In conclusion, CCK, SP, and GRP induced a desensitization of smooth muscle cell contraction that, depending on the time of pre-exposure of the cell to the agonist, was either homologous or heterologous. After occupancy of specific membrane receptors, there is a rapid contraction that reaches its maximum after 30 s, and, simultaneously, homologous desensitization begins. Subsequently, the contractile response decreases and progression to the heterologous pattern occurs. The cell becomes refractory at first to the same agonist and then to other receptor agonists. Thirty minutes later, the efficacy of the contractile cascade machinery is fully restored.