Vasoconstrictor-mediated the perfused rat hindlimb

release of lactate from

MANTHINDA HETTIARACHCHI, KATHERINE KIM A. DORA, STEPHEN RATTIGAN, ERIC Department University

of Biochemistry, School of Medicine, of Tasmania, Hobart 7001, Australia

M. PARSONS, Q. COLQUHOUN,

Faculty

of Medicine

STEPHEN M. RICHARDS, AND MICHAEL G. CLARK and Pharmacy,

HETTIARACHCHI, MANTHINDA, KATHERINE M. PARSONS, STEPHEN M. RICHARDS, KIM A. DORA,STEPHENRATTIGAN, ERIC QXOLQUHOUN, ANDMICHAELG. CLARK. Vasocunstric-

hindlimb under resting conditions in vitro has parallels in vivo where release of this metabolite has been reported to occur from muscle beds of anesthetized rats (25) and tor-mediated release of lactate from the perfused rut hindlimb. from the forearm musculature of conscious humans (17). J. Appl. Physiol. 73(6): 2544-2551, 1992.~The effects of dif- Although not directly addressed, lactate release under ferent vasomodulators on lactate release by the constantis believed to originate from skeletal flow-perfused rat hindlimb were examined and comparedwith these conditions that by perfused mesenteric artery, incubated preparations of muscle fibers and to reflect basal glycolysis of either gluaortas, soleusand epitrochlearis muscles,and perifused so- cose or glucose 6-phosphate derived from glycogenolysis. Recently we noted that lactate production by the perleus muscles.Infusion of vasopressin(0.5 nM), angiotensin II was increased when perfusate flow (5 nM), norepinephrine (50 nM), and methoxamine (10 ,uM) fused rat hindlimb into the hindlimbs of 180- to 200-g rats increased the perfu- was increased (33) and also when vasoconstriction was sion pressureby 112-167% from 30.4 & 0.8 mmHg, 0, con- induced at constant flow by I-norephedrine (16) or by sumption by 26-68% from 6.4 of:0.2 pmol g-’ 9h-l, and lactate norepinephrine (10). Because vasoconstrictor-induced efflux by 148--380%from 5.41 -t 0.25 prnol. g-’ h-l. Hind- release of lactate was blocked by infusion of the nitrovalimbs of lOO- to 120-grats respondedsimilarly to angiotensin sodilator nitroprusside (10,16), it appeared possible that II. Isoproterenol (1 PM) had no effect on 0, uptake or perfuvascular tissue might be involved in controlling and/or sion pressure but increased lactate releaseby 118%. Nitrocontributing to lactate release by the hindlimb. prusside (0.5 mM) markedly inhibited the vasoconstrictorA direct contribution of vascular tissue to lactate promediated increasesin lactate release,perfusion pressure,and 0, consumption by the hindlimb but had no effect on isopro- duction by the hindlimb would be consistent with known terenol-mediated lactate efflux, Serotonin (6.7 PM) increased properties of vascular smooth muscle (22). Under aerobic lactate releasefrom the perfused mesenteric artery by 120% conditions, this tissue converts 60-80% of glucose taken from 5.48 mol g-l h-l. Lactate release by incubated aorta up to lactate (18), and lactate production is further stimuwas increased by angiotensin II (50 nM), isoproterenol (1 lated by various vasoconstrictors, such as epinephrine PM), and mechanicalstretch. The increasemediated by angio- and histamine (22). Thus in the present study we have tensin II was blocked by glycerol trinitrate (2.2 PM), which examined the hypothesis that lactate production during had no effect on lactate releaseby isoproterenol. Neither anaction in the perfused rat hindlimb at giotensin II (5 nM) nor vasopressin(0.5 nM) increasedlactate vasoconstrictor releasefrom incubated soleusand epitrochlearis muscles;how- constant flow is of vascular origin. l

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ever, lactate releasewas increased by isoproterenol, and this increasewas unaffected by glycerol trinitrate (2.2 PM). Similarly, neither norepinephrine (O-O.1PM) nor angiotensin (U0.1 PM) altered the 0, consumption and lactate efflux by perifused soleusmuscles.Taken together, these findings suggest that, in the perfused rat hindlimb, the increase in lactate efflux during agonist-mediated vasoconstriction is of vascular smooth muscleorigin and may constitute a significant fuel for whole body thermogenesis. vascular smooth muscle; vascular oxygen consumption; thermogenesis;vasodilator LACTATE RELEASE by perfused

hindlimb preparations under basal conditions and in the absence of hypoxia or exercise has been reported by numerous groups. Release occurs independently of whether the perfusate contains red blood cells (24,25) OFno red blood cells (l&16,28,33) and has been noted at 37°C (24, 25), 32°C (28, 29), and 25OC (10, 16, 33). In addition, the release of lactate by

2544

MATERIALS AND METHODS Chemicals and drugs. Arginine vasopressin, human angiotensin II, norepinephrine, epinephrine, methoxamine, serotonin, propranolol, isoproterenol, hydrazine hydrate, lactate dehydrogenase, and NAD+ were acquired from Sigma Chemical. Prazosin was a generous gift from Pfizer. Sodium nitroprusside was from Merck, and bovine serum albumin and pentobarbital sodium were from Boehringer. Glycerol trinitrate (GTN) for intravenous infusion, stabilized in 30% ethanol and 30% propylene glycol, was from David Bull. All other chemicals were of analytic grade from Ajax. Animals. All experiments were approved by the University of Tasmania Ethics Committee under the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Males of a local strain of hooded Wistar rats were kept at 22°C with free access to water and a commercial rat chow (Gibsons, Hobart) containing 21.4% protein, 4.6% lipid, 69% carbohydrate, and 6%

0161-7567192 $2.00 Copyright 0 1992 the American Physiological Society

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crude fiber, with added vitamins and minerals. Rats were anesthetized with pentobarbital sodium (5-6 mg/lOO g body wt ip) before all surgical procedures. Perfusions. To avoid uncontrolled lactate production by red blood cells, hindlimb perfusions were performed with erythrocyte-free medium and conducted at 25’C (8). For each experiment, one hindlimb from a 180- to 200-g male rat was perfused after anesthesia at a constant flow (4.1 t 0.1 ml/min) with Krebs-Ringer bicarbonate buffer [containing 1.27 mM CaCl,, 8.3 mM glucose, and 2% (wt/vol) bovine serum albumin that had been dialysed and freeze-dried] and gassed with 95% O,5% CO,. The forefoot, the contralateral iliac artery and vein, and the abdomen at approximately the level of L,L, vertebrae were tied off to exclude them from the perfusion. Venous PO,and perfusion pressure were continuously monitored. Arterial PO, was determined at the beginning and the end of the perfusion to allow calculation of 0, consumption. Hormones and drugs were continuously infused via a sidearm before a combined bubble trap and mixing chamber. Dose-response curves were cumulative with infusion of each agonist for 15 min. After the highest dose, the preparation was allowed to recover to show reversibility. For comparison, a number of hindlimbs of male rats of llO- to 120-g were perfused at 25*C at 2.3 t 0.1 ml/min. Mesenteric artery perfusions were performed as described in the study of Dora et al. (10). Briefly, the mesenteric artery was cannulated and the gut excised from the mesentery. The preparation was then immersed in a sealed buffer-filled chamber at 25*C and inverted, and the preparation was perfused with a constant flow of 1.5 ml/min from below, with the same perfusion and recording systems as those used for hindlimb perfusion. Perifusions. Six soleus muscles were quickly removed from three anesthetized male rats of 60-80 g and put into perfusion buffer at room temperature. They were then tied longitudinally tendon to tendon and tethered at each end with 3-O surgical silk in a water-jacketed cylindrical glass chamber (145 mm length and 5 mm internal diam). The chamber was purged of bubbles at a rapid flow rate that was then reduced to 3.19 t 0.09 ml/g. The perfusion medium and equipment were the same as for hindlimb perfusions except that the temperature was maintained at 37 & O.l”C. D rugs were dissolved in buffer and infused at O-10 &‘min (i.e., ~1% of flow); they had no detectable effect on apparent 0, consumption. Streamlining of perfusate and consequent failure of access of drugs to the soleus muscles were prevented by the use of an in-line preliminary mixing chamber and by passage of the medium through a l-cm layer of Ballotini soda glass beads (75-150 ,um diam) at the bottom of the muscle chamber to increase turbulence and mixing. Incubations. The method for incubation of rat thoracolumbar aortas was modified from that in Ref. 31. After intraperitoneal anesthesia with 60 mg/kg pentobarbitone sodium and 100 U heparin, aortas were excised from male rats of 100-120 g, dissected free of connective and brown adipose tissue, and then cut transversely into approximately equal halves. The aortas were initially incubated together at 37OC for 30 min in 1.5 ml of incubation medium (Krebs-Ringer bicarbonate containing 2.5 mM

PRODUCTION

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2545

CaC1, and 5 mM glucose without albumin) and continuously gassed with 95% O,-5% CO,. The aortic pieces were then transferred to an additional 1.5 ml of well-gassed fresh medium at 37”C, with or without agonist, incubated for an additional 120 min, and gassed with continuously humidified and heated O,-CO,. When required, aortas remained stretched throughout the experiments by the insertion of individual stainless steel 0.5-mm-diam wire “hair pins” into each aortic half before the initial incubation Skeletal muscles were incubated in a fashion similar to the method of Challis et al. (4), as follows. After anesthesia and heparinization as above, soleus muscles were excised from 60- to 80-g rats and then tied in parallel (4/ expt) to a stainless steel spring clip to maintain their approximate in vivo length. They were preincubated for 30 min in buffer, then transferred to fresh buffer, and incubated together for 2 h in an incubation volume of 2.5 ml of the same medium as the hindlimb perfusion buffer (Krebs-Ringer bicarbonate buffer containing 1.27 mM CaCl,, 8.3 mM glucose, and 2% albumin), which was continuously gassed on the surface of the medium with humidified and temperature-equilibrated O&O,. Epitrochlearis muscles were excised from anesthetized 100. to 120-g rats and treated and incubated as for soleus muscles except that the incubation volume was 2 ml. Lactate assay. Samples for lactate analysis were collected over timed periods (see RESULTS) from hindlimb perfusions and were centrifuged briefly to sediment the small number of red blood cells that sometimes emerged during vasoconstriction. Samples were also taken from the various incubation media at the end of the incubations. After collection, samples were immediately deproteinized with 2 M perchloric acid and placed on ice. After centrifugation to remove the protein pellet, the samples were neutralized with 2.5 M K&O,, and the KClO, precipitate (0°C) was removed by centrifugation. The supernatant was assayed for lactate by use of a standard lactate dehydrogenase enzymatic method (2). Statistical analysis. Values are means t SE. Differences between treatment and control groups were assessed by one-way analysis of variance or by Student’s t test. RESULTS

Ail hindlimbs were allowed to perfuse for 20-30 min, or longer, until steady-state conditions were reached, before measurements of basal pressure and venous PO, (Pv,,) were made. For perfusions of hindlimbs of 180. to 200-g rats, arterial PO, (Pa,J was 634.4 t 5.7 Torr and Pvo, was 377.3 t 6.1 Torr (n = 21); for hindlimbs from llO- to 120-g rats, Paoe was 641.1 & 21.2 Torr and Pvoz was 321.0 t 17.5 Torr (n = 5). The time courses for perfusion pressure, O2 consumption, and lactate release from perfused rat hindlimbs taken from rats of NO-200 g and loo-120 g during angiotensin II infusion are shown in Fig. 1. There was an increase to plateau values for both pressure and 0, consumption, but lactate production showed an initial peak before return to a plateau. The increased perfusion pres-

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The p-adrenergic agonist isoproterenol, a vasodilator in the hindlimb, slightly reduced 0, uptake and perfusion pressure but increased lactate release by 118% at 1 PM concentration (Fig. 2). The lactate production induced by each vasoconstrictor in the hindlimb was completely blocked by nitroprusside (0.5 mM), whereas that induced by isoproterenol was unaffected (Fig. 3). We previously reported that the perfused mesenteric artery responds to serotonin with a dose-dependent vasoconstrlction, as snown ray an Increase In perrusron pressure, together with an increase in 0, consumption (10). Figure 4 shows the dose-dependent increases in lactate efflux above a basal value of 5.48 pmol 8-l. h-l from this preparation, which have not previously been published. Isolated aortas and representative red (soleus) and white (epitrochlearis) skeletal muscles were incubated in vitro to compare their respective lactate production rates in response to various vasoconstrictors and vasodilators. The isolated unstretched aorta responded to the vasoconstrictor angiotensin II with a statistically significant increase (51%, P < 0.05) in lactate production over control (Fig. 5). The action of angiotensin II on lactate pro1

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1. Time COUPS~for effect of 5 nM angiotensin 11 and blocking effect of 0.5 mM sodium nitroprusside (NP) infused for periods indicated on perfused rat hindfimb 0, uptake, perfusion pressure, and lactate efflux. Perfusions were conducted at constant&w rate of 4.1 t 0.1 ml/min for hindlimbs (-15 g) from 180- to 200-g rats or 2.3 + 0.1 ml/min for hindlimbs (-9 g) from 1lC.Lto 120-g rats. Venous PO, (inline O2 electrode) and perfusion pressure (in-line transducer) were continuousfy monitored. Lactate concentration was determined enzymatitally on neutralized perchlorate samples of effluent perfusate. Values are means strSE; n = 5. FIG.

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sure, 0, consumption, and lactate release were substani ‘” tially blocked by 0.5 mM sodium nitroprusside. a The vasoconstrictors vasopressin, angiotensin II, norepinephrine, and methoxamine increased 0, consumpg‘r tion, perfusion pressure, and lactate efflux in a dose-dependent manner in the perfused hindlimb (Fig. 2). Infuz : 15 sion of the highest doses tested of vasopressin (0.5 nM), 3 & angiotensin II (5 nM), norepinephrine (50 nM), and me3 i 5 35 thoxamine (10 ,uM) into the hindlimb increased the perfusion pressure by 112-167% from 30.4 2 0.8 mmHg, 0, 45 -5 consumption by 26-68% from 6.4 t 0.2 pmol 9g-l h-l, -70 -8 -12 -10 -8 -6 -5 and lactate efflux by 14th380% from 5.41 -t 0.25 Log [Concentration] pmol 48-l h-l. The values shown for perfusion pressure FIG. 2. Dose-response curves for vasopressin, angiotensin 11,norepiand 0, consumption are the plateau values after 20 min, nephrine, isoproterenol, and methoxamine on changes (A) in 0, upwhereas those for lactate efflux are the average values fur take, perfusion pressure, and lactate efflux by constant-flow-perfused the first 20 min of infusion of each agonist. Perfusion of rat hindlimb of rats of 180-200 g. Agonists were infused for 15 min. For hindlimbs of lOO- to 120-g rats stimulated with angiotenrats of MO-200 g, control values (no addition) were 6.4 -t 0.2 sin II showed qualitatively similar responses of an 87% pmol g-l h-l (0, uptake), 30.4 & 0.7 mmHg (perfusion pressure), and increase in perfusion pressure from 19.0 t 0.7 mmHg, an 5.41 t 0.25 Arnold 8-l. h-l (lactate efflux). For comparison, data are shown on right for perfusion of hindlimbs from 100- to 120-g rats 0, consumption increase by 45% from 9.1 t 0.7 also stimulated with angiotensin II, for which control values were 9.1 t 0.7 pmol g-l h-l, and a lactate increase of 64% from 8.40 t pmol g-l . h-l (0, uptake), 19.0 -+ 0.7 mmHg (perfusion pressure), and 8.40 k 1.3 pmol g-l . h-l (lactate efflux). Values are means -t-SE; n = 5. 1.3 pmol g-l h-l. l

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Contfd 0.5 nM Vasopressin 5nM Angiotensin II 50nM Norepinephrine lO@i Methoxamine l@d isoproterenol

2547

OF LACTATE I Control D 50nM Ang II BSI Ang II + 2.2kM GTN rt;rza 2.2kM GTN e399 IpM Is0 Esl 1pM Is0 + 5@4l Prop I/.A 1pM Iso + 2,2kM GTN ELI Stretch IZZI Stretch

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FIG. 5. Lactate release from isolated rat aorta incubated for 2 h (details described in MATERIALS AND METHODS). Additions were angiotensin II (ANG II), isoproterenol (Iso), glycerol trinitrate (GTN), or propranolol (Prop). Values are means t SE, with no. of incubations in parentheses. Significant differences were determined using one-way analysis of variance (ANOVA). * Significantly increased lactate release compared with controls (P < 0.05).

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3. Effects of 0.5 mM nitroprusside (NP) on vasoconstrictorand vasodilator-mediated changes in 0, consumption, perfusion pressure, and lactate production by constant-flow-perfused rat hindlimbs taken from rats of 180-200 g. See legend of Fig. 1 for additional information. Values are means t SE; n = 5. *P < 0.02, **P < 0.01, ***P < 0.001 for treatment significantly different from corresponding treatment with NP (unpaired Student’s t test). FIG.

duction by the aorta was completely blocked by 2.2 ,uM GTN, yet this agent when used alone had no effect on aortic lactate production (Fig. 5). Nitroprusside was not used in tissue incubations because of the production of cyanide ions that occurs on standing or when exposed to tissue for long periods (9). Isoproterenol also led to signif-

icant increases in lactate production in the incubated aorta (Fig. 5). This increase was not blocked by GTN but was significantly blocked by propranolol, a P-adrenergic antagonist. Stretching of vascular smooth muscle is known to induce vasoconstriction (30). In the present study, stretch was observed to significantly increase lactate production (-5976, P < 0.05, Fig. 5). Angiotensin II further increased lactate release of the stretched aortas, but the increase above stretch alone was not statistically significant. In contrast to their effects on vascular smooth muscle, the vasoconstrictors angiotensin II and vasopressin had no effect on lactate production by incubated soleus muscle (Fig. 6) or epitrochlearis muscle (Fig. 7). However,

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Serotonin (PM) FIG. 4. Increase in lactate efflux from perfused rat mesenteric artery and its branches in response to serotonin. Values are means +: SE; IZ = 4-12. Basal lactate efflux was 5.48 4 0.61 pmol *g-l. h-l. For data on perfusion pressure and 0, consumption see Ref. 10.

FIG. 6. Lactate release by isolated and continually gassed incubated rat soleus muscles (details described in MATERIALS AND METHODS). Additions were ANG II, vasopressin (VP), Iso, GTN, Prop, norepinephrine (NE), and prazosin (Praz). Values are means t SE, with no. of incubations in parentheses. Significant differences were determined using one-way ANOVA. * Significantly increased lactate release compared with controls (P < 0.05).

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2548

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isoproterenol stimulated both muscles to significantly increase lactate release, and this was blocked by proprano101but not by GTN (Figs. 6 and 7). Interestingly, neither 50 nM norepinephrine, in the presence or absence of 5 PM propranolo& nor 100 nM prazosin caused a significant increase in lactate production by the incubated soleus muscle (Fig. 6), despite its significant effect on lactate efflux in the perfused hindlimb at this concentration. Because it was not technically possible to measure 0, consumption in a continually gassed skeletal muscle incubation, isolated soleus muscle 0, consumption was measured in a perifused system essentially similar in its arrangement to that of the perfusion system. Perifusate flow, although lower than for hindlimb perfusions to maximize any arteriovenous differences in Pvo2, was still similar in proportion to mass at 0.57 t 0.01 ml/min or 3.19 t 0.09 ml g-l min-l, the basal 0, consumption was 23.2 t 3.7 pmol g-l h-l, and the basal lactate efflux was 10.7 t 1.4 pm01 g-l h? Only small and nonsignificant changes in 0, consumption and lactate efflux were found with concentrations of norepinephrine and angiotensin II in the range of O-O.1 PM (Fig. 8). l

PRODUCTION

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strengthen the argument that the increased lactate production under stimulation by vasoconstrictors is linked either directly or indirectly to contraction of the resistance vessels of the perfused rat hindlimb. For the vasoconstrictors E-norephedrine (16), norepinephrine, vasopressin, and angiotensin II (Fig. 2), there is an approximate proportionality between the magnitude of increase in lactate efflux, 0, uptake, and perfusion pressure over the dose curve. Vasoconstriction in the perfused rat hindlimb, however, is not always associated with increased lactate release. In a recent study we noted that serotonin (0.5 PM) caused marked vasoconstriction but inhibited lactate production by 12% and 0, consumption by 19%. This action of serotonin appeared to be due to the operation of functional vascular shunts within the hindlimb (10). When tested on an isolated mesenteric artery preparation in which arteriovenous shunts were absent, serotonin increased perfusion pressure and 0, consumption (10) as well as lactate efflux (Fig. 4), indicating that isolated perfused arteries from the rat can produce significant quantities of lactate when stimulated to contract. Discrimination between skeletal muscle and other tissues as a source of lactate during vasoconstriction in the perfused rat hindlimb has not previously been made. Hence the enhancement of lactate release by epinephrine or epinephrine plus propranolol, either at rest or during skeletal muscle contraction, was believed to originate from striated muscle fibers (24). However, the rationale om-

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DIESCUSSION

The studies reported here from the constant-flow-perfused rat hindlimb extend our previous observations and show that, in addition to norepinephrine (10) and Z-norephedrine (16), the additional vasoconstrictors vasopressin, angiotensin II, and methoxamine (Figs. 1, 2, and 3) induce an increase in lactate production in the range of l48-380% in association with increased vascular resistance (112-167%) and increased 0, consumption (2668%) for hindlimbs from 180- to 200-g rats. Hindlimbs from rats of loo-120 g had a higher basal 0, consumption and lactate efflux and gave qualitatively similar, but lower, proportionate increases in perfusion pressure, 0, consumption, and lactate efflux when stimulated with angiotensin II. In addition, these effects were all blocked bv sodium nitroprusside (Figs. 1 and 3) and therebv

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Concentration ( n Ml FIG. 8. Effects of O-100 nM angiotensin II and O-100 nM norepinephrine on 0, uptake and lactate efflux by perifused soleus muscles (perifusion details described in MATERIALS AND METHODS). Values are means t SE; n = 5-7 (0, uptake); n = 3-4 (lactate efflux). There was no significant difference by two-tailed Student’s t test between stimulation and control of either vasoconstrictor at any concentration.

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Another possibility for increased lactate production with vasoconstrictor agonists is that the vasoconstrictors increase the rate of one or more forms of futile substrate cycling or ion pumping in the hindlimb, with the consequent increased hydrolysis of ATP, which then leads to increased lactate production and 0, consumption. Although this study does not directly address these possibilities, futile cycling in skeletal muscle is unlikely, because the force generated by calf muscle contraction in response to sciatic nerve stimulation is not diminished during infusion of angiotensin II or during control contractions in this perfused rat hindlimb model (7). Similar data were found by others with the infusion of epinephrine plus propranolol during induced contractions in the erythrocyte-perfused rat hindlimb (24). In the insect, flight muscle substrate cycling of fructose 6-phosphate is suppressed during contractions (5); thus, by analogy, substrate cycling could be expected to be inhibited during skelet,al muscle contraction. In isolated epitrochlearis muscle, estimates of heat production by phosphofructokinase futile cycling at rest are 0.1% of basal metabolic rate, increasing to 12% with ,& adrenergic stimulation (3). These rates are low compared with the increase in 0, consumption of 70 and 84% previously obtained with vasopressin (8) and with epinephrine plus propranolol(7) in the perfused rat hindlimb. A small decrease in 0, consumption was found with the ,&agonist isoproterenol in the perfused rat hindlimb. Challis et al. (3) found that the rate of glycolytic cycling was only 6% of basal lactate production but did increase with maximal ,&adrenergic (isoproterenol) stimulation by -30% and by 10% with maximal cu-stimulation (phenylephrine plus propranolol). This finding further suggests that substrate cycling or ion pumping could explain only a small fraction of the lactate production observed in this study with cu-adrenergic agonists and other vasoconstrictors. As discussed earlier for serotonin, the increased production of lactate is not always associated with a vasoconstriction action in the perfused rat hindlimb. A different example is provided by the vasodilating ,&adrenergic agonist isoproterenol, which increased lactate production by hindlimb (Figs. 2 and 3), by incubated skeletal muscles (Figs. 6 and 7), and by aortas (Fig. 5). This result was not unexpected, inasmuch as a P-adrenergic effect on the breakdown of glycogen in either skeletal muscle or smooth muscle would be predicted from in vitro studies by other researchers on skeletal (3, 4) and vascular smooth muscle (30). However, in the present study, the p-effect in each system tested was not blocked by the nitrovasodilators (Figs. 3, 5, 6., and 7). Thus, when taken cumulatively, the findings of this communication show that lactate production from vascular smooth muscle appears to be a general phenomenon associated with agonist-induced vasoconstriction and 0, uptake not occurring in response to hypoxia. The amount of lactate efflux from the constant-flow-perfused rat hindlimb of NO- to 200-g rats is considerable. Under nonstimulated conditions it is comparable to the amount of 0, consumed, with a ratio of lactate efflux to 0, uptake of -0.8-1.0. This ratio increases to a transient peak of -2- to $-fold under agonist stimulation (Figs. l-3) and then drops back to ~1.3~ to l.fi-fold (Fig. 1). Hindlimbs from lOO- to 120-g rats show a relatively

PRODUCTION

OF

LACTATE

greater 0, consumption than lactate efflux, with a ratio of unstimulated lactate efflux to 0, consumption of 0% 1.2, a transient peak ratio of ~1~6~ and a fall back to a plateau of 1.1-1.2 when stimulated with angiotensin II (Fig. 1). That the hindlimb lactate production in response to vasoconstrictors is originating from working vascular smooth muscle is consistent with evidence that vascular segments can produce large amounts of lactate. As reviewed by Somlyo and Somlyo (30) and Paul (22), such lactate values may reach up to five times that of 0, consumption. In the present study, other factors that are characteristic of the perfusion system used may be at work to increase the lactate production relative to 0, consumption. Ruderman et al. (25), in a recirculating erythrocyte-containing constant-flow rat hindlimb perfusion at 37”C, found a lactate production of 5.4 pmol 8-l. h-l, which is the same as that obtained in this report at 25°C. Shiota and Masumi (26), who perfused at 32OC in a nonrecirculating manner, obtained a basal value of 3.6 pm01 g-l h-l, which is less. However, their perfusion system differed significantly from that of most other investigat-ors by the lack of albumin in their perfusate. They obtained little or no increase in 0, consumption and lactate production in response to norepinephrine when perfusing the hindlimb from rats acclimated to 24OC. A marked increase in 0, consumption by the perfused rat hindlimb in response to either norepinephrine or t,o epinephrine plus propranolol is the more usual response (7, 8, 14, 24). Recirculation of the perfusate in the perfused hindlimb technique allows the possibility of lactate uptake and metabolism by muscle. The presence of pyruvate and its molar ratio to lactate appear to determine whether there is net uptake of lactate by perfused muscle (27) or by perfused liver (13). In the present system there is neither lactate nor pyruvate added to the perfusion buffer, nor does recirculation occur. Skeletal muscle uptake of lactate is possible. In the rabbit, differing thresholds for the uptake of arterial lactate, 2.5 mM for red and 3.8-3.9 mM for white or mixed muscles, were described by Pagliassotti and Donovan (21). It is possible that the perfusion temperature of 25OC can bring about a relative decrease in the O,-consuming tricarboxylic acid cycle when compared to the lactate-producing glycolysis in the formation of ATP. Seiyama et al. (26) showed that the lactate efflux to 0, uptake ratio is larger at 15 than at 35OC in the perfused rat hindlimb, with few differences in myoglobin saturation, suggesting that the speed of glycolysis in the cold is sustained relative to aerobic metabolism and that, unlike 0, consumption, it has a lower temperature sensitivity, i.e., &lo is low. When extrapolated to the in vivo situation, these factors may alter the absolute amount of lactate efflux that would occur, but it seems apparent nevertheless that significant lactate production would occur in response to agonist-mediated constriction. Lactate production by perfused vascular beds could have a facultative thermogenic role. Infused lactate has long been known to stimulate 0, consumption in vivo, and glucose can be resynthesized from lactate in the liver or other gluconeogenic tissues, such as the kidney and perhaps skeletal muscle, by the Cori cycle. Regardless of the exact site of the conversion of lactate to glucose or l

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VASCULAR

SMOOTH

MUSCLE

glycogen, the amount of high-energy phosphate required is equivalent to either 3 or 4 mol of ATP, depending on whether 1 mol of lactate is converted to glucose or glycogen, respectively. If 3 mol of ATP were consumed per 1 mol of lactate converted to glucose, -0.5 mol of 0, would be consumed by the mitochondria of the gluconeogenic tissue involved. Thus, if significant amounts of lactate were produced in vivo in response to the vasoconstrictors, a subsequently higher 0, consumption would occur in the gluconeogenic tissues than that observed directly. This observation may explain, in part, why various tissues such as liver (19) and skeletal muscle (1, 20) have been observed

to participate

in catecholamine-induced

thermogenesis and would complement the contribution by vascular thermogenesis recently suggested by our laboratory (7, 8, 33).

PRODUCTION

Received 15 July 1991; accepted in final form 8 July 1992. REFERENCES A., J. BULOW, J+ MADSEN, AND N. J. CHRISTENSON. Contribution of BAT and skeletal muscle to thermogenesis induced by ephedrine in man. Am. J. Physiol. 248 (Endocrinot. Metab. 11): E507-E515, 1985. BERGMEYER, H. U. Methods in Enzymatic Analysis. New York: Academic, 1974. CHALLISS, R. A. J., J. R. S. ARCH, AND E. A. NEWSH~LME. The rate of substrate cycling between fructose 6-phosphate and fructose 1,6bisphosphate in skeletal muscle. Biochem. J. 221: 153-161, 1984. CHALLISS, R. A. J., B. LEIGHTON, S. WILSON, P. L. THURLBY, AND J. R. S. ARCH. An investigation of the P-adrenoreceptor that mediates metabolic responses to the novel agonist BRL28410 in rat soleus muscle. Gen. Pharmucol. 37: 947-950, 1988. CLARK, M. G., D. P. BLOXHAM, P. C. HOLLAND, AND H. A. LARDY, Estimation of the fructose diphosphatase-phosphofructokinase substrate cycle in the flight muscle of Bombus affinis. Biochem. J.

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We thank John E. Jordan, Michael J. Glancy, and Sandi J. Warr for technical assistance. This work was supported in part by the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia. Present address of M. Hettiarachchi: Garvan Institute of Medical Research, Sydney, Australia. Address for reprint requests: E, Q. Colquhoun, Dept. of Biochemistry, Faculty of Medicine and Pharmacy, University of Tasmania, GPO Box 25X, Hobart, Tasmania 7001, Australia.

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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...
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