Introduction

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Desensitization, a process by which cells adapt to extended exposure to agonists, results in a diminished functional response despite continued presence of the agonist and/or the requirement for a greater agonist concentration to maintain a given response level. Among the G protein-coupled receptors desensitization has been most extensively investigated for the beta-2 adrenoceptor (Hausdorff et al., 1990; Hadcock and Malbon, 1993; Barnes, 1995; Premont et al., 1995). Thus, desensitization of the beta-2 adrenoceptor involves phosphorylation of the receptor, uncoupling from its G protein, sequestration into an intracellular compartment where it can no longer couple to the G protein and, finally, down-regulation of the receptor and/or components of its signaling machinery (e.g., G proteins). Agonist-promoted desensitization of beta-2 adrenoceptors involves two types of protein kinases, GRK and the second messenger kinase, cAMP-dependent protein kinase. Much less is known regarding the mechanisms of desensitization of other receptors, particularly those that activate other signaling mechanisms such as the phospholipase C/Ca⁺⁺/PKC pathway (e.g., the alpha-1 adrenoceptors).

alpha-1 Adrenoceptors typically act by activating phosphatidylinositolphosphate hydrolysis to form inositol-1,4,5-triphosphate and diacylglycerol (Summers and McMartin, 1993). This results in activation of PKC. Similar to beta-2 adrenoceptors, desensitization of alpha-1 adrenoceptors involves rapid processes such as sequestration and slower processes such as receptor down-regulation (Cotecchia et al., 1995; Garcia-Sainz, 1993). In recent years, some progress has been made in elucidating the mechanisms underlying rapid alpha-1 adrenoceptor desensitization by sequestration, and it has become clear that this phase of desensitization occurs independent of PKC activation (Lattion et al., 1994). On the other hand, slower forms of desensitization may be more relevant for pathophysiological desensitization that may occur in disease states associated with chronically elevated sympathoadrenal tone such as congestive heart failure (Packer, 1992) or chronic renal failure (Daul et al., 1987). However, little is known regarding the mechanisms underlying alpha-1 adrenoceptor down-regulation in response to agonists.

MDCK cells are a cell line derived from distal nephron segments. They contain alpha-1 adrenoceptors, which couple to a pertussis toxin-insensitive G protein, possibly G_q, to induce stimulation of phospholipase C, which results in activation of PKC and cytosolic phospholipase A₂ (Godson et al., 1990; Slivka et al., 1988; Terman et al., 1987; Xing and Insel, 1996). Some data indicate that the MDCK cell alpha-1 adrenoceptors may belong to the alpha-1B subtype (Blue et al., 1994). We have previously demonstrated agonist-promoted desensitization and down-regulation of alpha-1B adrenoceptors in MDCK cells (Meier et al., 1985). In the present study, we used MDCK cells to further investigate agonist-induced desensitization and down-regulation of the alpha-1B adrenoceptor and, in particular, a possible role of PKC in these events.

	Methods

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Cell culture. MDCK cells were grown in an atmosphere of 5% CO₂/95% air at 37° in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin and, unless otherwise indicated, 7.5% heat-inactivated horse serum and 2.5% heat-inactivated fetal calf serum. Subconfluent cells were subcultured every 3 to 4 days with a 0.5 g/liter trypsin and 0.2 g/liter Na₄EDTA solution. In some experiments, cell were treated for the indicated times and with the indicated concentrations of the alpha-1 adrenoceptor agonist phenylephrine and/or various activators and/or inhibitors of PKC. All treatment experiments involving phenylephrine were performed in the presence of 10 µM (±)-propranolol to prevent concomitant beta adrenoceptor activation.

Radioligand binding. Confluent cells were harvested, counted, resuspended into ice-cold binding buffer (50 mM Tris, 0.5 mM EDTA, pH 7.4) and homogenized by an Ultra-Turrax (Janke and Kunkel, Staufen, Germany) for 10 sec at full speed and thereafter twice for 20 sec each at two-thirds speed. The homogenate was centrifuged for 20 min at 4° at 50,000 × g. Alpha-1B adrenoceptor affinity and number were determined by saturation binding of [³H]prazosin as previously described (Michel et al., 1993). Briefly, aliquots of the membrane suspension were incubated with six concentrations of [³H]prazosin (10-300 pM) in a total volume of 1000 µl for 45 min at 25°. The incubations were terminated by rapid vacuum filtration of Whatman GF/C filters. Nonspecific binding was defined as binding in the presence of 10 µM phentolamine. Samples were counted in a liquid scintillation counter. Protein content was measured according to the method of Bradford (1976) using bovine IgG as the standard. Competition binding experiments were performed in the same manner but with a total volume of 2000 µl, a single concentration of [³H]prazosin (200 pM) and 21 narrowly spaced concentrations of competitors; agonist competition experiments were performed in the presence of 100 µM GTP to minimize GDP-dependent formation of agonist high affinity states of the receptor.

Immunoblotting. Quantification of immunoreactive alpha subunits of the G protein G_q was performed as previously described (Michel-Reher et al., 1993). Briefly, membrane preparations (100 µg of protein/sample) were diluted 4:1 with sample buffer, boiled for 5 min and separated on sodium dodecyl sulfate/polyacrylamide gels with 10% acrylamide in the running gel. The separated proteins were transferred to nitrocellulose membranes with an electric field (55 V) overnight. After the transfer, the blots were washed for 90 min in TBS (20 mM Tris, 100 mM NaCl, pH 7.5) at room temperature in the presence of 2% nonfat dry milk and twice for 10 min each in TTBS (TBS supplemented with 500 µl/liter Tween-20) and then incubated overnight at 4° in 15 ml of TTBS containing 1% nonfat dry milk and a 1:500 dilution of the QL antiserum. After removal of the antiserum, the blots were washed twice for 10 min each with TTBS and then incubated for 1 hr at room temperature in 100 ml of TTBS that had been supplemented with 1% nonfat dry milk and 70 µl of [¹²⁵I]protein A solution (8.5 µCi/µg, 129 µCi/ml). Finally, the [¹²⁵I]protein A was washed out four times for 10 min each with TTBS, and the blots were used for autoradiography at 80°. With the use of the autoradiograms, the molecular weights of the specific bands were identified, and corresponding sections were cut from the blots and counted in a scintillation counter.

Inositol phosphate experiments. Accumulation of inositol phosphates was assessed as previously described (Slivka and Insel, 1987) with minor modifications. Briefly, MDCK cells grown in poly-L-lysine pretreated 60-mm dishes were labeled for 48 hr with myo-[³H]inositol (3 µCi/ml) in inositol-free medium. Cell pretreatments were performed during the last 24 hr of labeling. After labeling and treatment, the cells (~85-95% confluence) were washed twice with medium containing 20 mM HEPES and 50 mM LiCl at pH 7.4 and 37°. Thereafter, 5 ml of medium with or without 100 µM phenylephrine was added, and cells were incubated at 37° for 45 min. Reactions were stopped by the addition of 1 ml of ice-cold methanol. The cells were removed by a cell scraper into vials, and 1.5 ml of chloroform and 0.5 ml of water were added. The mixture was vigorously vortexed twice, and thereafter the phases were separated by centrifugation at 820 × g for 10 min at 4°. Aliquots (450 µl) of the upper phase were placed on Dowex AG 1-X8 columns (230 mg/column). Free inositol was eluted twice each with 5 ml of water and 5 ml of 60 mM ammonium formate. Total inositol phosphates were eluted by the addition of 2× 1 ml of 1 M ammonium formate dissolved in 100 mM formic acid. Each data point was determined in triplicate in each experiment.

Chemicals. [³H]Prazosin (specific activity, 74 Ci/mmol) and the QL antiserum were purchased from New England Nuclear (Dreieich, Germany). myo-[³H]Inositol (specific activity, 74 Ci/mmol) was from Amersham (Braunschweig, Germany). ATPS, H-7 (1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine dihydrochloride), epinephrine bitartrate, indomethacin, phenylephrine HCl, methoxamine HCl, norepinephrine bitartrate, oxymetazoline HCl, PMA, (±)-propranolol HCl and staurosporine were from Sigma (Munich, Germany). 2-Methylthio-ATP and 5-methylurapidil were from Research Biochemicals (Natick, MA). Dowex AG1-X8 (formate form) was from BioRad Laboratories (Richmond, CA). W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl] and KT5926 [(8R*,9S*,11S*)()-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,/b,11a-triazadibenzo-[a,g]cycloocta-[c,d,e]trinden-1-one] were from Calbiochem (San Diego, CA). Phentolamine HCl and SDZ NVI-085 [()-(4aR,10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2H-naphth 2,3,b-1,4-oxazine HCl] were kind gifts of Novartis (Basel, Switzerland). Both ()- and (+)-tamsulosin were from Yamanouchi (Tokyo, Japan).

Data analysis. Radioligand binding saturation experiments were analyzed by fitting rectangular hyperbolic functions to the experimental data using the InPlot program (GraphPAD Software, San Diego, CA). Competition binding experiments were analyzed by fitting monophasic and biphasic sigmoidal functions to the experimental data; a biphasic fit was accepted only if it resulted in a significant improvement as judged by an F test. Because some of our treatments altered cell number and/or cell size, we determined the number of cells and the total protein content per flask in each experiment. Radioligand binding and inositol phosphate formation data were then calculated in parallel using cell number and protein content as denominators. Data are presented as mean ± SEM of n experiments. Statistical significance of differences was determined by paired two-tailed t tests when two groups were compared. If multiple groups were compared, one-way analysis of variance was performed; if this indicated that the variance between groups was significantly greater than that within groups, individual groups were compared with their respective control by Dunnet's multiple comparison test. All statistical calculations were performed using the InStat program, and a value of P < .05 was considered significant.

	Results

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In untreated MDCK cells, [³H]prazosin bound to an apparently single class of sites with an affinity of 54 ± 3 pM and a density of 160 ± 7 fmol/mg protein, which was equivalent to 13,518 ± 627 sites/cell (n = 34). Competition binding experiments were performed with a panel nine subtype-selective agonists (in the presence of 100 µM GTP) and antagonists. All of them had steep and monophasic competition curves and their calculated affinities were consistent with the presence of a homogeneous population of alpha-1B adrenoceptors (table 1).

In the receptor regulation studies described below, receptor affinity for [³H]prazosin was not consistently altered by any of the treatments (data not shown); this indicates that receptor density estimates in the radioligand binding experiments were not affected in a relevant manner by phenylephrine, which might have been retained from the pretreatments. On the other hand, some of the treatments affected MDCK cell number or size (assessed as protein content/cell); to avoid misinterpretation of the data due to such effects, we present our data in parallel using both protein content and cell number as denominators. Unless otherwise indicated, all further data on receptor number are expressed as percent of the receptor density measured in paired control cells.

Phenylephrine treatment (0.01-100 µM for 24 hr) in the presence of 10% serum did not significantly affect cell number or size, assessed as protein content per cell number (table 2). Under these conditions, phenylephrine treatment concentration-dependently reduced the number of alpha-1B adrenoceptors with a threshold concentration of 1 µM; a reduction of 49% was achieved by 100 µM phenylephrine. At all agonist concentrations, the extent of the down-regulation was similar when analyzed relative to protein content or cell number (fig. 1). In contrast, phenylephrine concentration-dependently increased cell number (control, 44 ± 3 × 10⁶ cells/175-cm² flask; n = 4) by 13 ± 2%, 24 ± 3% and 29 ± 4% at 1, 10 and 100 µM phenylephrine, respectively (P < .05 vs. control for 10 and 100 µM phenylephrine) in the presence of 0.5% serum. This was accompanied by a trend for reduced cell size (control, 142 ± 13 µg of protein/10⁶ cells) but failed to reach statistical significance with the given number of experiments. Under these conditions, phenylephrine also caused a concentration-dependent down-regulation of alpha-1B adrenoceptors (fig. 1). However, in 0.5% serum-containing medium, the extent of down-regulation was somewhat smaller and the threshold concentration for phenylephrine was higher than in standard (10%) medium. Moreover, in 0.5% serum-containing medium, the extent of down-regulation was somewhat greater when data were analyzed relative to cell number (40%) compared with relative to protein content (27%). Therefore, all further regulation studies were conducted in medium containing 10% serum. In time course experiments with 100 µM phenylephrine, statistically significant down-regulation of alpha-1B adrenoceptors was detected after 2 hr, and down-regulation was maximal with 8 to 24 hr of agonist treatment (fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Down-regulation of MDCK cell alpha-1 adrenoceptors by phenylephrine treatment. Cells were treated for 24 hr in the absence (control) and presence of the indicated phenylephrine concentrations in the presence of 10% (top) or 0.5% serum (bottom). Thereafter, receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein () or sites/cell () and are expressed as mean ± SEM of percentage of control values of 4 experiments. * and **, P < .05 and < .01, respectively, vs. control in a repeated-measures one-way analysis of variance followed by Dunnett's multiple-comparison test.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time course of MDCK cell alpha-1 adrenoceptor down-regulation by phenylephrine treatment. Cells were incubated in the absence (control) and presence of 100 µM phenylephrine for the indicated times. Thereafter, receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein () or sites/cell () and are expressed as mean ± SEM of percentage of control values of 4 experiments. **, P < .01 vs. control in a repeated-measures one-way analysis of variance followed by Dunnett's multiple-comparison test.

Incubation of the cells for 24 hr with the PKC activator PMA (1-100 nM) reduced cell number but did not significantly affect the size of the remaining cells (table 2). PMA (1-100 nM) concentration-dependently down-regulated MDCK cell alpha-1B adrenoceptors with maximal reductions exceeding those achieved by phenylephrine (fig. 3). The concentration-response curve for PMA was much steeper than that for phenylephrine (i.e., no significant effects were detected with 1 nM PMA but a 70-80% down-regulation occurred with 10 nM PMA).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Down-regulation of MDCK cell alpha-1 adrenoceptors by PMA treatment. Cells were treated for 24 hr in the absence (control) and presence of the indicated PMA concentrations. Thereafter, receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein () or sites/cell () and are expressed as mean ± SEM of percentage of control values of 3-5 experiments. **, P < .01 vs. control in a repeated-measures one-way analysis of variance followed by Dunnett's multiple-comparison test.

Because the PKC activator PMA mimicked the effects of phenylephrine, we investigated whether the PKC inhibitors H7, staurosporine and KT5926 could block them. These experiments were limited by toxic effects of the PKC inhibitors. Thus, in the presence of 100 µM H7, 100 nM staurosporine or 1 µM KT5926 the cell number was reduced and the remaining cells appeared to be larger (table 2). Accordingly, in cells treated with H7, staurosporine or KT5926 basal alpha-1B adrenoceptor density was reduced to 45 ± 7%, 48 ± 2% and 83 ± 3%, respectively, of values in control cells (n = 4 each; P < .01). The presence of H7 (fig. 4), staurosporine (fig. 5) or KT5926 (fig. 6) markedly attenuated the 100 nM PMA-induced down-regulation of MDCK cell alpha-1 adrenoceptors. On the other hand, neither H7 nor staurosporine significantly altered phenylephrine-induced down-regulation regardless of whether data were expressed relative to cell number or protein content (figs. 4 and 5). KT5926, which also can inhibit Ca⁺⁺/calmodulin-dependent protein kinase, enhanced phenylephrine-promoted down-regulation if data were expressed as sites/cells (P < .05) but not if expressed as fmol/mg protein (fig. 6). The calmodulin antagonist W-7 (30 µM) did not significantly affect cell number and size (table 2). Treatment with W-7 did not significantly affect the alpha-1B adrenoceptor down-regulation by PMA or phenylephrine (fig. 7).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Effects of H7 on down-regulation of alpha-1 adrenoceptors induced by phenylephrine (PE) or PMA. Cells were incubated with vehicle, 100 µM phenylephrine or 100 nM PMA in the absence (control; open bars) or presence of 100 nM H7 (hatched bars) for 24 hr. The receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein (top) and sites/cell (bottom) and are expressed as mean ± SEM of percentage of control values of 4 experiments. ***, P < .001 vs. vehicle in a paired two-tailed t test.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effects of staurosporine on down-regulation of alpha-1 adrenoceptors induced by alpha-1 agonist phenylephrine (PE) or PMA. Cells were incubated with vehicle, 100 µM phenylephrine or 100 nM PMA in the absence (control; open bars) or presence of 100 nM staurosporine (hatched bars) for 24 hr. The receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein (top) and sites/cell (bottom) and are expressed as mean ± SEM of percentage of control values of 4 experiments. ** and ***, P < .01 and <.001, respectively, vs. vehicle in a paired two-tailed t test.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effects of KT 4926 on down-regulation of alpha-1 adrenoceptors induced by alpha-1 agonist phenylephrine (PE) or PMA. Cells were incubated with vehicle, 100 µM phenylephrine or 100 nM PMA in the absence (control; open bars) or presence of 1 µM KT5926 (hatched bars) for 24 hr. The receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein (top) and sites/cell (bottom) and are expressed as mean ± SEM of percentage of control values of 4 experiments. * and ***, P < .05 and <.001, respectively, vs. vehicle in a paired two-tailed t test.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effects of W-7 on down-regulation of alpha-1 adrenoceptors induced by alpha-1 agonist phenylephrine (PE) or PMA. Cells were incubated with vehicle, 100 µM phenylephrine or 100 nM PMA in the absence (control; open bars) or presence of 30 µM W-7 (hatched bars) for 24 hr. The receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein (top) and sites/cell (bottom) and are expressed as mean ± SEM of percentage of control values of 4 experiments. Differences in down-regulation by either agonist in the absence and presence of W-7 were not statistically significant in a paired, two-tailed t test.

The addition of the cyclooxygenase inhibitor indomethacin (10 µM) to the culture medium did not change the density of MDCK cell alpha-1B adrenoceptors or the ability of phenylephrine to down-regulate them (fig. 8). In contrast, a combination of the P_2y1 (P_2Y) purinergic receptor agonist 2-methylthio-ATP (300 µM) and the P_2y2 (P_2U) purinergic receptor agonist ATPS (300 µM) caused a small but statistically significant reduction of alpha-1B adrenoceptor expression, regardless whether data were expressed based on protein content or on cell number (fig. 8). The combined treatment with phenylephrine and the purinergic agonists did not cause additive down-regulation (fig. 8).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Effects of indomethacin and purinergic agonists on MDCK cell alpha-1 adrenoceptors and their regulation by phenylephrine (PE). Cells were incubated with vehicle, 10 µM indomethacin (indo) or 300 µM each of ATPS and 2-methythio-ATP (purinergic) for 24 hr in the absence (control; open bars) or presence of 100 µM phenylephrine (+PE; hatched bars). The receptor number was determined by [3H]prazosin saturation binding experiments. Receptor numbers were calculated as fmol/mg protein (top) and sites/cell (bottom) and are expressed as mean ± SEM of 4 experiments. *, ** and ***, P < .05, .01 and < .001, respectively, vs. control in a paired two-tailed t test. + and **, P < .05 and .01, respectively, vs. vehicle in a paired two-tailed t test.

In the immunoblot experiment, the QL antiserum, which is directed against a carboxyl-terminal sequence of G_q and G₁₁, recognized a band with an apparent molecular mass of ~40 kDa. The amount of [¹²⁵I]protein A bound to this band was not significantly affected by a 24-hr pretreatment of MDCK cells with 100 µM phenylephrine or 100 nM PMA (fig. 9).

View larger version (39K): [in this window] [in a new window]   Fig. 9.   Effects of phenylephrine and PMA treatment on immunodetectable Gq/11 alpha subunits. Cells were treated with vehicle (control), 100 µM phenylephrine (PE) or 100 nM PMA for 24 hr. G protein alpha subunit abundance was determined by quantitative Western blotting. Data are mean ± SEM of 7 experiments and are expressed as cpm [125I]protein A bound mg protein loaded to the respective lane. Values do not significantly differ by one-way analysis of variance.

Finally, we studied the effects of a 24-hr treatment of MDCK cells with vehicle, 100 µM phenylephrine or 100 nM PMA on the subsequent ability of phenylephrine to stimulate inositol phosphate formation. After vigorous washout of the pretreatments, cells were exposed for 45 min to 100 µM phenylephrine or vehicle (basal). Phenylephrine-induced inositol phosphate formation, expressed as percentage over basal values, was significantly reduced in phenylephrine-pretreated cells and even more so in PMA-pretreated cells when compared with control conditions (fig. 10).

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Effects of phenylephrine and PMA pretreatment on phenylephrine-induced inositol phosphate formation. Cells were treated with vehicle (control), 100 µM phenylephrine (PE) or 100 nM PMA for 24 hr. After washout, inositol phosphate formation was determined in the absence (basal) and presence of 100 µM freshly added phenylephrine. Data are mean ± SEM of 4 experiments and expressed as percentage increase of inositol phosphate formation in the presence of phenylephrine relative to basal levels. Basal inositol phosphate formation was 5055 ± 992 cpm/dish, 1374 ± 89 cpm/106 cells and 3035 ± 419 cpm/mg protein; (n = 4). This was not significantly affected by a 24 hr treatment with 100 µM phenylephrine when analyzed per dish (5261 ± 1107 cpm/dish) or cell number (1536 ± 194 cpm/106 cells) but significantly decreased when analyzed according to protein content (2179 ± 419 cpm/mg protein, P < .01). In contrast, basal inositol phosphate formation was significantly reduced by a 24 hr pretreatment with 100 nM PMA when analyzed per dish (2227 ± 646 cpm/dish, P < .01) or cell number (949 ± 89 cpm 106 cells, P < .05) but not when analyzed according to protein content (2811 ± 374 cpm/mg protein). **, P < .01 vs. control in a repeated-measures one-way analysis of variance followed by Dunnett's multiple-comparison test.

	Discussion

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We used MDCK cells to study the agonist-induced down-regulation of alpha-1 adrenoceptors. A panel of nine subtype-selective agonists and antagonists had steep and monophasic competition curves indicating the presence of a homogeneous receptor population. The absolute drug affinities were consistent with those we previously reported using similar methods for alpha-1B adrenoceptors in rat spleen (Büscher et al., 1994; Michel et al., 1993) and rat liver (Büscher et al., 1994; Büscher et al., 1996) and with cloned rat alpha-1B adrenoceptors (Büscher et al., 1994; Michel and Insel, 1994). Thus, these and previous data (Blue et al., 1994) unequivocally demonstrate that MDCK cells express a homogeneous population of alpha-1B adrenoceptors. The down-regulation of the MDCK cell alpha-1B adrenoceptor was concentration- and time-dependent requiring 8 to 24 hr and 10 to 100 µM phenylephrine for maximum effects (i.e., a 50% down-regulation). This could be observed under conditions where the alpha-1B adrenoceptor stimulation enhances MDCK cell proliferation (0.5% serum) and under conditions where it does not (10% serum). Thus, agonist-induced down-regulation is not dependent on cellular quiescence.

The receptor down-regulation promoted by phenylephrine was accompanied by functional desensitization (i.e., a reduced ability of the agonist to stimulate inositol phosphate formation). The extent of functional desensitization corresponded to the extent of reduction in receptor number, indicating that the loss of receptors alone is sufficient to explain the observed desensitization. Data in other systems suggest that expression of alpha-1B adrenoceptors closely parallels maximal ability of agonists to promote inositol phosphate formation (Theroux et al., 1996). This idea was further confirmed by our immunoblotting experiments that failed to detect alterations of G_q/11 expression on phenylephrine treatment. On the other hand, a reduced expression of G_q/11 alpha subunits in response to extended agonist stimulation has been reported with TRH-receptor transfected HEK 293 cells (Svoboda et al., 1996) or with angiotensin II and vasopressin endogenously expressed in cultured rat vascular smooth muscle cells (Kai et al., 1996). Studies on beta-2 adrenoceptor-induced down-regulation of G_s alpha subunits have demonstrated that the loss of G_s may quantitatively depend on the expression density of the receptor coupled to this G protein (Adie and Milligan, 1994). This may explain the divergent results between the present study with natively expressed receptor and those with high densities of transfected receptors. Additionally, alterations of G_q/11 expression on extended agonist exposure may depend on cell type under investigation and/or the degree of effectiveness with which the receptor couples to its effector mechanisms.

MDCK cell alpha₁-adrenoceptors not only couple to phospholipase C activation but also can activate phospholipase A₂ to enhance arachidonic acid release (Xing and Insel, 1996). This results in the formation of prostaglandins, which might then act on prostanoid receptors to cause heterologous down-regulation of alpha-1B adrenoceptors. To investigate this possibility, experiments were performed in the presence of the cyclooxygenase inhibitor indomethacin. Because indomethacin did not affect the density of MDCK cell alpha_1B-adrenoceptors or the ability of phenylephrine to cause their down-regulation, formation of cyclooxygenase-derived arachidonic acid metabolites does not appear to contribute to agonist-induced alpha-1B adrenoceptor down-regulation. This hypothesis is further supported by our previous observation that alpha-1B adrenoceptor-stimulated arachidonic acid release is PKC mediated (Xing and Insel, 1996), whereas phenylephrine-induced alpha_1B-adrenoceptor down-regulation is not (present study).

The possibility that PKC may be involved in alpha-1B adrenoceptor down-regulation was indicated by several pieces of evidence. First, stimulation of MDCK alpha-1B adrenoceptors can activate PKC (Godson et al., 1990). Second, purinergic agonists, which also activate the phospholipase C/PKC cascade in MDCK cells (Firestein et al., 1996; Yang et al., 1997), at least partly mimicked alpha-1B adrenoceptor down-regulation. Third, and most important, several previous studies have demonstrated that PKC-activating phorbol esters can desensitize alpha-1B adrenoceptors. Short-term (10-30 min) incubation with PMA can uncouple the receptors from inositol phosphate formation in DDT₁ hamster smooth muscle cells (Leeb-Lundberg et al., 1985). In rat liver, short-term PMA treatment can inhibit formation of the agonist high affinity state of the alpha-1B adrenoceptor (Beeler and Cooper, 1993; Corvera et al., 1986). This is accompanied by a rapid, staurosporine-sensitive reduction of cell surface alpha-1B adrenoceptors and may involve receptor sequestration to intracellular compartments (Beeler and Cooper, 1995). Phorbol ester-induced alpha-1B adrenoceptor sequestration has also been observed in stably transfected HEK 293 cells (Fonseca et al., 1995). Some of these effects may result directly from receptor phosphorylation because PKC activation can cause alpha-1B adrenoceptor phosphorylation (Bouvier et al., 1987). Down-regulation of alpha₁-adrenoceptors promoted by longer activation of PKC may involve other mechanisms. For example, in rabbit vascular smooth muscle cells, it may partly result from phorbol ester-induced destabilization of alpha-1 adrenoceptor mRNA (Izzo et al., 1994). On the other hand, PKC activation in hamster DDT₁ MF-2 cells can increase both mRNA accumulation and the number of alpha-1B receptors after 1-hr treatment, and these phenomena can continue up to 24 hr (Hu et al., 1993).

In the present study, the PKC activator PMA concentration-dependently down-regulated MDCK cell alpha-1B adrenoceptors. This occurred with a very steep concentration-response relationship, and maximum reductions in receptor number exceeded those produced by the receptor agonist phenylephrine. PMA-promoted down-regulation was accompanied by functional desensitization, which quantitatively matched the down-regulation and was not accompanied by alterations in immunodetectable G_q/11. The alpha-1B adrenoceptor down-regulation by PMA was indeed PKC mediated because three distinct PKC inhibitors, H-7, staurosporine and KT5926, markedly inhibited it. Although down-regulation was not fully prevented with the applied inhibitor concentrations, higher concentrations could not be tested due to toxic effects of the compounds. It is intriguing that PMA treatment down-regulates MDCK cell alpha-1B adrenoceptors via PKC, although it concomitantly down-regulates several PKC isoforms in MDCK cells (Godson et al., 1990). This could be explained if alpha-1B adrenoceptor down-regulation by PMA is a very rapid event preceding down-regulation of PKC and/or if it involves PKC isoforms that are resistant to down-regulation on PMA treatment.

Despite these effects of the antagonists on PMA-promoted down regulation of alpha-1B adrenoceptors, none of these three kinase inhibitors prevented phenylephrine-induced down-regulation. Although none of these inhibitors is very specific for PKC, all three were used in concentrations in which they effectively inhibit PKC. Although we cannot exclude that the effects of the PKC inhibitors alone on MDCK cells and their alpha-1B adrenoceptors contribute to the lack of inhibition of phenylephrine-induced down-regulation, we do not consider this likely because PMA-induced down-regulation was markedly inhibited in parallel experiments. Thus, our results strongly suggest that PKC does not play a major role in phenylephrine-promoted down-regulation of MDCK cell alpha-1B adrenoceptors. This is consistent with evidence in other cell systems that PKC activation can mimic the rapid agonist-induced alpha-1B adrenoceptor sequestration but that PKC is not involved in agonist-induced alpha-1 adrenoceptor sequestration (Lattion et al., 1994). Similarly, PKC activation can cause down-regulation of delta-opioid receptors expressed in HEK 293 cells or of angiotensin AT₁ receptors found in rat aortic smooth muscle cells, but PKC does not mediate agonist-induced down-regulation in these receptor systems (Lassegue et al., 1995; Pei et al., 1995).

Kagaya et al. (1993) demonstrated that the calmodulin antagonist, W-7, prevents the desensitization of 5-hydroxytryptamine₂ receptors in C6BU-1 cells produced by a 4-hr incubation with serotonin. They concluded that Ca⁺⁺/calmodulin protein kinase may participate in the homologous desensitization of phospholipase C-coupled receptors, such as the serotonin 5- hydroxytryptamine₂ receptors. Therefore, we tested whether Ca⁺⁺/calmodulin protein kinase might mediate agonist-induced alpha-1B adrenoceptor down-regulation. However, our data with two distinct inhibitors of this pathway, W-7 and KT5926, demonstrate that this second messenger kinase is not involved in agonist-induced down-regulation of MDCK cell alpha-1B adrenoceptors.

In conclusion, we demonstrated that MDCK cell alpha-1B adrenoceptors can be down-regulated by the receptor agonist phenylephrine in a concentration- and time-dependent manner. Receptor down-regulation is accompanied by functional desensitization but does not involve altered expression of G_q/11 alpha subunits and is independent of formation of cyclooxygenase products. The alpha-1B adrenoceptor down-regulation also occurs in response to stimulation of purinergic receptors and by direct PKC activation. The latter treatment is more effective than phenylephrine in causing down-regulation. Nevertheless, PKC activation does not appear to mediate agonist-induced down-regulation, nor does the Ca⁺⁺/calmodulin protein kinase mediate such down-regulation. Whether other kinases are involved in agonist-induced down-regulation of alpha-1B adrenoceptors and what their identity may be are not clear from the present study. A member of the family of GRK should be considered because alpha-1B adrenoceptors are substrates for several members of this group of kinases, including GRK2, GRK3 and GRK6 (Premont et al., 1995). Moreover, a role for GRK has been demonstrated in the rapid uncoupling and sequestration of alpha-1B adrenoceptors (Diviani et al., 1996). The GRK are also involved in agonist-induced down-regulation of delta-opioid receptors (Pei et al., 1995) and of angiotensin II receptors (Oppermann et al., 1996). Whether GRK indeed are involved in agonist-induced down-regulation of alpha-1B adrenoceptors remains to be studied.