EFFECTS OF ANOXIA ON FORCE, INTRACELLULAR CALCIUM AND LACTATE PRODUCTION OF URINARY BLADDER SMOOTH MUSCLE FROM CONTROL AND DIABETIC RATS

0022-5347/00/1634-1357/0 THE JOURNAL OF UROLOGY® Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 163, 1357–1363, April 2000 Printed in U.S.A.

EFFECTS OF ANOXIA ON FORCE, INTRACELLULAR CALCIUM AND LACTATE PRODUCTION OF URINARY BLADDER SMOOTH MUSCLE FROM CONTROL AND DIABETIC RATS J. V. WARING*

AND

I. R. WENDT

ABSTRACT

Purpose: To examine the effects of inhibiting oxidative metabolism on lactate production (JLac), force and [Ca2⫹]i in longitudinal smooth muscle from urinary bladders of control and diabetic rats. Materials and Methods: Strips of longitudinal smooth muscle were isolated from urinary bladders of diabetic rats and their age-matched controls. Force and [Ca2⫹]i were measured simultaneously in muscle strips loaded with the calcium indicator, fura-2. Separate muscle strips were used to determine JLac by standard enzymatic assay. The muscles were stimulated to contract with 65 mM K⫹ or 1 ␮M carbachol (CCh) in the presence of 2.5 mM Ca2⫹ and either 5, 10 or 25 mM glucose. Oxidative metabolism was inhibited either by replacing O2 in solution with N2, or by addition of 2 mM NaCN. Results: JLac was significantly less in diabetic muscles than control muscles under both normoxic and anoxic conditions. During stimulation under anoxic conditions, the diabetic muscles were less able to maintain force than the controls. Despite a marked decline in force in both diabetic and control muscles under anoxic conditions, [Ca2⫹]i remained elevated to levels that were in fact higher than those observed during stimulation under normoxic conditions. Increasing the glucose concentration had no significant effect during normoxia, however, under anoxic conditions, the higher concentration improved force maintenance in both control and diabetic muscles. There were no apparent effects of the glucose concentration on [Ca2⫹]i in either diabetic or control muscles. Conclusion: The results reveal that urinary bladder smooth muscle from diabetic rats has a reduced ability to maintain contraction under anoxic conditions. This most likely reflects a greater energy limitation as evidenced by the reduced JLac in diabetic muscles. In both diabetic and control muscles there was a marked dissociation between force and [Ca2⫹]i when oxidative metabolism was inhibited. This may indicate preferential use of glycolytically produced ATP for maintenance of [Ca2⫹]i homeostasis under these conditions. KEY WORDS: glycolysis, contraction, cyanide, smooth muscle, streptozotocin

Urinary bladder smooth muscle requires an adequate supply of adenosine triphosphate (ATP) to sustain its contractile function and glucose metabolism plays a major role in the supply of this ATP.1–3 Glucose can be metabolized through oxidative pathways, being converted to pyruvate that then enters the tricarboxylic acid cycle or it can be converted to lactate. The glycolytic breakdown of glucose to lactate is of major importance during oxygen deficiency; however, a common characteristic of smooth muscle is the production of substantial amounts of lactate even when fully oxygenated.4, 5 This conversion of glucose to lactate under normoxic conditions has been termed aerobic glycolysis and has been suggested to reflect glycolytic support of specific cellular functions such as ATP-dependent membrane ion pumps.4, 6 Indeed, Paul et al4, 7 have proposed that metabolism in smooth muscle is functionally compartmentalised such that the ATP requirements of ion pumps are primarily met by glycolytic enzymes in the vicinity of the cellular membranes, while the ATP utilized by the contractile proteins is supplied predominantly from oxidative metabolism. This raises the possibility that the energetics of intracellular calcium ho-

meostasis and force production may be independently regulated. The decreased insulin levels associated with diabetes mellitus result in decreased glucose uptake by several tissues including skeletal muscle and adipose tissue. While glucose transport in smooth muscle is brought about by facilitative diffusion that is only moderately increased by insulin,1 glucose uptake and metabolism to CO2 has been reported to be reduced in vascular smooth muscle from diabetic animals.8 Impaired glucose metabolism was also found in colonic smooth muscle from alloxan-diabetic rabbits.9 Since glucose metabolism is likely to be disturbed in diabetes mellitus, the possibility exists that processes of glycolysis may also be compromised and that this may have consequences for the function of smooth muscle in the diabetic state. Furthermore, this may be exacerbated under anoxic conditions where smooth muscle becomes solely reliant on glycolysis for energetic support. Urinary bladder disturbances are found in diabetes mellitus and include large atonic bladders with residual urine in humans,10 and increases in bladder mass and capacity in animal models.11 In addition the bladder, like any organ, may experience hypoxic conditions under circumstances where blood flow is impaired or compromised. The purpose of the present study was to examine the effects of inhibiting oxidative metabolism on lactate production (JLac), force and intracellular calcium concentration

Accepted for publication October 12, 1999. * Requests for reprints: Department of Physiology, Monash University, Clayton, Victoria 3168, Australia. Supported by Grant No. 960093 from the National Health and Medical Research Council of Australia. 1357

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2⫹

([Ca ]i) in longitudinal smooth muscle from urinary bladders of control and streptozotocin-induced diabetic rats. MATERIALS AND METHODS

Experimental animals. Diabetes was induced in male Wistar rats weighing approximately 250 gm. by a single injection of 60 mg./kg. streptozotocin (STZ) into a tail vein. The STZ was dissolved in 0.1 M citrate buffer (pH 4.5). All animals were allowed free access to food and water and each diabetic rat was housed with an age- and weight-matched control animal. Non-fasted blood glucose concentrations were measured in all animals prior to STZ administration and again at the time of the experiments. All experiments conducted had the approval of the Monash University Standing Committee on Ethics in Animal Experimentation and complied with the guidelines of the National Health and Medical Research Council of Australia on the use and care of animals in scientific research. Tissue dissection. Eight weeks after treatment with STZ, the rats were killed by chloroform anesthesia and decapitation. The bladder was removed and placed in carbogenated Krebs-Henseleit solution at room temperature. It was opened by a longitudinal incision through the wall and any residual urine washed out prior to determining the wet weight. The tissue was then pinned out in a dissecting dish with the mucosal surface uppermost. The mucosa and inner circular smooth muscle layer were removed by careful dissection under a binocular dissecting microscope. This exposed the underlying longitudinal smooth muscle. The tissue was pinned out under some stretch, to give improved definition of the smooth muscle layers. A strip of the longitudinal muscle was cut, tied at each end with 4/0 non-capillary silk and removed. The length and weight of the muscle strips from control (n ⫽ 14) and the STZ-treated animals (n ⫽ 13) were 10.7 ⫾ 0.6 mm. and 4.0 ⫾ 1.0 mg., and 11.8 ⫾ 0.8 mm. and 5.9 ⫾ 1.2 mg. respectively. Each muscle was mounted in the appropriate apparatus for simultaneous measurement of force and either JLac or [Ca2⫹]i as described below. The muscle was then left to equilibrate for 20 minutes in nominally Ca2⫹ -free solution prior to the commencement of the experimental protocol. Measurement of lactate production. For the experiments examining contractility and JLac, the muscle was mounted vertically on a stainless steel holder with the lower tie fixed to the base of the holder and the upper tie connected via a stainless steel rod to a lever system equipped with force and length transducers. To determine JLac, the muscle was immersed in a small glass vial containing 1.5 ml. of the appropriate solution for a specified period of time. The muscle was then removed from the vial, the vial sealed and the solution stored at ⫺40C for subsequent lactate analysis by standard fluorometric assay as previously described.12 JLac was determined under unstimulated basal conditions and during stimulation with either carbachol (CCh) or high K⫹ solution with the muscle maintained in either a normoxic or anoxic environment. Sample collection times were 30 minutes for basal conditions and 15 minutes for stimulation. Measurement of [Ca2⫹]i. In the experiments where force and [Ca2⫹]i were monitored simultaneously, the muscle was mounted horizontally between two stainless steel pins, one of which was attached to a force transducer. The steel pins and force transducer were fixed to a set of micromanipulators that allowed movement of the muscle in all three planes as well as adjustment of muscle length. The muscle was placed in a tissue bath located on the stage of an inverted fluorescence microscope. The bath, which had a coverslip as its base, had a volume of 3 ml. and was continuously superfused with Krebs-Henseleit solution at a flow rate of 30 ml. per minute. The muscle strip, mounted on the force recording apparatus, was then positioned to lie over the microscope objective, thus allowing the simultaneous recording of force and tissue flu-

orescence. [Ca2⫹]i was monitored using the Ca2⫹-sensitive fluorescent dye fura-2. The fluorescence of the tissue was recorded using a spectrophotometer system (Cairn Research Limited, Kent, UK) coupled to the fluorescence microscope. The autofluorescence of each preparation was measured at the start of the experiment, immediately before loading the tissue with fura-2. The muscle strips were loaded with fura-2 by incubation in a solution containing 5 ␮M of the acetoxymethyl ester form of the indicator (fura-2/AM) for 3 hours at room temperature (approx. 21C). Following the incubation, the preparations were washed for 15 minutes with Ca2⫹-free Krebs-Henseleit solution. The muscle strips were exposed to excitation light via a Xenon lamp (75 W) and a rotating filter wheel containing 340 and 380 nm filters. The light was focused onto the muscle using a ⫻ 40 UV objective and the emitted fluorescent light at 510 nm was collected back through the objective and detected by a photomultiplier tube. During non-recording periods a solenoid driven shutter prevented the excitation light from reaching the preparation. The simultaneously acquired tension and fluorescence signals were digitized and captured on-line with a personal computer. All fluorescence signals were corrected by subtracting the autofluorescence, and the ratio of the corrected fluorescence at 340 nm excitation to that at 380 nm excitation (R340/380) was taken as an indication of the [Ca2⫹]i. Solutions and chemical agents. The standard KrebsHenseleit solution was prepared nominally Ca2⫹-free and had the following composition (mM): NaCl 118; KCl 4.75; MgSO4 1.18; KH2PO4 1.18; NaHCO3 24.8; CaCl2 2.5. Glucose was added to this to achieve the desired solution glucose concentration. The solution was continuously aerated with 95% O2 - 5% CO2, and had a pH of 7.36. The temperature was maintained at 32C for all the experiments reported. The rate of loss of fura-2 from loaded tissues is temperature dependent and is considerably reduced by lowering the experimental temperature from 37C to 32C. High K⫹ solution was prepared by equimolar substitution of KCl for NaCl in the above solution. The solution used during the fura-2 loading procedure was buffered with N-[2-hydroxyethyl] piperazineN⬘-[2-ethanesulfonic acid] (HEPES) rather than bicarbonate, and had the following composition (mM): NaCl 135.5; KCl 5.9; MgCl2 1.2; HEPES 11.6; glucose 10; pH 7.4. Pluronic F127 (0.01%) was added to this solution to aid dispersal of the fura-2/AM. Inhibition of oxidative metabolism. Oxidative metabolism was inhibited either by vigorously bubbling the KrebsHenseleit solution with 95% N2 - 5% CO2 or by the addition of 2 mM NaCN. NaCN was used in the majority of the experiments involving measurement of fura-2 fluorescence since it proved difficult to control the O2 level in solutions flowing through the tissue bath on the stage of the microscope. This was because the surface of the solution in the bath was exposed to room air and direct bubbling of the solution within the bath produced unacceptable movement of the tissue. The effects on force production were identical irrespective of which method was used to inhibit oxidative metabolism. Statistics. Data are presented as mean values ⫾ the standard error of the mean (SEM). In all cases, the n values quoted refer to the number of experimental animals represented in that data set. Statistical analyses for between group comparisons, employed either the student’s two-tailed t test for paired or unpaired data as appropriate. p values of less than 0.05 were considered significant. RESULTS

Some of the physical characteristics of the animals used in this study are shown in table 1. Following the STZ injection, the animals developed diabetes as illustrated by the subse-

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TABLE 1. Body weights, bladder weights and blood glucose levels of control and STZ treated diabetic rats Initial Body Weight (gm.)

Final Body Weight (gm.)

Bladder Weight (gm.)

Bladder Weight/Body Weight (gm./kg.)

Control (n ⫽ 14) 305 ⫾ 9 468 ⫾ 9 0.11 ⫾ 0.01 0.24 ⫾ 0.01 0.29 ⫾ 0.02§ 0.89 ⫾ 0.08§ STZ (n ⫽ 13) 330 ⫾ 9 339 ⫾ 13§ § Significantly different from corresponding values in control rats (p ⬍ 0.001, independent two-tailed t test).

quent rise in blood glucose concentration. These animals also clearly exhibited polyuria and polydipsia. Other symptoms of the condition that were apparent included a lack of increase in body weight and urinary bladder hypertrophy. This hypertrophy is emphasized when bladder weight is expressed relative to the body weight which reveals a four-fold increase over that of the control animals. Hypertrophy of the bladder is consistently reported in studies with diabetic animals and most likely results from the increased volume load since it also occurs in other models of high volume urine production such as sucrose feeding.13, 14 Lactate production. JLac of control and diabetic muscle strips provided with 10 mM glucose under unstimulated and stimulated conditions in either the presence or absence of oxygen are shown in fig. 1. It is notable that the diabetic muscles produced significantly less lactate than control muscles under all experimental conditions. Under each condition both control and diabetic muscles exhibited a strong Pasteur effect, significantly increasing their JLac when deprived of oxygen. There was no significant difference in the proportional extent of the Pasteur effect between control and diabetic muscles stimulated by either high K⫹ or CCh, with JLac increasing approximately 3 fold under anoxic conditions in each case. Force development. Unstimulated muscles bathed in 2.5 mM Ca2⫹-containing solution exhibited spontaneous contractile activity and there was no apparent difference in the nature of the spontaneous contractions (amplitude or frequency) between control and diabetic muscle strips. When placed in the anoxic environment spontaneous activity decreased in amplitude in both control and diabetic muscles and in 4 of 5 diabetic muscles all spontaneous activity had ceased within 30 minutes. In contrast, all control muscles (n ⫽ 5) continued to exhibit spontaneous activity after 30 minutes anoxia and although this was considerably reduced in amplitude the frequency remained essentially unchanged. The force profiles of control and diabetic muscles during 15 minutes stimulation with 1 ␮M CCh in the presence of 10 mM glucose under normoxic and anoxic conditions are shown

FIG. 1. JLac in control and diabetic muscle strips in unstimulated and stimulated conditions with 65 mM K⫹ and 1 ␮M CCh during normoxia and anoxia. * denotes statistically significant difference between control and diabetic tissues.

Blood Glucose (mmol./l.) 6.1 ⫾ 0.6 26.3 ⫾ 1.5§

in fig. 2. Although there was some decline in force over the 15 minutes of stimulation under normoxic conditions, there was no difference between control and diabetic muscle strips in their ability to maintain contraction under this circumstance. Under anoxic conditions, the ability of both control and diabetic muscles to sustain force was compromised, however, force declined more severely in the diabetic muscles. When expressed as a percentage of the peak force developed by each muscle under anoxic conditions, force was significantly less in the diabetic muscles at 5, 10 and 15 minutes of stimulation. Peak force in the anoxic contractions was reduced compared with the normoxic conditions by 18 ⫾ 5% and 17 ⫾ 1% in control (n ⫽ 5) and diabetic (n ⫽ 8) muscles respectively. Actual values for peak force in the two muscle groups under normoxic and anoxic conditions are presented in table 2. It should be noted in this context that exposure to the anoxic environment did not commence until the beginning of the period of stimulation (that is, the muscles had not been rendered anoxic prior to stimulation). When the force level maintained at 15 minutes stimulation in anoxia was expressed as a percentage of that maintained at 15 minutes stimulation in normoxia for each muscle, the respective values for the control and diabetic groups were 35 ⫾ 5% and 10 ⫾ 1% (p ⫽ 0.002). Effect of glucose concentration. The effect of varying the solution glucose concentration on the ability of the muscles to sustain force during stimulation under anoxic conditions is illustrated in fig. 3. In both control and diabetic muscles, raising the glucose concentration from 5 to 25 mM significantly increased the force the muscles were able to sustain after 15 minutes stimulation with high K⫹ under anoxic conditions (see also fig. 5). Again the impaired capacity of the diabetic muscles to sustain force under anoxic conditions as compared with the control muscles is evident at both glucose concentrations. Under normoxic conditions, there were no significant effects of the glucose concentration on the force responses of either control or diabetic muscles.

FIG. 2. Force generation of muscle strips from control and diabetic rats in response to 15 minutes exposure to 1 ␮M CCh in presence of 10 mM glucose with both normoxic and anoxic conditions. Force is expressed as percentage of peak response of each contraction. * indicates statistically significant difference between control and diabetic tissues while in presence of N2.

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ANOXIA IN DIABETIC RAT URINARY BLADDER SMOOTH MUSCLE TABLE 2. Mean force responses expressed in mN/mm.2 for KCl and CCh stimulation under the different conditions KCl Stimulation

CCh Stimulation

5 mM Glucose

Control Diabetic

25 mM Glucose

O2

N2

131 ⫾ 15 (10) 152 ⫾ 11 (10)

109 ⫾ 13 (10) 112 ⫾ 10 (10)

O2

10 mM Glucose N2

120 ⫾ 12 (9) 143 ⫾ 11 (10)

117 ⫾ 13 (10) 108 ⫾ 9 (10)

O2

N2

166 ⫾ 34 (5) 221 ⫾ 11 (7)

144 ⫾ 40 (5) 184 ⫾ 10 (7)

FIG. 3. Force at end of 15 minutes of stimulation with 65 mM K⫹ during anoxic conditions while in presence of 5 mM or 25 mM glucose for control and diabetic muscle strips. Force is expressed as percentage of end force at end of stimulation during normoxia. * represents statistically significant difference between control and diabetic tissues. § indicates significant difference between two glucose concentrations.

As shown in fig. 4, under normoxic conditions there was no significant effect of raising the glucose concentration on JLac of high K⫹-stimulated muscles, although a tendency toward a slight increase appeared to be evident. During stimulation under anoxic conditions, however, JLac was significantly higher in 25 mM glucose than 5 mM glucose. This was observed in both control and diabetic muscles. Intracellular calcium concentration. Simultaneous recordings of force and fura-2 fluorescence ratio during stimulation with high K⫹ under normoxic conditions and following inhibition of oxidative metabolism with NaCN are shown in fig. 5. Under normoxic conditions, [Ca2⫹]i and force rise to a peak

FIG. 5. Original recordings of force and fura-2 fluorescence ratio illustrating effect of stimulation with 65 mM K⫹ for 15 minutes while in presence of 5 mM glucose and normoxia (A). Panel B illustrates effect oxidative inhibition with 2 mM NaCN has on force and R340/380. Response when glucose concentration is elevated to 25 mM is illustrated in panel C.

FIG. 4. Effect of 5 and 25 mM glucose on JLac in control (n ⫽ 5) and diabetic (n ⫽ 6) tissues while stimulated with 65 mM K⫹ in presence of fully oxygenated or anoxic conditions. * denotes statistically significant difference between 5 and 25 mM glucose.

following which force declines somewhat. In most muscles [Ca2⫹]i also declined slightly over the 15 minutes of stimulation (fig. 5, A and 6). In the presence of NaCN, force decays to very low levels, however, there is no decline in [Ca2⫹]i which remains elevated throughout the period of stimulation. Raising the glucose concentration to 25 mM resulted in slightly improved maintenance of force in the presence of NaCN but was without effect on the changes in [Ca2⫹]i (fig. 6). The pattern of these responses was similar in control and diabetic muscles although, consistent with the previous results, the diabetic muscles were less able to sustain force in NaCN than the control muscles (data not shown). In both the control and diabetic muscles, [Ca2⫹]i after 15 minutes of

ANOXIA IN DIABETIC RAT URINARY BLADDER SMOOTH MUSCLE ⫹

FIG. 6. Fura-2 fluorescence ratio at peak and end of contractions elicited by 65 mM K⫹ while exposed to 5 and 25 mM glucose during normoxic and anoxic conditions in control and diabetic tissues. * represents statistically significant difference between oxygenated and anoxic conditions.

stimulation was significantly higher in the presence of NaCN than under normoxic conditions (fig. 6) revealing an apparent marked dissociation between force and [Ca2⫹]i at this point. DISCUSSION

The results of this study showed that longitudinal smooth muscle from the urinary bladder of diabetic rats produced less lactate than comparable muscle strips from control animals. This was observed under all conditions tested, including when the tissues were unstimulated as well as when they were stimulated with high K⫹ or CCh under both normoxic and anoxic conditions. Even though smooth muscle is not largely reliant on insulin-stimulated glucose uptake,1 this may be indicative of a reduced ability of the diabetic smooth muscle to utilize glucose. This is supported by similar observations in rat aortic tissue.8 It is characteristic of many smooth muscles to produce substantial quantities of lactate even when fully oxygenated.4, 5, 15, 16 Despite this high aerobic glycolysis smooth muscle nevertheless exhibits a strong Pasteur effect with JLac increasing significantly under anoxic conditions.17–20 Given the apparent reduced ability of the diabetic muscle strips to utilize glucose, it might be expected that the Pasteur effect would also be diminished in these muscles. This appears not to be the case since both control and diabetic muscle strips responded to anoxia with a similar proportional increase in JLac of approximately 3 fold. However, while the increase was proportionately similar, the absolute JLac of the diabetic tissues under anoxic conditions remained significantly less than that of control tissues. The diabetic muscle strips were less able to sustain force when challenged to contract during anoxia. Force development by diabetic muscles was significantly less than that of control muscles after 5, 10 and 15 minutes of stimulation with CCh under anoxic conditions. Similar results were ob-

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tained with high K stimulation. In addition, spontaneous activity ceased in the diabetic muscles during anoxia while in control muscles spontaneous activity, although reduced in amplitude, was not abolished. These observations seem to correlate with the decreased rate of JLac in these tissues and may reflect a larger energy limitation under anoxic conditions in the diabetic tissues, than in the controls. To assess whether glucose availability may be a limiting factor, the effects of raising the solution glucose concentration from 5 to 25 mM were examined. Both the control and diabetic tissues exhibited improved force maintenance under anoxic conditions at the higher glucose concentration. This was accompanied by a significant increase in anaerobic JLac in both groups. This shows that bladder smooth muscle of both control and diabetic animals is capable of increasing its rate of anaerobic glycolysis if provided with more glucose (at least in the range 5 to 25 mM). The increased ability of these tissues to maintain force under anoxic conditions at the higher glucose concentration, together with the increased JLac, is consistent with an improved energy supply. It has been reported previously that increasing the glucose concentration increases lactate production and partially restores ATP content and contraction in hypoxic guinea-pig taenia caecum.18, 19 It seems likely that the extent to which force can be maintained under hypoxic conditions will depend on both the rate of energy usage by the muscle and its capacity for anaerobic glycolysis. For example, under similar experimental conditions, rabbit aorta, which has a relatively low rate of energy usage, is able to maintain contraction and cellular ATP levels under hypoxia whereas guinea-pig taenia caecum and rat portal vein, which have high rates of energy use, are unable to.21, 22 It is interesting to note, that anoxic force maintenance of the diabetic tissues with 25 mM glucose is similar to that of controls with 5 mM glucose and these glucose concentrations approximate to the blood glucose concentrations in vivo in the respective animals. Under fully oxygenated conditions, the glucose concentration had no significant effect on JLac in either control or diabetic tissues, although it did tend to increase slightly in 25 mM glucose. Arner et al23 also found a slight but non significant increase in aerobic JLac of bladder smooth muscle when the glucose concentration was raised from 11 to 32 mM. In addition, under normoxic conditions the glucose concentration had no effect on the contractile responses in either group of animals. Longhurst et al24 also found that under normoxic conditions, raising glucose to 30 mM had no effect on the contractile responses of bladder smooth muscle from either control or diabetic rats and concluded that glucose availability was not a limiting factor for contractile function under normal aerobic circumstances. Strikingly, the decreased force seen in the control and diabetic muscle strips during anoxia was not accompanied by a decrease in the [Ca2⫹]i. This suggests that during anoxia there is a marked dissociation between force and [Ca2⫹]i. Several mechanisms have been proposed to account for the decrease in the force of contraction under anoxia in smooth muscle (see Taggart and Wray25 for review). In some smooth muscles, an attenuation of [Ca2⫹]i seems to occur during phasic contractions26 –28 and therefore could explain the decrease in force. However, many of these studies26 –29 also show that during anoxia or inhibition of oxidative metabolism, [Ca2⫹]i remains elevated during tonic contractions, even though force declines dramatically. In these tonic contractions, it appears that a decrease in [Ca2⫹]i cannot account for the decreased force. The present study has shown that this is also the case in bladder smooth muscle. The fluorescence signals recorded in the present study represent the spatially averaged fluorescence obtained from all the cells in the window of measurement and therefore may not reveal changes in localization or redistribution of Ca2⫹ within the cells if these were to occur. It could be

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possible that the apparent high [Ca2⫹]i observed under anoxic conditions represents a redistribution of Ca2⫹ to a noncontractile compartment30 within the cells such that it is not effective in activating the contractile filaments. This seems unlikely, however, since others have shown that myosin light chain phosphorylation levels remain elevated during anoxia,27, 29 suggesting that [Ca2⫹]i in the vicinity of the contractile apparatus remains elevated. Obara et al29 have suggested that the diminished force under anoxic conditions in guinea-pig taenia caecum simply related to energy limitation. The results of the present study could be considered to be consistent with this hypothesis. That is, the poorer force maintenance in the diabetic tissue could be related to the reduced JLac, signifying diminished anaerobic ATP production such that the diabetic tissues are more energy limited. Others, however, have argued that energy supply to the myofilaments under anoxic conditions is not limiting for force production (see Taggart and Wray25 for review) and propose that some mechanism(s) of desensitization of the contractile filaments to Ca2⫹ must occur. The data provided by this study do not provide any direct evidence on this matter, but if some form of anoxia-induced desensitization is involved it clearly exists in both the control and diabetic muscles. With oxidative metabolism inhibited, the muscles become solely reliant on glycolysis for metabolic support. Even though ATP production through glycolysis increases under anoxic conditions in urinary bladder smooth muscle, this cannot compensate fully for the loss of oxidative ATP production.5, 31 The observation that [Ca2⫹]i remains elevated during stimulation under anoxia suggests that the mechanisms for Ca2⫹ entry/mobilization are not inhibited by anoxia. It may then be that the limited energy supply available to the smooth muscle cells is preferentially directed to maintaining [Ca2⫹]i homeostasis at the expense of energy availability for the contractile elements. This would also be consistent with the notion that glycolytically produced ATP preferentially fuels membrane ion pumps.4, 7, 32 It is interesting to note that not only did [Ca2⫹]i remain high during stimulation under anoxic conditions but it was in fact significantly higher at the end of 15 minutes of stimulation during anoxia when compared with normoxia. This was observed in both control and diabetic tissues. Similar observations have been reported in other smooth muscles.27 At present, it is not clear what might be responsible for this increase in [Ca2⫹]i under anoxic conditions. One possibility is simply that energy limitation is reducing the ability of the Ca2⫹ uptake and extrusion processes to keep the [Ca2⫹]i stable in face of the continuing Ca2⫹ entry during stimulation. In conclusion, the results indicate that urinary bladder smooth muscle of diabetic animals has a reduced ability to maintain contraction under anoxic conditions when compared with control tissues. This is associated with a decreased rate of lactate production by the diabetic muscles as compared with the controls, which may suggest that during anoxia there is a greater energy limitation in the diabetics. Force maintenance during stimulation under anoxic conditions was improved in both control and diabetic muscles by elevating the glucose concentration to 25 mM. This was associated with an increase in JLac and so may be due to an improvement in energy supply under these conditions. The reduced force of contraction under anoxic conditions was not associated with a decrease in [Ca2⫹]i which remained unchanged or even elevated. This suggests a dissociation of the relationship between force and [Ca2⫹]i when oxidative metabolism is inhibited in both normal and diabetic urinary bladder smooth muscle and may reflect preferential use of the available glycolytically produced ATP for maintaining [Ca2⫹]i homeostasis.

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