Introduction

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The identification of endothelial ₂-adrenoceptors involved in a vasorelaxant response to catecholamines was made in the late 1980s (Vanhoutte and Miller, 1989). Conversely, vascular postjunctional ₁-adrenoceptors have been widely considered as vasoconstrictor receptors since their stimulation by sympathomimetic amines causes contraction of resistance arteries in most vascular beds. However, two reports (Zschauer et al., 1997; Boer et al., 1999) have produced evidence for the functional presence of vasorelaxant ₁-adrenoceptors in the brachial and pulmonary arteries isolated from the rabbit and rat, respectively. According to these reports, the pharmacological stimulation of ₁-adrenoceptors located on endothelial cells, is able to generate nitric oxide (NO), whereas the stimulation of ₂-adrenoceptors releases a relaxing prostanoid (Zschauer et al., 1997; Boer et al., 1999). According to a recent report (Nishina et al., 1999), ₂-adrenoceptors are involved in a vasorelaxant response induced by noradrenaline in conduit arteries of the neonatal rat. The release of endothelium-derived relaxing factors from endothelial cells, by activation of ₁- or ₂-adrenoceptors has also been previously demonstrated in other vascular beds (Kaneko and Sunano, 1993; Hu et al., 1994). Endothelial vasorelaxant adrenoceptors may represent a local control mechanism, which is, at least in part, involved in the modulation of the vasoconstrictor response to sympathomimetic amines. In fact, it is well known that the vascular response to sympathetic stimulation is enhanced by endothelium removal and NO synthase inhibitor administration (Greenberg et al., 1989; Bennet et al., 1992; Amerini et al., 1995; Zschauer et al., 1997; Boer et al., 1999). The aims of the present study were to test the functional presence of putative relaxant ₁-adrenoceptors in a preconstricted vascular preparation in vitro and to identify the cellular mechanisms involved in the vasorelaxant response. Moreover, since at least three subtypes of ₁-adrenoceptors (_1A, _1B, and _1D) are coexpressed in many tissues (Zhong and Minneman, 1999), we also determined the subtype(s) of ₁-adrenoceptors involved in the relaxant effect. For these purposes the effects of noradrenaline, of the selective ₁-adrenoceptor agonist phenylephrine and of pharmacological tools able to interfere with their actions were investigated in the MVB preparation isolated from the rat. Moreover, the transduction mechanisms were tested in cultured endothelial cells isolated from the bovine coronary vascular bed.

	Materials and Methods

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Indomethacin, 9,11-dideoxy-11, 9-epoxymethano-prostaglandin F₂ (U46619), N-nitro-L-arginine methyl ester hydrochloride (L-NAME), bradykinin, Dowex 50WX8-400 resin, Dulbecco's modified Eagle's medium (DMEM), lithium chloride, thapsigargin, L-arginine, HEPES (sodium salt), HEPES (free acid), acetylcholine (ACh), noradrenaline hydrochloride, and phenylephrine hydrochloride were purchased from Sigma (St. Louis, MO); (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxyl]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551 hydrochloride), (±)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino] ethoxy]-benzamide methanesulfonate (CGP 20712A methanesulfonate), and [2,6-dichloro(N--chloroethyl-N-methyl)-4-aminomethyl] phenylimino-2-imidazolidine dihydrochloride (chloroethylclonidine dihydrochloride) were purchased from Research Biochemicals International (Boston, MA); 1-(6((17-3-methoxy-estra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl)-2,5-pyrrolidine-dione (U73343) were purchased from Biomol (Plymouth Meeting, PA); ((S)-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid, 3-(4,4-diphenyl-1-piperidinyl) propylmethyl ester), (S)-(+)-niguldipine hydrochloride; and (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione), BMY 7378 dihydrochloride were purchased from Tocris Cookson Ltd. (Bristol, UK); bovine calf serum was purchased from Hyclone (Logan, UT); [³H]arginine, myo-2-[³H]inositol were purchased from Amersham Life Science Ltd. (Buckinghamshire, UK); and anion exchange columns were prepared with AG-K8 (200-400 mesh, formate form) from Bio-Rad Laboratories (Richmond, CA). A stock solution of indomethacin (10 mM) was prepared in ethanol; U73122, thapsigargin, and CGP 20712A were made in dimethyl sulfoxide; chloroethylclonidine was made in methanol; and the other drugs were dissolved daily in double distilled water and further dilutions to the final concentrations were made in Krebs' solution. Control experiments showed that the concentrations of dimethyl sulfoxide used modified neither the vasoconstrictor response to U46619 nor the relaxation induced by different agents.

Isolated MVB of the Rat. MVB were isolated from Wistar rats weighing 200 to 250 g and the superior mesenteric artery was cannulated with a stainless steel cannula. To eliminate the blood from the vessels, the preparations were flushed with Krebs' solution of the following composition: 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 25 mM NaHCO₃, 2.4 mM CaCl₂, and 5 mM glucose. The preparations were placed in an organ bath warmed to 37°C on a piece of titanium steel mesh and perfused with the same solution at a constant rate (4 ml/min) with a peristaltic pump. The solution (pH 7.3-7.4) was prewarmed and oxygenated with a gas mixture (5% CO₂, 95% O₂). Changes in the perfusion pressure were measured with a pressure transducer and recorded on a polygraph. To evaluate a drug-induced vasorelaxant effect, the preparation was precontracted by perfusion with a concentration of the thromboxane mimetic U46619 (0.3 µM) able to induce a submaximal vasoconstrictor response. The presence of functional endothelium was assessed by testing the vasodilator effect of ACh; the preparations in which ACh (1 µM) reduced the perfusion pressure by less that 30% were not used. Cumulative concentration-response curves to phenylephrine were obtained in preconstricted preparations; the effect of each concentration of the agonist was followed for 10 min. Blocking drugs were administered 30 min before testing their effects on the response to phenylephrine. The endothelium removal was performed by the method of perfusion with distilled water for 10 min, which is able to selectively destroy endothelial cells and to induce effects similar to those obtained by rubbing off the endothelium (Bolton et al., 1984; Criscione et al., 1984). The lack of a relaxation response to ACh in preconstricted vessels indicated that the procedure was successful. The baseline perfusion pressure after perfusion with U46619 was taken as 100%, and the reduction in perfusion pressure induced by relaxing agents was compared with this value.

Cell Cultures. Bovine coronary venular postcapillary endothelial cells (CVECs) were obtained and maintained in culture as previously described (Schelling et al., 1988). The endothelial cells were characterized by immunofluorescent staining for factor VIII antigen and uptake of acetylated low-density lipoproteins. Cells between passages 15 and 25 were used in the experiments.

Inositol Phosphate Metabolism. The method used in these experiments has been described in Ziche et al. (1993). Endothelial cells were seeded onto six multiwell plates (8 × 10⁴ cells/well) and, after overnight incubation, were labeled with [³H]myo-inositol (2 µCi/ml) in DMEM containing 10% bovine calf serum, without cold inositol for 48 h. Tritiated myo-inositol excess was removed by three washes with cold DMEM, followed by 4-h incubation with cold DMEM at 37°C. After washing, cells were incubated for 10 min with 20 mM LiCl to block myo-inositol-1-phosphatase, and were then exposed to test compounds for the designated times. Reaction was stopped by ice-cold methanol for 30 min. Cells were scraped, and cell-associated inositols were recovered by chloroform/methanol (1:1) extraction. Water-soluble fractions were applied to anion exchange columns and water-soluble inositols were eluted by successive washes (Ziche et al., 1993). Inositol monophosphate (IP₁) levels were measured as recovered radioactivity and expressed as dpm per well or as percentage over basal. Each experiment was performed in duplicate.

NO Synthase Activity. NO synthase activity was tested in CVEC monolayers according to the previously described method (Ghigo et al., 1995). The enzyme activity was evaluated by measuring the amount of L-[³H]citrulline produced after administration of L-[³H]arginine. Cells were seeded onto 60-mm culture dishes. Equilibration for 20 min at 37°C with HEPES buffer was followed by cell incubation for 30 min with 10 µM L-arginine and 20 min with 1 µCi of L-[³H]arginine. Cells were exposed to the tested drug for 5 min at 37°C, and then cold HEPES buffer was added to stop the reaction. Following the addition of ethanol and of 10 mM HEPES-sodium at pH 5.5, the amount of L-[³H]citrulline produced was assayed by liquid scintillation counting after elution through a resin column (Dowex AG50WX-8 activated sodium-form). NO synthase activity was measured as recovered radioactivity and expressed as cpm per milligram protein. Each experiment was performed in duplicate.

Statistical Analysis. All results are means ± S.E.M. of n experiments. Differences between groups were tested for significance by Student's t test for paired or unpaired data, and p < 0.05 was taken as significant. The pA₂ value was calculated using a statistical package for an IBM personal computer.

	Results

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Relaxant Effect of Noradrenaline and Phenylephrine on the Preconstricted MVB of the Rat. The exposure of isolated MVB of the rat to the thromboxane analog U46619 (0.3 µM) induced a stable increase in vascular tone: the perfusion pressure rose from 21.9 ± 1.5 to 62.2 ± 5.9 mm Hg after U46619 (n = 35). The addition of increasing concentrations of noradrenaline to preconstricted preparations induced a vasorelaxant effect at a concentration of 0.1 nM. Higher concentrations of noradrenaline did not produce any further increase in the relaxant effect (Fig. 1). Pretreatment of the preparations with both the ₁-adrenoceptor-selective antagonist CGP 20712A (1 µM) and the ₂-adrenoceptor antagonist ICI 118,551 (50 nM) did not affect the relaxant response induced by 0.1 nM noradrenaline. The vasorelaxant effect induced by noradrenaline was not detectable in preparations denuded of endothelium (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Vasorelaxant effect (percentage of decrease in perfusion pressure) induced by increasing concentrations of phenylephrine (n = 5) and noradrenaline (n = 5) in preparations preconstricted by 0.3 µM U46619. Points represent the mean values; vertical bars indicate S.E.M. values.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   A, changes in perfusion pressure induced by increasing concentrations of phenylephrine in intact preparations and in endothelium-denuded preparations. The preparations were preconstricted by exposure to 0.3 µM U46619. B, effect of 100 µM L-NAME and 100 µM L-NAME plus 3 µM indomethacin on the relaxant response to increasing concentrations of phenylephrine in preparations preconstricted by 0.3 µM U46619. Points represent the mean values of at least four experiments; vertical bars indicate S.E.M. values. *Statistically significant difference from control (p < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of different concentrations of prazosin on the vasorelaxant response to phenylephrine in preparations preconstricted by 0.3 µM U46619. Points represent the mean values of at least five experiments; vertical bars indicate S.E.M. values. *Statistically significant difference from control (p < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Vasoconstrictor effect (increase in perfusion pressure) induced by increasing concentrations of phenylephrine in preparations with intact endothelium. The influences of 1 nM (+)-niguldipine, 0.3 µM BMY 7378, and 10 µM CEC on the contractile response to the agonist are also shown. Points represent the mean values of at least four experiments; vertical bars indicate S.E.M. values. *Statistically significant difference from control (p < 0.05).

Effect of U73122 and Thapsigargin on the Relaxant Response to Phenylephrine. The relaxant response to phenylephrine was inhibited by 30-min pretreatment with 0.1 to 1 µM of the phospholipase C inhibitor U73122 (Fig. 5A). Conversely, a concentration of 1 µM U73343, a molecule structurally similar to U73122 that does not inhibit phospholipase C (Jin et al., 1994), did not significantly influence the pattern of response to phenylephrine (Fig. 5A). The relaxation also remained unaffected after exposure to a greater concentration (3 µM) of U73343 (data not shown). Moreover, exposure of the preparations for 30 min to the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (1 µM) was also able to completely block the vasorelaxant effect of phenylephrine (Fig. 5B).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   A, effect of a 30-min period of exposure to U73122 (0.1-1 µM) or 1 µM U73343 on the relaxing response to increasing concentrations of phenylephrine in preparations preconstricted by 0.3 µM U46619. B, effect of a 30-min period of exposure to 1 µM thapsigargin (TG) on the relaxing response to increasing concentrations of phenylephrine in preparations preconstricted by 0.3 µM U46619. Points represent the mean values of at least four experiments; vertical bars indicate S.E.M. values. *Statistically significant difference from control (p < 0.05).

Effect of Phenylephrine on Inositol Phosphate Metabolism in Cultured Endothelial Cells. To identify the cellular events involved in the mechanism of the phenylephrine relaxant effect, the effect of the exposure of stabilized cultures of CVECs to the drug was investigated. CVECs were chosen among the available endothelial cell models, since we have previously demonstrated that these cells possess only the Ca²⁺ calmodulin-dependent constitutive NO synthase (cNOS) that is sensitive to endothelium-dependent vasorelaxant agents (Parenti et al., 1998). The cellular level of the inositol trisphosphate metabolite IP₁ was measured following endothelial cell contact with different concentrations of phenylephrine for 15 min. Phenylephrine at a concentration of 0.03 nM significantly increased by 190 ± 17.6% the basal IP₁ level (dpm = 400 ± 40); the maximum increase in IP₁ was observed in response to 0.1 nM phenylephrine (Fig. 6A). Higher agonist concentrations did not produce any greater increase in IP₁ levels. The accumulation of IP₁ in endothelial cells induced by 0.1 nM phenylephrine was antagonized by 10 nM prazosin, whereas it was unaffected by 1 µM yohimbine (Fig. 6B), thus confirming the involvement of ₁-adrenoceptors in this response.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   A, effect of exposure to different concentrations of phenylephrine on IP1 levels in cultured endothelial cells. The effect of 100 nM bradykinin (BK) is shown for comparison. B, effects of the 1-adrenoceptor antagonist prazosin (10 nM) and of the 2-adrenoceptor antagonist yohimbine (1 µM) on IP1 accumulation in endothelial cells induced by 0.1 nM phenylephrine. Columns indicate the mean values of four experiments in duplicate; vertical bars indicate S.E.M. values. Statistically significant differences from control, **p < 0.01; ***p < 0.001.

Effect of Phenylephrine on NO Synthase Activity in Cultured Endothelial Cells. A 5-min time of contact with 0.03 and 0.1 nM phenylephrine induced an increase in the cNOS activity of the cells that rose from a control value of 11,487.5 cpm/mg of protein to 17,936.5 and 22,678, respectively (Fig. 7A). The increase in phenylephrine concentration to 1 nM did not produce any additional effect on cNOS activity. The effect of phenylephrine was mediated by NO synthase activation, since blockade of NO synthase activity by 100 µM L-NAME completely inhibited the effect of phenylephrine (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   A, effect of exposure to different concentrations of phenylephrine on cNOS activity of cultured endothelial cells. Columns indicate the mean values of two experiments in duplicate; vertical bars indicate S.E.M. values. B, influence of cell pretreatment with 0.3 µM BMY 7378 on the increase in cNOS activity induced by 0.1 nM phenylephrine. Columns indicate the mean values of three experiments in duplicate; vertical bars indicate S.E.M. values. *Statistically significant difference from control (p < 0.05).

Characterization of Subtypes of ₁-Adrenoceptors Involved in the Observed Responses. To characterize the ₁-adrenoceptor subtype involved in the relaxant response to phenylephrine, experiments with subtype-selective antagonists were carried out. Three antagonists were selected for this purpose: the selective _1A-adrenoceptor antagonist (+)-niguldipine (Boer et al., 1989), the selective _1D-adrenoceptor antagonist BMY 7378 (Goetz et al., 1995), and the irreversible _1B- and _1D-adrenoceptor antagonist chloroethylclonidine (Docherty and O'Rourke, 1997). The decrease in perfusion pressure induced by nanomolar concentrations of phenylephrine was reduced in a concentration-dependent manner either by 1 to 10 µM chloroethylclonidine (Fig. 8A) or by 0.01 to 0.3 µM BMY 7378 (Fig. 8B), but was not influenced by 1 nM (+)-niguldipine (data not shown). The pretreatment of cultured cells with 0.3 µM BMY 7378 also inhibited the increase in cNOS activity induced by 0.1 nM phenylephrine (Fig. 7B). Conversely, the vasoconstrictor response induced by micromolar concentrations of phenylephrine was blocked by 1 nM (+)-niguldipine, but was unaffected by 0.3 µM BMY 7378 and by 10 µM chloroethylclonidine (CEC) (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   A, effect of different concentrations of CEC on the relaxant response to increasing concentrations of phenylephrine in preparations preconstricted by 0.3 µM U46619. B, effect of different concentrations of BMY 7378 on the relaxant response to increasing concentrations of phenylephrine in preparations preconstricted by 0.3 µM U46619. Points represent the mean values of at least four experiments; vertical bars indicate S.E.M. values. *Statistically significant difference from control (p < 0.05).

	Discussion

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The functional significance of the presence of different subtypes of ₁-adrenoceptors in vascular tissues has been previously investigated mainly with regard to their role in mediating the contractile response to sympathomimetic amines (Muramatsu et al., 1998; Yousif et al., 1998; Daniel et al., 1999; Satoh et al., 1999; Zhu et al., 1999). The present study shows that two different subtypes of ₁-adrenoceptors are able to mediate opposite effects on vascular tone in the same preparation. In fact, although the _1A-adrenoceptors mediated a contractile response, the _1D-adrenoceptor was involved in an endothelium-dependent relaxant effect. Our findings also suggest that, at least in the rat MVB, ₁-adrenoceptor agonists may have a greater affinity for the endothelial _1D-receptors, since the relaxant effect occurred at concentrations lower than those able to induce a vasoconstrictor response. The cellular mechanism causing the relaxant response was studied using a variety of pharmacological tools. The relaxant effect of phenylephrine was clearly dependent on the ability of endothelium to generate NO, since it was not detectable in endothelium-denuded preparations, and was blunted by pretreatment of the preparations with the NO synthase inhibitor L-NAME, at a concentration that antagonizes the endothelium-dependent vasodilatory effect of ACh in the same preparation (Mantelli et al., 1995). Moreover, the observation that the effect of phenylephrine was effectively inhibited by U73122 suggested that phospholipase C and the following cellular steps represent the possible targets of the agonist activity, since this aminosteroid compound has an inhibitory effect on the enzyme (Bleasdale et al., 1990; Yule and Williams, 1992; Jin et al., 1994; Grierson and Meldolesi, 1995). Finally, the inhibition of the relaxant effect of phenylephrine by the inhibitor of the Ca²⁺-ATPase of the sarcoplasmic reticulum thapsigargin (Thastrup et al., 1990; Lytton et al., 1991; Dolor et al., 1992) suggested the involvement of inositol trisphosphate (IP₃)-sensitive Ca²⁺ stores of sarcoplasmic reticulum in the mechanism of the relaxant effect of phenylephrine. By emptying and preventing the refilling of IP₃-sensitive cellular calcium stores, thapsigargin is able to suppress the agonist induced release of NO by isolated vascular preparations, and to prevent the increase in intracellular free calcium levels induced by vasorelaxant endothelium-dependent agents (Macarthur et al., 1993; Amerini et al., 1996).

The results found in the rat MVB were confirmed by experiments in cultured endothelial cells where the selective ₁-adrenoceptor agonist phenylephrine caused an increase in NO synthase activity. The effect of phenylephrine on NO synthase was associated with an increase in IP₁ cellular levels, showing that the pattern of the postreceptor transduction mechanism triggered by phenylephrine includes inositol phosphate stimulation, which is followed by Ca²⁺ mobilization from IP₃-sensitive stores in the sarcoplasmic reticulum (Minneman, 1988). An increase in free Ca²⁺ has previously been demonstrated in endothelial cells after ₁-adrenoceptor stimulation (Tuttle and Falcone, 1997). It is conceivable that the two effects of phenylephrine detected in cultured endothelial cells (i.e., IP₃ generation and NO synthase activation) are linked, since it is known that the synthesis of NO is a calcium-dependent process (Luckhoff et al., 1988) and that constitutive NO synthase in endothelial cells is a calcium/calmodulin-dependent enzyme (Berdeaux, 1993). In fact, the release of endothelium-derived relaxing factor induced by ACh, as well as by bradykinin, adenosine diphosphate, and Substance P, is triggered by an increase in cellular free calcium concentration (Busse et al., 1988; Berdeaux, 1993; Ziche et al., 1993). The main characteristics of the effects observed with phenylephrine in endothelial cells were the following: 1) the effects were induced by surprisingly low agonist concentrations (in the nM range), and 2) they were concentration-dependent in a narrow range of concentrations (0.03-0.1 nM). The same pattern of response was observed in the experiments in which the relaxant effect of phenylephrine was tested in a preconstricted vascular preparation. In fact, in these experiments, a slight decrease in perfusion pressure was detected with the same small concentrations of phenylephrine, which were devoid of any contractile effect. However, neither the functional studies in isolated MVB preparations nor the findings in cultured endothelial cells gave any useful information about the subtype of ₁-adrenoceptor involved in the relaxant response, since it is known that all subtypes are able to activate phospholipase C and to release calcium from intracellular stores (Zhong and Minneman, 1999). Thus, the characterization of the ₁-adrenoceptor subtype mediating the relaxing response was carried out by the use of subtype-specific antagonists. The finding that the relaxant effect of phenylephrine was completely antagonized by both chloroethylclonidine, an irreversible antagonist for the _1B- and _1D-adrenoceptor subtypes (Docherty and O'Rourke, 1997) and the selective _1D-adrenoceptor antagonist BMY 7378 (Goetz et al., 1995) strongly suggested that the receptor subtype involved in the relaxation is the _1D-adrenoceptor subtype. This hypothesis was reinforced by the observation that BMY 7378 was able to antagonize the increase in cNOS activity induced by phenylephrine in endothelial cells. Conversely, the blocking effect exerted by the selective antagonist (+)-niguldipine (Boer et al., 1989) on the vasoconstrictor response to micromolar concentrations of phenylephrine showed that the _1A-adrenoceptor subtype is responsible for the increase in vascular tone induced by ₁-adrenoceptor stimulation.

In conclusion, the present study has shown that different subtypes of adrenoceptors can mediate opposite effects on vascular tone in the mesenteric vascular bed of the rat. In particular, the findings indicate that the endothelium-dependent relaxant response induced by nanomolar concentrations of phenylephrine, which are devoid of any contractile effect, is caused by the stimulation of _1D-adrenoceptors, which act through phospholipase C stimulation, followed by IP₃ generation, Ca²⁺ mobilization, and NO synthase activation. Conversely, the vasoconstrictor response induced by micromolar concentrations of phenylephrine is caused by stimulation of the _1A-adrenoceptor subtype.