Introduction

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Alpha-1 adrenergic signal transduction in the aorta, a conduit vessel, undergoes desensitization after prolonged exposure to catecholamines or ₁AR agonists in vivo (Maze et al., 1985; Rosenbaum et al., 1986; Tsujimoto et al., 1987; Hiremath et al., 1991; Johnson et al., 1991a, b) or in vitro (Lurie, et al., 1985; Hu et al., 1992a,b, 1994). The desensitization of the contractile response extends to responses mediated by other G protein-coupled receptors (Maze et al., 1985; Rosenbaum et al., 1986; Tsujimoto et al., 1987; Hu et al., 1994). Ligand binding studies with aortic membranes (Lurie et al., 1985; Seasholtz et al., 1997) or DDT₁ MF₂ smooth muscle cell membranes (Leeb-Lundberg et al., 1987) have shown that ₁AR density is not affected by treatments that elicit receptor desensitization. These results have previously led us to postulate that ₁AR desensitization may be associated with alterations at a site(s) distal to the receptor. In studies performed on aorta, we have demonstrated that chronic NE infusion results in reduced ₁AR-mediated functions and that this desensitization is mediated by a reduction in receptor/G protein coupling (Johnson et al., 1991b; Seasholtz et al., 1997).

Very little is known concerning the effects of prolonged catecholamine or ₁AR agonist exposure on resistance vessels such as the rat tail artery, despite evidence that resistance artery tone has a greater impact on blood pressure than does the tone of larger vessels such as the aorta. In addition, results have shown that tail arteries from spontaneously hypertensive rats have an increased contractile response to receptor stimulation when compared with tail arteries from normotensive Wistar-Kyoto rats (Vila et al., 1993). Increased contractility is not seen in aortas of spontaneously hypertensive rats (Vila et al., 1993), which indicates that resistance arteries may play a role in the pathophysiology of some forms of hypertension. In the present study, we investigate whether the tail artery does in fact undergo desensitization, and if so, identify the possible mechanisms involved in resistance artery desensitization.

	Materials and Methods

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In Vitro Phenylephrine Exposure

Male Sprague-Dawley rats (200-300 g) were decapitated. Tail arteries were dissected, cut into 5-mm rings and placed in conical tubes containing sterile DPBS on ice. Arteries were then transferred to 25-cm² tissue culture flasks containing 10 ml Dulbecco's modified Eagle's medium with 250 U/ml penicillin/streptomycin and placed in a 37°C incubator containing 5% CO₂. Vessels were permitted to equilibrate for 1 to 2 hr before addition of either 50 µl DPBS for the control vessels or the appropriate concentration of PE in 50 µl DPBS for experimental vessels. Antagonists were added 2 hr before agonists. After 22 hr vessels were washed six times with PSS of the following composition (mM): NaCl, 120; KCl, 4.7; MgCl₂, 1.2; NaH₂PO₄, 1.0; NaHCO₃, 25; CaCl₂, 1.8; glucose, 11; EDTA, 0.024; and bubbled with 95%0₂/5%CO₂.

Tail Artery Contraction

Tail arteries were placed into a Petri dish containing PSS and bubbled with 95%O₂/5%CO₂ while two stainless steel hooks were placed through each ring. Tail artery ring segments were mounted at 37°C in 20-ml organ baths by attaching the stainless steel hooks to gold chain connected to a force transducer at the top and to the bottom of the organ bath. Contraction was measured by force displacement transducers (Grass model FT.03) and a polygraph (Grass model 7D). Preparations were equilibrated in PSS for 60 min at a tension of 800 mg, which was previously determined to be optimal. Antagonists were administered 15 to 20 min before agonist.

Inositol Phosphate Accumulation

The method for measuring [³H]inositol metabolism was described previously (Kendall and Hill, 1990; Gurdal et al., 1995). Tail artery rings were prepared as described above and preincubated in oxygenated buffer of the following composition (mM): NaCl, 118; KCl, 4.7; MgSO₄, 1.2; KH₂PO₄, 1.2; NaHCO₃, 25; CaCl₂, 13 mM; glucose, 10; HEPES, 15; pH 7.4 at 37°C for 1 hr. Subsequently, arterial segments were incubated for 1.5 hr in 20 µCi/ml of [³H]myo-inositol (17 Ci/mmol, Dupont, NEN Research Products, Boston, MA) containing buffer under the same conditions. Labeled arterial segments were washed four times and placed in individual tubes containing buffer with 10 mM LiCl (total assay volume, 300 µl). Tail artery rings were incubated with agonist for 6 min, which was previously determined to be in the linear range of IP accumulation and a time where precursor availability was not depleted. Termination of the reaction was carried out by addition of 250 µl of ice-cold 30% trichloroacetic acid. Tubes were then left on ice for 20 min. The tubes were then centrifuged (1500 × g 10 min) and aliquots (350 µl) of supernatant were added to 125 µl of 10 mM EDTA in 1.5-ml microcentrifuge tubes, followed by 500 µl of 1:1 Freon tri-n-octyl-amine. The samples were vortexed and allowed to stand for 10 min before centrifugation (12,000 × g 10 min), and 300 µl of aqueous phase was taken for analysis of IPs. Samples were loaded on Dowex-1(X8) ion exchange columns (formate form, 100-200 mesh, 1 ml). The columns were washed with 20 ml myo-inositol (5 mM); the IPs were eluted with 4 ml of 0.1 M formic acid/1 M ammonium formate. Radioactivity was measured by liquid scintillation spectrometry.

Preparation of Membranes

Tail arteries were placed in 20 mM NaH₂PO₄-Na₂HPO₄ buffer (pH 7.6) containing 154 mM NaCl, 0.04 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, homogenized glass-to-glass with use of a motor-driven homogenizer and centrifuged at 500 × g for 10 min at 4°C. The supernatant was centrifuged at 100,000 × g for 60 min at 4°C. The resulting pellet was resuspended in PBS and used for binding assays.

Radioligand Binding

Saturation binding of [¹²⁵I]HEAT (2200 Ci/mmol) was used to measure ₁AR density. Tail artery membranes were incubated with different concentrations of [¹²⁵I]HEAT in 200 µl PBS containing 0.01 mg/ml bovine serum albumin for 60 min at room temperature (20-30 µg protein/tube). The reaction was terminated by rapid filtration with a Brandell cell harvester and Whatman GF/C filters. Filters were washed four times with 4 ml of ice-cold PBS. The filter-bound radioactivity was determined using a Beckman gamma counter. Nonspecific binding is defined as binding in the presence of 1 µM prazosin or 1 mM NE, with equivalent results.

Agonist-Stimulated [³⁵S]GTPS Binding and Immunoprecipitation

All procedures were carried out at 4°C unless otherwise indicated. Crude tail artery membranes were prepared by homogenizing tissues in 10 volumes of ice-cold homogenization buffer containing 20 mM HEPES (pH 7.4), 100 mM EGTA, 0.2% 2-mercaptoethanol, 50 µg/ml pepstatin A and 0.04 mM phenylmethylsulfonyl fluoride with a glass-to-glass homogenizer. The homogenate was centrifuged for 10 min at 48,200 × g. The pellet was resuspended in oxygenated KRB: 25 mM HEPES (pH 7.4), 118 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 0.2% 2-mercaptoethanol, 50 µg/ml leupeptin, 0.01 U/ml soybean trypsin inhibitor, 25 µg/ml pepstatin A and 0.04 mM phenylmethylsulfonyl fluoride. Protein values were determined by the method of Lowry et al. (1951). Membranes (200 µg protein) were incubated with 2 nM [³⁵S]GTPS for 5 min followed by an additional 5-min incubation with agonists (total incubation volume, 250 µl). The reaction was terminated by diluting with 750 µl of ice-cold Mg⁺⁺-free, KRB containing 1 mM EGTA, mixed, and immediately centrifuged at 16,000 × g for 5 min. The obtained pellets were solubilized in 500 µl IMP containing 100 mM Tris, pH 7.4, 1.25% (vol/vol) Nonidet P-40, 200 mM NaCl, 10 mM EDTA, 50 µg/ml leupeptin, 0.01 U/ml soybean trypsin inhibitor, 25 µg/ml pepstatin A and 0.04 mM phenylmethylsulfonyl fluoride with 0.2% (w/v) SDS by brief sonication and an additional 500 µl IMP was added to the suspension. The [³⁵S]GTPS-bound G proteins were immunoprecipitated by the method described by Friedman et al. (1993). The pellets containing [³⁵S]GTPS were resuspended in KRB by brief sonication and radioactivity was measured by liquid scintillation spectrometry. The radioactivity precipitated by the normal rabbit serum was considered background and was subtracted from all agonist-stimulated values.

Immunoblot Analysis

Tail artery membranes were prepared as described above in the GTPS binding assay and protein concentrations were determined by the method of Lowry et al. (1951). Twenty-five micrograms of membrane proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and then transferred electrophoretically to nitrocellulose. Immunoblotting was performed with antisera to G_q/11, G_i, G_s and G_o (NEN, dilutions 1:2000) and to common G (0.25 µg/ml, Santa Cruz) as described previously (Carlson et al., 1989; Spiegel, 1990). Nitrocellulose membranes were incubated overnight with 10% nonfat dry milk in PBS containing 0.1% Tween-20 (0.1% TBS) at 4°C. Blots were washed four times with 0.1% TBS (10 min each) and then incubated with horseradish peroxidase-labeled donkey anti-rabbit IgG (Amersham, Arlington Heights, IL) for 1 hr at room temperature. Blots were washed once with 0.3% TBS for 15 min, followed by four 5-min washes with 0.1% TBS, then incubated with enhanced chemiluminescence Western blotting reagent (Amersham) for 1 min and exposed to x-ray film for 15 to 30 sec.

Statistical Analysis

Results from the contraction, IP accumulation and GTPS binding studies were analyzed by two-factor ANOVA followed by Neuman-Keuls test or Dunnett's test where appropriate or by planned comparison with Student's t test. Radioligand binding data were analyzed by the LIGAND program (Munson and Rodbard, 1980; Munson, 1983).

Materials

Normal rabbit serum and Pansorben were purchased from Calbiochem (La Jolla CA). Antisera to G_s(RM/1), G_i(1,2) (AS/7), G_o (GC/2) and G_q (QL) were purchased from the New England Nuclear Corp. (Boston, MA). [¹²⁵I]HEAT (2200 Ci/mmol) and [³H]myo-inositol (17Ci/mmol) were purchased from the New England Nuclear Corp. (Boston, MA). 5-HT creatinine sulfate, pepstatin A, phenylmethylsulfonyl fluoride and soybean trypsin inhibitor were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco's PBS was purchased from GIBCO BRL Life Technologies (Gaithersburg, MD). PE, clonidine, UK 14,304, prazosin HCl, rauwolscine, were purchased from Research Biochemicals Inc. (RBI; Natick, MA).

	Results

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Control experiments demonstrated that maintaining tail artery rings in culture media for up to 72 hr did not adversely affect contractile responses (data not shown). Exposure of tail artery rings to 7.5 µM or 75 µM PE for 22 hr shifted the concentration-response curve of NE-stimulated contraction to the right. The pD₂ values for NE-stimulated contraction were 6.63 ± 0.15, 6.11 ± 0.12 and 5.85 ± 0.26 for DPBS-exposed, 7.5 µM PE-exposed and 75 µM PE-exposed tail artery rings, respectively (P < .05) (fig. 1). The maximal NE-stimulated contractions in the treated arteries were 1.74 ± 0.11 g, 1.28 ± 0.17 g and 1.02 ± 0.09 g, respectively (P < .05) (fig. 1a). Prazosin (1 µM) treatment beginning 2 hr before the addition of 7.5 µM PE completely abolished the shift in pD₂ values and maximal response to NE (data not shown), which indicates that PE is acting at ₁AR to produce desensitization.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   NE-stimulated contraction of tail artery rings exposed to PE (7.5 or 75 µM) or DPBS for 22 hr in vitro. Data represent the means ± S.E.M. from four experiments; 12 rings per group. Exposure to both concentrations of PE significantly reduced NE-induced contraction (P < .01, two-factor ANOVA).

Alpha-1 and alpha-2 adrenoceptors are found in the rat tail artery, and both contribute to the contractile response elicited by NE. To investigate whether contractile responses mediated by ₁AR and ₂AR were desensitized after incubation with PE, the selective ₁AR agonist PE, and the ₂AR agonists clonidine and UK 14,304 were tested. The pD₂ values (control: 6.25 ± 0.18 vs. PE: 5.30 ± 0.11, P < .01) and maximal responses (control: 1.49 ± 0.14 g vs. PE: 0.78 ± 0.14 g, P < .01) for PE-stimulated contraction were significantly lower for tail artery rings exposed to 75 µM PE for 22 hr when compared with control, DPBS-exposed, artery rings (fig. 2a). Clonidine, a partial ₂AR agonist, produced no detectable contraction in 75 µM PE-exposed tail artery rings, whereas control vessels exposed to DPBS responded with a maximal contraction of 1.00 g (fig. 2b). Tail artery rings exposed to 75 µM PE also exhibited decreases in the contractile responses to the full ₂AR agonist, UK 14,304, at each of the three concentrations (3 × 10⁷ M, 70%; 10⁶ M, 55%; and 10⁵ M, 33%) examined (fig. 2c). To test further the selectivity of the desensitization which is produced by in vitro exposure to PE, the contractility of the vessels to 5-HT, KCl and ionomycin were also assessed. Although the contractile response to 10 µM 5-HT was decreased by 28% after exposure to 75 µM PE, the responses to 60 mM KCl and 10 µM ionomycin did not differ from those obtained in control tail artery rings (fig. 2d). The data suggest that in vitro exposure to PE results in desensitization of ₁AR-, ₂AR- and 5-HT receptor-stimulated contractions of the vessels without a loss in vascular smooth muscle contractility.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effect of 22 hr exposure to 75 µM PE on tail artery contraction in response to PE (a), clonidine (b), UK 14,304 (c), 5-HT (d), KCl (d) and ionomycin (d). Each bar represents the means ± S.E.M. from three to five experiments; 6 to 15 rings per group. *P < .05, **P < .01 indicate the differences from corresponding contractile response in DPBS-exposed group (Neuman-Keuls test for multiple comparison following two-factor ANOVA).

One mechanism that mediates receptor-stimulated vascular contraction involves activation of PLC and the production of IP₃ (Alexander et al., 1985; Fox et al., 1985; Smith et al., 1985). To determine whether a reduction in receptor-stimulated activation of PLC occurs in the tail artery during desensitization, agonist-stimulated IP accumulation was measured after 22 hr exposure to PE (75 µM). Although basal IP formation in DPBS- and PE-exposed tail artery rings did not differ (257 ± 28 cpm and 327 ± 41 cpm, respectively), NE (1-30 µM)-stimulated IP accumulation was significantly reduced (61-75%) in the desensitized vessels (fig. 3a). Stimulated PI hydrolysis in response to the specific ₁AR agonist, PE (10 µM), was markedly diminished in tail artery rings exposed to 75 µM PE for 22 hr (519 ± 70% vs. 164 ± 54%; fig. 3b). Serotonin (10 µM)- and endothelin (0.3 µM)-mediated PI responses were also reduced (51% and 52% of control, respectively) in tail artery rings which were pretreated with 75 µM PE for 22 hr (fig. 3b). In contrast, 60 mM KCl- or 10 µM ionomycin-induced IP formation was unaffected by the same treatment conditions (fig. 3b). The ₂AR agonist, clonidine (10 µM), was unable to stimulate measurable IP accumulation in DPBS- or PE-exposed tail artery rings (data not shown). The latter finding is consistent with the results of previous studies (Aburto et al., 1995).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of 22 hr exposure to 75 µM PE on IP accumulation in response to NE (a), PE (b), endothelin (b), 5-HT (b), KCl (b) and ionomycin (b). Basal IP accumulation was 257 ± 28 cpm and 327 ± 41 cpm for DPBS- and PE-exposed vessels, respectively. Data represent the means of % control (basal IP accumulation) and S.E.M. from three to five experiments; 9 to 15 rings per group. *P < .05, **P < .01 indicate the differences from corresponding response in DPBS-exposed group (Neuman-Keuls test for multiple comparison after two-factor ANOVA).

[¹²⁵I]HEAT binding to ₁AR was performed to determine whether alterations in ₁AR number contribute to the desensitization produced by PE exposure. Saturation [¹²⁵I]HEAT binding revealed no difference in ₁AR density in membranes of DPBS- or PE (75 µM)-exposed tail arteries (45.6 ± 15.0 and 37.3 ± 5.8 fmol/mg protein, respectively) (fig. 4), which suggests that PE-mediated desensitization may result from changes at a site(s) distal to the receptor. We therefore considered that both the homologous and heterologous desensitizations that were observed after 22 hr of PE-exposure may be mediated via changes at the level of the G proteins that subserve the responses to the tested G protein-coupled receptors. Experiments were therefore performed to determine whether these receptors are associated with common G protein(s) in the rat tail artery as well as to assess if alterations in receptor/G protein coupling occur during desensitization. Receptor-stimulated binding of [³⁵S]GTPS to specific membrane G proteins was determined in membranes of DPBS- or PE (7.5 µM PE or 75 µM)-exposed rat tail artery rings. In control tissue, PE increased [³⁵S]GTPS binding to G_q/11 and G_i, whereas stimulation of ₂AR with UK 14,304 enhanced [³⁵S]GTPS binding only to G_i (fig. 5a). The results also demonstrate that both PE- (10 µM) and UK 14,304 (10 µM)-mediated responses were diminished after 22 hr exposure of the tail arteries to either 7.5 or 75 µM PE (fig. 5b). Immunoblot analyses with antibodies directed against G_s, G_i, G_o, G_q/11 and G proteins revealed decreases in G_q/11 (51% and 84%, respectively) and G_i (32% and 70%, respectively) levels in membranes from PE-treated tail arteries, whereas no changes in G_s, G_o or G were noted (fig. 6).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   [125I]HEAT binding to rat tail artery membranes after exposure to 75 µM PE in vitro for 22 hr remained unchanged when compared with membranes from DPBS-exposed rings. Receptor densities in membranes from DPBS- and PE-exposed tail arteries were 45.6 ± 19.0 and 37.3 ± 7.1 fmol/mg protein, respectively. The figure shows results from a representative experiment; five tail arteries were pooled for each of four experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effects of 22 hr exposure to 7.5 µM PE or 75 µM PE on PE- (a) and UK 14,304-stimulated (b) [35S]GTPS binding to G proteins. Basal [35S]GTPS binding to Gs, Gi, Go and Gq in DPBS-exposed tail artery membranes (in counts per min) were 374.0 ± 18.0, 422.3 ± 14.6, 364.7 ± 18.1 and 380.8 ± 14.4, respectively. Exposure to either concentrations of PE did not affect the basal [35S]GTPS binding to all G proteins examined. Each bar represents the data expressed as percent stimulation above the basal binding from four individual experiments. *indicates difference from corresponding control (DPBS-exposed) levels: P < .01, Dunnett's test after one-factor ANOVA. Data represent the means and S.E.M. from four different experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Immunoblot analysis of Gq, Gi, Gs, Go and G in membranes from tail arteries exposed to 7.5 µM PE or 75 µM PE for 22 hr in vitro. The blots are representative of six experiments. Exposure to 7.5 and 75 µM PE decreased the levels of Gi by 32.0 ± 1.9% and 70.0 ± 7.7% (P < .01, one-factor ANOVA followed by Dunnett's test), and Gq by 51.0 ± 4.5% and 83.7 ± 8.7% (P < .01, one-factor ANOVA followed by Dunnett's test), respectively.

	DISCUSSION

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The current studies demonstrate that in vitro exposure of the tail artery to prolonged ₁AR stimulation results in decreased receptor-mediated muscle contraction and IP accumulation. The development of this functional desensitization is mediated via direct stimulation of ₁AR by PE, because it can be completely prevented by co-treatment of the tail artery with the ₁AR antagonist, prazosin. The desensitization to alpha receptor-stimulated contractile and IP responses appear to be mediated at a site proximal to the receptor because KCl- and ionomycin-activated contraction were not affected by PE treatment. Moreover, the latter observations suggest that changes in voltage-operated Ca⁺⁺ channels or PLC activity per se are probably not involved in the desensitization of the rat tail artery, which develops during exposure to PE.

The results also indicate that desensitization is not related to alterations at the level of the ₁AR because the density and affinity of [¹²⁵I]HEAT binding was not altered by prolonged incubation of vascular rings with PE. The current results in the rat tail artery, as well as previous findings in rat aorta, demonstrate that desensitization induced by ₁AR stimulation results in a loss of function that is not restricted to ₁AR agonists (Maze et al., 1985; Rosenbaum et al., 1986; Tsujimoto et al., 1987; Hu et al., 1994). In this study, tail artery contraction in response to clonidine, UK 14,304 and 5-HT and IP accumulation in response to endothelin and 5-HT was reduced after PE exposure. Similarly, heterologous desensitization was observed previously in rat aorta showing that contraction and IP accumulation in response to NE and AII are reduced after in vivo NE-infusion (Seasholtz et al., 1997). In the rat aorta, a lack of alteration in ₁AR receptor density during desensitization was noted in both in vitro and in vivo studies (Lurie et al., 1985; Seasholtz et al., 1997). Thus, the desensitization resulting from protracted stimulation of ₁AR appears to be heterologous and is likely the result of an alteration at a site common to the responsive G protein-coupled receptors that were examined.

A decrease in receptor-activated G proteins was noted after sustained incubation of the rat tail artery with PE. In membranes obtained from PE-exposed tail arteries, [³⁵S]GTPS binding to G proteins in response to ₁-AR and ₂-AR stimulation was blunted. In contrast to the results obtained in aorta which indicate that in vivo NE infusion fails to affect G levels (Seasholtz et al., 1997) or results in a small reduction in G protein (Zhou et al., 1995), reductions in receptor-stimulated G protein activation in the tail artery appear to be mediated by marked decreases in levels of G_q and G_i proteins which are associated with prolonged stimulation of the ₁AR. Thus, although uncoupling of receptors from their associated G proteins is responsible for the functional desensitization in aorta after in vivo infusion of NE (Johnson et al., 1991b; Seasholtz et al., 1997), decreased G subunits, which subsequently results in uncoupling of vasoactive receptor/G protein, may account for the reduced receptor-mediated contraction and IP accumulation observed in the tail artery after in vitro PE exposure. In this regard, these findings are similar to a recent study that demonstrated decreased levels of G_q/11 subunits in ₁AR-transfected rat fibroblasts after prolonged exposure to PE (Wise et al., 1995). Similarly, sustained (6 hr) stimulation of rat aortic cells with arginine-vasopressin or AII also resulted in reduced cellular G_q/11 levels and heterologous desensitization of the IP responses to AII and vasopressin (Kai et al., 1996). In the current study, these changes in G protein tissue content appear to underlie the loss of ₁AR-mediated functions as well as the reduced responses to the other tested agonists of the G protein-coupled receptors, 5-HT, alpha-2 and endothelin. These receptors, therefore, appear to share common pools of membrane G proteins (G_q and G_i) with those that couple to ₁AR in the tail artery. Although it is possible that the relatively high concentrations of the tested agonists may induce contraction via activation of the ₁AR, the fact that these agents activate different subsets of G proteins and that the alpha-2 agonist, clonidine, did not increase IP accumulation suggest that contractions induced by the alpha-1 and alpha-2 agonists and by 5-HT or endothelin are mediated by specific receptors that are coupled to different G proteins and effectors. Furthermore, 5-HT-induced contraction in the tail artery was shown to be mediated by 5-HT_2A receptors because contraction was blocked specifically by ketanserin at concentrations that were selective for the 5-HT_2A receptor (Watts, 1996).

In agreement with previous results in the rabbit saphenous vein (Aburto et al., 1995), we observed no ₂AR-stimulated IP response in the rat tail artery. Activation of ₂ARs appears to elicit contraction solely via the influx of extracellular Ca⁺⁺ (Nichols and Ruffolo, 1988; Nichols et al., 1988, 1989; Aburto et al., 1995). In contrast, the contractile response mediated by ₁AR involves both Ca⁺⁺ influx as well as intracellular Ca⁺⁺ release mediated by IP₃ receptors (Nichols and Ruffolo, 1988; Nichols et al., 1989; Ruffolo et al., 1991). Thus, a direct relationship between receptor-stimulated blood vessel contraction and IP accumulation is not always observed. Moreover, ₁AR-stimulated contraction is only partially pertussis toxin-sensitive, whereas ₂AR-stimulated contraction can be completely abolished by pertussis toxin (Nichols et al., 1989; Ruffolo et al., 1991). These relationships between the adrenoceptor subtypes and the functional response are supported by the present direct evidence showing that the ₂ARs are coupled exclusively to G_i protein, whereas ₁ARs are coupled to both G_q and G_i proteins in the rat tail artery. Because ₂AR-stimulated GTPS binding to G_i is reduced after prolonged ₁AR stimulation, it appears that ₁ and ₂ARs share the same pool of G_i protein and this action underlies the cross desensitization noted in the present work.

In summary, the present studies show that the responses of various vasoactive G protein-coupled receptors are reduced after in vitro exposure to PE for 22 hr. These changes are mediated by reductions in G protein levels which result in impaired receptor/G protein/effector coupling. The studies furthermore suggest that in the tail artery, G protein-coupled receptors share membrane G proteins and that this leads to the heterologous desensitization which is produced by prolonged exposure to an ₁ receptor agonist.