Molecular and Cellular Biochemistry 117: 159-163, 1992. (~) 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder

Ahmet Bilgen, Alan J. Wein, Niels Haugaard, David Packard and Robert M. Levin Division of Urology, University of Pennsylvania School of Medicine and the Philadelphia Veterans Administration Medical Center, Philadelphia, Pennsylvania, USA

Received 19 March 1992; accepted 5 July 1992

Abstract

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose.

The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed, In these studies we determined: 1) the rate of 14C-pyruvate metabolism to 14CO2 in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase.

The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the Kd for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction. (Mol Cell Biochem 117: 159-163, 1992)

Key words: urinary bladder, obstruction, pyruvate oxidation, smooth muscle contractility

Introduction

Partial outlet obstruction of the bladder results in a significant contractile dysfunction which is associated with a marked decrease in the metabolism of glucose to CO2, and an increase in lactic acid formation [1-4]. The contractile dysfunction is observed within i day follow- ing partial outlet obstruction, and is associated with an initial period of overdistention. The ability of the blad-

der to empty remains significantly reduced through the first 7 days following the creation of the partial obstruc- tion [2, 5]. All previous metabolic studies utilized 7 days of partial obstruction, as this was the point of the great- est increase in bladder mass [2, 4, 5]. These studies provide evidence for decreased mitochondrial function

Address for offprints: R.M. Levin, Division of Urology, 3006 Ravdin Courtyard Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia. PA 19104, USA

160

in association with the bladder hypertrophy induced by outlet obstruction.

Prior studies demonstrated that pyruvate was a more effective substrate than glucose for the maintainance of the contractile response of the bladder to field stim- ulation [6]. In this regard, pyruvate is immediately available for mitochondrial oxidation whereas glucose must first be metabolized by the glycolytic enzymes to pyruvate. In addition, glucose can be metabolized to lactic acid and other metabolites which are not available to mitochondria.

The current study was designed to correlate in terms of time the decrease in mitochondrial oxidation in the urinary bladder following outlet obstruction with the contractile dysfunction observed. In these studies we determined: 1) the rate of 14C-pyruvate metabolism to 14CO2 in control and obstructed tissue (at 1, 3, 5, and 7 days following partial outlet obstruction), and 2) the mitochondrial enzymatic activities of malate dehydro- genase and citrate synthase.

Material and methods

Mature male New Zealand White rabbits weighing ap- proximately 2.5 kg were used for the experiments. The rabbits were separated into two groups: 1) Controls, 2) Obstructed (1, 3, 5, and 7 days).

For the obstructions, each rabbit was sedated with an intramuscular injection (0.7 ml/kg) of a ketamine/xyla- zine mixture (29.2mg/ml ketamine, 8.3mg/ml xyla- zine). Surgical anesthesia was maintained with 0.5 ml pentobarbital (50mg/ml iv). The bladder was cathe- terized with an 8 Fr Foley catheter. The urinary bladder was exposed through a midline lower abdominal in- cision. A 2-0 silk suture was tied loosely around the bladder neck of the catheterized rabbit. The catheter was removed and the incision closed, thereby creating a partial outlet obstruction [2].

At 1, 3, 5, or 7 days following obstruction, each rabbit was anesthetized as previously described and the uri- nary bladder removed. The bladder was dissected free of mucosa, fat tissue, blood vessels and serosa. The body and base were separated at the level of the ureteral orifices. The bladder tissue was either frozen and stored at - 85 ° C for enzymatic assays, or utilized immediately for the determination of pyruvate metabolism.

Pyruvate metabolism

Longtidinal strips of bladder body weighing 100mg were prepared and placed into cold Tyrode's-Hepes solution. Each strip of tissue was placed in a vial con- taining 2 ml of Tyrode's-Hepes solution with the follow- ing composition: 0.126M NaC1, 0.0027M KCI, 0.0005 M MgC12, 0.0024 M CaC12, 0.0003 M Na-phos- phate and 0.020M Hepes buffer (pH = 7.2) and var- ying concentrations of 14C-pyruvic acid. Each vial was gassed for 30 seconds with 100% Oz and tightly fitted with rubber stoppers equipped with plastic wells con- taining 0.2 ml phenylethylamine for the absorbtion of a4CO 2. Each vial was then incubated in a shaking water bath at 37°C for 1 h. As controls for each experiment vials without tissue were incubated with the same con- centrations of pyruvate as were used in the actual ex- periments.

At the end of the 1 h incubation period, the plastic wells containing phenylethylamine and absorbed 14CO2 were placed into scintillation vials containing 10 ml Cy- toscint. The radioactivity of the content of the vials were determined by liquid scintillation spectrometry. The concentration of 14CO 2 generated was calculated from the specific activity of the pyruvate and the effi- ciency of the counter for carbon-14 (95%).

Enzymatic assays

Sections of frozen tissue weighing about 0.5 g were homogenized in ten volumes of 0.225M manni- tol-0.075 M fructose using a Polytron homogenizer. The homogenates were filtered through gauze and kept in ice until used for determination of enzyme activity.

Malate dehydrogenase activity was determined by a modification [7] of the method of Bergmeyer and Bernt [8]. In this procedure the disappearance of NADH is measured in a system in which the substrate, oxaloace- rate, is generated from aspartate and alpha-keto glut- arate by added glutamate-oxaloacetate transaminase. Preliminary studies demonstrated that identical results were obtained when oxaloacetate was added directly. The following simplified procedure was used to gener- ate the data presented in the results section. Aliquots of tissue homogenates containing 5 to 10 mg tissue were added to a cuvette ( lcm width) with 2.0ml 0.05M triethanolamine (pH 7.6) 50tzl 10mM oxaloacetate (freshly prepared), 100/xl 1.8 mM N A D H and 100/~1 0.1 M MgCI> The rate of oxidation of N A D H was mea-

161

EFFECT OF OBSTRUCTION ON BLADDER WEIGHT EFFECT OF OBSTRUCTION ON PYRUVATE METABOLISM

360. e - - e CONTROL *

4 * "5= t 12° "'x . . . . . . " - " -

/ 0 1 | t . . . .

0 0 1 2 5 4 5 CONTROL 1 3 5 7

OBSTRUCTION (DAYS) PYRUVATE CONCENTRATION (rnM)

Fig. 2. Effect of obstruction on bladder pyruvate oxidation using Fig. 1. Effect of obstruction on bladder mass. Each bar is the mean

increasing concentrations of pyruvate. Each point is the mean + + SEM of 4--6 individual experiments. *= significantly different SEM of 4-6 individual experiments. from control.

sured by recording the absorbancy at 340nm every 0.5 rain. After 5 min 240/zl 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 25 ° C.

Citrate synthase was determined as described by Robinson et al. [7, 9]. Aliquots of homogenate were added to a cuvette (0.5cm width) containing 1.0ml 0.05 M TRIS buffer (pH 7.6), 100/A 1 mM 5,5'-dithio- bis-2-nitrobenzoic acid (DTNB or Ellmans reagent), 50/xl 10mM oxaloacetate and 30/zl 12.3mM acetyl- coenzyme A. The free coenzyme A generated reacts with DTNB to form a yellow compound that is mea- sured at 412 nm. After 5 rain 120/~1 of 10% Triton-X-100 was added and the increased rate of reaction recorded during a subsequent 5 min 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 (Vmax).

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 activ- ity. In hornogenates from control rabbit bladders ma- late 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 tables are the enzyme activities in the presence of 1% Triton X-100.

Statistics

Statistical analysis utilized Analysis of Variance fol- lowed by Newman-Keuls post hoc analysis between individual groups. A value of p < 0.05 was used to indicate significance.

Results

Preliminary studies demonstrated that at the maximum pyruvate concentration, the metabolism to CO2 was linear with time up to 90 min (15, 30, 60, 90, 120 rain). Above 90rain, the rate of pyruvate metabolism de- creased. These time course studies were performed for both control and obstructed bladders.

Figure 1 presents the effect of partial obstruction on bladder mass. The maximum mass occurred 7 days after obstruction as has been found in previous studies.

Figure 2 displays the effect of 7-days obstruction on pyruvate metabolism. Figure 3 presents the kinetic analysis of the data presented in Fig. 2 (Hofstee plot). Pyruvate metabolism was saturable and followed linear kinetics. Outlet obstruction induced a significant de- crease in the Vma~ of pyruvate metabolism with no change in the Kd. Figure 4 displays the maximal rate of pyruvate metabolism for control bladders and for blad- ders 1, 3, 5, and 7 days following obstruction. There was a significant decrease in the maximal rate of metabolism already 1 day after the obstruction with no significant further change after 5 and 7 days. The Kd for pyruvate was the same for all tissues studied (similar to that shown in Fig. 3). It should be noted, that all data were normalized per mg tissue.

The activities of malate dehydrogenase and citrate

162

HOFSTEE PLOT

400-

300.

(D 0 200. _1 ILl >

lOO.

0 0

• CONTROL O/ma x = 375 nrnol / ,g /mM)*

~ . (K d = 1.05 mM)

• 7 DAY OBSTRUCTED O/max = 230 nmol /g /mln)

(K d - 1.10 raM)

1 O0 200 300 400

VELOCITY / [SUBSTRATE]

Fig. 3. Effect of obstruction on bladder pyruvate oxidation using increasing concentrations of pyruvate: Hofstee plot. For the control bladders, the Vm~x =375-+ 32nmol/min/g tissue; Kd= 1.05_+ 0.3mM. For the 7 day obstructed bladders, the Vmax = 230+ 18 nmol/mildg tissue; Ko = 1.10 _+ 0.25 raM.

synthase (two important mitochondrial enzymes) were significantly reduced by over 50% at 1 day following outlet obstruction, and remained low through 7 days (Fig. 5).

D i s c u s s i o n

Similar to all smooth muscles, the urinary bladder is dependent on the generation of ATP via mitochondrial oxidation of glycolytic substrates to maintain the in- tracellular concentrations of ATP and CP required to support contraction and bladder emptying [4, 5, 11, 12]. Studies on bladder energetics have demonstrated that the ability of the bladder to empty derives its energy

EFFECT OF OBSTRUCTION ON PYRUVATE METABOLISM

' ~ 400-

• ~, 300 * \ \ "

0 E 200. t'-

o4 100. o o

CONTROL 1 3 5 7

OBSTRUCTION (DAYS)

Fig. 4. Effect of obstruction (1-7 days) on bladder pyruvate oxidation. Each point is the Vma x +-- SEM of 4-6 individual experiments. * = Significantly different from control (p < 0.05).

. . 1ooo u ~ 800-

Z ~ 6 0 0 -

, o o

200- 0

h i

<

MITOCHONDRIAL ENZYME ACTIVITY

_L 7"2 / / / / / / / / / / / / / / / / / / / / / / / / d r '

CONTROL

MALIC OEHYDROGENASE

I- I - I CITRATE SYNTHASE

1 DAY 7 DAY OBSTRUCTED

-150 o

-90

-60 3

"1o

o ~

Fig. 5. Effect of obstruction on bladder mitochondrial enzyme activi- ty. Each point is the V,,ax + SEM of 6-8 individual experiments.

from oxidative metabolism. Evidence for this comes from studies demonstrating that anoxia selectively in- hibits the tonic response to receptor stimulation [13] and a decrease in the rate of substrate oxidation also selectively inhibits bladder emptying (tonic contrac- tion) [6].

Partial urinary outlet obstruction induces a signif- icant dysfunction in both the ability of the bladder to generate pressure and to empty [2, 5]. The degree of dysfunction is greatest at one day following outlet ob- struction. Although the ability of the in vitro bladder to generate pressure returns to normal by 7 days, the ability to empty remains significantly reduced. The physiological mechanism which is responsible for the inability of the bladder to empty has been shown to be an inability of the bladder to sustain an increased in- travesical pressure during continuous stimulation. If the bladder cannot sustain an increased pressure, the blad- der cannot empty efficiently or completely [5].

Although it is clear that at 7 days following partial outlet obstruction in the rabbit, the ability to oxidize pyruvate is reduced by 50%, as is the maximal rate of malate dehydrogenase and citrate synthase (two impor- tant mitochondrial enzymes), it had not been shown previously that mitochondrial metabolism is impaired at one day following obstruction [4, 7]. This is an impor- tant demonstration since at one day, bladder mass in- creases only slightly, and is primarily due to edema. The current study was designed to determine if the acute overdistension observed at one day (and the accompa- nying contractile dysfunction) induces any alteration in the ability of bladder tissue to metabolize pyruvate, and whether the rate of oxidation of pyruvate can be corre- lated with the mitochondrial changes as exemplified in

the decreases in activities of malate dehydrogenase and citrate synthase.

The results clearly demonst ra te that at 1 day, the rate of pyruvate metabol ism is reduced by over 50%, as are the maximal rates of activity of malate dehydrogenase and citrate synthase. This indicates that the decrease in mitochondrial function is not secondary to bladder hy- per t rophy, but is probably directly induced by the struc-

tural damage caused by the acute overdistension. One factor which may part icipate in this process is the initial edema which is observed in the tissue at 1 day following obstruction. This edema accounts for the initial increase in mass at this t ime period. Although the decreased

metabolic activity at 1 and 3 days may in par t be ac- counted for by edema, the magni tude of the decreased enzymatic activity (50% decrease) is certainly far greater than the degree of edema present. By 5 days following obstruction the edema is completely resolved.

The duration of the decrease in mitochondrial oxidation is consistant with the decreased ability of the bladder to

empty. Future studies will be directed at correlating mitochondrial activity with the recovery of bladder

function. Our observations can be summarized as follows: 1)

there is a progressive increase in bladder weight over the first 7 days following obstruction, 2) the ability of the in vitro whole bladder to both generate pressure and empty is markedly impaired within 1 day following par- tial outlet obstruction, 3) the ability to generate pres-

sure returns to control values within 7 days, whereas the ability to empty remains low throughout the 7 days, 4) there is a 50% decrease in the rate of pyruvate oxidation of the bladder already 1 day following obstruction, the

low rate of pyruvate metabol ism remains constant throughout the 7 day study period (at a t ime when bladder mass increases substantially) 5) finally, the en- zymatic activities of the mitochondrial enzymes malate dehydrogenase and citrate synthase are decreased by 50% within 1 day, and remain constant throughout the 7 day study period.

Thus, the t ime-course for the reduction in the ability of the bladder to empty following partial outlet obstruc-

tion is similar to the t ime-course of the reduction in mitochondrial activity. Al though this does not prove a causal relationship between the decrease in bladder emptying observed following partial outlet obstruction and decreased mitochondrial function, it is consistant with the theory that the ability of the bladder to sustain increased pressure (and thus empty) is supported by

163

oxidative metabolism, and that it is the decreased mi-

tochondrial oxidative activity observed following par-

tial outlet obstruction that results (at least in part) in the decreased ability of the obstructed bladder to empty.

Acknowledgements

This work was supported in part by grants f rom the Veterans Administration, and N I H Grants RO-1-DK 26508, and RO-1-DK 33559.

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