INTRODUCTION

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MYOCARDIAL INFARCTION, ACUTE renal failure, adult respiratory distress syndrome, and organ transplantation are all characterized by a period of ischemia followed by reperfusion that results in increased regional vascular resistance and decreased organ perfusion (5, 9, 10, 13, 15, 31). Prolonged ischemia can result in a phenomenon termed "no reflow," where, during reperfusion, vascular resistance is elevated to the point of complete absence of blood flow (21, 22). Increase in vascular tone after ischemia-reperfusion (I/R) was shown to be related to damaged vascular smooth muscle and endothelial cells, resulting in a shift in balance between vasoconstrictor and vasodilator tone (3, 5, 13, 15, 26, 38). Numerous studies have demonstrated endothelial dysfunction after I/R that is characterized by reduction in release of endothelium-derived relaxing factor (26). Endothelium-dependent vasodilation in response to stimuli such as acetylcholine, bradykinin, A-23187, ADP, serotonin, and thrombin is impaired after I/R, whereas vasodilation due to nitric oxide, nitroglycerine, and nitroprusside remains unchanged (4, 19, 29, 30, 36, 39). Although many studies have examined the effects of I/R on endothelial function, the effect of I/R on vascular contractility has received little attention. The present study provides evidence for increased ₁-adrenergic receptor-mediated vascular contraction and receptor-mediated inositol phosphate production after I/R. The enhanced vascular responsiveness to norepinephrine (NE) after I/R is further shown to be associated with increased _1a-adrenergic receptor-G_q/11 protein coupling.

	METHODS

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Animals. All procedures using animals conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (no. 85-23, revised 1996). Male Sprague-Dawley rats (weighing 200-300 g) were purchased from the Harlan Teklad (Madison, WI). The rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (40 mg/kg). Lateral incisions, 3 cm in length, were made through the skin on the proximal ventral surface of the tail on either side of the caudal artery. A vertical cut was then made through the skin to expose the tail artery. Silk suture was tied around the tail and the exposed caudal artery to occlude the vessel. After 60 min of ischemia, the suture was cut to allow for 60 min of reperfusion. At the end of the reperfusion, the rats were decapitated and the tail arteries distal to the occlusion were dissected out. Sham-operated rats were anesthetized and underwent surgery identical to the experimental rats, but their tail arteries were not occluded.

Contraction. Rat tail arteries distal to the ligature were dissected, cut into rings 5 mm in length, and placed in ice-cold physiological saline solution composed of 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1 mM NaH₂PO₄, 25 mM NaHCO₃, 1.8 mM CaCl₂, 11 mM glucose, 0.024 mM EDTA, and 1.4 mM ascorbic acid and bubbled with 95% O₂-5% CO₂. Tail artery ring segments were mounted at 37°C in 20-ml organ baths using stainless steel hooks connected by fine gold chain to a force transducer at the top and to the bottom of the chamber. Contraction was measured using force-displacement transducers (model FT.03, Grass) and a polygraph (model 7D, Grass). Preparations were equilibrated in physiological saline solution for 60 min at a resting tension of 750 mg, which was previously determined to be optimal. In the initial experiments, tail artery rings were tested for responsiveness to acetylcholine to assess endothelium-dependent relaxation of KCl-induced contraction. Acetylcholine did not produce relaxation in tail arteries obtained from both sham-operated and I/R-exposed rats, indicating that the endothelium of these rings was damaged under the present preparation conditions.

Inositol phosphate accumulation. The method for measuring [³H]inositol metabolism has been described previously (11, 18). Tail artery rings were preincubated in oxygenated HEPES buffer, pH 7.4, containing 10 mM HEPES, 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1.2 mM KH₂PO₄, 3.6 mM NaHCO₃, 1.3 mM CaCl₂, and 11 mM glucose at 37°C for 60 min. Subsequently, arterial segments were incubated for 90 min in 20 µCi/ml of [³H]myo-inositol (17 Ci/mmol, NEN, Boston, MA) containing buffer under the same conditions. Labeled arterial segments were washed four times and placed in individual tubes of HEPES buffer additionally containing 10 mM LiCl (total assay volume, 300 µl). Tail artery rings were incubated with agonist for 6 min. The reaction was terminated by addition of 250 µl of ice-cold 30% trichloroacetic acid. Tubes were left on ice for 20 min and then centrifuged at 1,500 g for 10 min. Aliquots (350 µl) of supernatant were added to 125 µl of 10 mM EDTA followed by addition of 500 µl of 1:1 Freon-tri-n-octylamine. The samples were vortexed and allowed to stand for 10 min before centrifugation (12,000 g, 10 min), and 300 µl of the aqueous phase were taken for analysis of inositol phosphates. Samples were loaded on Dowex-1(X8) ion-exchange columns (100-200 mesh, Bio-Rad, Hercules, CA). The columns were washed with 20 ml of 5 mM myo-inositol, and the inositol phosphates were then eluted with 4 ml of 0.1 M formic acid-1 M ammonium formate. Radioactivity in collected samples was measured by liquid scintillation spectrometry.

Adenylyl cyclase assay. The adenylyl cyclase assay was carried out according to the method described by Salomon et al. (32). Vessels were homogenized by glass-glass homogenizer in buffer containing 10 mM Tris · HCl, 1 mM EDTA, and 1 mM dithiothreitol, pH 7.5. The homogenate was centrifuged at 800 g for 10 min, and the supernatant was centrifuged at 22,000 g for 20 min at 4°C. The pellet obtained was suspended in 100 mM Tris · HCl, pH 7.5, and the protein concentration was determined by the method of Bradford (1). The reaction mixture contained 100 mM Tris · HCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM 3-isobutyl-1-methylxanthine, 0.5 mM ATP, 10 µM GTP, 2 mM creatine phosphate, 5 U creatine phosphokinase, and 1 µCi [-³²P]ATP (800 Ci/mmol; NEN). Total volume was 250 µl. After preincubation at 30°C for 5 min, the reaction was initiated by addition of 50 µg protein membrane and the incubation was carried on for additional 20 min. The reaction was terminated by addition of 300 µl stopping solution containing 2% SDS, 25 mM ATP, and 1.3 mM cAMP. Formed [³²P]cAMP was separated from [³²P]ATP by sequential passage through Dowex and alumina columns. [³H]cAMP (20 Ci/mmol, American Radiolabeled Chemicals, St. Louis, MO) was added to each assay tube for calculation of column recovery.

Immunoprecipitation and immunoblot analysis. Rat tail arteries were homogenized at 4°C using a glass-glass homogenizer in PBS, pH 7.6, containing 20 mM NaH₂PO₄, 20 mM Na₂HPO₄, and 154 mM NaCl with addition of 0.2% 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 20 µg/ml aprotinin, 25 µg/ml pepstatin, and 0.01 U/ml soybean trypsin inhibitor. The homogenate was centrifuged at 500 g for 10 min, and the supernatant was centrifuged at 49,000 g for 30 min. The resulting pellet was resuspended, rehomogenized, and then recentrifuged under the same conditions. The final pellet was resuspended in PBS. For immunoprecipitation, tail artery membranes were then solubilized by modification of a previously described procedure (12). Briefly, tail arterial membranes obtained as described above were solubilized by gentle end-over-end shaking for 60 min in PBS containing 1.5% digitonin, 0.5 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 20 µg/ml aprotinin, 25 µg/ml pepstatin, and 0.01 U/ml soybean trypsin inhibitor. The sample was centrifuged at 49,000 g for 30 min, and the supernatant (200 µg) was incubated with antibodies directed against the _1a (1:250 dilution; Santa Cruz Biotechnology, Santa Cruz, CA)-, _1b-, or _1d-adrenergic receptors (1:250 dilution; a kind gift of Dr. R. D. Brown, Denver Health Medical Center) for 3 h followed by a 60-min incubation with 100 µl 10% suspension of protein A-bearing Staphylococcus aureus cells (Pansorbin cells, Calbiochem, San Diego, CA). The specificity of the antibodies raised against each of the ₁-adrenergic receptors have been extensively characterized and described in previous reports (8, 12). After centrifugation and washing, the immunoprecipitates were solubilized in sample preparation buffer containing 62.5 mM Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue.

Statistical analysis. All data are presented as means ± SE. Data obtained from vessel contraction experiments, inositol phosphate accumulation, and adenylyl cyclase assays were analyzed by two-factor ANOVA followed by Newman-Keuls test or Dunnett's test where appropriate or by comparison using the Student's t-test.

	RESULTS

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Effect of I/R on tail artery contractility. The efficacy and potency of NE-induced contraction were assessed in arteries subjected to I/R and compared with control tissue. Maximal NE-stimulated contraction was increased from 1.53 ± 0.07 to 1.86 ± 0.11 g (P < 0.05), whereas the negative logarithm of the concentration yielding half-maximal effect was increased from 6.16 ± 0.05 to 6.45 ± 0.09 (P < 0.05) after I/R (Fig. 1). The contractile response to KCl was also assessed to test whether alterations in the contractile machinery account for the enhanced vascular response to NE. KCl (60 mM) induced equivalent contractions in tail arteries obtained from sham-operated and I/R animals (0.80 ± 0.07 vs. 0.89 ± 0.15 g; P > 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of ischemia-reperfusion (I/R) on contractile response of tail artery rings. Rat tail arteries subjected to I/R or sham operation (Sham) were cut into rings and placed in organ baths, and the contractile response to norepinephrine (NE; brackets denote concentration) was assessed. Values are means ± SE summarized from 6 individual experiments. The contractile response to NE was increased after I/R (P < 0.05).

Effect of I/R on ₁-adrenergic-receptor-mediated phosphoinositide hydrolysis and -adrenergic-receptor-stimulated adenylyl cyclase activity. Stimulation of vascular ₁-adrenergic receptors activates phospholipase C, thus increasing membrane phosphoinositide hydrolysis. Inositol phosphate accumulation was measured after I/R to determine whether changes in vascular contractility may be related to alteration in this ₁-adrenergic-receptor-mediated transmembrane signaling. As shown in Table 1, NE-stimulated inositol phosphate accumulation was increased after I/R from 1,153 ± 209 to 1,951 ± 292% (P < 0.01), whereas basal inositol phosphate accumulation remained unchanged (214 ± 53 vs. 281 ± 74 counts/min; P > 0.05). In separate experiments, we compared -adrenergic-receptor-sensitive activation of adenylyl cyclase in tail artery membranes of animals subjected to I/R and sham controls. I/R did not affect basal or isoproterenol-stimulated adenylyl cyclase activities. Furthermore, the activation of cyclase by forskolin, or by stimulation of G proteins with sodium fluoride, was not altered by I/R (Table 2).

View this table: [in this window] [in a new window]   Table 1.   1-Adrenergic-receptor-mediated inositol phosphate accumulation in the tail artery after ischemia-reperfusion

Effect of I/R on coupling of ₁-adrenergic-receptor subtypes to associated G proteins. The immunoblot data showed that the three ₁-adrenergic-receptor subtypes, _1a, _1b, and _1d, are present in tail artery membranes (Fig. 2). Immunoblot analyses also revealed the presence of single bands for G_i (41 kDa), G_o (39 kDa), G_q/11 (41 kDa), and G (36 kDa) and two bands for G_s (45 and 52 kDa) in tail artery membranes (Fig. 3). The expression level of tail artery ₁-adrenergic-receptor subtypes, or G proteins, was not affected by I/R (Figs. 2 and 3). We therefore considered that a change in ₁-adrenergic receptor-G protein coupling may be responsible for the enhanced receptor-mediated responses that follow I/R. Coupling of ₁-adrenergic receptors to G proteins was assessed by monitoring the levels of G proteins in the immunoprecipitates of specific ₁-adrenergic-receptor subtypes. The present experiment demonstrated that G_q/11 protein coimmunoprecipitated with _1a-, _1b-, or _1d-adrenergic receptors and that G_i protein was detected solely in the immunoprecipitate of the _1b-adrenergic receptor. Furthermore, receptor stimulation with the ₁-adrenergic-receptor agonist, phenylephrine, enhanced the coupling of the three tested receptor subtypes with their G proteins (Fig. 4). The basal associations of the three receptor subtypes with their G proteins were not influenced by I/R. However, the association of _1a-adrenergic receptor with G_q/11 protein in receptor-stimulated membranes was increased by more than twofold in vessels obtained from rats subjected to I/R (Fig. 4A). Receptor-stimulated couplings of _1b- and _1d-receptors with their G proteins were, however, not different in I/R compared with control tissue (Fig. 4, B, C, and D).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of I/R on tail artery 1-adrenergic-receptor (1-AR)-subtype levels. Tail arteries obtained from I/R or Sham rats were homogenized and solubilized, and 20 µg of the solubilized membrane proteins were size fractionated on 10% SDS-PAGE, electrically transferred to nitrocellulose membranes, and blotted with 1-AR-subtype-specific antibodies. The immunoreactive signals were visualized with enhanced chemiluminescence, and the optical density for each of the protein bands was assessed by soft-laser densitometry. Values are means ± SE summarized from 6 individual experiments. Insets, representative immunoblots of the indicated specific 1-AR subtypes. No differences in expression of 1-AR subtypes were found between sham-operated and I/R-exposed tissues (P > 0.05).

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effect of I/R on tail artery G protein levels. Arteries obtained from I/R or Sham rats were homogenized, and 20 µg of membrane proteins were size fractionated on 10% SDS-PAGE, electrically transferred to nitrocellulose membranes, and blotted with specific G protein antibodies. The immunoreactive signals were visualized with enhanced chemiluminescence, and the optical density for each of the protein bands was assessed by soft-laser densitometry. Values are means ± SE summarized from 6 individual experiments. Insets, representative immunoblots of specific G protein subunits. No differences in expression of G protein subunits were found between sham-operated and I/R-exposed tissues (P > 0.05).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Effect of ischemia-reperfusion on tail artery 1-AR-G protein coupling. Rat tail arteries subjected to I/R or sham operation were cut into rings and incubated with 1 µM phenylephrine (P) or vehicle (C) for 10 min. The ring segments were homogenized and solubilized, and 200 µg of membrane proteins were immunoprecipitated (IP) with specific 1-AR antibodies. Immunoprecipitates were subjected to 10% SDS-PAGE, electrically transferred to nitrocellulose membranes, and immunoblotted (IB) with specific G protein antibodies. The immunoreactive signals were visualized with enhanced chemiluminescence, and the optical density for each of the protein bands was assessed by soft-laser densitometry. Values are means ± SE summarized from 6 individual experiments. Insets, representative immunoblots of G proteins that coimmunoprecipitated with the indicated specific 1-AR subtypes. **P < 0.01 compared with the respective vehicle-treated group. ++ P < 0.01 compared with sham-operated phenylephrine-treated group.

	DISCUSSION

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This study utilized the rat tail artery as model system in the investigation of the effects of I/R on vascular ₁-adrenergic-receptor function. Exposure of tail arteries to 60 min of ischemia followed by 60 min of reperfusion resulted in increased contractile responses to NE as evidenced by higher maximal contraction and increased sensitivity to NE. The results are consistent with those previously observed in rabbit aorta and canine femoral artery (33, 37). The present changes in contractile response to NE did not result from a generalized alteration in the contractile machinery, because the contractile response to KCl, which increases Ca²⁺ entry through voltage-operated channels, was unaltered by I/R.

Activation of tail artery ₁-adrenergic receptors elicits vascular smooth muscle contraction via stimulation of phospholipase C and mobilization of intracellular Ca²⁺ (23, 25) that are dependent on a G protein that is insensitive to pertussis toxin (24). The present data demonstrate that NE-stimulated phosphoinositide hydrolysis parallels the increased vascular contractile response that is observed after I/R, suggesting that this signaling cascade is evoked by stimulation of vascular ₁-adrenergic receptors and may mediate the increased contractile response. I/R-induced increase in ₁-adrenergic-receptor signaling may result from increased densities of receptors, G proteins, or effectors. Previous investigations have indicated that acute myocardial ischemia increases ₁-adrenergic-receptor density (2, 3, 27). In an earlier, in vitro, study, we reported that prolonged stimulation of tail artery ₁-adrenergic receptors elicits decreases in G_q and G_i protein levels that appears to be responsible for desensitization of the receptor (34). In the present study, however, no differences in levels of ₁-receptor subtypes or of G or G proteins were detected after I/R, suggesting that changes in these signaling membrane proteins are not responsible for the enhanced contractile response after I/R in the tail artery.

The functional consequences of G protein-coupled receptor activation depend not only on the levels of the signaling proteins involved but also on the efficiency of interaction between receptor and G protein. Using the coimmunoprecipitation technique to directly determine the association of specific ₁-receptor subtypes with G subunits, we found that _1a- and _1d- adrenergic receptors are coupled to G_q/11 protein, whereas the _1b receptor is linked to both G_q/11 and G_i proteins. Moreover, receptor stimulation enhances the coupling of all three ₁-adrenergic receptors to their G proteins. In the tail arteries obtained from rats subjected to I/R, receptor-stimulated coupling of the _1a-adrenergic receptor to G_q/11 protein was increased. This enhanced coupling appears to be specific, because I/R did not affect the association of _1b- or _1d-adrenergic receptors with their G proteins. The results, therefore, suggest that increased _1a-adrenergic-receptor-G_q/11 protein coupling may underlie the enhanced ₁-adrenergic-receptor-mediated contractility and phosphoinositide signaling in the rat tail artery after I/R. Interestingly, the _1a-adrenergic receptor has been shown to activate ras, phosphatidylinositol 3-kinase and mitogen-associated protein kinase. These intracellular signaling molecules are associated with the cellular growth response (16). I/R has also been associated with a variety of events involved in vascular remodeling, including accumulation of growth factors, protein tyrosine phosphorylation, and cell proliferation (7, 14, 20, 35, 40). Therefore, the possible role of enhanced _1a-adrenergic-receptor signaling in the enhanced vascular growth response after I/R warrants further investigation.

Activation of -adrenergic-receptor-regulated adenylyl cyclase mediates blood vessel relaxation. A reduction in -adrenergic-receptor-stimulated adenylyl cyclase activity may, in turn, contribute to enhance vascular contractility in response to vasocontractile agents. In the present study, I/R did not alter isoproterenol-, sodium fluoride- or forskolin-stimulated cAMP accumulations in rat tail artery membranes, indicating that a reduction in cellular cAMP formation is unlikely to be involved in mediating the enhanced contractile response to ₁-adrenergic-receptor stimulation. These results are in agreement with those found in isolated dog coronary arteries after I/R (29) but differ from those reported in pulmonary arteries of dogs that underwent autologous lung transplant (9, 10). This discrepancy may be linked to differences in the arteries compared or to the duration of ischemia to which the two vessels were subjected.

Although it is well documented that NE induces arterial contraction mainly by stimulating ₁-adrenergic receptors located on vascular smooth muscle cells, ₂-adrenergic receptors are found in blood vessels (17, 41) and therefore activation of ₂-adrenergic receptors by NE may also contribute to the enhanced contractile response after I/R. Furthermore, the ₂-adrenergic receptors on vascular endothelial cells promote nitric oxide release, resulting in vascular relaxation (6, 17, 28, 41). Previous studies have repeatedly demonstrated that I/R leads to endothelial dysfunctions (5, 13, 15, 26, 38). Thus an impairment in ₂-adrenergic-receptor-mediated endothelium-dependent relaxation may contribute to enhanced ₁-adrenergic-receptor-mediated contractile response. However, the tail artery segments obtained from both sham-operated and I/R-exposed rats, as shown in the initial experiments, failed to respond to acetylcholine, suggesting that the endothelium did not factor into the present results. Furthermore, the enhanced ₁-adrenergic responses measured in terms of phosphoinositide hydrolysis and receptor-G protein coupling do not support a role for ₂-adrenergic receptors or for the endothelium in the present context.

In summary, the rat tail artery provides a useful model to investigate the effects of I/R on vascular function and the mechanisms responsible for enhanced vascular reactivity. The present results demonstrate that exposure of the tail artery to I/R leads to enhanced ₁-adrenergic-receptor-stimulated vascular contractility and phosphoinositide signaling that appear to result from increased coupling of _1a-adrenergic receptors to G_q/11 protein. The results provide evidence in support of the concept that selective I/R-mediated alteration in vascular ₁-adrenergic receptor and/or associated G_q/11 protein leads to changes in coupling efficiency of these two membrane components that results in vascular dysfunction.