Effect of Partial Obstruction of the Rabbit Urinary Bladder on Malate Dehydrogenase and Citrate Synthase Activity

Effect of Partial Obstruction of the Rabbit Urinary Bladder on Malate Dehydrogenase and Citrate Synthase Activity

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0022-534 7 /92/14 75-1391$03.00/0 Vol. 147, 1391-1393,

THE JOURNAL OF UROLOGY AMERICAN UROLOGICAL ASSOCIATION, !NC.

Printed in

Copyright© 1992 by

EFFECT OF PARTIAL OBSTRUCTION OF THE RABBIT URINARY BLADDER ON MALATE DEHYDROGENASE AND CITRATE SYNTHASE ACTIVITY NIELS HAUGAARD,* LISA POTTER, ALAN J. WEIN

AND

ROBERT M. LEVIN

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

ABSTRACT

Previous studies have demonstrated that partial outlet obstruction in rabbits induced a significant decrease in oxidative metabolism in urinary bladder smooth muscle. The current experiments were designed to determine whether the decreased oxidative metabolism of obstructed bladder tissue is associated with alterations in the activities of specific mitochondrial enzymes. The activities of two important enzymes in the tricarboxylic acid cycle, malate dehydrogenase and citrate synthase, were measured in samples of bladder body and base from normal bladders and in bladders from rabbits in which partial outlet obstruction had been produced seven days prior to the experiments. The results can be summarized as follows: malate dehydrogenase activity was similar in. bladder body and base isolated from control rabbits; and decreased by approximately 40% in both segments of the bladder isolated from obstructed rabbits. In contrast to malate dehydrogenase, citrate synthase activity was significantly higher in the bladder body than in the base of normal rabbits. Outlet obstruction caused about a 50% decrease in activity of this enzyme in the bladder body, but had no significant effect on citrate synthase activity of the bladder base. These findings demonstrate that the deficiency in bladder function following partial outlet obstruction is associated with a marked decrease in the activities of two essential enzymes in oxidative metabolism: malate dehydrogenase and citrate synthase. This decrease in enzyme activity is consistent with the previously observed decrease in oxidative metabolism and would be expected to lead to an inability of the tissue to supply sufficient metabolic energy for proper contractile function. KEY WORDS:

bladder, rabbits, mitochondria, enzymes

The ability of the normal bladder to contract and empty is dependent on the availability of preformed ATP as well as on the continuous formation of high energy phosphate bonds by oxidative phosphorylation. 1 · 2 Studies from this laboratory using in vitro preparations have provided strong support for the concept that the initial contractile response to stimulation of the bladder (field stimulation or muscarinic) is mediated by intra-cellular ATP while the sustained contraction during the plateau phase is more directly related to the ability of the tissue to produce energy by oxidation of metabolites. 1 -'1 When outlet obstruction is produced in the experimental animal the bladder undergoes a dramatic increase in bladder mass and bladder function is significantly decreased. 2 • 4 • 0 The ability of bladder tissue to respond to field stimulation is decreased markedly with the maintenance of the contractile state being more strongly depressed than the initial contractile response. 2 • 4 • 0 The functional consequence of this is that the ability of the bladder to empty is decreased to a significantly greater degree than the ability of the bladder to generate pressure. 2 • 4 ' 5 In a previous study, we demonstrated that oxidative metabolism was significantly reduced in bladder tissue obtained from obstructed rabbits as compared to normal bladder tissue. 6 In view of these findings, the current study investigates whether certain key enzymes in oxidative metabolism were decreased in the obstructed bladder. We chose to study malate dehydrogenase, which has important functions in both the cytosol and in Accepted for publication October 30, 1991. *Requests for reprints: Division of Urology, H.U.P., 3400 Spruce Street, 3010 Ravdin Courtyard Bldg., Philadelphia PA 19104. Supported by Grants from the Veterans Administration and NIH Grants R0-1 DK-26508, R0-1 DK 33559, R0-1 DK-39086 and PSODK-39257.

mitochondria, and citrate synthase, a rate-limiting enzyme present only in mitochondria. METHODS

Animals. White New Zealand male rabbits (three to 3.5 kg.) were used in the experiments. Urinary outlet obstruction was produced by the procedure described by Malkowicz et al. 4 Surgery was performed under anesthesia using an initial intramuscular injection of ketamine-xylazine. Surgical anesthesia was maintained by injection of nembutal (25 mg./ml. i.v.) as needed. Each urinary bladder was catheterized with an 8 Fr Foley catheter and the bladder exposed through a midline incision. A 2-0 silk ligature was tied around the bladder neck of the catheterized rabbit to produce a partial outlet obstruction. The catheter was removed and the incision was closed. The animals were euthanized seven days after surgery. Bladders were removed from experimental and control animals, separated into bladder body and base segments, and the sections of tissue were frozen in liquid nitrogen for subsequent enzyme assays. The samples were kept at -84C until used. Tissue homogenates. Sections of frozen tissue weighing about 0.5 µg. were homogenized in ten volumes of 0.225 M mannitol0.075 M fructose using a Polytron homogenizer. The homogenates were filtered through gauze and kept in ice until used for determination of enzyme activity. Measurement of enzyme activity. Malate dehydrogenase activity was determined as described by Bergmeyer and Bernt. 7 In this procedure the disappearance of NADH is measured in a system in which the substrate, oxaloacetate, is generated from aspartate and alpha-keto glutarate by added glutamate-oxaloacetate transaminase. Preliminary studies demonstrated that identical results were obtained when oxaloacetate was added

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FIG. 1. Malate dehydrogenase activity of bladder body and base from control rabbits and from rabbits with seven days partial outlet obstruction. Results are means ± SEM of six to nine individual preparations. * = Significantly different from activity in control bladders, p <0.005.

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FIG. 2. Citrate synthase activity of bladder body and base from control rabbits and from rabbits with seven days partial outlet obstruction. Results are means ± SEM of six to nine individual preparations. t = Significantly different from activity in bladder body, p <0.01. * = Significantly different from activity in control bladders, p <0.01.

directly. The following simplified procedure was used to generate the data presented in the results section. Aliquots of tissue homogenates containing 5 to 10 mg tissue were added to a cuvette (one cm. width) with 2.0 ml. 0.05 M triethanolamine (pH 7.6), 50 µl. 10 mM oxaloacetate (freshly prepared), 100 µl. 1.8 mM NADH and 100 µl. 0.1 M MgCb. The rate of oxidation of NADH was measured by recording the absorbancy at 340 nm. every 0.5 minute. After five minutes 240 µl. of a 10% solution of Triton-X-100 was added and the increased rate of reaction recorded. In all measurements two amounts of tissue were used and the temperature was adjusted to be close to 25C. Citrate synthase was determined as described by Robinson et al. 8 Aliquots of homogenate were added to a cuvette (0.5 cm. width) containing 1.0 ml 0.05 M TRIS buffer (pH 7.6), 100 µl. one mM 5,5' -dithio-bis-2-nitrobenzoic acid (DTNB or Ellmans reagent) and 30 µl. 12.3 mM acetyl-coenzyme A. The free coenzyme A generated reacts with DTNB to form a yellow compound that is measured at 412 nm. After five minutes 120 µl. of 10% Triton-X-100 was added and the increased rate of reaction recorded during a subsequent five minute period of incubation. For both enzyme reactions the rates were completely linear during the period of observation. Preliminary studies were used

to determine the concentrations of reactants required to give the maximum rate of reaction (V max). Triton-X-100 was added to the reaction mixtures to induce lysis of mitochondria in order to fully expose the mitochondrial enzymes to the substrates of the enzyme reactions. With both enzymes the addition of Triton-X-100 caused an increase in measurable enzyme activity. In homogenates from control rabbit bladders malate dehydrogenase activity increased by 34.5% (N=9, p <0.05) and citrate synthase activity was elevated by 41.1 % (N=9, p <0.05). The values in the figures are the enzyme activities in the presence of 1 % Triton-X-100. Protein concentrations were measured by the procedure of Lowry et al. 9 and all results were calculated as nmols/min./mg. tissue and as nmols/min./mg. protein. Statistics. -Statistical. analysis-utilized Analysis -0-f Variance followed by Newman-Keuls post hoc analysis between individual groups. A value ofp <0.05 was used to indicate significance. RESULTS

The effect of bladder outlet obstruction on malate dehydrogenase activity of bladder tissue is presented in figure 1. There was no significant difference in activity of this enzyme between bladder body and bladder base. On the other hand, a significant and substantial decrease in activity of the enzyme in both the bladder body and base following obstruction is clearly demonstrated. Enzyme activity in these experiments was expressed as nmols/min./mg. tissue. When the results were expressed in units of nmols/min./mg. protein identical conclusions were reached. For example, malate dehydrogenase activity of bladder body from control rabbits was 814 ± 75 nmols/min./mg. protein and the activity in bladder base was 802 ± 95 nmols/min./mg. protein showing no difference in enzyme activity between the two sections of the bladder. Urinary outlet obstruction caused a 38 % reduction in malate dehydrogenase activity when activity was expressed per mg. tissue as compared to a 44% decrease when expressed per mg. protein. It is obvious that changes in protein content of the muscle associated with obstruction are of little importance in causing the changes in enzyme activity observed. The results with citrate synthase are presented in figure 2. In contrast to the findings with malate dehydrogenase there was a highly significant difference in citrate synthase activity between the bladder body and base with the bladder body having an enzyme activity about 60% higher than that of the base. Outlet obstruction caused a significant decrease in citrate synthase activity of the bladder body (-48%, p <0.001), but the decrease in activity of the bladder base was not statistically significant. As with malate dehydrogenase, activity was calculated per mg. protein as well as per mg. tissue. Citrate synthase activity was 132.6 ± 11.86 nmols/min./mg. protein in bladder body compared to 74.0 ± 9.82 nmols/min./mg. protein in bladder base again showing the marked difference between the two parts of the bladder. As with malate dehydrogenase the effect of obstruction was essentially the same when activity was expressed per mg. protein as it was when expressed per mg. tissue. DISCUSSION

The bladder, like other smooth muscle, 10- 13 is dependent on an adequate supply of metabolites for normal function. Glucose appears to be a preferred substrate, 14- 17 although fatty acids are also oxidized. 18 Hypolite and coworkers 19 made a systematic study of the ability of various metabolites to maintain contraction of rabbit bladder strips following electrical stimulation. The response of the isolated bladder strip to field stimulation consists of an immediate contractile effect leading to a peak tension followed by a plateau tension lasting for several minutes. The investigation by Hypolite et al. 19 showed that incubation of bladder

MITOCHONDRIAL ENZYME ACTIVITY AND OUTLET OBSTRUCTION

strips in the absence of substrate for up to 160 minutes led to a progressive ioss of the ability of the muscle to maintain tension while the peak tension reached after electrical stimulation was decreased to only a small extent. In the presence of glucose or pyruvate both peak and plateau tensions were well maintained for up to 160 minutes of incubation, in fact the mitochondrial substrate pyruvate maintained the contractile response to field stimulation to a greater degree than did glucose. These results are in full agreement with earlier studies from which it was concluded that the immediate contractile effect of electrical or muscarinic stimulation of the bladder is supported primarily by preformed ATP while the ability to maintain an increased contractile state is dependent on the continuous production of energy by oxidative phosphorylation. That bladder outlet obstruction in rabbits is associated with a marked depression of contractile function has been well demonstrated. 2 Simultaneously there is a significant decrease in the ability of the bladder to oxidize glucose. 6 Production of ischemia by ligating one vesical artery induced a similar decrease in the rate of glucose oxidation by the affected tissue. 20 The studies presented here were designed to establish whether key enzymes involved in the total oxidation of glucose were affected by bladder outlet obstruction. We chose to assay for two such enzymes, malate dehydrogenase, an enzyme existing both in the cytoplasm and in mitochondria, and citrate synthase, an enzyme that exists exclusively in the mitochondrial matrix. 8 Both of these enzymes are essential for the proper function of the tricarboxylic acid cycle and the concomitant formation of high-energy phosphate bonds by oxidative phosphorylation. The activity of these enzymes was high in the bladder homogenates and significantly increased by the addition of the detergent, Triton-X-100, showing that intact mitochondria are present in the preparations. The activity of malate dehydrogenase was much higher in all samples than citrate synthase. It is of considerable interest that citrate synthase, which is entirely mitochondrial in origin, showed a higher activity in bladder body than in base. It is the body of the bladder rather than the base that is primarily involved in the contractile events associated with emptying of the bladder. In contrast to citrate synthase, malate dehydrogenase activity was the same in the two segments of the bladder. Partial obstruction of the rabbit bladder, as produced in these experiments, clearly resulted in a marked depression of the activities of both malate dehydrogenase and citrate synthase. These findings then provide direct evidence that activities of enzymes essential for oxidative phosphorylation are decreased in the obstructed bladder. It is clear that the activity of all cellular enzymes is not decreased following partial outlet obstruction. In a previous the maximum activity of creatine kinase cytosolic enzyme which transfers a phosphate from creatine phosphate to ADP) was not altered by obstruction. 21 In addition, the metabolism of glucose to lactic acid (also regulated by cytosolic enzymes) was increased in obstructed tissues, indicating that there were no significant decreases in any of the rate-limiting enzymes in the glycolytic pathways. 2 • 6 Thus, the decreased enzyme activities observed thus far are specifically those which control mitochondrial function. This observation supports the conclusion that the functional deficiencies observed in the obstructed bladder may be related to decreased mitochondrial function. REFERENCES

L Levin, R M., Brendler, K, Van Arsdalen, KN. and Wein, A. J.: Functional response of the rabbit urinary bladder to anoxia and ischemia. NeurouroL Urodynam., 2: 233, 1983.

2. Levin, RM., Longhurst, P.A., M:onson, F. C., Kato, Kand Wein, A. J.: Effect of bladder outlet obstruction on the morphology, physiology and pharmacology of the bladder. Prostate (Supp.), 3: 9, 1990. 3. Levin, R. M., Ruggieri, M. R, Gill, H. S., Haugaard, N. and Wein, A. J .: Studies on the biphasic nature of urinary bladder contraction and function. NeurouroL Urodynam., 6: 339, 1987. 4. Malkowicz, S. R, Wein, A. J., Elbadawi, A., Van Arsdalen, K, Ruggieri, M. R and Levin, R M.: Acute biochemical and functional alterations in the partially obstructed rabbit urinary bladder. Urology, 16: 1324, 1986. 5. Kato, K, Wein, A. J., Longhurst, P.A., Haugaard, N. and Levin, R M.: The functional effects of longterm outlet obstruction on the rabbit urinary bladder. J. UroL, 143: 600, 1990. 6. Kato, K, Lin, A. T., Haugaard, N., Longhurst, P.A. and Levin, R M.: Effect of outlet obstruction on glucose metabolism of the rabbit urinary bladder. J. UroL, 143: 844, 1990. 7. Bergmeyer, H. U. and Bernt, E.: Malate dehydrogenase. UV assay. In: Methods of Enzymatic Analysis. Edited by U V. Bergmeyer. New York: Academic Press, vol. 2, 1974. 8. Robinson, KR, Jr., Brent, L. G., Sumegi, Rand Srere, P. A: An enzymatic approach to the study of the Krebs tricarboxylic acid cycle. In: Mitochondria, A Practical Approach. Edited by V. M. Darley-Usmar, D. Rickwood and M. T. Wilson. Oxford: IRL Press, chapter 6, pp. 153-170, 1989. 9. 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, 195L 10. Butler, R M., Siegman, J. J., Mooers, S. U and Davies, R E.: High energy phosphate utilization during force development and force maintenance in mammalian smooth muscle. In: Excitation Contraction Coupling in Smooth Muscle. Edited by Casteels, R, Godfraind, T., and Ruegg, J. C. Elsevier, Amsterdam, pp. 463469, 1977. lL Stephens, N. L. and Wrogemann, K: Oxidative phosphorylation in smooth muscle. In: Biochemistry of Smooth Muscle. Edited by Stephens, N. L. CRC Press, Inc. Boca Raton, Florida, pp. 119-126, 1983. 12. Siegman, M. J., Butler, T. M., Mooers, S. U. and Davies, R E.: Chemical energetics of force development, force maintenance, and relaxation in mammalian smooth muscle. J. Gen. PhysioL, 76: 609, 1980. 13. Paul, R J., Peterson, J. W. and Caplan, S. R: Oxygen consumption rate in vascular smooth muscle: relation to isometric tension. Biochem. Biophys. Acta, 305: 474, 1973. 14. Lundholm, L, Pettersson, G., Anderson, R G. G. and MohmeLundholm, E.: Regulation of the carbohydrate metabolism of smooth muscle; some current problems. In: Biochemistry of Smooth Muscle. Edited by N. L. Stephens. Boca Raton: CRC Press, voL 2, pp. 85-105, 1983. 15. Arnquist, H. J.: Glucose transport and metabolism in smooth muscle; action of insulin and diabetes. In: Biochemistry of Smooth Muscle. Edited by N. L. Stephens. Boca-Raton: CRC Press, pp. 109-118, 1983. 16. Bihler, L, Sawh, P. C. and Elbrink, J.: A specific sugar transport mechanism in smooth muscle and its regulation. Can. J. PhysioL PharmacoL, 54: 254, 1976. 17. Haugaard, N., Wein, A. J. and Levin, R IvL: In vitro studies of glucose metabolism of the rabbit urinary bladder. J. UroL, 137: 782, 1987. 18. Hypolite, J. A., Haugaard, N., Wein, A. J., Ruggieri, M. R and Levin, R M.: Comparison of palmitic acid and glucose metabolism in the rabbit urinary bladder. NeurouroL Urodynam., §: 599, 1989. 19. Hypolite, J. A., Wein, J. A, Haugaard, N. and Levin, RM.: Role of substrate in the maintenance of contractility of the rabbit urinary bladder. PharmacoL, 42: 202, 199L 20. Lin, AT., Monson, F. C., Kato, K, Haugaard, N., Wein, A. J. and Levin, R M.: Effect of chronic ischemia of glucose metabolism of rabbit urinary bladder. J. UroL, 142: 1127, 1989. 2L Levin, RM., Haugaard, N., Levin, S.S. and Wein, A. J.: Creatine kinase activity in normal and hypertrophied tissue. MoL CelL Biochem., 106: 143, 199L