Mechanisms of hypoxic vasodilatation of isolated rat mesenteric arteries: a comparison with metabolic inhibition

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1 Keywords: 8557 Journal of Physiology (1999), 516.1, pp Mechanisms of hypoxic vasodilatation of isolated rat mesenteric arteries: a comparison with metabolic inhibition Donna Otter and Clare Austin Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK (Received 26 July 1998; accepted after revision 11 December 1998) 1. Hypoxia (POµ <5mmHg) decreased vessel tone in isolated rat mesenteric arteries precontracted with either high [K ] or the thromboxane analogue U This response wasnotaltered byn-nitro- _arginine (L-NA) and indomethacin. 2. Simultaneous measurement of phé and tension showed that the decrease in vessel tone was accompanied by an intracellular acidification. Similar reductions in tone and phé were observed with the metabolic inhibitors 2,4-dinitrophenol (DNP) and sodium azide. 3. The presence of the lactate transport inhibitor á-cyano-4-hydroxy-cinnamic acid (CHC) increased the magnitude of the acidification and resulted in a significantly faster reduction in tone in response to hypoxia. Addition of CHC to normoxic tissues caused both a vasodilatationandareductionofphé. 4. A decrease in phé induced on washout of ammonium chloride (NHÚCl) resulted in an increase in tone. 5. Relaxation to hypoxia or metabolic inhibition was unaffected when the change in phé was neutralized by addition of the weak base trimethylamine (TMA). 6. It is concluded that severe hypoxia decreases tone in isolated rat mesenteric arteries by a mechanism which is independent of nitric oxide and prostaglandins. Both severe hypoxia and metabolic inhibition reduced phé, although this does not appear to be contributing to the changes in tone observed. Changes in the tone of vascular resistance vessels play an extremely important role in determining blood flow, and therefore oxygen supply, to various parts of the body. Restricted blood flow may result in local hypoxia which itself may then influence vascular tone, blood flow and oxygen supply to the region. Although it is well established that hypoxia may alter tone, the contractile responses of different tissues are varied, with both increases (Yuan et al (rat pulmonary); Toda et al (human, monkey and dog coronary)), decreases (Brown et al (guinea-pig coronary); Aalkjaer & Lombard, 1995 (rat cerebral and mesenteric)) and biphasic effects (Leach et al (rat pulmonary and mesenteric)) in response to a decreased POµ having been reported. These different responses may reflect not only differences between vessel type and size but may also depend on species and experimental conditions such as the degree of hypoxia, the degree of precontraction andïor the precontracting agent, etc. For example, previous studies have found that while resting rat pulmonary arteries contract (Yuan et al. 1990), vessels precontracted with either addition of agonists or depolarization exhibited a biphasic response (Leach et al. 1994) in response to severe hypoxia. Such experimental variations thus make between-study comparisons difficult. We have previously shown that severe hypoxia induces a relaxation of isolated rat mesenteric resistance arteries precontracted by high-k solution (Otter & Austin, 1997). This vasodilator response to hypoxia may be a protective mechanism increasing blood flow, and therefore Oµ supply, to the tissue, thus alleviating the hypoxia. The mechanisms by which hypoxia may dilate mesenteric arteries are, however, unclear. In other systemic arteries that also exhibit vasodilatory responses to hypoxia, it has been suggested that the decrease in tone may be due to the release of endothelium-derived relaxing factors (Graser & Rubanyi, 1992 (rat aorta); Jiang & Collins, 1994 (rabbit coronary)). However, in some studies, the responses have been found to be independent of the endothelium (Graser & Rubanyi, 1992 (canine coronary); Aalkjaer & Lombard, 1995 (rat cerebral, mesenteric)). Again, the responses observed may depend on the type and

2 250 D. Otter and C. Austin J. Physiol size of vessel under study and on the species. As indicated above, experimental conditions and protocols may also influence the results obtained. To date, only one study has investigated the role of the endothelium in the contractile responses of isolated rat mesenteric vessels during hypoxia. Aalkjaer & Lombard (1995) demonstrated that after > 30 min of severe hypoxia, mesenteric arteries exhibited a reduced contractile response to adenosine vasopressin (AVP), which was unaffected by removal of the endothelium. This study clearly demonstrates that established hypoxia reduces contractile responses to subsequently applied AVP by an endothelium-independent mechanism(s) but it cannot be assumed that the vasodilator response of precontracted tissues to hypoxia is also independent of the endothelium; indeed in other tissues, such as the canine coronary artery, hypoxic relaxations of depolarized tissues were prevented by inhibitors of endothelial prostaglandin synthesis (Graser & Rubanyi, 1992). The present study therefore initially investigated the role of the endothelial vasodilator substances nitric oxide (NO) and prostaglandins in the vasodilator responses of isolated rat mesenteric arteries to severe hypoxia. Hypoxia may be expected to increase the production of lactic acid due to stimulation of anaerobic glycolysis. It has previously been suggested that, during hypoxia, lactic acid may accumulate within the cells reducing intracellular ph (phé) and indeed, addition of metabolic inhibitors has been found to decrease phé in a number of isolated smooth muscle preparations (Wray, 1990; Smith et al. 1996). As alterations in phé are well known to alter vascular contractility (Austin & Wray, 1993a, 1994; Smith et al. 1998) it has been postulated that the change in tension associated with hypoxia may be a result of a change in phé and indeed in a preliminary study, one of us has previously found that metabolic inhibition with cyanide reduced both the tone and phé of depolarized mesenteric arteries. It is unclear, however, whether the acidifications observed with metabolic inhibition are responsible for the vasodilatation (Smith et al. 1996). The effect of hypoxia per se (i.e. a decrease in POµ), as opposed to metabolic inhibition, on phé in vascular smooth muscle has, however, been very poorly studied and we could find only one study in which phé was measured in vascular smooth muscle during hypoxia. In this study, addition of AVP to normoxic tissues resulted in a small decrease in phé and contraction. After tissues had been hypoxic for > 30 min, however, the contractile response to AVP was reduced although the decrease in phé was not altered (Aalkjaer & Lombard, 1995). It is thus unclear whether metabolic inhibition and hypoxia have different effects on phé or whether the differences are due to experimental variations and protocols. This study therefore examines, and directly compares, the effect of metabolic inhibition and hypoxia on phé of rat mesenteric arteries. The role of any changes in phé in effecting the vasodilatation of precontracted vessels in response to hypoxia was also studied. METHODS Preparation of vessels Male Wistar rats ( g) were killed by stunning and exsanguination. Mesenteric arteries ( ìm diameter) (3rd 4th order branches) were dissected out and mounted on a Mulvany Halpern myograph for measurement of isometric tension. The vessels were continuously perfused with a Krebs solution of the following composition (mò): NaCl, 119; KCl, 4 7; MgSOÚ.7HµO, 1 2; NaHCO, 25; KHµPOÚ, 1 17; EDTA, 0 03; glucose, 5 5; Ca, 2 5 at ph 7 4. The myograph chamber was covered and sealed and the solutions were heated to 37 C and gassed with 95 % air 5 % COµ. The volume of the chamber was 4 ml and the complete bath exchange time was approximately 70 s. Hypoxia was induced by changing the gassing mixture to 95 % N2 5 % COµ (i.e. a severe hypoxic state). The oxygen tension of the solutions was continuously measured by placing a small oxygen electrode (Instech; calibrated totwopointsusingairfollowedbyadditionoftheoµscavenger sodium dithionite) close to the tissue. After mounting and equilibration, the ratio of resting tension to internal circumference of the arteries was determined and the vessels set to a normalized internal circumference of l0 where lñ is 0 9l100 and l100 is the internal circumference of the vessel under an effective transmural pressure of 100 mmhg. Under these conditions maximum active tension is developed (Mulvany & Halpern, 1977). Following normalization, vessels were subjected to a routine run up procedure (3 ² 2 min exposures to 60 mò KCl) before experiments were begun. In all experiments the presence of a functional endothelium was tested by observing responses of precontracted tissues to acetylcholine (50 ìò) or carbachol (50 ìò). All vessels included in the study showed a relaxation to these endotheliumdependent agonists of > 60 % of precontraction, i.e. all had a functionally intact endothelium. In some experiments the effects of nitric oxide (NO) and prostaglandins on hypoxic responses were examined by prior incubation (for > 30 min) with both 50 ìò N_nitro- _arginine (l-na) and 10 ìò indomethacin (inhibitors of NO synthase and cyclo-oxygenase, respectively). These inhibitors were added to the tissues simultaneously. The effects of hypoxia (induced by gassing with 95 % N2 5 % COµ), in both the presence and absence of _NA and indomethacin, was investigated in vessels precontracted with either 60 mò KCl or 1 ìò of the thromboxane analogue U46619 (Sigma). Unless otherwise stated, all other experiments were carried out on vessels contracted with 60 mò KCl. In these experiments, KCl was isosmotically substituted for NaCl. The effects of severe hypoxia and metabolic inhibition were examined and compared in depolarized tissues. The metabolic inhibitors used were 2,4_dinitrophenol (DNP) (1 mò) and sodium azide (2 mò). These inhibitors act at different steps of oxidative ATP production, sodium azide inhibiting electron transport and DNP uncoupling electron transport from phosphorylation (see Rounds & McMurtry, 1981). In some experiments the effect of á-cyano-4-hydroxy-cinnamic acid (CHC; Sigma) (4 mò), an inhibitor of lactate transport, on responses was investigated. In other experiments, phé was selectively changed (at constant extracellular ph (phï)) by addition and washout of the weak bases ammonium chloride (NHÚCl) (40 mò) ortrimethlyamine (TMA) (30 40 mò), which were again isosmotically substituted for NaCl. We have previously shown that substitutions for Na (30 40 mò) have no significant effect on phé (Austin & Wray, 1993a). When the weak base TMA (40 mò) was used in an attempt to neutralize the change in phé observed with hypoxia or metabolic inhibition, the protocol was as follows. Depolarized vessels were made hypoxic (or metabolic inhibitors added) and responses,

3 J. Physiol Hypoxia and metabolic inhibition 251 i.e. decreases of both tension and phé, were allowed to plateau. TMA was then added, in the continued presence of hypoxia/ metabolic inhibition and depolarizing stimulus, and phé allowed to reach a new level (approximately 2 3 min). Vessels were then returned to a normoxic high-k solution and responses allowed to return towards pre-hypoxicïmetabolic inhibition levels before washing with normoxic Krebs solution. These experiments were carried out in the presence of either DIDS or SITS (150 ìò) to prevent phé regulation following an intracellular alkalinization, thus allowing any change in phé to be maintained and so any effect on tone to be determined more accurately. In another set of experiments, both the metabolic inhibitor DNP (1 mò) and the weak base TMA (40 mò) were added simultaneously to depolarized vessels and their effects on phé and tension noted. Any changes in phé and tension were allowed to stabilize before tissues were washed with normoxic Krebs solution. To investigate the possible involvement of camp in the vasodilator responses of isolated mesenteric arteries to hypoxia, vessels were incubated (for > 25min) with 25ìÒ of the RP stereoisomer of adenosine - 3', 5' - cyclic monophosphothionate (Rp-cAMPS; Calbiochem Novabiochem). Rp_cAMPS is an inactive stereoisomer of camp which competitively inhibits stimulation of protein kinase A (see McKinnon et al. 1996). Vessels were precontracted with high-k solution, in the continued presence of Rp-cAMPS, and the effects on hypoxic vasodilatation examined and compared with control responses to hypoxia obtained in the same system. Due to financial constraints, these experiments were performed without continual perfusion and therefore phé was not measured. We have previously shown, however, that the magnitude of the vasodilator responses observed to both hypoxia and metabolic inhibition in this experimental set-up are similar to those observed when vessels are constantly perfused. The effects of Rp-cAMPS on vasodilatory responses of depolarized tissues to isoprenaline (0 1 ìò) were also examined. Measurement of phé After mounting and equilibration, the vessel was placed on the stage of a Leica DM IRB inverted microscope where it was incubated with 5 10 ìò of the acetoxymethylester form of the phsensitive dye carboxy-snarf (Molecular Probes) for 2 3 h. After washing, vessels were excited at 340 nm and emissions collected at 570 nm and > 600 nm via photomultiplier tubes. The ratio of these emissions was calibrated in terms of absolute ph units using either the K H ionophore nigericin or, more usually, by an in vitro methodusingthefreeacidformofsnarf.wehavepreviously shown that there is a good agreement between these methods in vascular tissue (Austin & Wray, 1993b). Data collection and statistical analysis Vascular tone, phi (individual emissions and uncalibrated ratio) and POµ were all recorded continuously and simultaneously on both a chart recorder and computer for later detailed analysis. The sampling rates for all parameters in the experiments was 2 Hz. Using similar equipment, we have previously found that sampling rates of 1 2 Hz are adequate to detect differences in the speed and time course of changes in tension and phé in vascular smooth muscle even when the changes are fast, e.g. upon addition of weak acids and bases (see Austin & Wray, 1994; Austin et al. 1996). All results presented are expressed as means ± s.e.m. with n representing the number of experiments. All responses were expressed as changes in active wall tension (mn mm ) from resting levels and normalized as a percentage of the response to 60 mò KCl or 1 ìò U46619 as appropriate. Time courses of changes were determined from simultaneous recordings of the parameters with zero time being the addition of the experimental manoeuvre. Differences between groups were compared by ANOVA and Student s t test (paired or unpaired) and by the Student Newman Keuls test for multiple comparisons. RESULTS Resting parameters The vessels used in this study had a mean diameter of 288 ± 6ìm (n = 30). The POµ of the solutions bathing the tissues, when gassed with 95 % air 5 % COµ, was within the range mmhg. Tension responses to hypoxia and involvement of endothelial factors Additionof60mÒ KCl resulted in a rapid contraction of vessels followed by a relaxation to a smaller maintained level. The mean magnitude of the maintained contraction was 2 7 ± 0 2 mn mm (n = 4). Changing the gassing mixture from 95 % air 5 % COµ to 95 % N2 5 % COµ resulted in a decrease in POµ of the bathing solution (measured as close to the tissue as possible) to < 5 mmhg. This fall in POµ Figure 1. The effect of hypoxia on arterial tone A, exampleoftheeffectofhypoxiaonthetoneofan isolated rat mesenteric artery. The vessel was precontracted with 60 mò KCl. Simultaneous recordings of vascular tone and POµ are shown. B, effects of 50 ìò N-nitro-l-arginine (l-na) and indomethacin (INDO) (50 ìò) on the relaxation response to hypoxia. Vessels were precontracted with either KCl (60 mò) or U46619 (1 ìò). Values are means ± s.e.m. and expressed as a percentage of the maximum response.

4 252 D. Otter and C. Austin J. Physiol was accompanied by a relaxation of precontracted vessels of 99 5 ± 7 3 % of the KCl maximum to near baseline levels (Fig. 1A). Upon return to normoxic gassing solutions both the tension and the POµ returned to near pre-hypoxic levels. The magnitude of this hypoxic vasodilatation was unaffected by prior incubation with _NA and indomethacin, inhibitors of nitric oxide and prostaglandin synthesis, respectively (n = 4)(Fig. 1B). One micromolar U46619 contracted tissues by 2 9 ± 0 4 mn mm. This was not significantly different from the contraction observed with 60 mò KCl. Hypoxia resulted in a relaxation of all vessels by 94 0 ± 4 6 % to near baseline levels. The magnitude of this relaxation was not significantly altered by the presence of _NA and indomethacin (99 6 ± 1 9 %) (Fig. 1B). Simultaneous measurement of phé and vascular tone; addition of KCl In vessels loaded with carboxy-snarf, addition of KCl (60 mò) againresultedinarapidcontractionfollowedbya relaxation to a smaller maintained level. The mean magnitude of the contraction was 1 6 ± 0 1 mn mm (n = 36) which was not significantly different from that in unloaded tissues. When phé was simultaneously measured it was found that resting phé in these vessels was 6 95 ± 0 06 (n = 36). Addition of KCl resulted in a small transient intracellular alkalinization (+0 11 ± 0 01 ph units) in the majority of vessels (36Ï45) with phé quickly returning towards baseline andïor becoming more acidic ( 0 12 ± 0 02 ph units). In 9Ï45 tissues only an acidification ( 0 15 ± 0 03 ph units) was observed. A similar pattern of phé changes with depolarization has previously been reported in mesenteric vessels (Austin & Wray, 1993b). Effects of hypoxia on phé and tone Induction of hypoxia resulted in a decrease in both vascular tension and phé. Tension decreased by ± 3 8 % of KCl contraction and phé was decreased by 0 35 ± 0 03 ph units (n = 11). Upon return to normoxia, both tension and phé rose to near pre-hypoxic levels (67 9 ± 6 1 % and ± 0 04 ph units) (Fig. 2). The times taken for maximal (t) and half-maximal (t½) responses for the changes in tension and phé were similar to each other; t was 1462 ± 92 s and 1432 ± 76 s for tension and phé, respectively while t½ was 526±30s for tension and 570±49s for phé (Fig. 3). It should be noted that POµ began to decrease before any changes in tension or phé were observed (21 ± 10 s) and that the decrease in POµ was significantly faster than the decrease in tension and phé, t½ for POµ being 97 ± 19 s. Effects of metabolic inhibitors on phé and tone Addition of the metabolic inhibitors DNP and azide also caused relaxation and a decrease in phé of tissues precontracted with KCl. DNP caused tissues to relax by 96 5 ± 1 9 % and phé to fall by 0 38 ± 0 1 ph units (n = 5), both of which are not significantly different from those observed with hypoxia. Upon washout both tension and phé rose to near normal levels (84 3 ± 2 2 % and by ± 0 10 ph units, respectively). Addition of the metabolic inhibitor azide again caused both a relaxation of vessels and an intracellular acidification of 88 1 ± 4 0 % and 0 31 ± 0 07 ph units, respectively (n = 6). Upon washout, although phé increased by ± 0 13 units, i.e. to preazide levels, tension only returned by 36 1 ± 2 7 %. Examples of the responses observed to hypoxia, DNP and azide are shown in Fig. 2. Figure 2. Simultaneous measurement of tension, phé and POµ in an isolated rat mesenteric artery The vessel was precontracted with KCl (60 mò) and the effects of hypoxia (N2) and metabolic inhibition due to addition of either 2,4-dinitrophenol (DNP) (1mÒ) or sodium azide (2 mò) are shown.

5 J. Physiol Hypoxia and metabolic inhibition 253 Figure 3. Time courses of the changes in tension and phé in isolated rat mesenteric arteries in response to hypoxia (A), 2,4-dinitrophenol (B) or sodium azide (C) 1, change in tension; 0, change in phé. Vessels were precontracted with KCl (60 mò) and responses normalized to the maximum response. All data shown are mean ± s.e.m. values. Points were obtained by sampling simultaneous traces of tension and phé every 2 min for hypoxia and5sforazideanddnp. For both inhibitors there was no difference in the response times for tension and phé. For DNP, t was75 ± 3sand 70±4s andt½was35±4s and36±5s fortensionand phé, respectively: for azide, t was106 ± 15sand110 ± 17s, and t½ was 48±8s and 51±6s for tension and phé, respectively. Note that the responses to the metabolic inhibitors were significantly faster than those to hypoxia but were similar to each other (Fig. 3). Effects of CHC In paired experiments the effects of hypoxia in the absence and presence of 4 mò CHC (an inhibitor of lactate transport) were directly compared. In these experiments (n = 5) tension was reduced by 99 1 ± 7 7 % by hypoxia alone and by a similar amount, 93 7 ± 2 8 %, by hypoxia in the presence of CHC. It should be noted, however, that in the presence of CHC the fall in tension with hypoxia was significantly faster than with hypoxia alone; t = 815 ± 165sand1462±92s (P < 0 01) and t½ =262±28s and 526±30s(P < 0 01) in the presence and absence of CHC, respectively. In the presence of CHC, hypoxia resulted in a significantly greater intracellularacidification, phédecreasingby 0 78 ± 0 16 compared with a decrease of 0 34 ± 0 03 ph units in its absence (P < 0 05). Similarly the times for maximal and half-maximal responses were significantly faster in the presence of CHC; t = 812 ± 169 s and 1432 ± 76 s and t½ = 232 ± 79 s and 570 ± 49 s in the presence and absence of CHC, respectively (P < 0 01 for both) (Fig. 4). Again Figure 4. Comparison of the effects of hypoxia alone and hypoxia in the presence of 4 mò á_cyano-4-hydroxycinnamic acid (CHC) Simultaneous recordings of tension and phé are shown.

6 254 D. Otter and C. Austin J. Physiol Figure 5. Comparison of the effects of an intracellular acidification due to hypoxia and that due to washout of NHÚCl Simultaneous recordings of tension and phé are shown. there were no significant differences between t and t½ for the changes in tension and phé. Addition of CHC to normoxic vessels caused decreases in both tension (53 0 ± 6 7 %) and phé ( 0 47 ± 0 07 ph units, n = 4). In three of four vessels studied the effects of CHC were reversible, tension returning by 42 6 ± 14 3 % and phé returning by ± 0 05 units. Effects of decreases in phé due to removal of NHÚCl on tone Addition of the weak base NHÚCl resulted in an intracellular alkalinization ( 0 44 ± 0 03 ph units) and a biphasic contractile response consisting of a transient relaxation (13 4 ± 3 6 %) followed by a more prolonged contraction (44 2 ± 7 9 %) of isolated mesenteric vessels precontracted with 60 mò KCl (Fig. 5). In these vessels a change in phé was observed before a change in tension (latency = approximately 1 1 ± 0 4 s; n = 7) although as the responses were so rapid this was difficult to determine accurately. Washout of NHÚCl resulted in a rebound acidification of 0 42 ± 0 04 ph units and a further contraction (21 6 ± 2 9 %) of the vessels. Note that the same vessels relaxed (to > 90 % of KCl contraction) when a decrease in phé was observed with hypoxia. Effects of neutralization It was found that the decrease in phé due to hypoxia could be neutralized by addition of the weak base trimethylamine (TMA) (in the presence of SITS or DIDS to prevent regulation of phé) in the continued presence of hypoxia. Addition of mò TMA returned phé to > 100 % of Figure 6. Effects of 40 mò trimethylamine (TMA) on the decrease in phé and tension caused by hypoxia (N2), 1 mò 2,4-dinitrophenol (DNP) or 2 mò sodium azide in an isolated rat mesenteric artery Note that although TMA more than neutralizes the reduction in phé, tension is unaltered.

7 J. Physiol Hypoxia and metabolic inhibition 255 pre-hypoxic levels, but despite this, tension was unaltered i.e. remained at hypoxic levels (n =5)(Fig. 6). Similar results were also obtained with the metabolic inhibitors DNP and azide (n = 9), i.e. although the phé change was returned to, or above, resting levels, tension was unchanged by this. Upon washout of TMA and metabolic inhibitorsïreturn to normoxic KCl solutions, both tension and phi returned rapidly towards pre-hypoxicïpre-metabolically inhibited levels (see Fig. 6). In a further set of experiments (n = 5), DNP and TMA were added concomitantly to depolarized vessels. These additions had variable effects on phé. In two of five experiments, initially no effect was seen on phé although it later fell (after approximately 30 s) by between 0 20 and 0 25 ph units. In two of five experiments an initial alkalinization of ph units (taking about 15 s to peak) was observed, which was then followed by an acidification of 0 10 ph units. In a further experiment a maintained alkalinization of ph units was observed. In all these experiments, however, tension fell rapidly (t½ =26±3s)tonearbaseline levels (97 4 ± 0 2 % of KCl contraction). Thus, despite the variable effects on phé which were observed, the contractile response observed was always a fall in tension. This decrease in tone was similar in both speed and magnitude to that previously observed to DNP in the absence of TMA. Effects of Rp-cAMPS To determine whether an elevation of camp is responsible for the observed vasodilatation to hypoxia, the effect of Rp_cAMPS on responses was examined. Rp-cAMPS is a biologically inactive, membrane permeable isomer of camp which competes for the camp binding site on protein kinase A. In these experiments, severe hypoxia relaxed depolarized vessels by 95 1 ± 2 5 %. Incubation of tissues with Rp_cAMPS had no effect on either the resting tension of vessels or the contractions to 60 mò KCl (in the continued presence of Rp-cAMPS), contractions being 1 4 ± 0 4 mn mm in the absence and 1 0 ± 0 3 mn mm in the presence of the inhibitor. Relaxations to severe hypoxia were also unaffected by the presence of Rp-cAMPS, being 94 0± 4 2 % (n = 4). It was found, however, that in the presence of Rp-cAMPS the vasodilator responses of depolarized vessels to 0 1 ìò isoprenaline were attenuated (> 75 %), thus indicating that, in our experimental system, the camp system is being inhibited. DISCUSSION The results of the present study show that severe hypoxia causes a relaxation of isolated rat mesenteric resistance vessels, precontracted by either depolarization with KCl or by addition of U46619, to near baseline levels. A vasodilatation in response to hypoxia may be regarded as a protective or compensatory mechanism in systemic vessels, increasing blood flow and Oµ supply to the region, and therefore relieving the hypoxia. Hypoxia may modify the contractile behaviour of isolated blood vessels either by a direct effect on vascular smooth muscle andïor via an effect on the endothelium (see Introduction). As indicated previously, to date only one other study has investigated the effect of the endothelium on the hypoxic modulation of tone in isolated mesenteric arteries. In this study, it was found that the depressed responses to AVP observed during hypoxia were not altered by mechanical endothelium removal (Aalkjaer & Lombard, 1995). Although this study used similar sized vessels, which were mounted in the same way (i.e. as ring preparations on wire myograph) as those of the present study, an entirely different experimental protocol was adopted. Thus, although Aalkjaer & Lombard (1995) clearly showed that the mechanisms by which established hypoxia attenuates contraction to AVP are endothelium independent, it could not be automatically assumed that the mechanisms responsible for the vasodilator responses induced by hypoxia in precontracted vessels, as observed in the present study, are also independent of endothelial factors. This was therefore investigated. It was found that the magnitude of the vasodilatation induced by severe hypoxia was unaffected by the addition of _NA and indomethacin, demonstrating thatitwasnotduetoaninvolvementoftheendothelial factors nitric oxide (NO) andïor prostaglandins. It is now recognized that in certain tissues, including small mesenteric vessels, another, as yet unidentified factor, endotheliumderived hyperpolarizing factor (EDHF), may also contribute to vasodilator responses (Bolton et al. 1984; Kamata et al. 1996). Although we cannot completely rule out the possibility that EDHF may contribute to hypoxic vasodilatation we think this is unlikely. This is because the responses in depolarized vessels, where hyperpolarization could not occur, were not smaller than those in vessels constricted with U46619 (which produces a much smaller depolarization than KCl; Plane & Garland, 1996). In addition, we have previously shown (albeit on tissues gassed under resting conditions with hyperoxic solutions, i.e. 100 or 95 % Oµ) that mechanical removal of the endothelium had no effect on the magnitude or speed of the relaxation responses induced by severe hypoxia or by the metabolic inhibitor cyanide (Otter & Austin, 1998). The results of the present study therefore show that hypoxia dilates isolated precontracted small rat mesenteric arteries via a mechanism(s) which is independent of the endothelium and is therefore presumably due to a direct action on the vascular smooth muscle. Hypoxia may be expected to have two major effects on smooth muscle metabolism. It might (1) reduce [ATP] by inhibiting aerobic glycolysis and oxidative phosphorylation and (2) increase the production of lactic acid due to stimulation of anaerobic glycolysis. Although moderate hypoxia has not been associated with any measurable fall in [ATP] (Lovgren & Hellstrand, 1985), severe hypoxia, as used in the present study, and metabolic inhibition have been shown to moderately reduce [ATP] (Hellstrand et al. 1977; Wray, 1990). However, estimates from NMR studies of bulk

8 256 D. Otter and C. Austin J. Physiol MgATP in smooth muscle during metabolic inhibition show that although [ATP] falls, it remains at a concentration greater than 1 mò (Wray, 1990). As levels of myosin light chain (MLC) phosphorylation appear unaffected by metabolic inhibition (Taggart et al. 1997) and the Km of ATP for MLC kinase is < 100 ìò (Adelstein & Klee, 1981), it appears that even if a decrease in [ATP] does occur in severe hypoxia it is unlikely that it alters force production at the level of the myofilaments (for further discussion, see Taggart et al. 1997). It has previously been shown that addition of the metabolic inhibitor sodium cyanide decreases phé in a number of smooth muscles including rat mesentery (Smith et al. 1996), rabbit vascular (Spurway & Wray, 1987) and rat uterus (Wray, 1990). This acidification has been attributed to a build-up of lactic acid due to stimulation of anaerobic glycolysis (Wray, 1990). As it is well known that phé per se may alter vascular tone it has been postulated that the relaxation of these tissues in response to metabolic inhibition may be due to this intracellular acidification. This, however, has been poorly studied. The effects of metabolic inhibitors are often equated with those of hypoxia and indeed, due to the practical difficulty of reducing POµ to low and consistent levels, they are often used to study the effects of hypoxia on various parameters. Preliminary studies by one of us (Smith et al. 1996) have shown that metabolic inhibition with cyanide reduces both phé and tone in depolarized rat mesenteric arteries, although the relationship between the parameters was not investigated. In contrast, Aalkjaer & Lombard (1995) could demonstrate no significant effect of established hypoxia on phé in vessels from rat cerebral and mesenteric vascular beds although contractility was depressed (Aalkjaer & Lombard, 1995). As no studies have previously investigated the effects of hypoxia and metabolic inhibition on the same preparation it was unclear whether this disparity reflected differences between hypoxia and metabolic inhibition or whether they could be attributed to experimental or tissue differences. In the present study, to allow a more direct comparison between metabolic inhibition and hypoxia, we used severe hypoxia (i.e. POµ < 5 mmhg) throughout. We cannot exclude the possibility that more moderate levels of hypoxia, which would more usually be experienced physiologically, may have different mechanisms of action. We found that both severe hypoxia and addition of the metabolic inhibitors sodium azide or DNP resulted in a significant decrease in phé in KCl-contracted mesenteric arteries. In all cases phé was reduced to a similar extent and tension fell to near baseline levels. These results clearly contrast with the findings of Aalkjaer & Lombard (1995) although, as indicated above, their study investigated the effects of established hypoxia on the changes in contractility and phé evoked by addition of AVP. Nevertheless, it is interesting that Aalkjaer & Lombard (1995) demonstrated that when acid extrusion mechanisms were inhibited, the fall in phé evoked by AVP was enhanced during hypoxia. This suggests that although hypoxia enhances intracellular acid production, acid extrusion mechanisms normally act to maintain phé near control levels. Thus, differences in the activation of these mechanisms during the different experimental protocols may partly explain the differences in the effect of hypoxia on phi in the two studies. At physiological concentrations lactate, or lactic acid, leaves cells mainly via a specific H monocarboxylate cotransporter which is inhibited by agents such as CHC (Poole & Halestrap, 1993). In the present study, the magnitude and speed of the phé change with hypoxia was increased in the presence of CHC, suggesting that it was due to an accumulation of lactic acid. The concentration of CHC used in the present study was similar to those previously used in uterine smooth muscle to block lactate efflux (Wray, 1990) and was chosen to ensure that all monocarboxylate carrier isoforms were inhibited (Wang et al. 1996) although at these concentrations some inhibitory effects on mitochondrial enzymes may occur (Halestrap & Denton, 1975). The transporter has, however, not been characterized in smooth muscle and although a small contribution of other carrier substrates to the change in phé cannot be discounted the major effect would be expected to be due to lactate accumulation (see Poole & Halestrap, 1993). The net effect of any manoeuvre on phé reflects a balance between the rate of lactate production and the rate of lactate efflux, both of which may vary between tissues and preparations. It has previously been suggested that smooth muscle cells produce lactic acid even in conditions where oxygen is not limited (Paul, 1980). This idea was supported in the present study, which found that CHC reduced phé even in normoxic conditions. The addition of CHC also caused a relaxation of precontracted tissues under normoxic conditions. Thus we have demonstrated that both severe hypoxia and metabolic inhibition reduce phé and tone in our isolated vessels and that this acidification is probably due to an accumulation of lactic acid. If these changes in phé were responsible for the changes in tension then it would be expected that phé, in response to either hypoxia or metabolic inhibition, would change before tension. However, when the times taken for the changes in phé and tension to reach maximal and half-maximal values were examined, it was found that both phé and tension followed very similar time courses. Using similar equipment and sampling rates (1 2 Hz) we have previously been able to demonstrate temporal differences between changes in phé and tension in response to alterations of both intracellular and extracellular ph (phï) and therefore we do not believe that our equipment has prevented us from detecting any differences. In addition it should be noted that the responses observed to hypoxia are significantly slower than those previously observed to changes in phé and phï, produced by addition of weakïstrong acids and bases, and therefore any differences would be expected to be more easily detectable (Austin &

9 J. Physiol Hypoxia and metabolic inhibition 257 Wray, 1994; Austin et al. 1996). Thus, although it may be argued that the similarities of their time courses is consistent with the changes in phé and tension being causally related, the results discussed so far certainly do not show that hypoxia or metabolic inhibition alters phé, which in turn alters tension. If the decrease in phé that we observed with hypoxiaïmetabolic inhibition were, in fact, responsible for the vasodilatation observed then it may be expected that a selective reduction of phé produced by other means, e.g. additionïwashout of weak acids and bases, would also reduce tone. However, the responses of isolated vessels to changes in phé appear varied. While we have shown that a decrease in phé due to addition of the weak acid butyric acid causes relaxation of isolated strips of rat mesenteric vessels (Austin & Wray, 1994), others have shown that similar vessels mounted as ring preparations on a wire myograph show a biphasic and more complex response when phé is decreased by washout of NHÚCl (Aalkjaer & Poston, 1996). Thus, in the present study, for direct comparison we investigated the effects of an intracellular acidification induced by washout of NHÚCl with an intracellular acidification induced by hypoxia and metabolic inhibition. Washout of NHÚCl was used in preference to addition of butyrate as recent reports have suggested that butyrate may in fact relax vessels by ph-independent mechanisms (Aaronson et al. 1996). In the present study, washout of NHÚCl resulted in a contraction of depolarized arteries. In the same vessels, however, hypoxia or metabolic inhibition resulted in a large vasodilatation despite the fact that the magnitude of the acidification was similar in both cases. The way in which ph alters tone is poorly understood, although it is known that protons may interact at a number of different points in the excitation contraction pathway. These interactions may result in different effects on contractility (for review see Smith et al. 1998). Therefore it is possible that the protons produced during hypoxia or metabolic inhibition may interact with a different part of the contractile pathway than those produced by washout of NHÚCl. They may modulate contractility differently if, for example, the modulatory step affected was dependent on the speed of the phé change. However, it could also mean that the relaxations due to hypoxiaïmetabolic inhibition were purely causally associated with the change in phé, i.e. the acidification was itself not responsible for the change in tension. To investigate this further we carried out a series of neutralization experiments. We found that by adding the weak base TMA we were able to more than neutralize the acidification due to both hypoxia and metabolic inhibition, but still tension remained unchanged and at hypoxicïmetabolic inhibition levels. This therefore again supports the hypothesis that although an intracellular acidification may be produced by hypoxia or metabolic inhibition, it does not contribute to the changes in vascular tone observed in mesenteric artery. Although we have previously used neutralization-type experiments to reverse the effects of increases of phé on an increase in vascular tone (Austin & Wray, 1993a) it might be argued thatinthepresentstudy,whereseverelevelsofhypoxiaor metabolic inhibition are used, it may not have been possible to reverse the already established decreases in vascular tone. When neutralized preparations (i.e. those in the presence of high [K ], hypoxia or metabolic inhibitors and TMA), were washed with normoxic high-k solution, however, tension increased rapidly towards pre-hypoxicïpre-inhibited levels, suggesting that the tension change was readily reversible. To further investigate the role of phé in the observed vasodilatory responses, a small set of experiments were performed in which TMA (40 mò) and the metabolic inhibitor DNP (1 mò) were added simultaneously to depolarized preparations. This was done in an attempt to prevent any phé changes, resulting from metabolic inhibition, from occurring. In these experiments DNP was used in preference to hypoxia as the time course of the change in phé due to its addition more closely matched that previously described for TMA (Austin & Wray, 1993a; Austin et al. 1994). The effects these additions had on phé were variable; in the majority of cases no initial change in phé andïor a reduced intracellular acidification compared with that due to addition of DNP alone was observed, yet in another experiment a maintained increase in phé was observed. The reasons for these differing results are unclear but may represent differences between intracellular buffering andïor rate of lactate efflux in different experiments. However, irrespective of the effects on phé, tension always fell to levels similar to those observed when DNP was added to vessels in the absence of TMA. Therefore these experiments reinforce the idea that the decrease in vascular contractility observed during hypoxia or metabolic inhibition is independent of any changes in phé. Similar results to these have also been found in uterine smooth muscle (Taggart & Wray, 1995). Thus the results of the present study show that severe hypoxia relaxes isolated rat mesenteric arteries by a mechanism(s) that is independent of the endothelial factors NO and prostaglandins. Although phé decreases during hypoxia or metabolic inhibition, this change does not appear to be responsible for the changes in tension observed. Therefore the mechanisms involved are still unclear and require further investigation. Previous studies on coronary arteries have suggested that hypoxic vasodilatation may be a result of a hyperpolarization due to activation of KATP channels as a result of a decrease in [ATP] (Daut et al. 1990). Although [ATP] may fall in severe hypoxia, activation of KATP channels is unlikely to be involved in the vasodilatation induced by hypoxia in the present study as relaxations to hypoxia in depolarized tissues were similar to those precontracted with U On the other hand, the present study showed that addition of CHC resulted in an acidificationandarelaxationoftissuesinbothhypoxicand normoxic tissues, suggesting that intracellularly produced

10 258 D. Otter and C. Austin J. Physiol lactate can relax mesenteric vessels. If the mechanism of hypoxic relaxation is independent of phé (see above), it is reasonable to suggest that lactate may dilate vessels by a mechanism that is independent of phé. One possible mechanism that we investigated is an effect of lactate on the camp system. It has previously been shown that vasodilatation of noradrenaline-contracted isolated mesenteric arteries (mounted as ring preparations on a wire myograph) to exogenously applied _lactate was significantly reduced in the presence of Rp-cAMPS, suggesting an involvement of camp in the reduction in tone (McKinnon et al. 1996). Rp_cAMPS is an inactive stereoisomer of camp which competes for the camp binding site on protein kinase A and may therefore, if present in high enough concentration, prevent its activation (van Haastert et al. 1984). In our experiments, 25 ìò Rp-cAMPS inhibited the vasodilatation observed to the â-adrenergic agonist isoprenaline, which relaxes smooth muscle via activation of camp-dependent protein kinase (Silver et al. 1982), thus confirming that Rp_cAMPS was inhibiting the camp system. In similar concentrations Rp-cAMPS has also been shown to reduce the effects of forskolin, which increases camp by directly stimulating adenylate cyclase (McKinnon et al. 1996). In the present experiment, however, Rp-cAMPs had no effect on the vasodilatation of tissues in response to hypoxia, suggesting that the decrease in contractility was not due to an elevation of camp. Thus, we can conclude that in depolarized rat mesenteric arteries, hypoxia has a vasodilator effect which is not due to effects on the camp system. Other ph-independent effects of lactate have previously been reported, including effects on Ca affinity (Hester et al. 1980) and on Ca -activated ion channels (Mori et al. 1998) and may have contributed to the results seen in the present experiment. In conclusion, we have shown that severe hypoxia relaxes isolated rat mesenteric resistance vessels by an endotheliumindependent mechanism. We have shown that both severe hypoxia and metabolic inhibition cause an intracellular acidification, which appears to be associated with an accumulation of lactic acid. Although it is well established that alterations in phé may alter vascular tone, it appears that the vasodilator responses of depolarized mesenteric arteries in response to hypoxia or metabolic inhibition are independent of phé. The mechanisms responsible therefore remain to be elucidated. Aalkjaer, C. & Lombard, J. H. (1995). 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