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.

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.

Acute and chronic effects of capsaicin in perfused rat muscle: the role of tachykinins and calcitonin gene-related peptide.

In perfused rat skeletal muscle (hindlimb), capsaicin either stimulates (submicromolar concentrations) or inhibits (micromolar concentrations) oxygen consumption (VO2). Both VO2 effects are associated with vasoconstriction, evident as an increase in perfusion pressure (PP), under constant flow. We have proposed that these effects are mediated by two vanilloid receptor subtypes: VN1 (stimulation of VO2) and VN2 (inhibition of VO2) (; ). In the present study, the role of capsaicin-sensitive neurons and sensory neuropeptides in the VN1/VN2 receptor actions of capsaicin was investigated. The observed maximum stimulation of VO2 by capsaicin (0.4 microM; DeltaVO2, 1.35 +/- 0.14 micromol g-1 h-1) was accompanied by mild vasoconstriction (DeltaPP, 5.8 +/- 0.6 mm Hg). In contrast, 2 microM capsaicin produced strong inhibition of VO2 (DeltaVO2, -2.25 +/- 0.23 micromol g-1 h-1) with pronounced vasoconstriction (DeltaPP, 28.0 +/- 1.3 mm Hg). VO2 stimulation was significantly inhibited (P <.05) by the selective NK1 receptor antagonist CP-99994 (1 microM) and the NK2 receptor antagonist SR 48968 (1 microM) (by 42% and 51%, respectively), but PP was not altered. Infused SP and neurokinin A (NKA) stimulated VO2 (observed maximum DeltaVO2, 0.52 +/- 0.06 and 0.53 +/- 0.08 micromol g-1 h-1, respectively; EC50 values, 269 +/- 23 and 21.2 +/- 3.0 nM, respectively) and induced mild vasoconstriction (4.30 +/- 0.33 and 6. 75 +/- 1.18 mm Hg, respectively; EC50 values, 352 +/- 25.7 and 25.5 +/- 2.7 nM, respectively). Neurokinin B (NKB) also stimulated VO2 (maximum not determined) and vasoconstriction (maximum DeltaPP, 3.40 +/- 0.25 mm Hg; EC50, 34.4 +/- 5.2 nM). The rank order of potency for the tachykinins in this preparation was NKA > NKB > SP, which suggests stimulation primarily of NK2 receptors. Although infused calcitonin gene-related peptide (CGRP) did not alter hindlimb VO2 or PP, the selective CGRP antagonist CGRP(8-37) markedly potentiated the inhibition of VO2 produced by 1 microM capsaicin (84%) and the maximum capsaicin-induced vasoconstriction (57%), which indicates that endogenously released CGRP may act as a vasodilator. Hindlimbs perfused 1 day after capsaicin pretreatment showed attenuation of capsaicin-induced (0.4 microM) stimulation of VO2 (92%) (P <.05) and vasoconstriction (64%), but this returned to normal after 7 days. The inhibition of VO2 by 1 microM capsaicin was significantly (P <. 05) enhanced 7 and 14 days after pretreatment (66% and 140%, respectively), as was the maximum vasoconstriction (64% and 68%, respectively). These data suggest that capsaicin-sensitive neurons, presumably via release of SP and NKA, are involved in VN1 responses and that capsaicin pretreatment potentiates VN2 responses, either by depletion of CGRP reserves or by upregulation of putative VN2 receptors.

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).

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.

Effects of noradrenaline and flow on lactate uptake in the perfused rat hindlimb.

Skeletal muscle can release or take up lactate depending on the lactate concentration gradient across the cell membrane. In the perfused rat hindlimb without arterial lactate, both noradrenaline (NA) infusion and increased flow promote lactate release and oxygen consumption (VO2). However, it is unclear whether NA or increased flow rate have similar effects on lactate uptake. The present study compares these effects in the rat hindlimb perfused at a basal flow rate of 0.33 mL min-1 g-1 and 25 degrees C in the presence of added arterial lactate. When 10 mmol L-1 L-(+)-lactate was added to the arterial perfusate, lactate was taken up (16 +/- 1.0 mumol g-1 h-1, n = 13) by the hindlimb with a 35% higher VO2 than that without added lactate. Doubling perfusion flow rate enhanced lactate uptake and VO2 by 120% and 40%, respectively. Glucose uptake was also increased (by 253%) with increased flow. Infusion of NA increased perfusion pressure, VO2 and glucose uptake similarly to those induced by increased flow rate. However, lactate uptake was inhibited by NA. This inhibition was not altered by the beta-adrenergic antagonist propranolol. Vasopressin also showed similar effects to NA to decrease lactate uptake associated with increased VO2 and vasoconstriction. These data indicate that in the presence of a high arterial lactate concentration, NA has opposite effects from increased flow rate on skeletal muscle lactate uptake although both have similar effects on lactate release in the absence of arterial lactate. Inhibition of lactate uptake may relate to the vasoconstrictive action of NA.

Serotonin inhibition of 1-methylxanthine metabolism parallels its vasoconstrictor activity and inhibition of oxygen uptake in perfused rat hindlimb.

The effect of serotonin (5-HT) on the metabolism of infused 1-methylxanthine (1-MX), a putative substrate of capillary endothelial xanthine oxidase (XO), and on the distribution of infused fluorescent microspheres (15 microns) by the artificially constant-flow perfused rat hindlimb preparation was investigated. 1-MX (5-100 microM) caused a slight inhibition of oxygen uptake (Vo2) but was not vasoactive, either alone or with 5-HT. 1-MX was converted to 1-methylurate (1-MU) and this conversion was inhibited by allopurinol and xanthine. 5-HT (0.35 microM), which caused vasoconstriction and decreased Vo2, also inhibited the conversion of 1-MX, indicated by a lowered venous perfusate steady-state 1-MU:1-MX ratio from 1.14 +/- 0.02 to 0.71 +/- 0.02 (P < 0.001), which is equivalent to the rate of conversion decreasing from 0.83 +/- 0.03 to 0.63 +/- 0.05 nmol min-1 g-1. This change closely followed the time course for changes in Vo2 and perfusion pressure and all three changes reversed in parallel when 5-HT was removed. Recoveries of 1-MU plus 1-MX at all times were high (100 +/- 5%). 5-HT did not act to inhibit XO. When compared with vehicle alone, 5-HT had either no effect (plantairs, gastrocnemius white, tibialis, extensor digitorum longus, vastus and thigh), or increased microsphere content (soleus and gastrocnemius red, P < 0.05) of muscles with only bone showing a significant decrease (P < 0.05). Since 5-HT did not inhibit XO or alter the net flow to individual muscles in this constant-flow model, the inhibition of conversion of 1-MX to 1-MU is concluded to be the result of a 5-HT-mediated decrease in the access of 1-MX to capillary XO within individual muscles. Possibilities include the redirection of flow to capillaries either in muscle or in connective tissue closely associated with muscle, where resistance is low and effective surface area is less. 1-MX has potential as a marker for muscle nutritive flow.

Potential for non-shivering thermogenesis in perfused chicken (Gallus domesticus) muscle.

The humoral modulation of resting muscle heat production of chickens (Gallus domesticus) was investigated in vitro. The resting distal lower limb was perfused via the popliteal artery at 25 degrees C without erythrocytes at constant flow. The preparation was stable for at least 3 hr, showing a constant oxygen uptake (MO2) and perfusion pressure as well as adequately maintaining muscle energy charge and creatine phosphate: creatine ratio. Noradrenaline (NOR), adrenaline (ADR) and serotonin (5-HT) each caused a dose-dependent rise in perfusion pressure. NOR and ADR evoked increased MO2 at low doses eventually followed by decreased MO2 at higher agonist concentrations. 5-HT gave smaller but qualitatively similar MO2 effects. The actions of 50 nM NOR were blocked by prazosin (10 microM) and nitroprusside (0.5 mM), but not altered by propranolol (10 microM). NOR-induced stimulatory MO2 changes in the presence of pharmacological concentrations (1 microM) of glucagon were more pronounced and the thermogenic concentration range of NOR was increased. Taken together, these in vitro findings demonstrate a potential for vasoconstrictor-controlled muscle nonshivering thermogenesis in birds as in marsupials and mammals, suggesting that vascular control of muscle MO2 may be a widespread biological mechanism. The possible implications of these findings for avian nonshivering thermogenesis are discussed.

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.

Alpha-adrenergic stimulation of thermogenesis in a rat kangaroo (Marsupialia, Bettongia gaimardi).

The Tasmanian bettong (Bettongia gaimardi) is a small rat kangaroo without detectable brown adipose tissue (BAT). In view of our previous findings of norepinephrine-mediated increase in O2 consumption (Vo2) in the perfused hindlimb of this species, the present study examined the effect of alpha-adrenoceptors on the thermogenesis of conscious bettongs at rest by infusing adrenergic agents via an indwelling catheter in the tail vein. The resting Vo2 was 22.9 +/- 1.9 mmol.kg-1.h-1. Norepinephrine (10-80 micrograms.kg-1.min-1) stimulated Vo2 in a dose-dependent manner with the maximal increment of 46.7%. Naphazoline (an alpha 1,alpha 2-adrenergic agonist) and phenylephrine (an alpha 1-adrenergic agonist) also elicited increases in Vo2 with maximal values of 29.6 and 34.8%, respectively. In contrast, the alpha 2-adrenergic agonist clonidine had no significant effects. Both alpha- and beta-adrenergic blockers were used to antagonize the submaximal increase in Vo2 elicited by norepinephrine. As a dose of 10 micrograms.kg-1.min-1, the alpha-adrenergic blocker phentolamine abolished the effects of naphazoline and phenylephrine and reduced norepinephrine-induced Vo2 by 45.5%. The beta-adrenergic blocker propranolol inhibited the norepinephrine-induced Vo2 by 58.8% at 20 micrograms.kg-1.min-1. A combination of the two antagonists blocked 82.5% of the norepinephrine-induced Vo2. Pretreatment of the animal with indomethacin (1 mg/kg), a known inhibitor of prostaglandin cyclooxygenase, had no effect on phenylephrine-elicited Vo2. Taken together, these results indicate that alpha 1-adrenoceptors are directly involved in norepinephrine-induced thermogenesis in non-BAT tissue(s).

Purine and pyrimidine nucleotide metabolism of vascular smooth muscle cells in culture.

1. Cultures of vascular smooth muscle cells accumulate extracellular breakdown products of purine and pyrimidine nucleotides that, over 9 hr, represent 60 +/- 7 and 78 +/- 17%, respectively, of the intracellular nucleotide content. 2. The accumulation is stimulated during contracture with 20 mM KCl or 70 microM carbachol, consistent with the notion that both pyrimidine and purine nucleotides are involved in the energetics of smooth muscle contracture. 3. Because the intracellular levels of pyrimidine and purine nucleotides remain constant, it appears likely that rates of synthesis match the rates of release. 4. Ectonucleotidases are present that can degrade ATP, UTP, and CTP. High-energy nucleotides may be the primary products released.

Norepinephrine and serotonin vasoconstriction in rat hindlimb control different vascular flow routes.

The vasoconstrictors, norepinephrine at low dose ( < or = 0.1 microM; LDNE) and serotonin (5-HT), produce opposing metabolic effects in the constant-flow perfused rat hindlimb characterized by increased and decreased oxygen uptake, respectively. In the present study, the effects of each vasoconstrictor are compared in the red blood cell-free buffer-perfused hindlimb on postequilibration endogenous red blood cell efflux, vascular entrapment of fluorescein-labeled dextran (Fx), and vascular corrosion casting by use of 30-micron spheres of methyl methacrylate (MM). A marked transient washout of red blood cells occurred immediately in association with vasoconstriction induced by LDNE that was not apparent when a similar extent of vasoconstriction was induced by 5-HT. Fx perfusions indicated that LDNE recruited a new vascular space that was reaccessed by a second exposure to the vasoconstrictor. 5-HT closed off a previously perfused vascular space that was reaccessed when the vasoconstrictor was removed. Corrosion casting of the arterial tree with MM showed no increase in cast weight, but more vessels filled because of LDNE. Higher doses of NE (2.5 microM) or 5-HT caused a marked decrease in cast weight with fewer vessels filled. The data suggest that LDNE and 5-HT, in association with vasoconstriction at different sites, control different capillary flow routes in the hindlimb that in turn may influence metabolism by increasing or decreasing nutrient access, respectively.

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.

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.

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.

Vascular and endocrine control of muscle metabolism.

Important differences exist between perfused and incubated (or perifused) skeletal muscle preparations with regard to their metabolism and control. A growing body of evidence suggests that the differences may be due to the role played by the vascular system. In the constant-flow perfused rat hindlimb preparation, a group of vasoconstrictors has been identified that enhance muscle metabolism and aerobic contractility. Another group of vasoconstrictors decrease muscle metabolism and aerobic contractility even though perfusate flow remains constant. All effects of both groups of vasoconstrictors are opposed by vasodilators. Because none of the vasoconstrictor effects is evident when isolated muscles are incubated or perifused, involvement of an active vascular system is indicated. Although some hormones may act directly on muscle by purely endocrine effects, a vascular component of their actions is now emerging. Mechanisms to account for vascular control of perfused skeletal muscle metabolism may involve 1) functional vascular shunts where the proportion of flow processed by these is regulated by site-specific vasomodulators, 2) a direct response to a change in the rate of supply of nutrients and removal of products, and 3) a signal substance released by vascular tissue in association with vasoconstriction that interacts with surrounding skeletal muscle cells. Impaired control at the level of the vascular system may have implications for long-term access of nutrients and hormones and therefore the control of skeletal muscle metabolism and contractile performance.

Vasoconstrictors alter oxygen, lactate, and glycerol metabolism in the perfused hindlimb of a rat kangaroo.

The Tasmanian bettong (Bettongia gaimardi) is a small marsupial rat kangaroo without detectable brown adipose tissue (BAT). The hindlimb was perfused with constant flow at 25 degrees C after cannulation under anesthesia of the femoral artery and vein to one hindlimb. Norepinephrine (NE, 25 nM-2.5 microM) and vasopressin (VP, 10 nM-0.1 microM) each increased perfusion pressure, oxygen consumption (VO2), and lactate and glycerol efflux of the perfused hindlimb. NE-mediated increases in VO2 and the efflux of lactate and glycerol were unaffected by propranolol (10 microM) but were completely blocked by the further addition of phentolamine (10 microM). In contrast, serotonin (5-HT; 0.1-2.5 microM) inhibited VO2 and inhibited lactate efflux. The changes induced by NE, VP, and 5-HT were all rapidly reversed by nitroprusside. These results suggest that resting thermogenesis in bettong hindlimb can be differentially controlled by the vasculature, which may also contribute to the induced VO2. This vascular control of skeletal muscle VO2 appears widespread in homeotherm evolution.

Treatment with the thiazolidinedione (BRL 49653) decreases insulin resistance in obese Zucker hindlimb.

Hindlimbs of mature age obese fa/fa Zucker rats were perfused and found to be markedly insulin-resistant when compared to the hindlimbs of age-matched lean Fa/? animals. Hindlimb analysis also showed a greater content of fat and a lower content of muscle in the obese. Treatment of the obese animals for 7 days with the thiazolidinedione, BRL 49653 (3 mumol/kg/day) significantly decreased the insulin resistance of the hindlimb and significantly increased the rate of weight gain in the whole rat. However, the decreased insulin resistance due to BRL 49653 could not be accounted for by an increase in the proportion of hindlimb muscle to fat or by an increase in the hindlimb muscle mass perfused.

Inhibition of insulin-mediated glucose uptake in rat hindlimb by an alpha-adrenergic vascular effect.

The vasoconstrictor norepinephrine, at high doses, inhibits oxygen uptake (VO2) in the perfused hindlimb, possibly by opening vascular shunts and reducing nutrient access. Thus, in the present study, the effect of norepinephrine on insulin-mediated glucose uptake (IMGU) was assessed. Rat hindlimbs were perfused at constant flow with medium containing 8.3 mM glucose and a tracer amount of 2-deoxy-D-[1-3H]glucose (2-DG) with and without 15 nM insulin, 10 microM norepinephrine (NE), and combinations of the adrenergic blockers propranolol (Prop) and prazosin. Perfusions were also conducted at a lower dose of 1 microM NE where VO2 is stimulated. NE (10 microM) inhibited IMGU > 80%, and this inhibition, when measured by 2-DG uptake, was most pronounced in muscles rich in white fibers. The inhibitory effect of NE on IMGU comprised a beta-adrenergic component also partly evident at lower concentrations of NE (i.e., 1 microM) and an alpha-adrenergic component only evident at 10 microM NE. In contrast to the results for the hindlimb, 10 microM NE plus Prop (alpha-adrenergic combination) had no significant effect on insulin-mediated 2-DG uptake by isolated incubated soleus or extensor digitorum longus muscles. It is concluded that NE, at doses likely to occur at sympathetic vasoconstrictor synapses in muscle, impairs IMGU by a vascular effect to cause shunting and reduce access.