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Journal of Physiology (1990), 423, pp. 411-423 With 6figures Printed in Great Britain

THE EFFECTS OF METABOLIC INHIBITION ON UTERINE METABOLISM AND INTRACELLULAR pH IN THE RAT

BY SUSAN WRAY

From the Department of Physiology, University College London, Gower Street, London WC1E 6BT

(Received 24 August 1989) SUMMARY

1. Uterine metabolism was studied in pregnant and non-pregnant rats by measuring high energy phosphates and intracellular pH (with 31-phosphorus nuclear magnetic resonance (31P NMR) spectroscopy) and lactic acid efflux. Isolated, superfused uteri were investigated during control conditions (30TC) and in the presence of metabolic inhibitors and high [K+]. 2. In control conditions the ratio of phosphocreatine and ATP concentrations ([PCr]/[ATP]) was higher in the pregnant (0-88 + 0 09) than the non-pregnant uterus (0'52 + 0-04). When oxidative phosphorylation was inhibited by cyanide, there was a significant (P < 001) decrease in [PCr] and [ATP] and an increase in the concentration of inorganic phosphate ([Pi]). These changes were greater in the nonpregnant than the pregnant uterus. 3. There was no difference in the mean value of resting pHi found in pregnant and non-pregnant uterus (7-19+0-04 and 7-17+0-03, external pH 7 4, n = 10 and 12 respectively). There was a significant intracellular acidification in both pregnant (0-31 + 0-04 pH unit) and non-pregnant (0-27 + 0-02 pH unit) uterine tissue in the presence of cyanide. These effects of cyanide on metabolites and pHi were fully reversed upon return to control solutions. 4. When both aerobic and anaerobic glycolysis were blocked by iodoacetate, there was a rapid disappearance of high energy phosphates from the 31P NMR spectrum and a large increase in the phosphomonoester spectral region, where sugar phosphate intermediates of glycolysis resonate. These changes were seen in both pregnant and non-pregnant uteri and were irreversible. 5. Lactate production was detected, in the presence of oxygen, in both pregnant and non-pregnant preparations (0 43 + 0 07 and 0-25 + 0 09 ,imol g-1 min-, respectively). In both preparations the rate of lactate production was markedly increased in the presence of cyanide. The increase was much more marked in non-pregnant (- 10-fold) than pregnant (- 5-fold) uteri, resulting in a very similar rate of lactate efflux in cyanide. 6. When lactate efflux in non-pregnant uteri was blocked by a-cyano-4hydroxycinnamate, there was a significant acidification (0-21 + 0-04 pH unit, n = 6). The addition of cyanide produced a more pronounced acidification (0-34 + 0-04 pH unit) than that seen with either cyanide or a-cyano-4-hydroxycinnamate alone. MS 7906

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7. When the non-pregnant uterus was perfused with a solution containing 70 mMK+, there was a significant fall of [PCr] to around half its initial concentration, while [ATP] fell just 10%. These changes were less marked than those found in the presence of cyanide. The metabolite changes were accompanied by a large acidification, 0-38 + 0-06 (n = 5). The changes were reversed upon return to control perfusate. 8. The possible functional significance of these results to pregnancy and parturition is briefly discussed. INTRODUCTION

During pregnancy there are many changes in the uterus. Some of these changes are related to metabolism; for example, there are increases in glycogen content, oxygen extraction and glycolytic enzymes (Wynn, 1977). There is also an increase in myometrial ATP and phosphocreatine (PCr) concentrations (Dawson & Wray, 1985). It may be postulated that these changes occur to ensure adequate contractile activity at labour. However, it has also been reported that uterine contractions during labour are so powerful that the blood supply to the active tissue is occluded (Greiss, 1965). This leads to a reduction in oxygen and glucose supply to the tissue. Since ATP production is largely via oxidative phosphorylation and ATP is directly required for muscle contraction, this reduction of oxygen may impair contractile ability. It is not known what happens to uterine contractions under conditions of reduced oxygen availability. There have been reports that lactate dehydrogenase (LDH) increases both in amount and activity in the rat with pregnancy. Furthermore, there are changes in the isoenzyme profile of LDH to that favouring anaerobic conditions (Battellino, Sabulsky & Blanco, 1971). Thus it may be that anaerobic glycolysis can maintain [ATP] and hence uterine contractions even when the oxygen supply is reduced. On the other hand, the lactic acid produced under anaerobic conditions may lead to an intracellular acidification which per se could diminish contractile performance, as has been demonstrated for example in cardiac muscle (Fabiato & Fabiato, 1978; but see Wray, 1988 a). Thus it is not clear what happens to metabolites and force during anaerobic glycolysis. It has been demonstrated that vascular smooth muscles utilize anaerobic glycolysis for ATP production even when oxygen is not limited (Paul, 1980). This is in marked contrast to cardiac muscle where anaerobic glycolysis does not occur under normal conditions. Thus the uterus may be producing lactate even in the presence of oxygen. The purpose of the experiments described in this paper therefore was to investigate the following points. (i) Can normal metabolite levels be maintained by anaerobic glycolysis alone? (ii) Does uterine pHi change when oxidative phosphorylation is inhibited? (iii) What is the effect on uterine metabolism of inhibiting both aerobic and anaerobic glycolysis? (iv) Does an increase in contractile activity as produced by high-K+ solution metabolically stress the uterus? (v) Is lactate produced by the uterus in the presence of an adequate oxygen supply and how much extra lactate is produced during inhibition of oxidative phosphorylation? (vi) Are there any differences in the above results between pregnant and non-pregnant uterus? The measurements of metabolites and pHi were made using 31-phosphorus nuclear

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magnetic resonance (31P NMR) spectroscopy as this permits simultaneous and sequential measurements of ATP, phosphocreatine (PCr), inorganic phosphate (Pi) and pHi in intact tissues. A preliminary account of some of these results has been presented to the Physiological Society (Wray, 1988b). METHODS

Animals. Sprague-Dawley rats were used, either virgin (200-250 g) or day 20-21 pregnant (parturition occurs on day 22). They were fed on SDS-3 pellets (Witham, Essex) and tap water ad libitum. Tissue preparation and maintenance. Under chloroform anaesthesia the uterus was removed from the animal, dissected free of any fat, placental sites or fetuses and placed in a 15 mm NMR tube. The tissue was superfused (rate 4 ml min-') with oxygenated solution while inside the NMR spectrometer (composition in mM: Na', 155; Cl-, 161; K+, 6; glucose, 7-5; HEPES, 10; Ca2", 0 3; pH 7 4.) In some experiments the [K'J was raised to 70 mm by isosmotic substitution for Na'. The temperature was maintained at 30 'C. Previous work had demonstrated that the tissue was stable under these conditions for many hours (Dawson & Wray, 1985). In addition, when faster flow rates were used (12 ml min-') no differences in spectra were found. 31P NMR spectroscopy. A vertical spectrometer (Bruker WM200) operating at 81 MHz for phosphorus was used. Spectra were obtained using a pulse duration of 30 ,us with a 2 s interval between pulses. The number of pulses varied from 300 to 600 and hence spectra were obtained every 10-20 min. Peak assignments were based on resonance positions as previously described (Dawson & Wray, 1985). Adenosine triphosphate is the major nucleotide in the uterus (> 75%), but the peaks from ATP contain contributions from the other nucleotides (e.g. CTP) which coresonate at the field strength used. As adenosine is by far the major component of this peak it will be referred to as 'ATP' throughout. Metabolite concentrations were obtained from peak areas by cutting and weighing (see Spurway & Wray, 1987 for details) and correcting for the saturation effects of pulsing more rapidly than the time taken for all the nuclei to return to their ground state (see Dawson & Wray, 1985, for full details). Intracellular pH was calculated from the resonance position of inorganic phosphate (Pi) using the following rearrangement of the Henderson-Hasselbalch equation:

pH = pK+log 85X, where a is the observed chemical shift difference between Pi and PCr and 81 and 82 are the chemical shifts of H2PO4- and HPO42- respectively. A pK value of 6-65 and chemical shift values 8 = 3-14 and 82 = 5-61 were used (Dawson & Wray, 1985). Metabolic inhibition. Oxidative phosphorylation was inhibited by adding 2 mM-NaCN to the superfusate and adjusting pH. Glycolysis was inhibited by the addition of 1-0 mM-iodoacetate. In either case NMR spectra were collected after 2-5 min perfusion with the new medium. Spectra were obtained at 10 min intervals for up to an hour. Upon return to control solution the same protocol was followed. Lactate production was measured enzymatically on the effluent at 340 nm, using an NADH-LDH linked assay. Lactate efflux was blocked using 2 mM-ax-cyano-4-hydroxycinnamate in the perfusate. Statistics. Figures given represent means + standard errors of the mean (S.E.M.). Significant differences were tested for by unpaired t tests. RESULTS

The effect of inhibiting oxidative phosphorylation When oxidative phosphorylation was blocked by the application of 2 mM-cyanide to the perfusate, normal concentrations of phosphorus metabolites were not maintained, in neither the pregnant nor the non-pregnant uterus. This is illustrated

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in Fig. 1, which shows 31P NMR spectra before, during and after the application of cyanide to pregnant uterine tissue (figure is representative of ten experiments). There was a marked decrease in [PCr] and an increase in [Pi] in going from control solution (top spectrum, Fig. 1) to one containing cyanide (middle spectrum, Fig. 1). The mean fall in PCr from control values was 54+11 % (n = 10) and Pi increased to 3-16 + 0-26 times its original value. There was a small but significant fall in the [ATP] of 20 + 6%. These effects of cyanide were fully reversible upon return to control solution, as can be seen in the spectra of Fig. 1. Control PME

p,

P r

ATP

Cyanide

Recovery

10

0

p.p.m.

-10

-20

Fig. 1. 31P NMR spectra from pregnant uteri. The spectra were obtained during perfusion with control bathing solution (top), 2 mM-cyanide added (middle) and return to control bathing solution (bottom). The dashed line shows the initial resonance position of inorganic phosphate (Pi). The shift to the right seen in the presence of cyanide indicates an intracellular acidification. In this and the following figures PCr = phosphocreatine; PME = phosphomonoesters, largely phosphoethanolamine; ATP = nucleotide triphosphates, overwhelmingly ATP; p.p.m. = parts per million.

Figure 2 shows typical results (representative of twelve experiments) obtained from non-pregnant uteri. It can be seen that the changes are more pronounced than those seen in the pregnant uterus; PCr almost disappears and ATP shows a more marked fall. As a percentage of control values ATP fell to 41 + 6 % and PCr to just 12 + 4%. Inorganic phosphate rose to 5-3 + 0-7 times its original value (n = 12). Despite such marked changes reperfusion with control solution led to full recovery of metabolites (bottom spectrum, Fig. 2). Using previously published data the above changes can be quantified in terms of metabolize concentrations. Dawson & Wray (1985) reported that the sums of uterine [PCr] + [NTP] + [Pi] in non-pregnant and pregnant rats were 6-9 and 8-9 mM respectively at 4 'C. The present work was performed at 30 'C; thus the relative amounts of PCr :NTP: Pi may be different. However, it is unlikely that their sum will

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change. If this assumption is made, then the peak areas can be quantified. When this was done the result were as shown in Fig. 3. The ratio of [PCr]/[NTP] in the pregnant uterus was higher than in the non-pregnant: 0O88 + 009 and 0-52 + 004 respectively, in agreement with previous reports (Dawson & Wray, 1985). Figure 3 shows that there was no significant effect on the phosphomonoester (PME) peak of inhibitory oxidative phosphorylation. Control PME Pi

ATP

PCr

~~~~Cyanide

^ 2|

Recovery

.1

10

I

0

I

p~pm.

I

-10

-20

Fig. 2. 31p NMR spectra from non-pregnant uteri; before, during and after cyanide application. Metabolite peaks as for Fig. 1.

The effect of cyanide on intracellular pH There was a significant intracellular acidification in both pregnant and nonpregnant uterine tissue in the presence of cyanide. These changes are shown in Table 1. It can be seen that resting pHi was similar in pregnant and non-pregnant uteri. The acidification following application of cyanic was very slightly larger in pregnant tissue, but this difference was not significant at the 5 % level. There was complete recovery of pHi upon return to control bathing solution.

Lactate production The perfusate was assayed for lactate produced by the uterus. Under control conditions, i.e. in the presence of oxygen, lactate was produced by both the pregnant and non-pregnant uterus. There was significantly (P < 001) more lactate produced by pregnant than non-pregnant uteri (043 + 007 jmol g'l min-, n = 11, and 025 + 0O09,mol g-1 min-', n = 6, respectively). Figure 4 shows the very marked increase in lactate production which occurred when cyanide was added to the solution. These increases from control values were significant for both the pregnant

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416

and non-pregnant uterus (P < 001), but the rate of lactate production in the presence of cyanide was not significantly different between the two. In other words, the increase in lactate production which occurred in the presence of cyanide was about twice as great in the non-pregnant than in the pregnant uterus. Upon return 10 Pregnant

Non-pregnant 8-

E C

0 C C

0

P

60) 4 TT

UT 2

-T

0

ATP

PCr

Pi

PME

ATP

PCr

Pi

PME

Fig. 3. The mean concentrations of ATP, PCr, PME and Pi in the non-pregnant and pregnant uterus. El, control; *, effect of cyanide on the metabolite concentrations. For details of quantification see text.

TABLE 1. Intracellular pH changes Non-pregnant Pregnant uterus uterus (n) 12 7-19+0-04 7-17+003 12 6-88+004 6-90+0-02 6 6-96+0-04

Condition (n) 10 Control 10 Cyanide Lactate efflux blocked 4 Cyanide and 6-83+0 04 lactate efflux blocked The mean values of uterine intracellular pH (+S.E.M.) during perfusion with control bathing solution, 2 mM-cyanide. 2 mM-a-cyano-4-hydroxycinnamate (CHC, a lactate blocker) or CHC and cyanide. n = number of observations.

to control solution lactate production rapidly declined to values only slightly higher than those found initially.

The effect of inhibitory lactate efflux on intracellular pH The drug a-cyano-4-hydroxycinnamate (CHC) blocks lactate efflux from cells (Halestrap & Denton, 1974). As lactate production was found to occur in the oxygenated uterus (see above) the effect of this lactate transport blocker was examined in the uterus. (As CHC is a coloured substance lactate efflux was not

UTERINE METABOLISM 417 assayed in these experiments.) In six non-pregnant preparations CHC was applied and caused an intracellular acidification each time (mean 0 21 + 004 pH unit). Upon return to control solution the pHi returned to initial values. As cyanide had been found to markedly increase lactate production by the uterus (see above), the effect upon pHi of cyanide in the presence of CHC was examined. The resulting intracellular acidification was more marked than with CHC or cyanide alone (see Table 1). Pregnant

Non-pregnant

2.57

2E2 0 0

IX

FE

1.5 1.0

0.5-

C

CN

R

R C CN Fig. 4. Lactate efflux (4umol g-' min') from the uterus measured before, during and after cyanide application. The values are means of eleven pregnant and six non-pregnant uteri, with the lines indicating S.E.M. Lactate was measured at 340 nm via NADH. C, control;

CN, cyanide; R, recovery.

The effect on metabolites of blocking glycolysis Glycolysis was prevented by the application of iodoacetic acid. The effects of iodoacetate were the same in the non-pregnant as the pregnant uterus and will therefore be discussed together. The application of 1 mM-iodoacetate led to a rapid disappearance of PCr and ATP. There was a very large increase in the peaks at around 6-7 p.p.m. This is where sugar phosphate intermediates of glycolysis resonate. There was a small increase in Pi in some preparations, but usually Pi remained unchanged in intensity. These changes are illustrated in Fig. 5A, which shows a typical set of spectra (representative of ten experiments), obtained before and after iodoacetate application to the uterus. Intracellular pH showed an acidic change in four preparations and in six others was either unchanged or no clear effect was seen. All the effects of iodoacetate were irreversible. In three preparations iodoacetate and cyanide were applied simultaneously to the uterus. This resulted in large Pi and PME peaks appearing in the spectra as all other metabolites disappeared. Intracellular pH became acidic. This is shown in Fig. 5B. The effect of high K+ When the uterus was placed in 70 mM-K' it produced a sustained contracture rather than periodic spontaneous contractions (S. Wray, unpublished observations). To investigate the effects of this increase in mechanical activity on the uterus, 14

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418

S. WRAY A Pregnant uteri

Control

ATP

lodoacetate

III

0

10 B

p.p.m.

-20

-10

Non-pregnant uteri

Control

Cyanide and iodoacetate

L 10

I

I

0

-10

p.p.m.

-20

Fig. 5. A, 31P NMR spectra from pregnant uteri, before (top) and after (bottom) the addition of 1 mM-iodoacetate to the bathing solution. Phosphate intermediates of glycolysis resonate in the PME region of the spectrum. B. 31P NMR spectra obtained from non-pregnant uteri under control conditions (top) then following the addition of iodoacetate and 2 mM-cyanide (bottom) to the bathing medium.

metabolites were measured in the presence of 70 mM-K+. (To ensure that the contracture was maintained, the [Ca2+] in the perfusate was elevated to 3 mm throughout these experiments. This increase in [Ca2+] had no effect upon resting metabolite concentrations or pHi.) Figure 6 shows spectra (representative of six experiments) obtained during control

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(top) and high-K+ conditions (bottom). A marked fall in PCr can be seen. There is also a smaller decrease in ATP and a small rise in Pi. In six experiments on nonpregnant uteri, the mean falls from control levels in [ATP] and [PCr] were 12 and 58% respectively, while [Pi] increased 1-41-fold. The changes in metabolites during the contracture were also accompanied by a large fall in intracellular pH, from a mean resting value of 7 18 + 0-07 to 6-78 + 007 (n = 5) and a mean acidification of 0-38 + 0 07 pH units. Control PCr

ATP

70 mM-K+

10

-10

0

-20

p.p.m.

Fig. 6. The 31P NMR spectra obtained under control conditions (top) and then following perfusion with bathing medium containing 70 mM-K+ (isosmotically substituted for Na+). Perfusion with this solution causes maintained contracture of the uterus.

DISCUSSION

Marked changes in the concentration of uterine metabolites and intracellular pH occurred when the normal metabolism or mechanical activity of the uterus were altered. Since intact tissues rather than tissue extracts can be studied by 31P NMR, artificial changes in labile metabolites (such as PCr) are avoided. Furthermore, a series of measurements can be followed in a single preparation. In the uterus the overwhelming (> 90 %) cellular constituent is the myometrial cell (Wynn, 1977) and so the results reported here can be taken as arising from uterine smooth muscle. It was found, in agreement with earlier findings (Dawson & Wray, 1985), that the pregnant uterus contains more PCr and ATP than the non-pregnant uterus. A recent NMR study of human myometria also reported higher PCr values in pregnant compared to non-pregnant tissue (Noyzewski, Raman, Trupin, McFarlin & Dawson, 1989). The effects of cyanide on metabolites Blocking aerobic glycolysis led to significant decreases in [ATP] and [PCr] and an increase in [Pi] in the pregnant uterus. Thus, despite the increased concentration of 14-2

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lactate dehydrogenase and the changes in its isoenzyme profile to favour anaerobic conditions (Battellino et al. 1971), anaerobic glycolysis cannot maintain normal concentrations of ATP and PCr. In the non-pregnant uterus the changes in these metabolites with application of cyanide were even more marked (see Fig. 2). This difference can probably be explained by the lower initial concentrations of ATP and PCr. The lower LDH levels compared to the pregnant uterus may also have been a reason (but see below). Thus, in the non-pregnant uterus [PCr] falls to very low levels (< 1 mM), largely removing its buffering action on [ATP]. In the pregnant uterus the fall in PCr is less ([PCr] > 1 mM) and consequently the fall in [ATP] is much less (20 cf. 40 %). Thus, on the basis of the [ATP] changes, the functions of the pregnant uterus should be less affected by cyanide than the non-pregnant uterus (see below).

The effect of cyanide on uterine pH Cyanide application led to an increased uterine lactate production reflecting an increased acid load on the uterus. In addition, the ATP break-down which occurred in the presence of cyanide would be associated with H' production (e.g. Wilkie, 1979). However, cyanide application also led to a decrease in [PCr] and when PCr is hydrolysed protons are absorbed. The fact that cyanide produced a significant acidification of the uterus indicates that an increased acid load was the predominant process. The uterus has been shown to regulate pHi in the face of changes in external pH (Wray, 1988c). However, further analysis of the pH change is limited by the lack of measurements of buffering power in myometrial cells, or indeed in smooth muscle in general (see Aickin, 1984). The effects of iodoacetate Iodoacetate inhibits glyceraldehyde 3-phosphate dehydrogenase and thus glycolytic ATP production. In the uterus the effect of iodoacetate was to rapidly deplete high energy phosphates. Inorganic phosphate did not markedly increase in contrast to the result obtained in the presence of cyanide (cf. Figs 1 and 5). This is presumably because phosphate from ATP break-down is taken up by sugars which then accumulate proximal to the iodoacetate block (glucose was present throughout). There was a marked increase in the peak in the PME spectral region when iodoacetate was applied. In control conditions the major contributor to this peak in smooth muscle is phosphoethanolamine (Kushmerick, Dillon, Meyer, Brown, Krisanda & Sweeny, 1986). It is unclear why this substance should be present in such concentrations (1-3 mM) in smooth muscles. It is also present in brain spectra (e.g. Tofts & Wray, 1985), but not in striated muscle spectra. The increase in the PME peak with iodoacetate is probably due to phosphate intermediates of glycolysis (e.g. fructose 6-phosphate) accumulating proximal to the iodoacetate block. Lactate production by the uterus Lactate efflux from the uterus rather than uterine lactate content was measured in this study to allow sequential measurements to be made. It is expected that in the steady state the lactate efflux will equal intracellular lactate production. The finding of lactate production by the uterus in the presence of oxygen is in contrast to studies of striated muscle but in agreement with several reports on vascular smooth muscle

421 UTERINE METABOLISM (see Paul, 1980) and earlier reports on the pregnant uterus (Kroeger, 1976; Rubanyi, Toth & Kovach, 1982). The results reported here demonstrated lactate production by both non-pregnant and pregnant uteri and found differences between the two. Lactate production would be expected under anaerobic conditions and so the question arises as to whether the uterus was adequately oxygenated. In an attempt to answer this the critical tissue thickness (t) above which the [02] becomes zero can be calculated from the equation:

t = 2 (VI2)(vCDs/A).

Oxygen consumption (A) in the pregnant rat uterus has been measured to be 17 /tmol g-1 h-1 (Kroeger, 1976). The external oxygen (C) was 100% which is approximately 100 kPa. The diffusion coefficient of 02 through muscle (D) was assumed to be 13 8 x 10-4 mm2 s-1 and s, the solubility of oxygen in muscle, to be 0-00018 mm3 02 mm-3 (Allen, 1983). The resulting critical thickness for the uterus is found to be 1P4 mm. This is greater than either the pregnant or non-pregnant uterus and hence oxygenation should have been adequate. Cyanide caused around a 5-fold increase in lactate production by the pregnant uterus and around 10-fold by the non-pregnant uterus. These changes presumably reflect the markedly increased anaerobic glycolytic rate. However, this increase in rate was still not sufficient to maintain [ATP], as ATP fell in the presence of cyanide. This increased lactic acid production in cyanide may well account for the intracellular pH acidification found under these conditions (see above). The rate of lactate production in cyanide was not significantly different between pregnant and nonpregnant uteri and the intracellular acidification was very similar in the two preparations (0-31 +0 04 and 0-27 + 0-02 pH units, respectively). When lactate efflux was blocked by CHC in non-pregnant uteri in the presence of cyanide, the acidification was greater (0 34 + 0 04) than when lactic acid was able to leave the cell. The functional significance of aerobic glycolysis in smooth muscle is unclear, but Rubanyi et al. (1982) concluded that aerobic glycolysis was tightly coupled to Na+-K+ transport in the myometrium. Functional significance At parturition the uterus has to perform a series of sustained contractions to expel the young. In order to perform this vital task efficiently, the contractile ability of the uterus should not be compromised. ATP is an important part of that functional ability. It is probably for this reason that [ATP] is elevated in pregnancy. The uterus cannot maintain normal [ATP] in the presence of cyanide, application of which mimics the decrease in oxygen occurring when uterine blood flow is stopped. However, as shown above, the fall in ATP is much less in the pregnant uterus than the non-pregnant. This must be partly due to the increased starting levels of ATP and possibly to an increased efficiency of anaerobic ATP production. Thus ATP levels in the pregnant uterus may not fall to levels which are unable to sustain contractile ability. Preliminary results measuring spontaneous force production showed that whereas in the non-pregnant uterus force is not produced in the presence of cyanide, in the pregnant rat uterus force can be produced although at a reduced level (Nyman, Osman & Wray, 1988). As pHi falls in the presence of cyanide,

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additional experiments should be performed to determine what effect cytoplasmic acidification per se has on uterine force production, since in smooth muscle it may increase or decrease force (Wray, 1988a). In conclusion, uterine metabolism involves lactate production in aerobic conditions. Normal metabolite levels cannot be maintained without oxygen, although the changes are less in the pregnant than the non-pregnant uterus. Without glycolysis high energy phosphates cannot be maintained at all. There is a fall in [PCr] during prolonged K+-evoked contractures. This work was supported by the MRC. It is a pleasure to thank Dr C. Bauer and the staff of the MRC Biomedical NMR Centre at Mill Hill for all their help. I would also like to thank Ms L. Nyman and Ms V. Osman for performing some of the lactate measurements and Dr D. Eisner for commenting on an earlier version of this manuscript. REFERENCES

AICKIN, C. C. (1984). Direct measurement of intracellular pH and buffering power in smooth muscle cells of guinea-pig vas deferens. Journal of Physiology 349, 571-586. ALLEN, D. G. (1983). Techniques in Cardiovascular Research, pp. 1-21. Elsevier, Amsterdam. BATTELLINO, L. J., SABULSKY, J. & BLANCO, A. (1971). Lactate dehydrogenase isoenzymes in rat uterus: changes during pregnancy. Journal ofReproduction and Fertility 25, 393-399. DAWSON, M. J. & WRAY, S. (1985). The effects of pregnancy and parturition on phosphorus metabolites in rat uterus studied by 31P nuclear magnetic resonance. Journal of Physiology 368, 19-31.

FABIATO, A. & FABIATO, F. (1978). Effects of pH

on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. Journal of Physiology 276, 233-255. GREISS, F. C. (1965). Effect of labor on uterine blood flow: observations of gravid ewes. American Journal of Obstretics and Gynecology 93, 917-923. HALESTRAP, A. P. & DENTON, R. M. (1974). Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by a-cyano-4-hydroxycinnamate. Biochemical Journal

138,313-316. KROEGER, E. A. (1976). Effect of ionic environment on oxygen uptake and lactate production of myometrium. American Journal of Physiology 230, 158-162. KUSHMERICK, M. J., DILLON, P. F., MEYER, R. A., BROWN, T. R., KRISANDA, J. M. & SWEENEY,

H. L. (1986). 31P NMR spectroscopy, chemical analysis and free Mg2+ of rabbit bladder and uterine smooth muscle. Journal of Biological Chemistry 261, 14420-14429. NoYZEWSKI, E. A., RAMAN, J., TRUPIN, S. R., McFARLIN, B. L. & DAWSON, M. J. (1989). 31Phosphorus nuclear magnetic resonance examination of female reproductive tissues. American Journal of Obstretics and Gynecology 161, 282-288. NYMAN, L., OSMAN, V. A. & WRAY, S. (1988). The effect of metabolic inhibition on force and lactate production in pregnant and non-pregnant isolated rat uterus. Journal of Physiology 403, l25P. PAUL, R. J. (1980). Chemical energetic of vascular smooth muscle. In Handbook ofPhysiology. vol. 11, section 2, ed. BOHR, D. F., SOMYLO, A. P. & SPARKS, H. V., pp. 201-235. American Physiological Society, Bethesda, MD, USA. RUBANYI, G., TOTH, A. & KOVACH, A. G. B. (1982). Distinct effect of contraction and ion transport on NADH fluorescence and lactate production in uterine smooth muscle. Acta physiologica Academiae scientiarum hungaricae 59, 45-58. SPURWAY, N. C. & WRAY, S. (1987). A phosphorus nuclear magnetic resonance study of metabolites and intracellular pH in rabbit vascular smooth muscle Journal of Physiology 393, 57-71. TOFTS, P. & WRAY, S. (1985). Changes in brain phosphorus metabolites during post-natal development of the rat. Journal of Physiology 359, 417-429. WILKIE, D. R. (1979). Generation of protons by metabolic processes other than glycolysis in muscle cells. Journal of Molecular and Cellular Cardiology 11, 325-330. WRAY, S. (1988a). Smooth muscle intracellular pH: measurement, regulation and function. American Journal of Physiology 254, C213-225.

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WRAY, S. (1988b). The effects of cyanide on intracellular pH and metabolites in isolated rat uterus. Journal of Physiology 401, 82P. WRAY, S. (1988c). Regulation of intracellular pH in rat uterine smooth muscle, studied by 31p NMR spectroscopy. Biochimica et biophysics acta 972, 299-301. WYNN, R. M. (1977). Biology of the Uterus. Plenum Press, London.