Relationship of MTT reduction to stimulants of muscle metabolism.

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na(+) channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 microM) of (+/-)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na(+) channels and the Na(+)/K(+) pump, energy required for Na(+) pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.

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.

Vascular control of nutrient delivery by flow redistribution within muscle: implications for exercise and post-exercise muscle metabolism.

There is evidence for non-nutritive flow routes within, or associated with, skeletal muscle. Large capillary-like structures are possible candidates. The proportion of flow distributed between nutritive and non-nutritive routes appears to be tightly regulated and can control muscle metabolism and contraction by regulating delivery and product removal. The portion of flow that is carried by the non-nutritive routes at rest affords a flow reserve for amplifying nutrient delivery as muscle begins to work and may determine post-exercise metabolism. Inappropriate signals, however, may diminish nutritive flow to the detriment of muscle performance and post-exercise recovery. New technologies should allow the identification of the non-nutritive routes.

Hormonal control of thermogenesis in perfused muscle of Muscovy ducklings.

Endocrine stimulation of muscle nonshivering thermogenesis (NST) in ducklings was investigated in vitro using a perfused hindlimb preparation maintained at 25 degrees C. Effects of flow rate, norepinephrine (NE), epinephrine, and glucagon on perfused muscle oxygen consumption (MO2) and perfusion pressure were studied. Control ducklings (Cairina moschata, 5 wk old) reared at thermoneutrality (25 degrees C, TN) were compared with two age-matched groups exhibiting muscle NST in vivo: cold-acclimated ducklings (4 degrees C, 4 wk, CA) and glucagon-treated ducklings (103 nmol/kg twice-daily, intraperitoneally, GT). Basal MO2 was higher in CA than in TN or GT ducklings and increased in all groups with elevated flow rates. Catecholamines increased both MO2 and perfusion pressure. The maximal effect on MO2 was higher in CA (+ 36%) and GT ducklings (+ 43%) than in controls (+ 31%), but was associated with reduced vasoconstriction. Flow rate did not consistently potentiate the NE response. At high doses, catecholamines became inhibitory on MO2 while a monotonous increase of pressure was still observed. Glucagon, by contrast, slightly decreased both MO2 and pressure. This vasodilatory effect was greater in CA ducklings than controls in preconstricted preparations. In vivo, low-dose epinephrine induced a modest thermogenic effect (+ 10%) in CA ducklings. These findings showed that duckling muscle thermogenesis is directly stimulated in vitro by catecholamines but not by glucagon. Higher in vitro thermogenic effects of NE in ducklings that were expected to exhibit muscle NST in vivo suggests catecholamine involvement in muscle NST in vivo. Potential vascular control of avian muscle NST is discussed.

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.

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.

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.

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.

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.

Potential defect in the vascular control of nonshivering thermogenesis in the obese Zucker rat hind limb.

Vascular control of nonshivering thermogenesis in the perfused hind limb of obese and lean Zucker rats were compared using two vasoconstrictors, norepinephrine and serotonin. For hind limbs of both phenotypes, norepinephrine infusions resulted in a dose-dependent uninterrupted increase in perfusion pressure and a biphasic change in oxygen uptake (VO2), characterized by a stimulation at low concentrations, and an increasing inhibition at higher concentrations that gradually overcame the stimulation in a dose-dependent manner. At concentrations of norepinephrine greater than 1 microM, the inhibitory effect predominated and gave rise to values for VO2 less than basal. The obese hind limb had a lower basal VO2 and a lower maximal VO2 mediated by norepinephrine than the lean rat, but these differences appeared to relate largely to the lower muscle mass and higher content of fat of the obese hind limb. Serotonin infusions resulted in dose-dependent increase in perfusion pressure and an accompanying decrease in VO2. Pressure changes were identical for the obese and lean hind limbs, but the decrease in VO2 due to serotonin was greater in the hind limbs from the lean rats, and this difference remained when the data were expressed in terms of muscle mass perfused. It is concluded that the relatively lower content of muscle of the obese hind limb accounts for its lower basal and lower maximal norepinephrine-mediated thermogenesis. In addition, an intrinsic defect in obese hind limb muscle response to serotonin is present, which may be indicative of a decrease in the potential for vasoconstrictor-regulated thermogenesis that could have implications for whole-body energy balance by the obese phenotype.

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.

Pungent principles of ginger (Zingiber officinale) are thermogenic in the perfused rat hindlimb.

Crude extracts of both fresh and dry ginger induced the perfused rat hindlimb to consume oxygen in association with increases in perfusion pressure and lactate production. The principles responsible for these observations, the gingerols and shogaols, were isolated and tested for relative thermogenic activity. The gingerol homologues possessed greater molar potency than their shogaol counterparts. (6)-Gingerol was the most potent principle isolated, causing a mean maximal increase in oxygen consumption of 1.4 +/- 0.1 mumol/g/h (21%), an increase in lactate efflux of 4.7 +/- 0.6 mumol/g/h (87%) with a perfusion pressure increase of 7.7 +/- 0.7 mmHg (30%). Increases in alkyl chain length within each homologous series led to decreased molar potency. Specific nitro-vasodilation using glyceryl trinitrate demonstrated that thermogenesis was at least partly associated with vasoconstriction. Concurrent infusion of alpha or beta antagonists showed that neither adrenergic receptors nor secondary catecholamine release were responsible for the observed effects. Increasing doses of the ginger principles ultimately led to inhibition of steady state oxygen consumption, although perfusion pressure continued to increase. Removal of high ginger principle doses was followed by apparent increases in oxygen uptake unaccompanied by elevated perfusion pressure. As a consequence, the effective concentration ranges of the ginger principles were relatively narrow. The cause of high dose effects is as yet undetermined but may have been due in part to disruption of mitochondrial function.