Introduction

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Studies in humans and animals have indicated a close link between hypertension and obesity. While one of the major causes for hypertension is an increased peripheral vascular resistance, obesity is due to either an excessive energy intake, a decreased energy expenditure or both. Both peripheral vascular resistance and energy metabolism are regulated by the sympathetic nervous system (Reaven, 1995).

In skeletal muscle, the largest and potentially most important thermogenic tissue, the sympathetic nervous system controls thermogenesis through both  and  ARs by different mechanisms. Whereas -ARs directly mediate VO₂ (an indirect measure of thermogenesis) in muscle cells, -ARs appear to control muscle VO₂ by hemodynamic mechanisms (Ye et al., 1995). In the constant-flow perfused hindlimb of Wistar rats, a reliable muscle vascular preparation with many characteristics similar to those in vivo (Bonen et al., 1994), administration of 1-AR agonists or sympathetic nerve stimulation elicits either positive or negative changes in VO₂ during a sigmoidal increase in perfusion pressure, an indicator of vasoconstriction in this model (Clark et al., 1995; Hall et al., 1997). One of the major features of -AR mediated VO₂ is its bell-shaped dose-response curve characterized by increases (the ascending limb) at low concentrations of norepinephrine (LNE, <1 µM) and decreases from the maximum to a value below the basal level (the descending limb) at high concentrations of NE (HNE > 1 µM) (Dora, 1993; Rattigan et al., 1995; Clark et al., 1995). Similarly, sympathetic nerve stimulation raises VO₂ at low frequencies (<4 Hz) but reduces VO₂ at high frequencies (>4 Hz) during vasoconstriction (Hall et al., 1997). Both the increase and the decrease in VO₂ are reversed when the vasoconstriction is blocked by either -1-AR antagonists or by vasodilators such as nitroprusside (Dora, 1993; Rattigan et al., 1995; Ye et al., 1995, Hall et al., 1997). These findings strongly suggest a close link of muscle VO₂ to vasoconstriction mediated by -ARs in the perfused rat hindlimb.

In the perfused hindlimb of SHR, NE is known to cause stronger vasoconstriction compared to that in their genetically normotensive counterparts, WKY (Cheng and Shibata, 1980). Similar results were obtained with the 1-AR agonist methoxamine (Adams et al., 1989). These data suggest that functional changes in -1-ARs may occur in the resistance blood vessels of muscle vascular beds in SHR. If so, the muscle VO₂ (and therefore thermogenesis) controlled by -1-AR-mediated vasoconstriction is also likely to be affected.

At least three -1-ARs have so far been identified in vascular tissues, namely alpha-1A-, alpha-1B- and alpha-1D-subtypes (Hieble et al., 1995a). Functional characterization of these subtypes has been made possible now by using subtype selective antagonists. For instance, all these three subtypes show a high affinity for prazosin and a low affinity for yohimbine (Bylund et al., 1994). Both 5 MU (Perez et al., 1994) and KMD (Shibata et al., 1995) have a higher affinity for the -1A-AR than for the other two subtypes, whereas BMY (Goetz et al., 1995) and SK&F (Hieble et al., 1995b) each possess a higher affinity for the -1D-AR. The -1B-AR is most sensitive to alkylation by CEC (Minneman et al., 1988, Bylund et al., 1994). We hypothesized that the increased sensitivity of SHR muscle vascular bed to NE may be mediated by differently altered -1-AR subtypes and these alterations may then lead to changes in NE-elicited thermogenesis in this tissue. Therefore, we compared the effects of seven selective -AR antagonists on NE-induced vasoconstriction and associated VO₂ in the perfused hindlimb of SHR and their age-matched WKY in our study.

	Materials and Methods

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Animals. Age-matched (11 wk) male SHR (277.7 ± 1.0 g, n = 36) and WKY (276.2 ± 0.9 g, n = 36) used for the experiments were purchased from the Animal Resources Center of Australia. The animals were housed on arrival at 20°C with a 12 hr light/12 hr dark cycle and allowed free access to food and water. The diet consisted of 2% protein, 4.6% lipid, 69% carbohydrate, 6% crude fiber with added vitamins and minerals (Gibson, Hobart, Tasmania). All experiments were approved by the Ethics Committee of the University of Tasmania under the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1990). Blood pressure determined in the anesthetized state (pentobarbital, 60 mg/kg, i.p) from a cannulated carotid artery by the use of a manometer (ALPK2, Japan) was much higher in SHR compared with that in WKY (182 ± 3.0 vs. 94 ± 3.0 mmHg, P < .001, n = 8).

Hindlimb perfusion. The rats were anaesthetized with sodium pentobarbital (60 mg/kg i.p.). A midline incision was made to expose the abdominal cavity and the cut edges of the abdominal wall were ligated, where necessary, with sutures. Gut, seminal vesicles and testicles were removed after appropriate ligations. The right common iliac artery and vein were ligated so that only the left hindlimb was perfused. To prevent any perfusate spillover when perfusion pressure was increased, blood vessels connected with tissues other than the left hindlimb were carefully tied off. These included the right hypogastric vessels, the left inferior and superficial epigastric vessels, the inferior mesenteric and superior vesicle vessels, and the iliolumbar and spermatic vessels on both sides. Before cannulation, heparin (0.5 ml, 1000 U/ml) was injected into the vena cava between the right and left renal veins. Two cannulae (Ohmeda, Sweden) were then inserted caudally into the abdominal aorta (16G) and vena cava (18G) between the left renal and iliolumbar vessels. The rat was then immediately placed on perspex platform for perfusion followed by an overdose injection of the anesthetic to kill the animal. Ligatures were then placed firmly around: the lumbar trunk between L₃-L₄ vertebrate, right thigh (near the inguinal ligament), and the genitalia (above the penis), respectively.

1

1

Experimental protocols. After commencing the perfusion, 50 min was allowed to elapse before constructing the dose-response curve for NE. The basal values for perfusion pressure and VO₂ were obtained between 40 and 50 min. Three sets of experiments were performed in both SHR and WKY as follows. Set 1 was designed to assess the involvement of -ARs in NE-induced changes in vasoconstriction and associated VO₂ by comparing the effects of NE in the absence and presence of 10 µM propranolol. In set 2, experiments were divided to three groups and conducted in the presence of 10 µM propranolol to evaluate the role of ₁- and ₂-ARs in altered vasoconstriction and VO₂ induced by NE: control (from set 1), prazosin (2.5 nM) and yohimbine (0.1 µM). Assessments of the contribution of each ₁-AR subtype to the altered vasoconstriction and VO₂ were conducted in set 3 in the presence of propranolol (10 µM) plus yohimbine (0.1 µM). The experiments were assigned to the following groups: control (from set 2), 5 MU (3.3 nM), KMD (1 nM), CEC (10 µM), BMY (0.1 µM) and SK&F (2 µM). The doses of these -AR antagonists were chosen to maximize the differentiation of differences between SHR and WKY according to our preliminary experiments within the range of their selectivity. The experiments on SHR and WKY were interspersed randomly. After obtaining the results from set 3, additional experiments were performed with low doses of BMY (10 nM) and SK&F (0.33 µM) in SHR to further examine the role of -1D-AR subtype on the descending limb of the VO₂ response curve. Each antagonist was infused 30 min before and during the period of NE infusion. NE and -AR antagonists were infused from a port in the arterial line at a rate less than 1% of the perfusion flow rate and mixed by a magnetic stirrer in a small bubble trap before entering the hindlimb. In experiments with CEC, the alkylating agent was infused for a period of 30 min and then washed out for 20 min before the infusion of NE. Dose-response curves were constructed in a cumulative fashion. Each hindlimb was used for constructing the dose-response curves only once to ensure that the metabolic characteristics of the preparation were valid within the period of time previously established (Colquhoun et al., 1990). The perfusion flow rate was checked (corrected if necessary) each time after changing NE dose.

Chemicals. [-]-NE bitartrate, dl-propranolol hydrochloride, prazosin hydrochloride and yohimbine hydrochloride were obtained from Sigma (St. Louis, MO). 5-Methylurapidil, BMY 7378 dihydrochloride, chloroethylclonidine dihydrochloride were purchased from RBI (Natick, MA). KMD-3213 ((-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2,2-trifluroroethoxy)phenoxyl]ethyl]amino]propyl]indoline-7-carboxamide) was a gift from Dr. Y. Kurashina (Kissei Pharmaceutical Co., Matsumoto, Japan) and SK&F 105854 (furo-3-ben-zazenpine) a gift from Dr. J. P. Hieble (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). NE was dissolved freshly in .9% NaCl containing 0.1% ascorbic acid. Prazosin was initially dissolved in dimethylsulfoxide in a stock solution and then diluted to an appropriate concentration with 0.9% NaCl before use. Other antagonists were dissolved in the normal saline. Bovine serum albumin (fraction V) was obtained from Boehringer Mannheim Corp. (Indianapolis, IN). Other chemicals were analytical grade from Ajax Chemicals (Sydney, Australia).

Calculation and statistical analysis. EC₂₅, EC₅₀ and IC₇₅ (designated here as the inhibitory effect of NE on VO₂) were calculated individually from the best fit dose-response curves by Sigma Plot for Windows on the basis of fractional changes (Kenakin, 1993). The regression coefficient closest to 1 was used to determine the best fitness of a curve. Because NE-induced changes in VO₂ were bell-shaped, EC₂₅ and IC₇₅ were calculated instead of EC₅₀ and IC₅₀ to avoid, where possible, the influence of one side of the response on the other (Szabadi, 1977). The negative log values of EC₂₅, EC₅₀ and IC₇₅ were expressed as pEC₂₅, pEC₅₀ and pIC₇₅, respectively (Jenkinson et al., 1995). pK_B values were calculated according to the following formula (Kenakin, 1993) using EC₅₀ of perfusion pressure: pK_B = log (DR-1)-log[B], where DR is the ratio of EC₅₀ in the presence of a given antagonist to EC₅₀ in absence of antagonist, and [B] is the concentration of the antagonist. Because DR could not be obtained from individual hindlimb preparations, pK_B was calculated using the mean EC₅₀ and was without a S.E. Data are presented as means ± SE. Dose-response curves were determined to differ (P < .05) using ANOVA (Startview SE, Abacus Concept, Berkeley, CA) with dose as a repeated measure. Bell-shaped dose-response curves were firstly tested using all points. Then each side of the curves was further analyzed based on the model of two functionally antagonistic receptor populations activated by the same agonist (Szabadi, 1977). Student's t tests were used for the comparison between two means values with P < .05 as statistically significant.

	Results

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Effects of adrenergic antagonists on basal perfusion pressure and VO₂. None of the antagonists used, when infused alone, had any significant effect on either the basal perfusion pressure or VO₂ in SHR or WKY (table 1). The calculated basal perfusion pressure and VO₂ from the pooled data were 37.3 ± 0.3 and 7.0 ± 0.1 µmol g¹ hr¹, respectively, for WKY (n = 36). In SHR, the basal perfusion pressure (45.5 ± 0.6 mmHg) and VO₂ (7.9 ± .1 µmol g¹ hr¹) were both significantly higher (P < .01, n = 36).

Effects of NE on perfusion pressure and VO₂. NE induced a dose-dependent sigmoidal increase in perfusion pressure in both SHR and WKY (fig. 1). Compared with WKY, the perfusion pressure was displaced to the left for more than 2-fold in SHR as indicated by the value of pEC₅₀ (table 2). The maximal increase in pressure (P_max) was greater in SHR (235.7 ± 7.5 mmHg, P < .01) than in WKY (199.3 ± 3.1 mmHg). During vasoconstriction, NE-elicited bell-shaped changes in VO₂ in both strains. Compared with WKY, the change in VO₂ was altered in SHR with the ascending side increased (P < .05) and descending side depressed (P < .01, ANOVA). Furthermore, LNE-induced maximal increment in VO₂ (VO_{2 max}) was smaller (3.65 ± 0.11 vs. 4.41 ± 0.14 µmol g¹ hr¹, P < .01) and HNE-induced maximal inhibition of VO₂ (VO_{2 min}) was greater (-3.51 ± 0.37 vs. -0.87 ± .43 µmol g¹ hr¹, P < .01) in SHR.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of NE on perfusion pressure and VO2 in the perfused hindlimb of SHR and WKY. Dose-response curves were constructed by an accumulative infusion of NE. Basal values of perfusion pressure and VO2 are shown in table 1. Open circles, WKY; closed circles, SHR. Data are means ± S.E. (n = 4). P < .01 for the pressure curves; P < .05 for the ascending side and P < .01 for the descending side of the VO2 curves (ANOVA).

View this table: [in this window] [in a new window]   TABLE 2 Effect of - and -AR antagonists on the pEC50 for perfusion pressure and the pEC25 and pIC75 for VO2 produced by NE in the perfused hindlimb of SHR and WKY

Effects of -AR antagonist on NE-induced perfusion pressure and VO₂. In the presence of propranolol, NE-induced perfusion pressure was shifted to the left in WKY (fig. 2 A and B; table 2). Although VO_{2 max} was smaller in WKY (5.64 ± 0.18 vs. 4.40 ± 0.20 µmol g¹ hr¹, P < .01, t test), the entire VO₂ curve was not significantly different (P > .05, ANOVA). Neither pressure nor VO₂ produced by NE was significantly altered by propranolol in SHR (fig. 2 C and D).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effects of propranolol on NE-induced perfusion pressure and VO2 in the perfused hindlimb of SHR and WKY. NE-induced dose-response curves were constructed in the absence (squares) or presence (circles) of 10 µM dl-propranolol in both WKY (A and B) and SHR (C and D). Basal values of perfusion pressure and VO2 are shown in table 1. Data are means ± S.E. (n = 4). P < .01 for the pressure curves in A and P > .05 for the curve comparisons in B, C and D (ANOVA).

Effects of ₁-AR and ₂-AR antagonists on NE-induced perfusion pressure and VO₂. In WKY, both prazosin (2.5 nM) and yohimbine (0.1 µM) markedly shifted NE-induced dose-response curves of perfusion pressure and VO₂ to the right without changing P_max, VO_{2 max}, or VO_{2 min} (fig. 3 A and B; table 2). In SHR, the antagonistic effect of prazosin was much bigger and that of yohimbine was significant only within the range of LNE (fig. 3 C and D). NE-induced perfusion pressure was shifted to the right more in SHR by prazosin compared with WKY and the differences in associated changes in VO₂ between SHR and WKY disappeared (fig. 4). The pK_B values for prazosin and yohimbine of NE-induced perfusion pressures for WKY were 9.24 and 7.34, respectively. In comparison, the pK_B value was higher for prazosin (9.81) and lower for yohimbine (6.89) in SHR.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of prazosin and yohimbine on NE-induced perfusion pressure and VO2 in the perfused hindlimb of SHR and WKY. Dose-response curves were constructed in the presence of 10 µM dl-propranolol. Basal values of perfusion pressure and VO2 are shown in table 1. A and B represent WKY and C and D represent SHR. Control (circles with solid lines), 2.5 nM prazosin (squares with dash lines), 0.1 µM yohimbine (circles with dash lines). Data are means ± S.E. (n = 4). A, P < .01 for both prazosin and yohimbine; B, P < .01 for prazosin in both sides, P < .01 for the ascending side and P > .05 for the descending side of yohimbine; C, P < .01 for prazosin and P > .05 for yohimbine; D, P < .01 for prazosin in both sides, P < .01 for the ascending side and P > .05 for the descending side of yohimbine Analyses were performed using ANOVA (vs. control).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Comparison of NE-induced perfusion pressure and VO2 in the presence of prazosin or yohimbine in the perfused hindlimb of SHR and WKY. The perfusions were performed in the presence of 10 µM propranolol. Open symbols represent WKY and close symbols represent SHR. 2.5 nM prazosin (A and B), 0.1 µM yohimbine (C and D). Data are means ± S.E. (n = 4). A, P = .056; B, P > .05; C, P < .01; D, P < .05 for both sides (ANOVA).

Effects of -1A-AR antagonists on NE-induced perfusion pressure and VO₂. At 3.3 nM, 5 MU had no significant effect on NE-induced changes in either pressure or VO₂ in WKY (fig. 5 A and B). In contrast, NE-induced dose-response curve of pressure was shifted to the right by 5 MU more than 3-fold in SHR (fig. 5 C and D; table 3) with a pK_B value of 8.94. Associated with the inhibition of vasoconstriction, HNE-inhibited VO₂ in SHR was markedly attenuated. Similar attenuation of HNE-inhibited VO₂ was also observed with KMD at 1 nM. However, the P_max values were significantly reduced in both WKY and SHR by KMD.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of -1A-antagonists on NE-induced perfusion pressure and VO2 in the perfused hindlimb of SHR and WKY. Dose-response curves were constructed in the presence of 10 µM dl-propranolol plus 0.1 µM yohimbine. Basal values of perfusion pressure and VO2 are shown in table 1. A and B represent WKY and C and D represent SHR. Control (circles with dash lines), 3.3 nM 5 MU (hexagons with solid lines), 1 nM KMD (squares with solid lines). Data are means ± S.E. (n = 4). A, P > .05 for 5 MU and P < .01 for KMD; B, significance was only found for the descending side of KMD (P < .01); C, P < .01 for both 5 MU and KMD; D, P > .05 for the ascending sides and P < .01 for the descending side of both 5 MU and KMD. Analyses were performed using ANOVA (vs. control).

View this table: [in this window] [in a new window]   TABLE 3 Effect of 1-AR subtype antagonists on the pEC50 for perfusion pressure and the pEC25 and pIC75 for VO2 produced by NE in the perfused hindlimb of SHR and WKY

Effects of -1D-AR antagonists on NE-induced perfusion pressure and VO₂. Neither BMY (0.1 µM) nor SK&F (2 µM) significantly altered NE-induced dose-response curves of perfusion pressure in WKY (fig. 6 A and B), although NE-induced perfusion pressure and the associated increases in VO₂ were both slightly reduced at 33 and 100 nM (P < .05). However, BMY and SK&F markedly shifted dose-response curves of pressure to the right by 3.4- and 2.9-fold respectively in SHR (table 3). Associated with the shift of the pressure curve, LNE-elicited ascending limb of the VO₂ curve was markedly reduced and the HNE-elicited descending limb was moderately attenuated (fig. 6 C and D). Pretreatment with 10 µM CEC had only small inhibitory effect on perfusion pressure and VO₂ in SHR at one or two low concentrations of NE without affecting the overall dose-response curves (P > .05, ANOVA). In WKY, pretreatment with 10 µM CEC was without effect (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of -1D-antagonists on NE-induced perfusion pressure and VO2 in the perfused hindlimb of SHR and WKY. Dose-response curves were constructed in the presence of 10 µM dl-propranolol plus 0.1 µM yohimbine. Basal values of perfusion pressure and VO2 are shown in table 1. A and B represent WKY and C and D represent SHR: control (circles with dash lines), 0.1 µM BMY (triangles with solid lines) and 2 µM SK&F (reversed triangles with solid lines). Data are means ± S.E. (n = 4). A, P > .05 for BMY and SK&F; B, P < .05 for the ascending side of both BMY and SK&F only; C, P < .01 for both BMY and SK&F; D, P < .01 for the ascending side of both BMY and SK&F and P < .05 for the descending side of both BMY and SK&F. Analyses were performed using ANOVA (vs. control).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effects of low dose -1D-antagonists on NE-induced perfusion pressure and VO2 in the perfused hindlimb of SHR. Dose-response curves were constructed in the presence of 10 µM dl-propranolol plus 0.1 µM yohimbine. Basal values of perfusion pressure and VO2 were 44.2 ± 1.3 mmHg and 7.9 ± 0.1 µmol g hr for BMY and 47.0 ± 1.1 mmHg and 7.4 ± 0.3 µmol g hr for SK&F, respectively. Control (circles with dash lines), 10 nM BMY (triangles) and 0.33 µM SK&F (reversed triangles). Data are means ± S.E. (n = 4). A, P > .05 for BMY and SK&F (however, when expressed as a fractional change using percentage, P < .05 for the both; data not shown); B, P < .01 for the ascending side of both BMY and SK&F, and P > .05 for the descending side of both BMY and SK&F. Analyses were performed using ANOVA (vs. control).

	Discussion

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Basal perfusion pressure and VO₂. SHR have been shown to have a higher vascular resistance in skeletal muscle in the absence (Cheng and Shibata, 1980) or presence (Adams et al., 1989) of a vasodilator presumably due to morphological changes of the resistance vessels in muscle vascular bed. The increased muscle vascular resistance in SHR was confirmed in the present experiment. Interestingly, the basal VO₂ of the hindlimb was also found to be 13% higher in SHR. The reason for this may be associated with increased numbers of the sarcolemmal Na⁺-K⁺-ATP pump and its overall compensatory activity to expel accumulated intracellular Na⁺ at this age (Pickar et al., 1994). Such an explanation is supported by recent findings showing an elevated ATP turnover in skeletal muscle from patients with untreated primary hypertension (Ronquist et al., 1995).

Roles of -ARs in altered vasoconstriction and VO₂ induced by NE in SHR. -ARs have been shown to be widely distributed in skeletal muscle of normal rats in radioautography (Summers et al., 1995). We have previously found blockade of ₁/₂-ARs by 1 µM propranolol in perfused hindlimb of Wistar rats leads to a leftward shift of vasoconstriction produced by adrenaline (Colquhoun et al., 1990). Consistent with this earlier finding, blockade of ₁/₂-ARs by 10 µM propranolol in our study also enhanced NE-induced vasoconstriction in WKY with a small inhibition of VO_{2 max}. In comparison, propranolol did not show any significant effect on either vasoconstriction or VO₂ induced by NE in SHR, pointing to a reduced role of -ARs in the muscle vascular bed. Coincidentally, a loss of -AR-mediated vasodilation has been noted in portal veins (Doggrell and Surman, 1995) and mesenteric arteries (Blankesteijn et al., 1996) of SHR. Nonetheless, NE-induced bell-shaped VO₂ is not attributable to -ARs because it was present in the presence of 10 µM propranolol.

Roles of ₁- and ₂-ARs in altered vasoconstriction and VO₂ induced by NE in SHR. Compared with -ARs, -ARs are sparse in skeletal muscle (Rattigan et al., 1986) and predominantly located on small arteries with high affinity for prazosin (Martin et al., 1990). In the constant-flow perfused hindlimb of Wistar rats performed earlier in this laboratory, NE-induced biphasic changes in VO₂ were both completely reversed by prazosin at concentrations more than 1000-fold lower than by yohimbine (Dora, 1993). In our study, the predominant role of -1-ARs in NE-induced vasoconstriction and associated bell-shaped changes of VO₂ in the perfused rat hindlimb is supported by a high pK_B for prazosin (9.24) and low pK_B for yohimbine (7.34) in WKY. These results are consistent with our recent findings that the -1-AR agonist phenylephrine produces bell-shaped VO₂ with a strong vasoconstriction whereas the -2-AR agonist UK-14,304 elicits only a small and monophasic increase in VO₂ with a much weaker vasoconstriction in the perfused rat hindlimb (Hall et al., 1997). Compared with WKY, -1-ARs in SHR are functionally up-regulated with decreases of the role of -2-ARs in NE-induced changes in vasoconstriction as suggested by a larger rightward shift of perfusion pressure by prazosin at a lower dose in comparison to the effect of yohimbine. Subsequent results showing antagonism by 5 MU, BMY and SK&F of NE-induced changes in vasoconstriction and VO₂ in SHR but not in WKY in the presence of propranolol (10 µM) and yohimbine (0.1 µM) further support this notion.

Roles of ₁-AR subtypes in the altered vasoconstriction and associated VO₂ in SHR. The effects of -1A-AR subtype on NE-induced vasoconstriction and descending limb of the VO₂ curve were first suggested by a blockade by 5 MU at 0.25 µM in Wistar rats (Dora, 1993). However, this dose appeared to be too high because the P_max at 20 µM of NE was only a quarter of the control. In our study, 5 MU clearly showed antagonistic effects on vasoconstriction in SHR without suppressing the P_max at 3.3 nM which had no effect in WKY. The pK_B value of 8.94 is similar to those reported for its action on the -1A-AR subtype in mesenteric, carotid and caudal arteries of SHR (Villalobos-Molina and Ibarra, 1996). Intriguingly, HNE-elicited descending limb of the VO₂ dose-response curve was markedly attenuated, but LNE-induced ascending limb of VO₂ was unaffected. Similar attenuating effects on HNE-elicited descending limb of the VO₂ curve were also found with 1 nM KMD. These results suggest that HNE-elicited descending limb of the bell-shaped VO₂ dose-response curve appears to be predominantly mediated by the _1A-AR subtype.

Mechanisms for -1-AR-mediated biphasic changes in VO₂. The mechanism for -1-AR mediated changes VO₂ in muscle is not fully understood. As previously reviewed by us (Clark et al., 1995 and references therein), experiments using muscle preparations where nutrients are delivered through diffusion, -1-AR agonists are unable to show any marked stimulation of VO₂. However, -1-AR agonists cause remarkable changes in VO₂ via -1-ARs in similar ways to other vasoconstrictors in the perfused rat hindlimb where nutrients are delivered through the vascular system. These vasoconstrictor-controlled changes in VO₂ seem to be determined by the ratio of nutritive to nonnutritive routes in the hindlimb presumably because of the heterogeneous distribution and affinities of different receptors or receptor subtypes in the vascular tree (Clark et al., 1995). For instance, heterogeneous distribution of -1-AR subtypes has been shown in rat skeletal muscle bed (Leech and Faber, 1996). In the context of the present study, we speculate that the -1D-AR subtype may be predominantly distributed on the precapillary arterioles before the nonnutritive route, so that stimulation of this subtype may direct the flow from nonnutritive to nutritive routes, leading to rises in VO₂. In contrast, the -1A-subtype may be predominantly located in arterioles controlling nutritive routes and stimulation of this subtype closes these nutritive routes, causing functional vascular shunting. However, further experiments are needed to reveal the distribution of -1A- and -1D-subtypes in relation to nutritive and nonnutritive routes in the microvascualture.

	Conclusion

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Two major findings have emerged from our study regarding alterations of ARs in the SHR muscle vascular bed. First, the role of -2- and -ARs in NE-elicited changes in vascular function and VO₂ in SHR muscle are impaired whereas the effects of -1-ARs are markedly exacerbated. Second, both -1A- and -1D-subtypes are functionally up-regulated in SHR muscle vascular bed where increases in VO₂ seem to be predominantly mediated by the -1D- at a 100-fold lower concentration of NE than decreases in VO₂ which appear to be predominantly mediated by the -1A-subtype. The results may provide some clue for the possible role of -1-AR subtypes in the syndrome of hypertension and obesity. If similar changes also occur in vivo, the hypertension mediated by -1A-AR subtypes might be more likely to be associated with obesity as they inhibit thermogenesis. Hence, highly selective -1A-AR antagonists may offer better control of obesity than other -1-AR antagonists during the treatment of hypertension.