Lysosomal thyroid hormone 5'-deiodinase.

Lysosomes prepared from rat liver and kidney after loading with the detergent Triton WR-1339 show membrane-bound 5'-deiodinase activity with marked specificity for 3,3',5'-triiodothyronine (reverse T3), lesser activity with respect to thyroxine (T4) and almost none towards 3,3',5'-triiodothyronine (T3). The enzyme is thiol dependent and shows maximal catalysis at pH 7.2. As many of the states known to alter thyroid hormone levels also affect lysosomal function, inhibition of the lysosomal 5'-deiodinase leading to an increase in intracellular reverse T3 may be an initiating mechanism for thyroid hormone change.

Functional and desensitizing effects of the novel synthetic vanilloid-like agent 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) in the perfused rat hindlimb.

1. In the present study, the effects of the novel vanilloid agonist, 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), on oxygen consumption (VO(2)) and vascular resistance (perfusion pressure, PP) were investigated in the constant flow, perfused rat hindlimb. The acute desensitizing properties of this novel synthetic agent were also examined. 2. Maximum stimulation of VO(2) was produced by 0.2 microM PPAHV (delta VO(2), 0.83+/-0.06 micromol g(-1) h(-1)) and was accompanied by mild vasoconstriction (increase in PP; 8.0+/-1.1 mmHg). The highest concentration of PPAHV tested (2 microM) caused inhibition of VO(2) (delta VO(2), -2.73+/-0.51 micromol g(-1) h(-1)) and strong vasoconstriction (delta PP, 42.0+/-1.2 mmHg). 3. Capsazepine (10 microM) caused a parallel shift to the right of both VO(2) and PP concentration-response curves for PPAHV (pK(b)=5.00), indicative of competitive binding to vanilloid receptors. 4. The stimulation of VO(2) produced by 0.2 microM PPAHV decreased, but was not completely abolished, after repeated infusion of PPAHV (change in VO(2), first infusion, 0.66+/-0.18 micromol g(-1) h(-1); sixth infusion, 0.29+/-0. 08 micromol g(-1) h(-1), P<0.05), an acute tachyphylactic response not previously seen with the repeated infusion of other vanilloid analogues. Conversely, the PP response to repeated PPAHV infusion increased (delta PP, first infusion, 5.8+/-0.7 mmHg; sixth infusion, 9.0+/-0.6 mmHg, P<0.05). 5. In conclusion, PPAHV produces vasoconstriction and a biphasic effect on VO(2) in the perfused rat hindlimb very similar to that induced by naturally occurring vanilloids. Both effects are blocked by the competitive antagonist capsazepine. Since, the metabolic response to low concentrations of PPAHV (stimulation of VO(2)) undergoes tachyphylaxis, the present data suggest that PPAHV desensitizes putative vanilloid receptors in the hindlimb.

Aerobic muscle contraction impaired by serotonin-mediated vasoconstriction.

Vasoconstriction mediated by serotonin (5-HT) inhibits muscle metabolism in resting constant-flow-perfused rat hindlimb and may do so by vascular shunting. In the present study, the effects of 5-HT on tension development and contraction-induced oxygen uptake by the sciatic nerve-stimulated gastrocnemius-plantaris-soleus muscle group of the perfused rat hindlimb and tension development by electrically stimulated isolated incubated soleus and extensor digitorum longus muscles were examined. In both erythrocyte and erythrocyte-free perfusions, 0.25 microM 5-HT increased perfusion pressure and markedly decreased contraction-induced tension, oxygen uptake, and lactate release. The release of metabolic vasodilators from exercising skeletal muscle did not appear to affect 5-HT-mediated vasoconstriction; rather, vascular resistance increased during the period of muscle contraction. In contrast, vasoconstriction during muscle contraction mediated by alpha-adrenoceptor stimulation did not impair tension and was partially overcome by metabolic vasodilators. In addition, contraction of isolated incubated soleus and extensor digitorum longus muscles was not affected by 5-HT addition to the incubation medium. We conclude that 5-HT impairs contractility of working muscle during the aerobic phase by limiting oxygen delivery through redistributing perfusate flow. The results are consistent with a vasoconstrictor action of 5-HT on larger vessels, perhaps at feed arteries external to the working muscle. When constricted by 5-HT, these vessels are apparently insensitive to metabolic vasodilatation.

Vasoconstrictor-mediated release of lactate from the perfused rat hindlimb.

The effects of different vasomodulators on lactate release by the constant-flow-perfused rat hindlimb were examined and compared with that by perfused mesenteric artery, incubated preparations of aortas, soleus and epitrochlearis muscles, and perifused soleus muscles. Infusion of vasopressin (0.5 nM), angiotensin II (5 nM), norepinephrine (50 nM), and methoxamine (10 microM) into the hindlimbs of 180- to 200-g rats increased the perfusion pressure by 112-167% from 30.4 +/- 0.8 mmHg, O2 consumption by 26-68% from 6.4 +/- 0.2 mumol.g-1 x h-1, and lactate efflux by 148-380% from 5.41 +/- 0.25 mumol.g-1 x h-1. Hindlimbs of 100- to 120-g rats responded similarly to angiotensin II. Isoproterenol (1 microM) had no effect on O2 uptake or perfusion pressure but increased lactate release by 118%. Nitroprusside (0.5 mM) markedly inhibited the vasoconstrictor-mediated increases in lactate release, perfusion pressure, and O2 consumption by the hindlimb but had no effect on isoproterenol-mediated lactate efflux. Serotonin (6.7 microM) increased lactate release from the perfused mesenteric artery by 120% from 5.48 mol.g-1 x h-1. Lactate release by incubated aorta was increased by angiotensin II (50 nM), isoproterenol (1 microM), and mechanical stretch. The increase mediated by angiotensin II was blocked by glycerol trinitrate (2.2 microM), which had no effect on lactate release by isoproterenol. Neither angiotensin II (5 nM) nor vasopressin (0.5 nM) increased lactate release from incubated soleus and epitrochlearis muscles; however, lactate release was increased by isoproterenol, and this increase was unaffected by glycerol trinitrate (2.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertension in obesity may reflect a homeostatic thermogenic response.

Systemic hypertension of mild to moderate degree is often associated with obesity. The hypothesis is that over-eating leads to increased sympathetic activity targeted at the peripheral vasculature as well as other tissues in an attempt (that in many cases may be futile) to stimulate facultative thermogenesis and burn-off the excess energy. This hypothesis represents an important modification of one proposed by Landsberg and is supported by: 1) recent observations that carbohydrate feeding to humans specifically increases muscle sympathetic vasoconstrictor activity in the peroneal nerve, and 2) studies with animal models in which active vasoconstriction in the limbs and elsewhere is associated with marked increases in oxygen consumption (energy expenditure).

Serotonin-mediated acute insulin resistance in the perfused rat hindlimb but not in incubated muscle: a role for the vascular system.

We have recently shown that the vasoconstrictor serotonin (5-HT) inhibits oxygen uptake in perfused hindlimb possibly due to vascular shunting. Thus in the present study the effect of 5-HT on insulin-mediated glucose uptake was assessed. Rat hindlimbs were perfused at constant flow with medium containing 8.3 mM glucose and a tracer amount of 2-deoxy-D-[1-3]glucose (2DG) with and without 10 microM 5-HT, 15 nM insulin and a combination of the two. 5-HT inhibited insulin-mediated stimulation of glucose uptake by 30.4% when added after insulin and 34.4% when added before insulin. In addition, 5-HT inhibited insulin-mediated 2DG uptake by perfused muscles with inhibition ranging from 32% (soleus) to 80% (extensor digitorum longus). The effects of 5-HT on insulin-mediated glucose uptake were partially reversed by vasodilation with carbachol. In contrast to the results for the hindlimb, 10 microM 5-HT had no significant effect on either basal glucose uptake or the stimulation of glucose uptake mediated by 15 nM insulin by isolated incubated soleus or extensor digitorum longus muscles. It is concluded that 5-HT impairs insulin-mediated glucose uptake in the perfused rat hindlimb that may derive from vascular shunting not apparent when muscles are incubated with 5-HT in vitro. These findings may have implications for the link between hypertension and diabetes.

Capsaicin-induced biphasic oxygen uptake in rat muscle: antagonism by capsazepine and ruthenium red provides further evidence for peripheral vanilloid receptor subtypes (VN1/VN2).

Previous studies with the vanilloid spice principle capsaicin have demonstrated a biphasic VO2 response, with vasoconstriction, in the perfused rat hindlimb that has led to suggestions of vanilloid receptor subtypes (VN1/VN2) in this preparation (1). In the present study, the known competitive vanilloid antagonist capsazepine inhibited the above capsaicin-mediated effects in a manner that was indicative of binding at specific vanilloid recognition sites. Low concentrations of capsazepine selectively inhibited the increased VO2 produced by the putative VN1 receptor at submicromolar concentrations of capsaicin, while the inhibition of VO2 produced by high concentrations of capsaicin (putative VN2) was enhanced. These observations, showing different susceptibilities to blockade by capsazepine, further support the presence of two vanilloid receptor subtypes in the rat hindlimb. Schild plots of the data yielded variable slopes that approach unity at greater responses to capsaicin (mean KB = 8.44 +/- 2.08 microM and 7.28 +/- 0.78 microM for VO2 and perfusion pressure curves, respectively). Low concentrations of the capsaicin antagonist ruthenium red selectively blocked the putative VN2 receptor-mediated effects produced by high concentrations of capsaicin. The noncompetitive nature of this inhibitor suggests an operation through separate receptor-coupled ion channel complexes at high and low concentrations of the vanilloid. Tetrodotoxin failed to attenuate any changes produced by capsaicin, suggesting that the mechanism of action of capsaicin in the rat hindlimb may differ from other tissues.

The apparent absence of serotonin-mediated vascular thermogenesis in perfused rat hindlimb may result from vascular shunting.

Vasoconstriction by norepinephrine, angiotensin II and vasopressin in the constant-flow perfused rat hindlimb is associated with increased oxygen uptake and has given rise to the concept of vascular thermogenesis. In the present study serotonin (5-hydroxytryptamine, 5HT) was found to inhibit oxygen uptake by up to 40% in a dose dependent manner whilst inducing vasoconstriction in this model, whereas norepinephrine increased oxygen consumption by up to 100% during vasoconstriction. This contrasted with the perfused isolated rat mesenteric artery arcade in which serotonin stimulated oxygen uptake by up to 130% in association with vasoconstriction in a dose dependent manner similar to the previously described norepinephrine induced vascular thermogenesis in this arterial preparation. In both perfusion systems, changes in pressure and oxygen uptake mediated by serotonin were completely blocked by ketanserin. These results and evidence from dye washout studies suggest that serotonin-mediated vascular thermogenesis, if it occurs in the constant-flow hindlimb, is masked by vascular shunting.

Resiniferatoxin and piperine: capsaicin-like stimulators of oxygen uptake in the perfused rat hindlimb.

The naturally occurring capsaicin-like molecules, resiniferatoxin (RTX, Euphorbia spp.) and piperine (Piper nigrum), each stimulated oxygen uptake (VO2) in association with increased vascular resistance in a concentration-dependent manner when infused into the perfused rat hindlimb. 5 microM glyceryl trinitrate (GTN, a nitrovasodilator) significantly blocked the oxygen and pressure responses to both RTX and piperine, indicating a close relationship between changes in VO2 and the vasoconstriction. Concentrations greater than those required for maximal VO2 resulted in an inhibition of VO2, although perfusion pressure continued to increase. Time course studies showed that both RTX and piperine at high doses resulted in a tri-phasic response. An initial phase of transient VO2 stimulation was followed by a second phase of inhibition. A third phase involving an often larger but transient stimulation of VO2 followed removal of the agents and continued after the pressure returned to basal. The actions of RTX and piperine were similar to those of other active capsaicin-like molecules tested previously in this system, including capsaicinoids (Capsicum spp.), gingerols (Zingiber officinale), and shogoals (Zingiber officinale). RTX was the most potent, and piperine the least potent of this series. Although receptor involvement has yet to be unequivocally established, the data are consistent with the presence of a functional capsaicin-like (vanilloid) receptor in the vasculature of the rat hindlimb that mediates vasoconstriction and oxygen uptake. These findings may have implications for the future development of thermogenic agents.

Vasopressin and angiotensin II stimulate oxygen uptake in the perfused rat hindlimb.

Vasopressin and angiotensin II markedly stimulated oxygen uptake in the perfused rat hindlimb. The increase due to each agent approached 70% of the basal rate, and was greater than that produced by a maximal concentration of norepinephrine. Half-maximal stimulation occurred at 60 pM vasopressin, 0.5 nM angiotensin II and 10 nM norepinephrine. Angiotensins I and III were less potent than angiotensin II. For each agent, the dose-dependent increase in oxygen uptake coincided with a dose-dependent increase in perfusion pressure. The effects of both vasopressin and angiotensin to increase oxygen uptake and pressure were not inhibited by either phentolamine, propranolol or a combination of the two, but were completely inhibited by the vasodilator, nitroprusside. Nitroprusside also inhibited flow-induced increases in hindlimb oxygen uptake and perfusion pressure. The findings indicate a key role for the vascular system in the control of hindlimb oxygen uptake.

Functional and metabolic evidence for two different vanilloid (VN1 and VN2) receptors in perfused rat hindlimb.

Vanilloid spice principles, including capsaicin, stimulate vasoconstriction in the rat hindlimb perfused at constant flow and, depending on dose, either stimulate or inhibit oxygen consumption by this vascular bed. We now present metabolic and functional evidence for two different vanilloid (VN1 and VN2) receptor types. These receptors can be distinguished on the basis of their differing agonist affinity for capsaicin, their different calcium and oxygen dependencies for inducing vasoconstriction, and whether they stimulate, or inhibit, oxygen consumption. The higher affinity vanilloid receptor, VN1 can be distinguished on the basis of initiating vasoconstriction at low doses of capsaicin and simultaneously stimulating oxygen consumption. Its apparent biological function is dependent on the presence of oxygen and external calcium. In contrast, the lower affinity receptor, VN2 induces vasoconstriction associated with inhibition of oxygen consumption. Its vasoconstriction action can occur independently of either external calcium ions, or the presence of oxygen in the perfusate.

Spicy meal disturbs sleep: an effect of thermoregulation?

Tabasco sauce and mustard taken with the evening meal markedly disturbed sleep of six, young, healthy male subjects; reducing slow wave and stage 2 sleep, increasing total time awake and tending to increase sleep onset latency. Whilst post meal effects on temperature and oxygen consumption were not significantly different from control meals the spicy food condition elevated body temperature during the first sleep cycle. The possibility that the spice principle capsaicin affects sleep via changes in body temperature is discussed.

Nonshivering thermogenesis in a marsupial (the tasmanian bettong Bettongia gaimardi) is not attributable to brown adipose tissue.

The Tasmanian bettong (Bettongia gaimardi, a marsupial) is a rat-kangaroo that increases nonshivering thermogenesis (NST) in response to norepinephrine (NE). This study attempted to assess whether brown adipose tissue (BAT), a specialized thermogenic effector, is involved in NST in the bettong. Regulatory NST, indicated by resting oxygen consumption (Vo2) of the whole body, was measured under conscious conditions at 20 degrees C with various stimuli: cold (4 degrees -5 degrees C) or warm (25 degrees C) acclimation, NE injection, and the beta3-adrenoceptor agonist (BRL) 37344. In line with the functional studies in vivo, the presence of BAT was evaluated by examining the expression of the uncoupling protein 1 (UCP1) with both rat cDNA and oligonucleotide probes. Both NE and BRL 37344 significantly stimulated NST in the bettong. After cold acclimation of the animals (at 4 degrees -5 degrees C for 2 wk), the resting Vo2 was increased by 15% and the thermogenic effect of NE was enhanced; warm-acclimated animals showed a slightly depressed response. However, no expression of UCP1 was detected in bettongs either before or after cold exposure (2 wk). These data suggest that the observed NST in the marsupial bettong is not attributable to BAT.

Altered muscle metabolism associated with vasoconstriction in spontaneously hypertensive rats.

In the rat muscle vascular bed, vasoconstrictors either increase or decrease oxygen consumption (VO2). The present study compared the effects of norepinephrine (NE), angiotensin II (ANG II), and 5-hydroxytryptamine (5-HT) on vasoconstriction-associated metabolism in the constant-flow perfused hindlimb of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in the absence of insulin. Basal perfusion pressure, VO2, glucose uptake, and lactate production were increased by 21.4, 11.9, 46.4, and 44.9% (P < 0.05 for all), respectively, in SHR, which also had higher blood pressure and metabolic rate (P < 0.05) in vivo. Dose-response curves for NE-induced perfusion pressure, VO2, and lactate production in SHR were shifted to the left compared with WKY. Associated with the increased perfusion pressure, NE-induced VO2 and glucose uptake were both decreased (P < 0.01), particularly at high concentrations. These differences were unaffected by 10 microM propranolol but were all diminished by further addition of prazosin (2.5 nM). ANG II stimulated VO2, glucose uptake, and lactate production in both strains, but the increased lactate production was smaller in SHR (P < 0.05) with a proportional decrease (P < 0.05) in glucose uptake. Conversely, 5-HT decreased VO2 in both strains (P < 0.01), and this effect was greater in SHR (P < 0.01). These data suggest that SHR muscle thermogenesis and glucose uptake are impaired during vasoconstriction, especially in response to NE.

Changes in functional expression of alpha-1 adrenoceptors in hindlimb vascular bed of spontaneously hypertensive rats and their effects on oxygen consumption.

Norepinephrine (NE) induces a sigmoidal dose-response curve for perfusion pressure and a bell-shaped curve for oxygen consumption (VO2) in the constant-flow perfused hindlimb of Wistar rats. These effects are now described in spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY). In SHR, the pressure curve was shifted left- and upward whereas the VO2 curve was shifted left- but downward, when compared with WKY. In the presence of 10 microM propranolol, prazosin (2.5 nM) shifted the pressure and VO2 curves much more than yohimbine (0.1 microM) to the right in both strains and its effects were greater in SHR, suggesting that these effects were mediated largely by alpha-1 receptors, particularly in SHR. In the presence of propranolol plus yohimbine, the pressure curve was markedly shifted to the right by both the selective alpha-1A-antagonist 5-methylurapidil (3.3 nM), and by the alpha-1D antagonist BMY 7378 (0.1 microM) or SK&F 105854 (2 microM) in SHR but not in WKY. With respect to the VO2 curve, 5-methylurapidil attenuated the descending limb without affecting the ascending limb. Similar effects were also obtained with another alpha-1A antagonist 1 nM KMD-3213 in both SHR and WKY. In contrast, BMY and SK&F markedly inhibited the ascending limb of the VO2 curve. These results indicate that both alpha-1A- and alpha-1D subtypes are functionally up-regulated in SHR muscle vascular bed where the ascending limb of VO2 is predominantly mediated by the alpha-1D at a much lower concentration for NE than the descending limb which is predominantly mediated by the alpha-1A subtype.

Creatine phosphate as the preferred early indicator of ischemia in muscular tissues.

Changes in creatine compounds, especially the creatine phosphate to creatine ratio (CrP/Cr), are more sensitive indicators than changes in other metabolites for early ischemia in the different muscular tissues of heart, small intestine, skeletal muscle, and aorta. Changes in adenine nucleotide ratios are buffered by CrP reserves and the absolute concentration of adenine nucleotides can vary greatly between different muscular tissues. Accumulation of lactate is indicative of ischemia, but is not as sensitive as the ratio of CrP/Cr, but may better indicate the duration of ischemia. Glycerol also accumulates in muscular tissues during prolonged ischemia, so that consideration of both lactate and glycerol levels together, might confer a better estimate of the duration of ischemia of different muscular tissues.

Constant-pressure perfusion of rat hindlimb shows alpha- and beta-adrenergic stimulation of oxygen consumption.

Isolated rat hindlimbs were perfused at 37 degrees C and constant physiological pressure (80 +/- 0.5 mmHg) while the flow rate that was allowed to freely self-adjust was monitored. Under these conditions, evidence was obtained for both alpha- and beta-adrenergic stimulation of oxygen consumption (VO2) in contrast to constant-flow perfusion, which has only convincingly shown alpha-adrenergic stimulation of VO2 in response to adrenergic agents. Addition of norepinephrine (NE; 1-33 nM) led to an increase in VO2 with a maximum of 29% above the basal value at 3.3 nM, even though the flow rate decreased. Phenylephrine (3.3-33 nM) and vasopressin (10-100 pM) also showed similar, but lesser in magnitude, vasoconstriction-associated stimulatory effects on VO2. Prazosin (an alpha 1-antagonist) completely reversed the NE-mediated decrease in flow rate and significantly blocked the increased VO2. In contrast, isoproterenol (10-1,000 nM) increased both flow rate (30%) and VO2 (32%). The isoproterenol-stimulated VO2 was not blocked by the beta 1-, beta 2-antagonist propranolol (10 microM), although the increased flow was reversed. In the presence of propranolol (1 or 10 microM), BRL-35135A (a beta 3-agonist) also stimulated VO2 (18%) without significant change in flow rate. These results lend further support to the role of the alpha 1-adrenoceptor in muscle VO2. In addition there is evidence for the presence of a functional beta 3-adrenoceptor as an additional subtype responsible for NE-mediated thermogenesis in the rat hindlimb.

A comparison of vasopressin and noradrenaline on oxygen uptake by perfused rat hindlimb, kidney, intestine and mesenteric arcade suggests that it is in part due to contractile work by blood vessels.

1. The rat hindlimb, kidney and intestine were each perfused in a nonrecirculating mode at 25 degrees C using an artificial perfusate (initial pressure 85 +/- 5 mmHg) and the effects of vasopressin and noradrenaline on oxygen uptake and perfusion pressure determined. 2. Both vasopressin (K0.5 = 0.1 nM) and noradrenaline (K0.5 = 2 nM) increased oxygen uptake as well as perfusion pressure by the perfused hindlimb; changes in oxygen uptake were closely matched by changes in pressure. The maximum increase in oxygen uptake was approx. 9 mumol/hr per g wet wt of hindlimb. 3. The perfused kidney also responded to vasopressin and noradrenaline with parallel increases in oxygen uptake and perfusion pressure for each agent. The largest increase in oxygen uptake was approx. 30 mumol/hr per g wet wt but this was not maximal. 4. Vasopressin increased oxygen uptake and pressure by the perfused intestine over the range 0.01-2 nM, but the changes in pressure only became significant at doses greater than 0.1 nM. 5. Noradrenaline inhibited oxygen uptake and increased perfusion pressure in a dose-dependent manner at pharmacological concentrations (greater than 30 nM) when shunting of perfusate may have contributed to unperfused regions. 6. A network of mesenteric blood vessels estimated to contain approx. 6% vascular tissue by weight, with the remainder white fat cells, lymphatics and connective tissue, was also perfused. 7. Vasopressin (K0.5 = 0.3 nM) and noradrenaline (K0.5 = 30 nM) each increased oxygen uptake and perfusion pressure in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of endothermy in a Tasmanian marsupial, Bettongia gaimardi and its response to cold and noradrenaline.

Marsupials at birth are ectothermic and gradually attain the ability to change their metabolic heat production during pouch life. How this process occurs in the bettong has been measured on 13 pouch young from week 1 until 3 weeks after pouch vacation (week 18). Oxygen consumption was measured at 35 degrees C (pouch temperature) and at 22 degrees C. The results at 35 degrees C showed an increase in metabolic rate from week 1 until week 12 when there was a decrease to near adult levels after pouch vacation. At 22 degrees C young bettongs had a lower metabolic rate (compared with measurements made at 35 degrees C) until week 9 after which there was an increase above measurements made at 35 degrees C. Noradrenaline had little effect until week 10 after which the metabolic rate (although measured at 35 degrees C) paralleled the levels measured at 22 degrees C. The free thyroxine level was low in early pouch life, increased to a peak at week 12 then decreased. Thermal conductance increased until week 10 after which it decreased, reaching values similar to those of adult bettongs by week 20. The results indicate that non-shivering thermogenesis occurs in this macropodoid marsupial. This phenomenon may be a phylogenetic difference between macropodid and non-macropodid marsupials as also suggested by Nicol et al. (1997).

Sympathetic stimulation elicits increased or decreased VO2 in the perfused rat hindlimb via alpha 1-adrenoceptors.

The effects of lumbar sympathetic nerve stimulation on oxygen uptake (VO2) in curarized muscle of the perfused rat hindlimb were investigated. Stimulation of sympathetic nerves elicited vasoconstriction at all frequencies. Importantly, this was associated with changes in VO2 that were generally stimulatory at low frequencies (0.5-2 Hz) and inhibitory at high frequencies (5-10 Hz). Both the pressor response and the changes in VO2 were almost completely blocked by the alpha 1/alpha 2-blocker phentolamine (1.0 microM) but were not affected by the beta 1/beta 2-blocker DL-propranolol (2.0 microM). The alpha 2-blocker yohimbine (0.1 microM) did not significantly affect either the pressor or VO2 response. The alpha 1-antagonist prazosin (0.1 microM) abolished the vasoconstriction with low-frequency stimulation and inhibited > 90% of the vasoconstriction with high-frequency stimulation. Intra-arterial infusion of phenylephrine (alpha 1-agonist), but not of UK-14304 (alpha 2-agonist), also elicited a similar biphasic response in VO2 during vasoconstriction. The changes in VO2 at both low- and high-frequency stimulation were fully reversed by prazosin. The vasodilator sodium nitroprusside also showed similar effects to prazosin in blocking both VO2 and vasoconstriction. Thus sympathetic control of VO2 in the perfused rat hindlimb appears to be initiated by activation of predominantly vascular alpha 1-adrenoceptors.