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EFFECTS OF STREPTOZOTOCIN-INDUCED DIABETES MELLITUS ON INTRACELLULAR CALCIUM AND CONTRACTION OF LONGITUDINAL SMOOTH MUSCLE FROM RAT URINARY BLADDER
0022-5347/00/1631-0323/0 THE JOURNAL OF UROLOGY® Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 163, 323–330, January 2000 Printed in U.S.A.
EFFECTS OF STREPTOZOTOCIN-INDUCED DIABETES MELLITUS ON INTRACELLULAR CALCIUM AND CONTRACTION OF LONGITUDINAL SMOOTH MUSCLE FROM RAT URINARY BLADDER J. V. WARING*
AND
I. R. WENDT
From the Department of Physiology, Monash University, Clayton, Victoria, Australia
ABSTRACT
Purpose: To examine the effect of diabetes on [Ca21]i and contractility in longitudinal smooth muscle of the urinary bladder. Materials and Methods: Longitudinal smooth muscle strips were isolated from the urinary bladders of rats with STZ-induced diabetes as well as age matched controls. Force and [Ca21]i were measured simultaneously in muscle strips loaded with the calcium indicator, fura-2. Contractions were initiated by electrical field stimulation (EFS) at various frequencies, as well as by high K1, carbachol (CCh) and cyclopiazonic acid (CPA) in the presence of varying concentrations of extracellular Ca21. Results: In unstimulated muscles, there was no significant difference in resting [Ca21]i between the control and diabetic groups. However, the muscle strips from the diabetic animals produced higher force levels in response to EFS, high K1, CCh and CPA than those from control animals. The higher force development in the diabetic muscles was not associated with greater increases in [Ca21]i, which in fact tended to be lower during stimulation in the diabetic tissues. When stimulated by CCh in the presence of nifedipine, both control and diabetic muscles exhibited a nifedipine-resistant component of contraction, however, this was significantly larger in the diabetic muscles. Conclusion: The results suggest that there are no major impairments in either intracellular calcium regulation or contractile function in bladder smooth muscle after 8-weeks of STZ-induced diabetes. However, a non-specific enhancement of force production was seen, which was not associated with increases in [Ca21]i. These changes imply that the apparent sensitivity to [Ca21]i is enhanced in bladder smooth muscle from diabetic rats. KEY WORDS: intracellular calcium, contraction, smooth muscle, diabetes mellitus, streptozotocin
Urinary bladder disturbances are found in long-term diabetes mellitus. It has been suggested that the condition remains asymptomatic for a significant period of time.1–3 The most common characteristics of bladder dysfunction in animal models of diabetes are increases in distension and bladder capacity.4 – 6 The presence of residual urine is also a manifestation of the condition especially in the later stages,2, 3 which suggests that there may be some compromise of the smooth muscle contraction. Whilst it is generally accepted that neuropathy plays a role in this organ’s impairment2, 7 there are reports to suggest that alterations may also exist at the level of the bladder smooth muscle itself.8 These include changes in postsynaptic muscarinic receptor function9, 10 and possible changes in intracellular second messenger systems.11 The possibility that there may be a myogenic component to diabetic bladder dysfunction has led to several studies of bladder smooth muscle contractility in various animal models of diabetes. The results of these studies are often conflicting. Some report that contractile responses of the diabetic bladder, especially to muscarinic agonists, are enhanced5, 9, 11, 12 while others suggest that the responses to a variety of contractile stimuli are diminished.6, 8, 13 These apparent discrepancies may be due, at least in part, to the different animal models and duration of diabetes employed as well as the data normalization pro-
cedures applied. Despite the variability in the results of these studies, it seems likely that contractile function of urinary bladder smooth muscle is altered in diabetes. Little is known, however, of the possible mechanisms that might underlie these alterations. Over recent years there has been considerable interest in the possibility that altered intracellular calcium regulation may be an underlying abnormality in many complications of diabetes mellitus.14 Indeed many studies have reported that the intracellular calcium concentration ([Ca21]i) is elevated in diabetes in a wide variety of cell types.14 Given that [Ca21]i plays a pivotal role in regulating the contractile activity of smooth muscle, it may be that altered contractile function of urinary bladder smooth muscle in diabetes is secondary to disturbances of [Ca21]i handling and/or homeostasis. This study has attempted to address this issue by measuring simultaneously [Ca21]i and contractile responses in urinary bladder smooth muscle of control and streptozotocininduced diabetic rats, the most commonly used animal model of diabetes mellitus. MATERIALS AND METHODS
Experimental animals. Diabetes was induced in male Wistar rats weighing 283 6 11 gm. (n 5 25) 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 initially weight-
Accepted for publication July 22, 1999. * Requests for reprints: Department of Physiology, Monash University, Clayton, Victoria 3168, Australia. Supported by project grant no. 960093 from the National Health and Medical Research Council of Australia. 323
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
matched control animal. Blood glucose concentrations were measured in control and diabetic 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 care and use of animals in scientific research. Tissue dissection. Eight weeks after treatment with STZ, the rats were killed by chloroform anaesthesia and decapitation. The bladder was removed and placed in carbogenated Krebs-Henseleit solution at room temperature. A longitudinal incision was made in the bladder and any residual urine washed out to allow for the weighing of the empty organ. The tissue was then pinned out in a dissecting dish with the mucosal surface uppermost. The mucosa and 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 muscle strips from control animals (n 5 24) had a length of 7.0 6 0.3 mm. and weighed 1.2 6 0.1 mg. while in the STZ treated animals (n 5 25) they were 8.2 6 0.3 mm. long and weighed 1.7 6 0.2 mg. The cross-sectional area, as determined by the length and mass, and assuming a density of 1.0 mg./mm.3, was 0.18 6 0.01 mm.2 and 0.21 6 0.02 mm.2 for the control and diabetic muscle strips respectively. Force measurements. In most instances two muscle strips were dissected, one of which was used for simultaneous measurement of [Ca21]i and force whilst the other was used for contractility studies only. Contractility studies were carried out on this second strip during the time the first strip was being loaded with the Ca21 indicator, fura-2 (see below). This allowed a greater range of studies to be carried out and confirmed that the contractile behavior of the fura-2 loaded muscle was not altered by the dye loading procedure. For the contractility only studies, 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. The muscle was immersed in a tissue bath containing 30 ml. of carbogenated Krebs-Henseleit solution. In the experiments where force and [Ca21]i were monitored simultaneously, the muscle was mounted in a 3 ml. tissue bath and superfused continuously with carbogenated KrebsHenseleit solution at a flow rate of 30 ml./min. The muscle strip was attached 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. Once attached to the transducer the muscles were stretched to a resting tension of 1 gm. and allowed to equilibrate in nominally Ca21-free solution for 15 minutes. Intracellular calcium measurements. [Ca21]i was monitored using the Ca21-sensitive fluorescent dye fura-2. The fluorescence of the tissue was recorded using a spectrophotometer system (Cairn Research Limited, Faversham, Kent, UK) combined with an inverted fluorescence microscope (Olympus IMT-2, Tokyo, Japan). The tissue bath, which had a coverslip as its base, was located on the microscope stage and the muscle strip, mounted on the force recording apparatus, was positioned to lie over the microscope objective, thus allowing the simultaneous recording of force and tissue fluorescence. 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 mM of the acetoxymethyl ester form of the indicator (fura-2/AM) for 3
hours at room temperature (approx. 21 C). Following the incubation, the preparations were washed for 15 min. with Ca21-free Krebs-Henseleit solution. The tissue 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 340 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 [Ca21]i. At the conclusion of each experiment, an internal Ca21 calibration was performed as previously described15 by exposing the tissues to the Ca21 ionophore, ionomyocin and measuring the Rmin and the Rmax in Ca21-free solution containing 3 mM EGTA and 1.5 mM Ca21 solution respectively. [Ca21]i was subsequently calculated from the experimental R340/380 assuming the KD of fura-2 for Ca21 to be 224 nM. Solutions and chemical agents. The Krebs-Henseleit solution was prepared nominally Ca21-free and had the following composition (mM): NaCl 118; KCl 4.75; MgSO4 1.18; KH2PO4 1.18; NaHCO3 24.8; glucose 10. CaCl2 was added to this to achieve the desired solution Ca21 concentration. The solution was continuously aerated with 95% O2-5% CO2 and had a pH of 7.35. It was maintained at 32 C 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 37 C to 32 C. High K1 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-[2hydroxyethyl]piperazine-N9-[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. Fura-2/AM and Pluronic F127 were obtained from Molecular Probes (Eugene, OR, USA) while carbachol (CCh), cyclopiazonic acid (CPA), ionomycin and nifedipine were from Sigma (St. Louis, MO, USA). Concentrated stock solutions of CCh (10 mM in distilled water), CPA (10 mM in DMSO) and nifedipine (10 mM in DMSO) were prepared and appropriate aliquots of these were added to the experimental solutions to obtain the desired final concentrations of these agents. Experimental protocol. Following the initial equilibration period in nominally Ca21-free solution, each muscle was then exposed to Krebs-Henseleit solution containing 2.5 mM Ca21. They remained in this solution for at least 20 min. to allow the development of any spontaneous activity. Following this, contractile responses were initiated by a variety of stimuli, including electrical field stimulation (EFS), CCh and high K1. For EFS, 10 V, 1 ms square pulses at frequencies ranging from 0.2 to 20 Hz were delivered for 20 s, via two platinum wire electrodes positioned on either side of the muscle. In most cases, responses to CCh and high K1 were elicited in the presence of a range of different solution Ca21 concentrations and each response was preceded by a period in Ca21-free Krebs-Henseleit solution which allowed the response to occur against a consistent (quiescent) baseline for each muscle. Statistics. Data are presented as mean values 6 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
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
t test for unpaired data or 2 way repeated measures analysis of variance; p-values of less than 0.05 were considered significant. RESULTS
Some of the characteristics of the animals used in this study are shown in the table. All control animals showed an increase in body weight while the diabetic animals failed to gain weight over the seven to eight week period following STZ injection. Observation of the diabetic animals clearly indicated polyuria and polydipsia and the blood glucose levels in this group were significantly elevated. Bladder weight in absolute terms was increased in the diabetic rats by approximately two times that of the controls. This tissue hypertrophy was further emphasized when the bladder weight was expressed relative to the body weight which revealed a four fold increase over that of the normal animals. Basal intracellular calcium concentration. While in the presence of Ca21-free solution, the resting [Ca21]i of the muscle strips was not different, being 74 6 30 nM (n 5 21) and 74 6 29 nM (n 5 23) in the control and diabetic groups respectively. As shown in figure 1, when 2.5 mM Ca21 was added to the superfusing solution, [Ca21]i increased, the muscles developed tone and, in most cases, spontaneous contractile activity. This occurred in both control and diabetic muscle strips. The average [Ca21]i in the presence of 2.5 mM Ca21-containing solution was 256 6 30 nM (n 5 21) in control muscles and 275 6 29 nM (n 5 23) in the diabetic muscles. These values were not significantly different. There was also no difference in the incidence of spontaneous activity between the control and diabetic muscles, it occurring in approximately 70% of preparations in each group. In addition, there were no apparent differences in the pattern or frequency of spontaneous contractions between the two groups of muscles. Response to electrical field stimulation. Peak force in response to EFS increased in both control and diabetic strips as the frequency of stimulation increased. In absolute terms, the diabetic strips produced more force per unit cross-sectional area than did the control strips. This was most evident at the higher frequencies (figure 2, top panel). However, as shown in the lower panel of figure 2, when the response at each frequency was expressed relative to the response at 20 Hz for each muscle, the diabetic strips actually produced proportionately less force at the lower frequencies than did the control strips. Response to carbachol with graded [Ca21]o. Typical responses of a control muscle strip to stimulation with 10 mM CCh at two different extracellular Ca21 concentrations are shown in figure 3. As can be seen, [Ca21]i rises upon exposure of the muscle to CCh and this is followed by a rise in force. At this reasonably high concentration of CCh, force and [Ca21]i both rise to attain steady levels that are relatively well maintained and the responses are graded with the extracellular Ca21 concentration. The responses of control and diabetic muscle strips were qualitatively similar in this regard. In both groups of muscles responses of this type were initiated at solution Ca21 concentrations ranging from 0.2 to 6.4 mM and the mean force levels and [Ca21]i attained at each [Ca21]o in control and diabetic muscles are presented in figure 4. In absolute terms the diabetic muscles developed higher levels of force at the higher Ca21 concentrations. Analysis of the data using 2 way repeated measures analysis
of variance revealed that the relationship between force and [Ca21]o was significantly different between the two groups. It is notable from figure 4 that [Ca21]i was actually somewhat lower during CCh stimulation in the diabetic muscles as compared to the control muscles; however, statistical analysis of the data failed to reveal a significant difference in [Ca21]i between the control and diabetic groups. Response to high potassium with graded [Ca21]o. Figure 5 shows the force and [Ca21]i in control and diabetic muscles stimulated with 65 mM K1 in the presence of varying concentrations of extracellular Ca21. The results are essentially the same as those obtained with CCh stimulation (fig. 4). Again, the diabetic muscles developed higher levels of force at the higher Ca21 concentrations and, paradoxically, at each level of stimulation [Ca21]i was actually somewhat lower in the diabetic muscles as compared to the control muscles. Effects of nifedipine. In an attempt to elucidate the sources of Ca21 contributing to the contraction, the effects of 3 mM nifedipine on CCh-induced responses was examined. Both control and diabetic muscle strips exhibited a residual response after nifedipine exposure as shown in figure 6. At [Ca21]o concentrations of 1.6 mM and above this nifedipine resistant contraction was significantly larger in the diabetic muscles. Responses to cyclopiazonic acid. Treatment of urinary bladder smooth muscle with CPA, an inhibitor of the sarcoplasmic reticulum Ca21-ATPase, has previously been reported to lead to an influx of Ca21 through a pathway that is not sensitive to nifedipine.15 As shown in figure 7, [Ca21]i increases in CPA-treated muscle as the extracellular Ca21 concentration increases. There were no differences in the CPA-induced increase in [Ca21]i between control and diabetic muscles; however, as shown in the upper panel of figure 7, the diabetic muscles developed significantly more force under these conditions. DISCUSSION
The primary purpose of this study was to investigate the effects of diabetes on [Ca21]i and contractile function of urinary bladder smooth muscle. Previous studies have suggested that [Ca21]i may be elevated in diabetes in a variety of cell types including vascular smooth muscle14 and that this may be an influential factor behind many complications associated with the disease. The results of the present study revealed that [Ca21]i in urinary bladder smooth muscles from diabetic animals showed no difference compared to control tissues strips when the preparations were unstimulated in the presence of either Ca21 free or 2.5 mM Ca21 containing solution. Qi et al16 also found that [Ca21]i in isolated rat bladder smooth muscle cells, was unchanged by diabetes. It would seem that, resting [Ca21]i of urinary bladder smooth muscle is not disturbed by diabetes, at least not following 8 weeks of STZ-induced diabetes in the rat. When placed in Ca21 containing solution, the majority of muscle strips developed tone and spontaneous contractile activity as would be expected from the associated increase in [Ca21]i. There was no apparent difference between the control and diabetic strips in this regard. Tammela et al11 reported that bladder strips from diabetic rats developed more spontaneous activity than those from control animals although they provided no quantitative data in support of this. In contrast, Hashitani et al17 found that diabetic rat detrusor
Body weights, bladder weights and blood glucose levels of control and STZ treated rats Initial Body Weight (gm.)
Final Body Weight (gm.)
Bladder Weight (mg.)
Bladder Weight/Body Weight (gm./kg.)
Control (n 5 24) 296 6 7 482 6 13 120 6 4 STZ (n 5 25) 283 6 11 289 6 10§ 265 6 10§ § Significantly different from corresponding values in control rats (p ,0.05, independent two-tailed t test).
0.25 6 0.01 0.95 6 0.06§
Blood Glucose (mmol./l.) 5.5 6 0.4 23.0 6 1.2§
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
FIG. 1. Original recordings of force and [Ca21]i illustrating effect of addition of 2.5 mM Ca21 to solution superfusing on unstimulated control muscle strip. In 2.5 mM Ca21 solution, muscle developed tone and exhibited spontaneous activity.
smooth muscle exhibited fewer spontaneous spike discharges than control muscles. Spontaneous contractile activity is notoriously difficult to quantitate and may be influenced by a number of experimental factors such as temperature and degree of muscle stretch. The present study found no evidence for a difference in spontaneous activity between control and diabetic bladder smooth muscle. The muscle strips from the diabetic animals developed higher force levels in response to each mode of stimulation when compared to control. This is consistent with a number of other reports of enhanced contractile responses in diabetic rat bladder.5, 9, 11, 12, 18 –20 It is important to note, though, that there is no universal agreement on the effects of diabetes on bladder smooth muscle contractility and some studies have also found diminished contractility.6, 8, 13 The urinary bladder from the diabetic animals exhibited clear hypertrophy as consistently reported in other studies from diabetic animals,5, 9, 11, 12, 18 –20 as well as in other models of high volume urine production such as sucrose feeding.5, 18 It has been suggested that some of the discrepancies in the literature on the effects of diabetes on bladder smooth muscle contractility may be attributable to whether or not contractile force data has been appropriately normalized.19 The control and diabetic muscle strips used in the present study had similar dimensions and all force data were normalized for the crosssectional area of the muscles. The increased force responses of the diabetic muscles are not, therefore, merely a consequence of the increased muscle mass due to the hypertrophy, but reflect an inherent change in the smooth muscle. The diabetic muscle strips exhibited higher absolute levels of force when stimulated with EFS, particularly at the higher frequencies. This has also been reported by others.11, 21, 22 The response of these longitudinal muscle strips to EFS is neurogenic in origin and involves both cholinergic and purinergic components.23, 24 The purinergic component has been suggested to contribute largely to the initial phasic portion of the response while the cholinergic component predominates in the tonic portion and is thought to be primarily responsible for bladder emptying.23 The purinergic component may, therefore, be proportionately greater at low frequencies of stimulation. Changes in cholinergic enzyme activities have been reported in the urinary bladder of rats after 14 days of STZ-induced diabetes25 and it has been postulated that the increased responsiveness of bladder strips from chronically diabetic rats could represent a form of denervation supersen-
FIG. 2. Peak force generation of muscle strips from control and diabetic rats in response to electrical field stimulation at different frequencies. At frequencies of 2 Hz and higher, peak force generally occurred early with some subsequent decay of force. At frequencies below 1 Hz, there were oscillations in force and peak level was attained more slowly with generally little decay over 20 sec period of stimulation. Upper panel shows absolute force values (mN/mm2) while in lower panel force is expressed as percentage of response at 20 Hz.
sitivity, possibly related to diabetic neuropathy.11 However, the enhanced response to EFS is evident as early as 7 days after induction of diabetes which would seem to make diabetic neuropathy unlikely as a causative factor.12, 22, 26 Luheshi and Zar21, 27 have suggested that bladder strips from diabetic rats have a reduced non-adrenergic non-cholinergic response and an increased cholinergic response, and that the latter is due to enhanced release of acetylcholine. In contrast, enhanced purinergic responsiveness has been observed in gastric fundus of diabetic rats with ATP release reported to be increased three-fold.28 In the present study the increased response, in absolute terms, of the diabetic strips to EFS was most evident at the higher stimulation frequencies which would seem to be consistent with an enhanced cholinergic component. Interestingly, when the responses were expressed relative to the maximum response at 20 Hz stimulation it was evident that, proportionately, the responses of the diabetics at frequencies lower than this (particularly in the range 2–10 Hz) were in fact less than those of the controls
[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
327
FIG. 3. Typical original traces of simultaneously recorded force and [Ca21]i responses evoked by 10 mM CCh in control (upper panel) and diabetic (lower panel) urinary bladder muscle strip in presence of either 0.8 or 3.2 mM Ca21.
resulting in the relationship between stimulation frequency and response (as a percent of maximum) being altered. Further studies are required to elucidate the underlying basis of this; however, it does suggest that there may be some subtle changes in the motor transmission. Perhaps the most striking observation made in the present study, was that the higher forces developed by the diabetic muscle strips in response to CCh, high K1 and CPA were not accompanied by similar enhanced increases in [Ca21]i. Indeed, [Ca21]i during stimulation with each of these agents, tended to be lower in the diabetic tissues than the controls. White et al29 have demonstrated enhanced responsiveness of vascular smooth muscle from diabetic rats to extracellular Ca21 in the presence of norepinephrine or KCl. Although they did not measure [Ca21]i, they proposed that the enhanced vascular contractions in STZ-diabetes resulted from increased Ca21 entry due to enhanced activity and/or number of Ca21 ion channels in the vascular smooth muscle membrane. It has similarly been speculated that enhanced Ca21 entry underlies the increased responsiveness of both vas deferens30 and urinary bladder31 smooth muscle in STZdiabetes. The present experiments clearly show, however, that the increased contractile responses of diabetic bladder smooth muscle are not associated with elevated levels of [Ca21]i. There appear to be two main possibilities that could account for this observation. One is that there has been some change in the sensitivity of the contractile apparatus to Ca21 in the diabetic tissues. There has been considerable interest over recent years in factors that can modify the apparent Ca21 sensitivity of the contractile apparatus in smooth mus-
FIG. 4. Force generation (upper panel) and associated increases in suprabasal [Ca21]i (lower panel) in control and diabetic urinary bladder smooth muscle stimulated with 10 mM CCh in presence of varying concentrations of extracellular Ca21.
cles and “Ca21-sensitisation” appears to be an important element in the physiological regulation of the contractile state of smooth muscle.32 Within this context, protein kinase C (PKC) has been suggested to be an important mediator of increases in Ca21 sensitivity in smooth muscle since activation of PKC has been shown to enhance contraction in a variety of smooth muscles in the absence of proportional changes in [Ca21]i.32, 33 It has been reported that the contractile responses of vascular smooth muscle to phorbol esters, which activate PKC, are enhanced in diabetes.29, 34 This would be consistent with an augmentation of the mechanisms mediating increases in Ca21 sensitivity in diabetic smooth muscle. The increased contractile responsiveness of diabetic urinary bladder reported in the present study, as well as in previous studies,5, 9, 11, 12, 18 –20 appears to be of a rather non-specific nature, making it unlikely to be the result of changes in specific receptors. An increase in the Ca21 sensitivity of the contractile apparatus would be expected to lead to a non-specific increase in contractile responsiveness. It is noteworthy that the enhanced force responses of the diabetic muscles to CCh and K1 are most evident at higher concentrations of Ca21 and this may indicate a more complex situation than just a general increase in Ca21 sensitivity in the diabetic muscles. It is important to recognize that the
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
FIG. 6. Force generated by control and diabetic bladder smooth muscle strips in response to 10 mM CCh in presence of varying extracellular Ca21 concentrations before and after addition of 3 mM nifedipine. Force has been expressed relative to response in 6.4 mM Ca21 in absence of nifedipine for each muscle.
FIG. 5. Force generation (upper panel) and associated increases in suprabasal [Ca21]i (lower panel) in control and diabetic urinary bladder smooth muscle stimulated with 65 mM K1 in presence of varying concentrations of extracellular Ca21.
[Ca21]i determined with fura-2 in these experiments represents the spatially averaged [Ca21]i in the cells within the recording field. Another possible explanation for the increased force of the diabetic muscles in the presence of an apparently unchanged, or even lower, spatially averaged [Ca21]i is that it reflects an altered intracellular distribution of Ca21. There is evidence that cytoplasmic Ca21 in smooth muscle cells may be distributed between a non-contractile compartment and a contractile compartment with only the latter contributing to contractile activation.33 Regulation of intracellular Ca21 movements by the peripheral SR is likely to be important in determining the intracellular Ca21 distribution.33 In this light, it is interesting that in the CPA treated tissue, where Ca21 uptake by the SR is inhibited and internal Ca21 stores become depleted, the enhanced force development of the diabetic tissues is evident at much lower extracellular Ca21 concentrations than in CCh or high K1 stimulated muscles. It may be that access of Ca21 to the contractile compartment is greater in the diabetic muscles and that this is facilitated further with CPA treatment. As with most smooth muscles, a significant proportion of the Ca21 that enters bladder smooth muscle during stimulation is believed to enter through voltage operated calcium channels (VOCCs).35 Nifedipine was utilized in an attempt to ascertain
the contribution of Ca21 entry through VOCCs during the contractile response of the control and diabetic muscles to CCh. Tissues from both the control and diabetic animals, exhibited nifedipine-resistant contractions, that were most evident at high [Ca21]o. The nifedipine-resistant component of the contractions was significantly larger in the diabetic muscles, suggesting that they may be less reliant on Ca21 entry through VOCCs. This observation is supported by work from Sakai et al30 showing that vas deferens smooth muscle from diabetic rats exhibits a greater VOCC-independent Ca21 uptake than control muscles. Kamata et al31 have also suggested that the enhanced response to acetylcholine in diabetic bladder smooth muscle is due to an increased Ca21 influx through channels other than VOCCs. Depletion of intracellular calcium stores, by CPA or thapsigargin for example, has been shown to activate Ca21 entry in a variety of non-excitable cells and smooth muscles, including bladder smooth muscle.15, 33, 36 Treatment of both control and diabetic muscle strips with CPA led to Ca21 entry and the development of force, indicating that this store release-activated Ca21 entry pathway is functional in diabetic as well as control muscle strips. The greater force response of the diabetic muscle to CPA could be consistent with the greater nifedipine-resistant component of the contraction of these muscles to CCh. Although it must be remembered that the increase in [Ca21]i induced by CPA was not significantly different between control and diabetic muscles. The fact that [Ca21]i was similar in control and diabetic muscles in the presence of CPA while the force developed by the diabetic muscles was substantially greater, again highlights that either the sensitivity of the contractile apparatus to [Ca21]i or the [Ca21]i distribution are likely to be altered in the diabetic muscle. In conclusion, the results indicate that there is no major impairment of either intracellular Ca21 homeostasis or contractile function in rat urinary bladder smooth muscle after 8 weeks of STZ-induced diabetes. Nonetheless, some alterations in the smooth muscle function were evident. An apparently non-specific enhancement of force generation was observed, which was surprisingly not accompanied by a corresponding enhancement of the stimulus-induced increases in [Ca21]i. This implies that the apparent [Ca21]i sensitivity of contraction is enhanced in the diabetic smooth muscle.
[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
FIG. 7. Force (upper panel) and suprabasal [Ca21]i (lower panel) in control and diabetic urinary bladder smooth muscle treated with 10 mM CPA and exposed to varying concentrations of extracellular Ca21.
Subtle changes in the Ca21 mobilizing pathways may also occur with the diabetic muscles exhibiting a significantly greater nifedipine-resistant component of contraction. The increased force generation of the smooth muscle from the diabetic animals may represent a compensatory mechanism to allow sufficient generation of wall tension and pressure in the substantially enlarged bladders of these animals. REFERENCES
1. Buck, A. C., Reed, P. I., Siddiq, Y. K., Chisholm, G. D. and Fraser, T. R.: Bladder dysfunction and neuropathy in diabetes. Diabetologia, 12: 251, 1976. 2. Buck, A. C., McRae, C. U., Reed, P. I. and Chisholm, G. D.: The diabetic bladder. Proc R Soc Med, 67: 81, 1974. 3. Ellenberg, M.: Development of urinary bladder dysfunction in diabetes mellitus. Ann Int Med, 92: 321, 1980. 4. Andersson, P. O., Malmgren, A. and Uvelius, B.: Cystometrical and in vitro evaluation of urinary bladder function in rats with streptozotocin-induced diabetes. J Urol, 139: 1359, 1988. 5. Eika, B., Levin, R. M. and Longhurst, P. A.: Comparison of urinary bladder function in rats with hereditary diabetes insipidus, streptozotocin-induced diabetes mellitus, and nondiabetic osmotic diuresis. J Urol, 151: 496, 1994.
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6. Santicioli, P., Gamse, R., Maggi, C. A. and Meli, A.: Cystometric changes in the early phase of streptozotocin-induced diabetes in rats: evidence for sensory changes not correlated to diabetic neuropathy. Naunyn-Schmiedeberg’s Arch Pharmacol, 335: 580, 1987. 7. Steers, W. D., Mackway-Gerardi, A. M., Ciambotti, J. and de Groat, W. C.: Alterations in neural pathways to the urinary bladder of the rat in response to streptozotocin-induced diabetes. J Auton Nervous Sys, 47: 83, 1994. 8. Longhurst, P. A. and Belis, J. A.: Abnormalities of rat bladder contractility in streptozotocin-induced diabetes mellitus. J Pharmacol Exp Ther, 238: 773, 1986. 9. Kolta, M. G., Wallace, L. J. and Gerald, M. C.: Streptozocininduced diabetes affects rat urinary bladder response to autonomic agents. Diabetes, 34: 917, 1985. 10. Malmgren, A., Andersson, P. O. and Uvelius, B.: Bladder function in rats with short- and long-term diabetes; effects of age and muscarinic blockade. J Urol, 142: 1608, 1989. 11. Tammela, T. L. J., Briscoe, J. A. K., Levin, R. M. and Longhurst, P. A.: Factors underlying the increased sensitivity to field stimulation of urinary bladder strips from streptozotocininduced diabetic rats. Br J Pharmacol, 113: 195, 1994. 12. Steers, W. D., Mackway, A. M., Ciambotti, J. and de Groat, W. C.: Effects of streptozotocin-induced diabetes on bladder function in the rat. J Urol, 143: 1032, 1990. 13. Malmgren, A., Uvelius, B., Andersson, K.-E. and Andersson, P-O.: Urinary bladder function in rats with hereditary diabetes insipidus; a cystometrical and in vitro evaluation. J Urol, 148: 930, 1992. 14. Levy, J., Gavin, J. R. and Sowers, J. R.: Diabetes Mellitus: A disease of abnormal cellular calcium metabolism? Am J Med, 96: 260, 1994. 15. Munro, D. D. and Wendt, I. R.: Effects of cyclopiazonic acid on [Ca21]i and contraction in rat urinary bladder smooth muscle. Cell Calcium, 15: 369, 1994. 16. Qi J., Curley, R. M. and Belis, J. A.: Cytosol-free calcium concentration in single bladder smooth muscle cells from normal and diabetic rats. Pharmacology, 45: 90, 1992. 17. Hashitani, H. and Suzuki, H.: Altered electrical properties of bladder smooth muscle in streptozotocin-induced diabetic rats. Br J Urol, 77: 798, 1996. 18. Kudlacz, E. M., Chun, A. L., Skau, K. A., Gerald, M. C. and Wallace, L. J.: Diabetes and diuretic-induced alterations in function of rat urinary bladder. Diabetes, 37: 949, 1988. 19. Longhurst, P. A., Kang, J., Wein, A. J. and Levin, R. M.: Lengthtension relationship of urinary bladder strips from streptozotocin-diabetic rats. Pharmacology, 40: 110, 1990. 20. Lincoln, J., Haven, A. J., Sawyer, M. and Burnstock, G.: The smooth muscle of rat bladder in the early stages of streptozotocin-induced diabetes. Br J Urol, 56: 24, 1984. 21. Luheshi, G. N. and Zar, M. A.: The effect of streptozotocininduced diabetes on cholinergic motor transmission in the rat urinary bladder. Br J Pharmacol, 103: 1657, 1991. 22. Tammela, T. L. J., Leggett, R. E., Levin, R. M. and Longhurst, P. A.: Temporal changes in micturition and bladder contractility after sucrose diuresis and streptozotocin-induced diabetes mellitus in rats. J Urol, 153: 2014, 1995. 23. Turner, W. and Brading, A.: Smooth muscle of the bladder in the normal and diseased state: pathophysiology, diagnosis and treatment. Pharmac Ther, 75: 77, 1997. 24. Munro, D. D. and Wendt, I. R.: Contractile and metabolic properties of longitudinal smooth muscle from rat urinary bladder and the effects of aging. J Urol, 150: 529, 1993. 25. Wahba, Z. Z., Soliman, K. F. A. and Kolta, M. G.: Effects of diabetes on the cholinergic enzyme activities of the urinary bladder and seminal vesicles of the rat. Exp Clin Endocrinol, 99: 26, 1992. 26. Kudlacz, E. M., Gerald, M. C. and Wallace, L. J.: Effects of diabetes and diuresis on contraction and relaxation mechanisms in rat urinary bladder. Diabetes, 38: 278, 1989. 27. Luheshi, G. and Zar, M.: Inhibitory effect of streptozotocininduced diabetes on non-cholinergic motor transmission in rat detrusor and its prevention by sorbinil. Br J Pharmacol, 101: 411, 1990. 28. Belai, A., Lefebvre, R. A. and Burnstock, G.: Motor activity and neurotransmitter release in the gastric fundus of streptozotocindiabetic rats. Eur J Pharmacol, 194: 225, 1991.
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29. White, R. E. and Carrier, G. O.: Vascular contraction induced by activation of membrane calcium ion channels is enhanced in streptozotocin-diabetes. J Pharmacol Exp Ther, 253: 1057, 1990. 30. Sakai, Y., Aihara, K., Honda, H. and Inazu, M.: Calcium mobilization and phosphatidylinositol turnover in vas deferens smooth muscle of diabetic rats. Eur J Pharmacol, 162: 475, 1989. 31. Kamata, K., Inoue, K. and Kasuya, Y.: Changes in muscarinic responsiveness, muscarinic receptor density and Ca21 mobilization of the urinary bladder in streptozotocin-induced diabetic rats. Res Commun Chem Pathol Pharmacol, 75: 143, 1992.
32. Somlyo, A. P. and Somlyo, A. V.: Signal transduction and regulation in smooth muscle. Nature, 372: 231, 1994. 33. Karaki, H., Ozaki, H., Hori, M., Mitsui-Saito, M., Amano, K.-I., Harada, K.-I., Miyamoto, S., Nakazawa, H., Won, K.-J. and Sato, K.: Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev, 49: 157, 1997. 34. Abebe, W. and MacLeod, K. M.: Protein kinase C-mediated contractile responses of arteries from diabetic rats. Br J Pharmacol, 101: 465, 1990. 35. Damaser, M. S., Kim, K-B., Longhurst, P. A., Wein, A. J. and Levin, R. M.: Calcium regulation of urinary bladder function. J Urol, 157: 732, 1997. 36. Berridge, M.: Capacitative calcium entry. Biochem J, 312: 1, 1995.
Vol. 163, 323–330, January 2000 Printed in U.S.A.
EFFECTS OF STREPTOZOTOCIN-INDUCED DIABETES MELLITUS ON INTRACELLULAR CALCIUM AND CONTRACTION OF LONGITUDINAL SMOOTH MUSCLE FROM RAT URINARY BLADDER J. V. WARING*
AND
I. R. WENDT
From the Department of Physiology, Monash University, Clayton, Victoria, Australia
ABSTRACT
Purpose: To examine the effect of diabetes on [Ca21]i and contractility in longitudinal smooth muscle of the urinary bladder. Materials and Methods: Longitudinal smooth muscle strips were isolated from the urinary bladders of rats with STZ-induced diabetes as well as age matched controls. Force and [Ca21]i were measured simultaneously in muscle strips loaded with the calcium indicator, fura-2. Contractions were initiated by electrical field stimulation (EFS) at various frequencies, as well as by high K1, carbachol (CCh) and cyclopiazonic acid (CPA) in the presence of varying concentrations of extracellular Ca21. Results: In unstimulated muscles, there was no significant difference in resting [Ca21]i between the control and diabetic groups. However, the muscle strips from the diabetic animals produced higher force levels in response to EFS, high K1, CCh and CPA than those from control animals. The higher force development in the diabetic muscles was not associated with greater increases in [Ca21]i, which in fact tended to be lower during stimulation in the diabetic tissues. When stimulated by CCh in the presence of nifedipine, both control and diabetic muscles exhibited a nifedipine-resistant component of contraction, however, this was significantly larger in the diabetic muscles. Conclusion: The results suggest that there are no major impairments in either intracellular calcium regulation or contractile function in bladder smooth muscle after 8-weeks of STZ-induced diabetes. However, a non-specific enhancement of force production was seen, which was not associated with increases in [Ca21]i. These changes imply that the apparent sensitivity to [Ca21]i is enhanced in bladder smooth muscle from diabetic rats. KEY WORDS: intracellular calcium, contraction, smooth muscle, diabetes mellitus, streptozotocin
Urinary bladder disturbances are found in long-term diabetes mellitus. It has been suggested that the condition remains asymptomatic for a significant period of time.1–3 The most common characteristics of bladder dysfunction in animal models of diabetes are increases in distension and bladder capacity.4 – 6 The presence of residual urine is also a manifestation of the condition especially in the later stages,2, 3 which suggests that there may be some compromise of the smooth muscle contraction. Whilst it is generally accepted that neuropathy plays a role in this organ’s impairment2, 7 there are reports to suggest that alterations may also exist at the level of the bladder smooth muscle itself.8 These include changes in postsynaptic muscarinic receptor function9, 10 and possible changes in intracellular second messenger systems.11 The possibility that there may be a myogenic component to diabetic bladder dysfunction has led to several studies of bladder smooth muscle contractility in various animal models of diabetes. The results of these studies are often conflicting. Some report that contractile responses of the diabetic bladder, especially to muscarinic agonists, are enhanced5, 9, 11, 12 while others suggest that the responses to a variety of contractile stimuli are diminished.6, 8, 13 These apparent discrepancies may be due, at least in part, to the different animal models and duration of diabetes employed as well as the data normalization pro-
cedures applied. Despite the variability in the results of these studies, it seems likely that contractile function of urinary bladder smooth muscle is altered in diabetes. Little is known, however, of the possible mechanisms that might underlie these alterations. Over recent years there has been considerable interest in the possibility that altered intracellular calcium regulation may be an underlying abnormality in many complications of diabetes mellitus.14 Indeed many studies have reported that the intracellular calcium concentration ([Ca21]i) is elevated in diabetes in a wide variety of cell types.14 Given that [Ca21]i plays a pivotal role in regulating the contractile activity of smooth muscle, it may be that altered contractile function of urinary bladder smooth muscle in diabetes is secondary to disturbances of [Ca21]i handling and/or homeostasis. This study has attempted to address this issue by measuring simultaneously [Ca21]i and contractile responses in urinary bladder smooth muscle of control and streptozotocininduced diabetic rats, the most commonly used animal model of diabetes mellitus. MATERIALS AND METHODS
Experimental animals. Diabetes was induced in male Wistar rats weighing 283 6 11 gm. (n 5 25) 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 initially weight-
Accepted for publication July 22, 1999. * Requests for reprints: Department of Physiology, Monash University, Clayton, Victoria 3168, Australia. Supported by project grant no. 960093 from the National Health and Medical Research Council of Australia. 323
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
matched control animal. Blood glucose concentrations were measured in control and diabetic 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 care and use of animals in scientific research. Tissue dissection. Eight weeks after treatment with STZ, the rats were killed by chloroform anaesthesia and decapitation. The bladder was removed and placed in carbogenated Krebs-Henseleit solution at room temperature. A longitudinal incision was made in the bladder and any residual urine washed out to allow for the weighing of the empty organ. The tissue was then pinned out in a dissecting dish with the mucosal surface uppermost. The mucosa and 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 muscle strips from control animals (n 5 24) had a length of 7.0 6 0.3 mm. and weighed 1.2 6 0.1 mg. while in the STZ treated animals (n 5 25) they were 8.2 6 0.3 mm. long and weighed 1.7 6 0.2 mg. The cross-sectional area, as determined by the length and mass, and assuming a density of 1.0 mg./mm.3, was 0.18 6 0.01 mm.2 and 0.21 6 0.02 mm.2 for the control and diabetic muscle strips respectively. Force measurements. In most instances two muscle strips were dissected, one of which was used for simultaneous measurement of [Ca21]i and force whilst the other was used for contractility studies only. Contractility studies were carried out on this second strip during the time the first strip was being loaded with the Ca21 indicator, fura-2 (see below). This allowed a greater range of studies to be carried out and confirmed that the contractile behavior of the fura-2 loaded muscle was not altered by the dye loading procedure. For the contractility only studies, 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. The muscle was immersed in a tissue bath containing 30 ml. of carbogenated Krebs-Henseleit solution. In the experiments where force and [Ca21]i were monitored simultaneously, the muscle was mounted in a 3 ml. tissue bath and superfused continuously with carbogenated KrebsHenseleit solution at a flow rate of 30 ml./min. The muscle strip was attached 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. Once attached to the transducer the muscles were stretched to a resting tension of 1 gm. and allowed to equilibrate in nominally Ca21-free solution for 15 minutes. Intracellular calcium measurements. [Ca21]i was monitored using the Ca21-sensitive fluorescent dye fura-2. The fluorescence of the tissue was recorded using a spectrophotometer system (Cairn Research Limited, Faversham, Kent, UK) combined with an inverted fluorescence microscope (Olympus IMT-2, Tokyo, Japan). The tissue bath, which had a coverslip as its base, was located on the microscope stage and the muscle strip, mounted on the force recording apparatus, was positioned to lie over the microscope objective, thus allowing the simultaneous recording of force and tissue fluorescence. 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 mM of the acetoxymethyl ester form of the indicator (fura-2/AM) for 3
hours at room temperature (approx. 21 C). Following the incubation, the preparations were washed for 15 min. with Ca21-free Krebs-Henseleit solution. The tissue 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 340 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 [Ca21]i. At the conclusion of each experiment, an internal Ca21 calibration was performed as previously described15 by exposing the tissues to the Ca21 ionophore, ionomyocin and measuring the Rmin and the Rmax in Ca21-free solution containing 3 mM EGTA and 1.5 mM Ca21 solution respectively. [Ca21]i was subsequently calculated from the experimental R340/380 assuming the KD of fura-2 for Ca21 to be 224 nM. Solutions and chemical agents. The Krebs-Henseleit solution was prepared nominally Ca21-free and had the following composition (mM): NaCl 118; KCl 4.75; MgSO4 1.18; KH2PO4 1.18; NaHCO3 24.8; glucose 10. CaCl2 was added to this to achieve the desired solution Ca21 concentration. The solution was continuously aerated with 95% O2-5% CO2 and had a pH of 7.35. It was maintained at 32 C 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 37 C to 32 C. High K1 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-[2hydroxyethyl]piperazine-N9-[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. Fura-2/AM and Pluronic F127 were obtained from Molecular Probes (Eugene, OR, USA) while carbachol (CCh), cyclopiazonic acid (CPA), ionomycin and nifedipine were from Sigma (St. Louis, MO, USA). Concentrated stock solutions of CCh (10 mM in distilled water), CPA (10 mM in DMSO) and nifedipine (10 mM in DMSO) were prepared and appropriate aliquots of these were added to the experimental solutions to obtain the desired final concentrations of these agents. Experimental protocol. Following the initial equilibration period in nominally Ca21-free solution, each muscle was then exposed to Krebs-Henseleit solution containing 2.5 mM Ca21. They remained in this solution for at least 20 min. to allow the development of any spontaneous activity. Following this, contractile responses were initiated by a variety of stimuli, including electrical field stimulation (EFS), CCh and high K1. For EFS, 10 V, 1 ms square pulses at frequencies ranging from 0.2 to 20 Hz were delivered for 20 s, via two platinum wire electrodes positioned on either side of the muscle. In most cases, responses to CCh and high K1 were elicited in the presence of a range of different solution Ca21 concentrations and each response was preceded by a period in Ca21-free Krebs-Henseleit solution which allowed the response to occur against a consistent (quiescent) baseline for each muscle. Statistics. Data are presented as mean values 6 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
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
t test for unpaired data or 2 way repeated measures analysis of variance; p-values of less than 0.05 were considered significant. RESULTS
Some of the characteristics of the animals used in this study are shown in the table. All control animals showed an increase in body weight while the diabetic animals failed to gain weight over the seven to eight week period following STZ injection. Observation of the diabetic animals clearly indicated polyuria and polydipsia and the blood glucose levels in this group were significantly elevated. Bladder weight in absolute terms was increased in the diabetic rats by approximately two times that of the controls. This tissue hypertrophy was further emphasized when the bladder weight was expressed relative to the body weight which revealed a four fold increase over that of the normal animals. Basal intracellular calcium concentration. While in the presence of Ca21-free solution, the resting [Ca21]i of the muscle strips was not different, being 74 6 30 nM (n 5 21) and 74 6 29 nM (n 5 23) in the control and diabetic groups respectively. As shown in figure 1, when 2.5 mM Ca21 was added to the superfusing solution, [Ca21]i increased, the muscles developed tone and, in most cases, spontaneous contractile activity. This occurred in both control and diabetic muscle strips. The average [Ca21]i in the presence of 2.5 mM Ca21-containing solution was 256 6 30 nM (n 5 21) in control muscles and 275 6 29 nM (n 5 23) in the diabetic muscles. These values were not significantly different. There was also no difference in the incidence of spontaneous activity between the control and diabetic muscles, it occurring in approximately 70% of preparations in each group. In addition, there were no apparent differences in the pattern or frequency of spontaneous contractions between the two groups of muscles. Response to electrical field stimulation. Peak force in response to EFS increased in both control and diabetic strips as the frequency of stimulation increased. In absolute terms, the diabetic strips produced more force per unit cross-sectional area than did the control strips. This was most evident at the higher frequencies (figure 2, top panel). However, as shown in the lower panel of figure 2, when the response at each frequency was expressed relative to the response at 20 Hz for each muscle, the diabetic strips actually produced proportionately less force at the lower frequencies than did the control strips. Response to carbachol with graded [Ca21]o. Typical responses of a control muscle strip to stimulation with 10 mM CCh at two different extracellular Ca21 concentrations are shown in figure 3. As can be seen, [Ca21]i rises upon exposure of the muscle to CCh and this is followed by a rise in force. At this reasonably high concentration of CCh, force and [Ca21]i both rise to attain steady levels that are relatively well maintained and the responses are graded with the extracellular Ca21 concentration. The responses of control and diabetic muscle strips were qualitatively similar in this regard. In both groups of muscles responses of this type were initiated at solution Ca21 concentrations ranging from 0.2 to 6.4 mM and the mean force levels and [Ca21]i attained at each [Ca21]o in control and diabetic muscles are presented in figure 4. In absolute terms the diabetic muscles developed higher levels of force at the higher Ca21 concentrations. Analysis of the data using 2 way repeated measures analysis
of variance revealed that the relationship between force and [Ca21]o was significantly different between the two groups. It is notable from figure 4 that [Ca21]i was actually somewhat lower during CCh stimulation in the diabetic muscles as compared to the control muscles; however, statistical analysis of the data failed to reveal a significant difference in [Ca21]i between the control and diabetic groups. Response to high potassium with graded [Ca21]o. Figure 5 shows the force and [Ca21]i in control and diabetic muscles stimulated with 65 mM K1 in the presence of varying concentrations of extracellular Ca21. The results are essentially the same as those obtained with CCh stimulation (fig. 4). Again, the diabetic muscles developed higher levels of force at the higher Ca21 concentrations and, paradoxically, at each level of stimulation [Ca21]i was actually somewhat lower in the diabetic muscles as compared to the control muscles. Effects of nifedipine. In an attempt to elucidate the sources of Ca21 contributing to the contraction, the effects of 3 mM nifedipine on CCh-induced responses was examined. Both control and diabetic muscle strips exhibited a residual response after nifedipine exposure as shown in figure 6. At [Ca21]o concentrations of 1.6 mM and above this nifedipine resistant contraction was significantly larger in the diabetic muscles. Responses to cyclopiazonic acid. Treatment of urinary bladder smooth muscle with CPA, an inhibitor of the sarcoplasmic reticulum Ca21-ATPase, has previously been reported to lead to an influx of Ca21 through a pathway that is not sensitive to nifedipine.15 As shown in figure 7, [Ca21]i increases in CPA-treated muscle as the extracellular Ca21 concentration increases. There were no differences in the CPA-induced increase in [Ca21]i between control and diabetic muscles; however, as shown in the upper panel of figure 7, the diabetic muscles developed significantly more force under these conditions. DISCUSSION
The primary purpose of this study was to investigate the effects of diabetes on [Ca21]i and contractile function of urinary bladder smooth muscle. Previous studies have suggested that [Ca21]i may be elevated in diabetes in a variety of cell types including vascular smooth muscle14 and that this may be an influential factor behind many complications associated with the disease. The results of the present study revealed that [Ca21]i in urinary bladder smooth muscles from diabetic animals showed no difference compared to control tissues strips when the preparations were unstimulated in the presence of either Ca21 free or 2.5 mM Ca21 containing solution. Qi et al16 also found that [Ca21]i in isolated rat bladder smooth muscle cells, was unchanged by diabetes. It would seem that, resting [Ca21]i of urinary bladder smooth muscle is not disturbed by diabetes, at least not following 8 weeks of STZ-induced diabetes in the rat. When placed in Ca21 containing solution, the majority of muscle strips developed tone and spontaneous contractile activity as would be expected from the associated increase in [Ca21]i. There was no apparent difference between the control and diabetic strips in this regard. Tammela et al11 reported that bladder strips from diabetic rats developed more spontaneous activity than those from control animals although they provided no quantitative data in support of this. In contrast, Hashitani et al17 found that diabetic rat detrusor
Body weights, bladder weights and blood glucose levels of control and STZ treated rats Initial Body Weight (gm.)
Final Body Weight (gm.)
Bladder Weight (mg.)
Bladder Weight/Body Weight (gm./kg.)
Control (n 5 24) 296 6 7 482 6 13 120 6 4 STZ (n 5 25) 283 6 11 289 6 10§ 265 6 10§ § Significantly different from corresponding values in control rats (p ,0.05, independent two-tailed t test).
0.25 6 0.01 0.95 6 0.06§
Blood Glucose (mmol./l.) 5.5 6 0.4 23.0 6 1.2§
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
FIG. 1. Original recordings of force and [Ca21]i illustrating effect of addition of 2.5 mM Ca21 to solution superfusing on unstimulated control muscle strip. In 2.5 mM Ca21 solution, muscle developed tone and exhibited spontaneous activity.
smooth muscle exhibited fewer spontaneous spike discharges than control muscles. Spontaneous contractile activity is notoriously difficult to quantitate and may be influenced by a number of experimental factors such as temperature and degree of muscle stretch. The present study found no evidence for a difference in spontaneous activity between control and diabetic bladder smooth muscle. The muscle strips from the diabetic animals developed higher force levels in response to each mode of stimulation when compared to control. This is consistent with a number of other reports of enhanced contractile responses in diabetic rat bladder.5, 9, 11, 12, 18 –20 It is important to note, though, that there is no universal agreement on the effects of diabetes on bladder smooth muscle contractility and some studies have also found diminished contractility.6, 8, 13 The urinary bladder from the diabetic animals exhibited clear hypertrophy as consistently reported in other studies from diabetic animals,5, 9, 11, 12, 18 –20 as well as in other models of high volume urine production such as sucrose feeding.5, 18 It has been suggested that some of the discrepancies in the literature on the effects of diabetes on bladder smooth muscle contractility may be attributable to whether or not contractile force data has been appropriately normalized.19 The control and diabetic muscle strips used in the present study had similar dimensions and all force data were normalized for the crosssectional area of the muscles. The increased force responses of the diabetic muscles are not, therefore, merely a consequence of the increased muscle mass due to the hypertrophy, but reflect an inherent change in the smooth muscle. The diabetic muscle strips exhibited higher absolute levels of force when stimulated with EFS, particularly at the higher frequencies. This has also been reported by others.11, 21, 22 The response of these longitudinal muscle strips to EFS is neurogenic in origin and involves both cholinergic and purinergic components.23, 24 The purinergic component has been suggested to contribute largely to the initial phasic portion of the response while the cholinergic component predominates in the tonic portion and is thought to be primarily responsible for bladder emptying.23 The purinergic component may, therefore, be proportionately greater at low frequencies of stimulation. Changes in cholinergic enzyme activities have been reported in the urinary bladder of rats after 14 days of STZ-induced diabetes25 and it has been postulated that the increased responsiveness of bladder strips from chronically diabetic rats could represent a form of denervation supersen-
FIG. 2. Peak force generation of muscle strips from control and diabetic rats in response to electrical field stimulation at different frequencies. At frequencies of 2 Hz and higher, peak force generally occurred early with some subsequent decay of force. At frequencies below 1 Hz, there were oscillations in force and peak level was attained more slowly with generally little decay over 20 sec period of stimulation. Upper panel shows absolute force values (mN/mm2) while in lower panel force is expressed as percentage of response at 20 Hz.
sitivity, possibly related to diabetic neuropathy.11 However, the enhanced response to EFS is evident as early as 7 days after induction of diabetes which would seem to make diabetic neuropathy unlikely as a causative factor.12, 22, 26 Luheshi and Zar21, 27 have suggested that bladder strips from diabetic rats have a reduced non-adrenergic non-cholinergic response and an increased cholinergic response, and that the latter is due to enhanced release of acetylcholine. In contrast, enhanced purinergic responsiveness has been observed in gastric fundus of diabetic rats with ATP release reported to be increased three-fold.28 In the present study the increased response, in absolute terms, of the diabetic strips to EFS was most evident at the higher stimulation frequencies which would seem to be consistent with an enhanced cholinergic component. Interestingly, when the responses were expressed relative to the maximum response at 20 Hz stimulation it was evident that, proportionately, the responses of the diabetics at frequencies lower than this (particularly in the range 2–10 Hz) were in fact less than those of the controls
[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
327
FIG. 3. Typical original traces of simultaneously recorded force and [Ca21]i responses evoked by 10 mM CCh in control (upper panel) and diabetic (lower panel) urinary bladder muscle strip in presence of either 0.8 or 3.2 mM Ca21.
resulting in the relationship between stimulation frequency and response (as a percent of maximum) being altered. Further studies are required to elucidate the underlying basis of this; however, it does suggest that there may be some subtle changes in the motor transmission. Perhaps the most striking observation made in the present study, was that the higher forces developed by the diabetic muscle strips in response to CCh, high K1 and CPA were not accompanied by similar enhanced increases in [Ca21]i. Indeed, [Ca21]i during stimulation with each of these agents, tended to be lower in the diabetic tissues than the controls. White et al29 have demonstrated enhanced responsiveness of vascular smooth muscle from diabetic rats to extracellular Ca21 in the presence of norepinephrine or KCl. Although they did not measure [Ca21]i, they proposed that the enhanced vascular contractions in STZ-diabetes resulted from increased Ca21 entry due to enhanced activity and/or number of Ca21 ion channels in the vascular smooth muscle membrane. It has similarly been speculated that enhanced Ca21 entry underlies the increased responsiveness of both vas deferens30 and urinary bladder31 smooth muscle in STZdiabetes. The present experiments clearly show, however, that the increased contractile responses of diabetic bladder smooth muscle are not associated with elevated levels of [Ca21]i. There appear to be two main possibilities that could account for this observation. One is that there has been some change in the sensitivity of the contractile apparatus to Ca21 in the diabetic tissues. There has been considerable interest over recent years in factors that can modify the apparent Ca21 sensitivity of the contractile apparatus in smooth mus-
FIG. 4. Force generation (upper panel) and associated increases in suprabasal [Ca21]i (lower panel) in control and diabetic urinary bladder smooth muscle stimulated with 10 mM CCh in presence of varying concentrations of extracellular Ca21.
cles and “Ca21-sensitisation” appears to be an important element in the physiological regulation of the contractile state of smooth muscle.32 Within this context, protein kinase C (PKC) has been suggested to be an important mediator of increases in Ca21 sensitivity in smooth muscle since activation of PKC has been shown to enhance contraction in a variety of smooth muscles in the absence of proportional changes in [Ca21]i.32, 33 It has been reported that the contractile responses of vascular smooth muscle to phorbol esters, which activate PKC, are enhanced in diabetes.29, 34 This would be consistent with an augmentation of the mechanisms mediating increases in Ca21 sensitivity in diabetic smooth muscle. The increased contractile responsiveness of diabetic urinary bladder reported in the present study, as well as in previous studies,5, 9, 11, 12, 18 –20 appears to be of a rather non-specific nature, making it unlikely to be the result of changes in specific receptors. An increase in the Ca21 sensitivity of the contractile apparatus would be expected to lead to a non-specific increase in contractile responsiveness. It is noteworthy that the enhanced force responses of the diabetic muscles to CCh and K1 are most evident at higher concentrations of Ca21 and this may indicate a more complex situation than just a general increase in Ca21 sensitivity in the diabetic muscles. It is important to recognize that the
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[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
FIG. 6. Force generated by control and diabetic bladder smooth muscle strips in response to 10 mM CCh in presence of varying extracellular Ca21 concentrations before and after addition of 3 mM nifedipine. Force has been expressed relative to response in 6.4 mM Ca21 in absence of nifedipine for each muscle.
FIG. 5. Force generation (upper panel) and associated increases in suprabasal [Ca21]i (lower panel) in control and diabetic urinary bladder smooth muscle stimulated with 65 mM K1 in presence of varying concentrations of extracellular Ca21.
[Ca21]i determined with fura-2 in these experiments represents the spatially averaged [Ca21]i in the cells within the recording field. Another possible explanation for the increased force of the diabetic muscles in the presence of an apparently unchanged, or even lower, spatially averaged [Ca21]i is that it reflects an altered intracellular distribution of Ca21. There is evidence that cytoplasmic Ca21 in smooth muscle cells may be distributed between a non-contractile compartment and a contractile compartment with only the latter contributing to contractile activation.33 Regulation of intracellular Ca21 movements by the peripheral SR is likely to be important in determining the intracellular Ca21 distribution.33 In this light, it is interesting that in the CPA treated tissue, where Ca21 uptake by the SR is inhibited and internal Ca21 stores become depleted, the enhanced force development of the diabetic tissues is evident at much lower extracellular Ca21 concentrations than in CCh or high K1 stimulated muscles. It may be that access of Ca21 to the contractile compartment is greater in the diabetic muscles and that this is facilitated further with CPA treatment. As with most smooth muscles, a significant proportion of the Ca21 that enters bladder smooth muscle during stimulation is believed to enter through voltage operated calcium channels (VOCCs).35 Nifedipine was utilized in an attempt to ascertain
the contribution of Ca21 entry through VOCCs during the contractile response of the control and diabetic muscles to CCh. Tissues from both the control and diabetic animals, exhibited nifedipine-resistant contractions, that were most evident at high [Ca21]o. The nifedipine-resistant component of the contractions was significantly larger in the diabetic muscles, suggesting that they may be less reliant on Ca21 entry through VOCCs. This observation is supported by work from Sakai et al30 showing that vas deferens smooth muscle from diabetic rats exhibits a greater VOCC-independent Ca21 uptake than control muscles. Kamata et al31 have also suggested that the enhanced response to acetylcholine in diabetic bladder smooth muscle is due to an increased Ca21 influx through channels other than VOCCs. Depletion of intracellular calcium stores, by CPA or thapsigargin for example, has been shown to activate Ca21 entry in a variety of non-excitable cells and smooth muscles, including bladder smooth muscle.15, 33, 36 Treatment of both control and diabetic muscle strips with CPA led to Ca21 entry and the development of force, indicating that this store release-activated Ca21 entry pathway is functional in diabetic as well as control muscle strips. The greater force response of the diabetic muscle to CPA could be consistent with the greater nifedipine-resistant component of the contraction of these muscles to CCh. Although it must be remembered that the increase in [Ca21]i induced by CPA was not significantly different between control and diabetic muscles. The fact that [Ca21]i was similar in control and diabetic muscles in the presence of CPA while the force developed by the diabetic muscles was substantially greater, again highlights that either the sensitivity of the contractile apparatus to [Ca21]i or the [Ca21]i distribution are likely to be altered in the diabetic muscle. In conclusion, the results indicate that there is no major impairment of either intracellular Ca21 homeostasis or contractile function in rat urinary bladder smooth muscle after 8 weeks of STZ-induced diabetes. Nonetheless, some alterations in the smooth muscle function were evident. An apparently non-specific enhancement of force generation was observed, which was surprisingly not accompanied by a corresponding enhancement of the stimulus-induced increases in [Ca21]i. This implies that the apparent [Ca21]i sensitivity of contraction is enhanced in the diabetic smooth muscle.
[Ca21]i AND CONTRACTION IN DIABETIC BLADDER SMOOTH MUSCLE
FIG. 7. Force (upper panel) and suprabasal [Ca21]i (lower panel) in control and diabetic urinary bladder smooth muscle treated with 10 mM CPA and exposed to varying concentrations of extracellular Ca21.
Subtle changes in the Ca21 mobilizing pathways may also occur with the diabetic muscles exhibiting a significantly greater nifedipine-resistant component of contraction. The increased force generation of the smooth muscle from the diabetic animals may represent a compensatory mechanism to allow sufficient generation of wall tension and pressure in the substantially enlarged bladders of these animals. REFERENCES
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