Metabolic and Contractile Effects of Anoxia on the Rabbit Urinary Bladder

0022-534 7/82/1281-0194$02.00/0

THE

Vol. 128, July

JOURNAL OF UROLOGY

Printed in U.S.A.

Copyright© 1981 by The Williams & Wilkins Co.

METABOLIC AND CONTRACTILE EFFECTS OF ANOXIA ON THE RABBIT URINARY BLADDER ROBERT M. LEVIN,* JANICE HIGH

AND

ALAN J. WEIN

From the Division of Urology, Department of Surgery, University of Pennsylvania School of Medicine, and the Veterans Administration Medical Center, Philadelphia, Pennsylvania

ABSTRACT

The in vitro metabolic and contractile effects of anoxia and glucose deprivation on the rabbit urinary bladder were studied. The exposure of isolated strips of rabbit urinary bladder to a glucose deficient medium equilibrated with nitrogen rather than with oxygen resulted in 1) a rapid decrease in baseline tension, 2) a progressive decrease in the intracellular concentration of adenosine 5' triphosphate (ATP) and progressive increases in the concentrations of adenosine 5'-monophosphate and adenosine 5' -diphosphate, 3) a rapid decrease in the ability of bethanechol to stimulate contractility, and 4) the inability of bethanechol to maintain a sustained increase in contractile force. Recovery from a 60-minute exposure of bladder strips to anoxia was characterized by a rapid recovery of the ability of the bladder to respond to bethanechol (50 per cent recovery occurring within 15 minutes) and a slightly slower recovery of the intracellular ATP level. Both the contractile response to bethanechol and the intracellular concentration of ATP returned to control levels within 60 minutes after the termination of anoxia. The proper functioning of the urinary bladder is dependent on the delivery of a normal supply of blood and nutrients to the tissue, and the partial or complete loss of blood flow is expected to have serious effects on bladder function. Clinically, blood flow to the bladder can be compromised by several mechanisms, including overdistention, atherosclerosis and arterial obstruction. In the case of overdistention, blood flow to the tissue has been shown to decrease progressively as the intravesical pressure increases. 1• 2 With bladder outlet obstruction, the bladder wall is subject to prolonged periods of overdistention, which is expected to seriously compromise bladder circulation, One of the primary consequences of reduced blood flow would be bladder hypoxia with a concomitant reduction in high energy phosphates, which are required for proper bladder smooth muscle contraction. 3 The bulk of our knowledge concerning the response of smooth muscle to hypoxia and anoxia comes from metabolic studies on vascular smooth muscle. 4- 6 Very few metabolic studies have been performed on the smooth muscle of the urinary bladder.1-9 The purpose of our study was to determine both the in vitro sensitivity of a bladder smooth muscle to anoxia and its rate of recovery from anoxia. MATERllALS AND METHODS

Muscle bath studies. The urinary bladders of 28 male New Zealand White rabbits were removed under Ketamine-Xylazine anesthesia. The bladder was rapidly dissected free of fat, and the body and base were separated at the level of the ureteral orifices. The bladder body was divided into 4 longitudinal strips of equal size, which were placed in individual muscle baths containing 30 ml. of Tyrode's solution containing glucose (1 mg./ml.) and equilibrated with a gas mixture of 95 per cent 0 2 and 5 per cent CO2, After 20 minutes of equilibration, 1 gm. of tension was placed on each strip, and the strips were maintained in the oxygenated buffer for an additional 10 minutes. At the end of the equilibration period the medium in 2 of the 4 Accepted for publication December 7, 1981. Supported in part by grants from the Veterans Administration, grant lROl-AM-26508-01 from the National Institutes of Health and the McCabe Fund. * Requests for reprints: 3010 Ravdin Courtyard Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Pennsylvania 19104.

chambers was changed to either Tyrode's solution containing no glucose and preequilibrated with a gas mixture of 95 per cent N and 5 per cent CO2, or oxygenated, glucose free medium. The tissues were maintained in the experimental buffer for variable periods of time. The medium in the remaining 2 chambers was not changed. At the end of the experimental procedure either the mechanical response of the tissues to bethanechol chloride (1 mM) was determined or the tissues were removed rapidly from the baths and frozen in liquid nitrogen for subsequent adenine nucleotide determinations. Recovery experiments were performed as follows. Isolated muscle strips of bladder body were equilibrated with oxygenated buffer containing glucose for 30 minutes. The medium was then changed to the anoxic buffer without glucose for 60 minutes. Following the 60-minute period of anoxia, the medium was again changed to the oxygenated buffer with glucose, and the tissues were equilibrated for variable periods. At the end of the recovery period the tissues were either stimulated by the addition of bethanechol or frozen for adenine nucleotide determinations. Metabolic studies, The frozen tissue was homogenized rapidly in 0.625 M perchloroacetic acid (50 mg./ml.) and centrifuged at 5000 X g for 20 minutes. The pellet was dissolved in 1 ml. 1 N NaOH. The supernatant was neutralized with half its volume of a mixture containing 2 parts 1.625 M K2C03 and 3 parts 0.5 M glycylglycine buffer. The neutralized supernatant was assayed for adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP), with the use of the luciferin-luciferase reaction, 10 The protein concentration of the dissolved pellet was determined by the method of Lowry and associates. 11 Statistical significance was determined by analysis of variance. RESULTS

Time course for the mechanical effects of anoxia. Figure 1 shows a representative tracing of the contractile response of strips of bladder body to bethanechol after 15 minutes of anoxia (in the absence of glucose), as well as a tracing obtained in the absence of anoxia. Anoxia produced an immediate, rapid decrease in basal tension. After 15 minutes bethanechol produced only a slight contraction compared with that observed in the absence of anoxia.

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utes in the absence of but in the presence of oxygen. At the end of this period the were either stimulated by the addition of bethanechol or frozen for adenine nucleotide determinations. Sixty minutes of glucose deprivation had no effect on either the mechanical response of the tissue to bethanecol (data not shown) or the intracellular concentration of adenine nucleotides (ATP, 13.2 ± 3 nmol./mg protein; ADP and AMP, 3.26 ± 0.5 nmol./mg protein). Recovery from anoxia. The recovery of the mechanical response to bethanechol after 60 minutes of anoxia is shown in figure 5. The contractile response returned to the control level over a 30-minute period. The metabolic recovery of the bladder tissue, as indicated by the intracellular ATP concentration,

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FIG. 3. Effect of anoxia (glucose present) on the sustained contraction produced by bethanechol: a representative tracing from experiments in which the aeration of the incubation medium was changed from 95 per cent 0 2 and 5 per cent CO2 to 95 per cent N and 5 per cent CO2 during a sustained bethanechol contraction. 140

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Fm. 2. Time course of the effect of anoxia on the contractile response to bethanechol: the contractile responses of strips of urinary bladder body to bethanechol mM) before and after various periods of anoxia. Each bar represents mean of 4 to 6 separate determinations. Vertical brackets represent SEM. Responses following 3.75, 7.5 and 15.0 minutes of anoxia are statistically different from the control value (p <0.01).

The progressive decrease in the maximal effect of bethanechol is shown in figure 2. The effectiveness of bethanechol decreased rapidly over a 30-minute period. In a separate experiment, the submaximal stimulation by bethanechol (50 µ.M) was characterized by a rapid increase in tension followed by a prolonged period of increased tension. Rapid change of the aeration from oxygen to nitrogen during the plateau phase produced a rapid fall in tension below the 1-gm. resting tension (fig. 3) (glucose was present throughout). Time course for the metabolic effects of anoxia. Figure 4 displays adenine nucleotide levels following various periods of anoxia. Over a 60-minute period, ATP decreased progressively from a control value of 14.0 nmol./mg. protein to near zero, whereas ADP and AMP increased from 2.2 nmol./mg. protein to more than 9.0 nmol./mg. protein. Effect of glucose deprivation in the absence of anoxia. As a control experiment, bladder strips were incubated for 60 min-

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FIG. 4. Time course of the effect of anoxia on adenine nucleotide levels: adenine nucleotide levels before and after various periods of anoxia. Each bar represents the mean of 5 to 7 separate determinations. Vertical brackets represent SEM. Adenine nucleotide concentrations for 7.5, 15.0, 30.0 and 60.0 minutes of anoxia are significantly different from the control values (p <0.01).

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occurred gradually over a 60-minute period, at the end of which the concentration of ATP was similar to that of the control tissue (fig. 6). DISCUSSION

The proper functioning of the urinary bladder (like that of any smooth muscle structure) depends on the delivery of adequate oxygen and substrate to meet its metabolic needs. The purpose of these studies was to determine the mechanical and metabolic effects of anoxia on the urinary bladder. Like all smooth muscle organs, the bladder is capable of producing ATP via the anaerobic metabolism of glucose. In the presence of anoxia, glycolysis and lactate production would be expected to increase significantly, thus protecting the bladder (to some extent) from the detrimental effects of anoxia on intracellular ATP levels. For this reason (i.e., to maximize the mechanical and metabolic effects of anoxia) glucose was removed from the anoxic medium. Anoxia produced an immediate, rapid decrease in resting tension, possibily by affecting membrane potentials, as described by Hellstrand and associates. 4 The contractile response to bethanechol decreased rapidly over a 30-minute period. A decrease of 50 per cent was observed after 4 minutes of anoxia, 85 per cent after 15 minutes and 100 per cent after 30 minutes. The concentration of ATP decreased by 50 per cent after approximately 15 minutes of anoxia and by 95 per cent after 60 minutes. The response to bethanechol decreased at a faster rate than did the concentrations of ATP; the initial rapid decrease in contractile response may be more a function of the effect of anoxia on basal tension than of decreased ATP. These results are consistent with the data presented by Hellstrand and associates4 on the effects of anoxia on the vascular smooth muscle of the rat. Anoxia completely prevented bethanechol from producing a sustained increase in bladder strip contraction. In addition, switching from oxygenated to anoxic buffer resulted in an immediate ablation of the sustained contraction produced by bethanechol. The observed effect of anoxia on both basal tension and bethanechol-induced contraction may be related, at least in part, to alterations in prostaglandin synthesis and release. Recent evidence demonstrated that alterations in prostaglandins can markedly affect both bladder tone and spontaneous activity.12-15 Additionally, the work ofBultitude and associates 16 and Laekeman and Herman 1 7 indicates that prostaglandins may play a role in modulating cholinergic stimulation of bladder contraction. The rapid decline in both basal tension and bethanechol induced contraction observed in these studies would be consistent with a decrease in prostaglandin synthesis and release that would occur in the presence of anoxia.

Preliminary studies demonstrated that the bladder was capable of rapid recovery following short exposure to anoxia. We used a 60-minute period of anoxia in the recovery experiments because after 60 minutes of anoxia in the absence of glucose the bladder was completely unresponsive to bethanechol and the intracellular concentration of ATP was less than 10 per cent of normal. When the anoxic tissue was placed in oxygenated buffer containing glucose, both the response to bethanechol and the intracellular ATP concentration increased progressively to control levels within 60 minutes. The possibility that the rapid recovery in bethanechol sensitivity is related to increased prostaglandin synthesis and release is presently under investigation. The ability of the urinary bladder to recover from chronic obstruction in vivo is well documented in the literature. 18 Our demonstration of its ability to recovery from 60 mintues of anoxia in vitro reflects the stable nature of the smooth muscle elements of the bladder. The recovery of both the response to bethanechol and the intracellular ATP concentration does not, however, exclude the possibility that cellular damage occurred during the anoxia. In vivo studies of the effects of hypoxia and anoxia on bladder function and cellular integrity are presently being carried out. REFERENCES

L Dunn, M.: A study of the bladder blood flow during distention in rabbits. Br. J. Urol., 47: 67, 1975. 2. Finkbeiner, A. and Lapides, J.: Effect of distention on blood flow in dog's urinary bladder. Invest. Urol., 12: 210, 1974. 3. Bozler, E.: Energetics of smooth muscle contraction. In: The Biochemistry of Smooth Muscle. Edited by N. L. Stephens. Baltimore: University Park Press, p. 3, 1977. 4. Hellstrand, P., Johansson, B. and Norberg, K.: Mechanical, electrical, and biochemical effects of hypoxia and substrate removal on spontaneously active vascular smooth muscle. Acta Physiol. Scand., 100: 69, 1977. 5. Detar, R.: Mechanism of physiological hypoxia-induced depression of vascular smooth muscle contraction. Am. J. Physiol., 238: H761, 1980. 6. Shoji, S. and Briggs, A. H.: Mechanical activity of vascular smooth muscle under anoxia. Am. J. PhysioL, 212: 981, 1967. 7. Rohner, T. J., Komins, J. I. and Schoenberg, H. W.: Utilization and glucose by normal defunctionalized and denervated bladder muscle. Invest. Urol., 5: 12, 1967. 8. Paton, D. M.: Effects of metabolic inhibitors on contraction of rabbit detrusor muscle. Br. J. Pharmacol., 34: 493, 1968. 9. Paton, D. M.: Effects of metabolic inhibitors on contractility of isolated rabbit detrusor muscle. Prof. Int. Union Physiol. Sci., 7: 340, 1968. 10. Lemasters, J. J. and Hackenbrock, C. R.: Firefly luciferase assay for ATP production by mitochondria. Methods Enzymol., 57: 36, 1978. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265, 1951. 12. Abrams, P.H. and Feneley, R. C. L.: The actions of prostaglandins on the smooth muscle of the human urinary tract in vitro. Br. J. Urol., 47: 909, 1976. 13. Andersson, K. E., Ek, A. and Persson, C. G. A.: Effects of prostaglandins on the smooth muscle of the human bladder and urethra. Acta Physiol. Scand., 100: 165, 1977. 14. Hills, N. Ii.: Prostaglandins and tone in isolated strips of mammalian bladder, Proc. B. P. S., April 1-2, p. 465, 1976. 15. Taira, N.: Mode of actions of prostaglandin F2 on the urinary bladder and its arterial bed in the dog. Eur. J. Pharmacol., 29: 30, 1974. 16. Bultitude, M. I., Hills, N. H. and Shuttleworth, K. E. D.: Clinical and experimental studies on the action of prostaglandins and their synthesis inhibitors on detrusor muscle in vitro and in vivo. Br. J. Urol., 48: 631, 1976. 17. Laekeman, G. M. and Herman, A. G.: Prostaglandins restore the hyoscine-induced inhibition of the guinea-pig ileum. Prostaglandins, 15: 829, 1978. 18. Wein, A. J. and Raezer, D. M.: Physiology of micturition. In: Clinical Neuro-Urology. Edited by R. Krane and M. Sirosky. Boston: Little, Brown & Co., p. 1, 1979. 0