0022-5347/95/1536-2014$03.00/0 THE JOURNAL. OF UROLOGY Copyright @ 1995 by AMERICAN UROmICAL ASSOCIATION, INC.

Vol. 153, 2014-2021, June 1995 Printed in U.S.A.

TEMPORAL CHANGES IN MICTURITION AND BLADDER CONTRACTILITY AFTER SUCROSE DIURESIS AND STREPTOZOTOCIN-

INDUCED DIABETES MELLITUS IN RATS TEWO L. J. TAMMELA, ROBERT E. LEGGETT, ROBERT M. LEVIN AND

PENELOPE A. LONGHURST" From the Division of Urology and Department of Pharmacology, University of Pennsylvania and Veterans Administration Medical Center, Philadelphia, Pennsylvania and the Division of Urology and Department of Clinical Medicine, University of Tampere, Tampere, Finland

ABSTRACT

Studies were done to compare the acute effects of streptozotocin-induced diabetes and sucrose consumption on micturition, bladder mass and contractile responses of bladder strips to field stimulation and contractile agonists. Micturition changes occurred gradually in diabetic rats, reached maximal values within 7 to 14 days, and were accompanied by significant increases in bladder mass after 7 days. Bladder strips from diabetics responded to field stimulation, carbachol and KC1 with significantly greater contractions than did those from controls within 7 days. Sucrose-drinking rats had maximal increases in fluid consumption and micturition frequency on the first night after starting treatment. Increases in micturition volumes were slower to develop than in diabetics. Bladder mass was significantly increased 30 and 60 days after starting sucrose treatment. Bladder strips from sucrose-drinking rats responded to field stimulation and carba- chol with significantly greater contractions than did those from controls only after 60 days. Monitoring of drinking and micturition patterns established that diabetic rats drink and urinate during both the dark and light cycles. In contrast, control and sucrose-drinking rats drink and urinate principally at night. The results demonstrate that differences in bladder function be- tween diabetic and sucrose drinking rats are apparent during the first month after treatment begins. The data suggest that the effects of diabetes and sucrose consumption on contractile bladder function are related to the diuresis-induced increases in bladder mass.

KEY WORDS: diabetes mellitus, sucrose, urination, drinking, muscle contraction

Urinary bladder function is significantly altered in pa- tients with diabetes mellitus. Many of these changes are also seen in the streptozotocin (STZ)-diabetic rat, the most com- monly used animal model of diabetes mellitus.1-8 The results of micturition studies show that STZ-induced diabetes is associated with significant increases in both fluid intake and urine output. These changes are accompanied by increases in the frequency of micturition and mean volume excreted per micturition.3.5.9 Diabetes-induced changes in micturition pattern occur within the first day after onset of diabetes, increase to maximal values within 2 weeks, and remain relatively stable for as long as 2 months.10 In addition to changes in the micturition pattern, STZ-induced diabetes also causes significant alterations in the contractile response of the bladder to field stimulation and contractile agonists.1-7 Most of these studies used rats 2 months after the onset of diabetes, but in one, bladder strips were examined 1 month after induction of diabetes.8

Polyuria induced by sucrose drinking in rats results in alterations in micturition and bladder contractility very sim- ilar to those observed following STZ-induced diabetes.4.9.11.12 Longhurst et al. showed that sucrose-drinking rats, like con- trol rats, urinated significantly smaller volumes than diabet- ics during the light cycle, but had an increased urination during the dark cycle with volumes similar to those in dia- betics.9 They postulated that these differences were due to different drinking patterns in diabetic and sucrose-drinking

Accepted for publication December 8, 1994. * Requests for reprints: Division of Urology, 3010 Ravdin Court-

yard Buildin Hospital of the University of Pennsylvania, 3400 Spruce St., Pkladelphia, Pennsylvania 19104.

This work was supported in part by grants from the Veterans Administration, N.I.H. Grant DK 41610 and the Finnish Academy of Sciences and the Paulo Foundation, Finland.

rats. In addition to effects on micturition, sucrose consump- tion, like diabetes, causes increases in bladder mass and increases in bladder strip contractile responsiveness com- pared with c0ntrols.4~9~ 11- 12

Previous studies of in vitro bladder contractility in diabetic and sucrose-drinking rats have examined responses almost exclusively after 2 months of treatment. The aim of the present study, therefore, was to examine the temporal effects of diabetes and sucrose-induced diuresis on micturition pat- terns and bladder strip contractility. Furthermore, the drink- ing patterns of control, sucrose-drinking and diabetic rats were observed after 2 months' treatment to establish whether micturition patterns changed in parallel with drink- ing patterns.

MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats (300 to 325 g.) ob- tained from Ace Animals Inc. (Boyertown, Pennsylvania.) were used throughout the study. All animals received free access to food and water, except when indicated.

Induction of diabetes. Rats were fasted for 18 to 24 hours. Diabetes was induced in approximately one-third of the rats with a single injection of streptozotocin (STZ, 60 mg./kg., intraperitoneally) in ice-cold 0.02 M. citrate saline. Success- ful induction of diabetes was assessed by decreases in rat weight and confirmed by measurement of serum glucose con- centration. The remainder of the rats were injected with vehicle. Rats were used 1 day to 8 weeks after the induction of diabetes.

Sucrose treatment. After injection of the vehicle, one-half of the control rats were given 5% sucrose in tap water to drink instead of water. This was continued until the day of exper- imentation. Rats were used 1 day to 8 weeks after beginning

2014

BLADDER FUNCTION AND DIURESIS 2015 sucrose treatment. The remaining group of control rats re- ceived tap water to drink.

Drinking and micturition. The rats were placed in individ- ual metabolic cages in a room maintained at 22C. After a 1-day period of acclimation, the drinking and micturition patterns were monitored for both the light and dark cycles. The light cycle was 07:OO to 19:OO, and the dark cycle was 19:OO to 07:OO. Cages were cleaned and water bottles were refilled between 07:OO and 08:OO each day. For the first 2 days in the cages, all rats were given water to drink. Then the rats in the sucrose group were switched to 5% sucrose solu- tion. Micturition data for the STZ diabetic rats used for comparison with control and sucrose-drinking rats at 1 to 30 days are taken from our previous publication.10

The rat cages were suspended over Ohaus GT400 balances, interfaced with a c ~ m p u t e r . ~ Plastic beakers were placed on the balances to collect urine, and the weight of the beaker was monitored every 2 minutes. Data were collected using Ohaus Profit Weigh and Lotus Measure. The frequency of drinking was monitored every 2 minutes using a drinking monitor (Columbus Instruments, Columbus, Ohio). At the end of the experiment, the volume of water or 5% sucrose remaining in the drinking bottles was measured, and the volume consumed was calculated.

Preparation of tissues. Before anesthesia, blood samples were collected from the tail artery, and the serum was sep- arated and analyzed for serum glucose using the ABTS method of Bergmeyer and Bernt.13 The rats were then anes- thetized with pentobarbital (50 mg./kg., intrapentoneally). The urinary bladder was removed from each rat and placed in ice-cold Krebs-Henseleit buffer of the following composi- tion (mM.): NaCl 113; KCl 4.8; CaC1, 2.5; KH,PO, 1.2; MgSO, 1.2; NaHCO, 25: dextrose 5.6. The bladder was sep- arated into bladder body and base at the level of the ureters. One longitudinal strip approximately 2 mm. X 10 mm. was cut from the center of the body.

Contractile studies. The bladder strips were suspended on %zero sutures between a pair of platinum ring electrodes 8 mm. apart and placed in 30 ml. organ baths containing Krebs-Henseleit solution equilibrated with 95% O,, 5% CO,, and maintained at 37C. The tissues were connected to Grass force displacement transducers (FT03, Grass Instruments, Quincy, Massachusetts) and adjusted to 2 g. resting ten- sion.14 Responses were recorded on a Grass Model 7E poly- graph (Grass Instruments). Following a 30-minute equilibra- tion period during which the tissues were washed and the resting tension was adjusted every 10 minutes, frequency- response curves were elicited by stimulating the tissues for 10 seconds with pulses of 0.05 msec. duration at a supra- maximal voltage every 2 minutes using a Grass S88 stimu- lator (Grass Instruments). These responses are neurogenic and sensitive to M. tetrodotoxin.12 Subsequently after resting periods of 15 minutes, dose-response curves to ATP, carbachol and KC1 were obtained using noncumulative addi- tions. Each concentration of agonist was left in contact with the bladders for 1 minute (ATP) or 3 minutes (carbachol and KCl), followed by 2 washes with drug-free buffer over a 7-minute period.

Drugs. Adenosine 5'-triphosphate (ATP) and carbamylcho- line chloride (carbachol) were obtained from Sigma Chemical Company, St. Louis, Missouri.

Statistical analysis. Data are presented as means ? the standard error of the mean. Comparisons between multiple groups were done using analysis of variance followed by the Bonferroni test.15 A probability of p <0.05 was taken as the criterion of significance.

RESULTS

Rat weight, bladder weight, and serum glucose concentra- tion. Diabetic rats weighed significantly less after induction of diabetes than did the control or sucrose-drinking rats (table 1). There were no differences between the weights of control and sucrose-drinking rats. Serum glucose concentra- tions were significantly higher in the STZ-diabetic rats than in the control or sucrose groups (data not shown). Sucrose consumption had no significant effect on serum glucose con- centrations. There were gradual increases in bladder weights in both the STZ-diabetic and sucrose-drinking rats com- pared with controls (table 1). Bladder weight increased faster in the diabetic group, but by 30 days there were no significant differences in bladder weights between the di- abetic and sucrose groups. There were no differences in the weights of control bladders, except that bladders from the 14-day group weighed significantly more than those of the 2-day group.

Fluid consumption and excretion. There were no changes in drinking or excretion patterns in control rats during the study period. There were gradual increases in water con- sumption and urine excretion in STZ-diabetic rats during the first 2 weeks after onset of diabetes (table 2). A different pattern was seen with sucrose-drinking rats, where maximal fluid consumption and excretion occured during the first day after consumption of the sucrose solution. The amounts of fluid consumed and excreted declined slightly during the next 2 weeks, but then gradually increased back to maximal values at 1 and 2 months. Diabetic and sucrose-drinking rats consumed and excreted significantly greater volumes of fluid than did controls, while the diabetics consumed and excreted significantly greater volumes of fluid than the sucrose-drink- ing rats after the first 4 days of treatment.

The frequency of micturition in controls was significantly greater during the dark cycle than the light cycle and did not change during the study period (day -1: light 0.47 % 0.04, dark 1.28 2 0.13 micturitions per hour; day 60: light 0.50 -C 0.03, dark 1.26 2 0.07 micturitions per hour). There were gradual increases in micturition frequency in diabetic rats (fig. 1). The differentiation between light and dark cycles was lost after 14 days of diabetes. Sucrose-drinking rats main- tained a reduced light cycle micturition frequency through- out the study period (fig. 1). After a small but significant increase in the sucrose group on the first day after treatment began, there were no differences between the micturition frequency of control and sucrose-drinking rats during the light cycle. However, there was a significant increase in micturition frequency on the first night, 12 hours after

TABLE 1. Changes in rat and bladder weight with time after induction of diabetes or beginning consumption of 5% sucrose on day 0 Prefast Day 1 Day 2 Day 4 Day 7 Day 14 Day 30 Day 60 Overall mean

Rat Weight (g.) Control 370 : 3 365 c 6 3682 10 385 t 5 403 2 6 433 : 11 455 t 7 523 2 13 STZ 365: 3 321 2 7 t 325 : 5t 312 2 7; Sucrose 373 : 3 362 : 8 383 : 4 363 It_ 4 384 2 6 420 2 6 467 : 7 513 2 17

Control 133.45 t 6.29 118.64 2 8.89 127.00 : 10.23 124.53 : 6.73 161.22 2 15.79 152.93 2 4.34 124.55 2 5.13 133.74 -C 3.51 STZ 120.88 2 8.27 123.07 : 3.49 134.75 : 5.62$ 160.37 : 5.83t 194.71 t 5.14$ 235.95 : 19.46* 195.22 2 11.89; Sucrose 136.04 2 13.35 129.01 : 7.41 112.76 : 6.72 131.99 t 6.28 161.72 2 7.76 187.55 t 13.44* 172.87 : 12.80*

Values represent mean 2 standard error of the mean of 7 to 12 individual observations, except for prefast and overall control mean values, where N=47 to 68. * significant difference compared with age-matched control rats, t significant difference compared with agematched control and sumse-drinking rats, $ significant difference compared with age-matched sucrose-drinking rats (p <0.05).

307 2 5t 300 2 It 270 2 17t 196 : 12t

Bladder Weight (mg.)

2016 BLADDER FUNCTION AND DIURESIS

TABLE 2. Changes in micturition patterns with time after induction of diabetes or beginning consumption of sucrose on day 0 Dav -1 Dav 0 Dav 1 Dav 2 Day 4 Dav 7 Day 14 Day 30 Day 60

0.0

~~

Volume Consumed (mlJhr.1 Control 1.80 i 0.09 1.65 t 0.11 2.04 t 0.13 1.84 -C 0.17 2.05 i 0.08 2.03 t 0.11 1.98 f 0.10 1.73 t 0.05 1.94 t 0.07 ST2 1.86 f 0.12 0.97 i 0.18 3.08 t 0.16' 5.09 % 0.39* 5.37 i 0.757 8.66 i 0.87t 10.06 -C 0.61t 6.47 2 0.57* 7.17 f 0.87'1 Sucrose 1.75 -C 0.08 1.95 f 0.05 4.51 i 0.50* 3.58 % 0.40* 3.15 i 0.20 3.61 i 0.35* 3.90 f 0.23* 4.57 % 0.31* 4.44 f 0.60*

Control 0.54 t 0.06 0.50 f 0.05 0.57 % 0.07 0.52 t 0.07 0.58 f 0.05 0.60 t 0.07 0.65 t 0.05 0 .58% 0.03 0.81 t 0.05 STZ 0.66 2 0.15 0.93 f 0.15 1 . 9 6 t 0.18* 4.24 t 0.30t 4.71 i 0.59t 7.44 t 0.65t 8.78 2 0.52t 5.49 f 0.471 5.91 2 0.61t Sucrose 0.42 f 0.06 0.41 i 0.04 2.40 t 0.39* 1.90 f 0.38* 1.55 f 0.23* 2.02 % 0.34* 2.20 2 0.24: 2.81 f 0.26* 2.87 t 0.55*

Control 1.61 f 0.21 1.48 f 0.17 1.27 t 0.14 1.18 f 0.20 1.44 t 0.15 1.42 -C 0.18 1.45 t 0.12 1.60 i 0.12 1.80 t 0.11

Volume Excreted ( m l h . )

Maximal Micturition Volume (ml.)

STZ 1.45 f 0.25 1.77 -C 0.16$ 2.59 % 0.12' 2.92 i 0.13'i 3.68 t 0.18t 4.59 % 0.36t 5.41 % 0.40t 4.14 t 0.32t 4.30 t 0.47t Sucrose 0.97 -C 0.25 0.80 % 0.17* 1.92 t 0.31 1.72 i 0.26 1.69 t 0.19 2.23 f 0.32 2.66 t 0.14* 2.98 2 0.18* 2.64 f 0.28' Values represent mean % standard error of the mean of 7 to 18 individual observations. * significant difference compared with age-matched control rats,

t significant difference compared with age-matched control and sucrose-drinking rats, $ significant difference compared with age-matched sucrose-drinking rats (p <0.05).

+ . . . .. . . .+

I I I I I I I I I

7 , c-a STZ - light H STZ-dark

Sucrose - light T A . 4 Sucrose - dark

0 1 I I I I I I I I I

-1 0 1 2 4 7 14 30 60

Days FIG. 1. Changes in micturition frequency with time after induc-

tion of diabetes or beginning consumption of 5% sucrose on day 0. Values represent mean ? standard error of mean of 7 to 9 individual observations.

switching to the sucrose solution (at 07:OO hours), which then declined slightly and was maintained at a similar level to that observed with the diabetics.

There was a small age-related increase in mean (day -1: light 1.11 2 0.17, dark 0.41 2 0.03 ml.; day 60: light 1.17 5 0.07, dark 0.66 2 0.06 ml.) and maximal (table 2) micturition volumes in control rats during the study period. Volumes were significantly greater during the light than the dark cycles (maximal micturition volumes reported in table 2 were obtained during the light cycle). After induction of diabetes there were gradual increases in mean and maximal micturi- tion volumes in diabetic rats, with loss of the 1ight:dark cycle differences (fig. 2, table 2). Significant differences compared with controls were apparent the first day after induction of diabetes. Similar increases in mean volume were observed in sucrose-drinking rats, but they were much slower to develop, and the 1ight:dark cycle differential was retained after the first few days. Volumes were not significantly increased com- pared with controls until 7 to 14 days after start of sucrose treatment. Maximal micturition volumes increased to a much lesser extent in the sucrose-drinking rats than in the diabetics, and were significantly greater than control values after 14 days of treatment.

G-0 STZ - light

H STZ-dark T - 2.5

A 4 Sucrose - light

2.0 L A Sucrose - dark

- 5

T 1.5

K 0 c. .-

Drinking profile. Typical drinking profiles for 3 individual rats 60 days after the start of treatment are shown in figure 3. The drinking profiles for the control and sucrose-drinking rats are biphasic. Rats in these groups drink mostly during the dark cycle. In addition, the sucrose group drank signifi- cantly more than the controls. In contrast, diabetic rats drink continually during both the light and dark cycles.

Licking and micturition frequency. Quantitatively, the fre- quency of drinking and micturitions per light and dark peri- ods is illustrated in figure 4, A and B. There were no differ- ences between control or sucrose-drinking rats for frequency of licking and micturitions during the light cycle. Changing from light to dark cycle induced a significant increase in frequency of licking and micturitions for both control (2.5-fold increase in micturitions and 5-fold increase in licking) and sucrose-drinking rats (&fold increase in micturitions and 9-fold increase in licking). The frequency of licking and of micturitions for the sucrose-drinking rats was significantly greater than for control rats during the dark cycle. The frequency of licking and micturitions for the STZ-diabetic rats was approximately the same during the dark and light cycles, was significantly greater than the respective values for control rats, and was similar to that of sucrose-drinking rats during the dark cycle.

BLADDER FUNCTION AND DIURESIS 2017

200 -

150-

100-

A

Y

6

0 3 II. 0

m I 3 z

B

In Y 0 3

k B m I 3 2

C

In Y 0 3 L 0

B m I 3 2

CONTROL

3o03 250 4 I

TIME

DIABETIC

2501

50

0 1000 14:oo 18:OO 22:oo 0200 0600 1o:oo

TIME

SUCROSE-DRINKING

150

100

50

1o:oo

1

TIME

FIG. 3. Typical drinking profiles of control (A), diabetic (B), and sucrose-drinking rats (C) &er 60 days of treatment. Each vertical line represents frequency of licks during a 2-minute period.

Contractile responses of urinary bladder body strips. There were no differences in the mass of the strips used for the studies described below. The maximal contractile responses of strips from 7-day control rats to field stimulation were significantly less than those of strips from 14- and 30-day controls (fig. 5, A). No other differences in responses of con- trol strips were found. Contractile responses of strips from 1 to 4 day diabetic rats to field stimulation were not different from those of age-matched controls (fig. 5, B). However, by 7 days there were significant increases in responses to field stimulation. The increases in contractility were much slower to develop in sucrose-drinking rats and were significantly greater than controls only after 60 days (fig. 5 , C).

300 6 3 8 200

0

0 3

$ 100

0 UGHT CYCLE DARK CYCLE

B T 5 0 Control

.a B msn ,,, 4 CQ Sucrose *

UGHT CYCLE OARK CYCLE

FIG. 4. Licking frequencies (top) and micturition frequencies (bot- tom) of control, diabetic and sucrose-drinking rats after 60 days of treatment. Each bar represents mean 2 standard error of mean of 8 to 12 individual rats. * significant difference compared with controls; + significant difference compared with sucrose-drinking and control rats (p (0.05).

There were no significant age-related changes in the re- sponses of strips from control rats to carbachol (fig. 6, A). Similar to the responses to field stimulation, there were no significant differences in the maximal responses of strips from 1 to 4 day diabetic rats to carbachol compared with controls (fig. 6, B). However, by 7 days the responses of strips from diabetic rats to carbachol were significantly greater than those of controls. Contractile responses of strips from sucrose-drinking rats were significantly greater than those of controls only after 60 days of treatment (fig. 6, C).

There were no significant age-related changes in the responses of strips from control rats to ATP. Maximal re- sponses to 10 mM. ATP are reported in table 3. Diabetes caused significant increases in maximal responses to ATP after 14, 30 and 60 days compared with age-matched con- trols. The responses of the strips isolated from 60-day sucrose rats were greater than the responses of strips from the con- trol rats at only 0.3 and 1 mM. (data not shown).

The maximal contractile responses of bladder strips from control rats to 120 mM. KCl were rather variable during the period of study (table 3). Responses of strips from STZ-dia- betic rats to KC1 were significantly greater than those of age-matched controls 7, 14 and 60 days after the onset of diabetes. Responses of strips from sucrose-drinking rats to KCl were also very variable and were significantly greater than those of controls only a t 7 days.

DISCUSSION

Streptozotocin-induced diabetes is associated with poly- uria, bladder hypertrophy and changes in the contractile responsiveness to nerve stimulation and pharmacological agents.*-5 Most studies have reported bladder strip contrac- tile data after 2 months of diabetes, and there are no data describing what happens to contractility at times earlier than 4 weeks after the onset of diabetes.8 Sucrose-drinking rats are often used as controls for diabetes-induced effects on the bladder because they develop polyuria and increases in

2018

A

14 -

12 -

10 - - 9 6 8 v1 - c I-"

6 -

4 -

2 -

CONTROLS

0 4 1 Day

H 2Days - 4 Days ** 7Days

H 14Days

W 30Days

* * ControlMea

e* 60Days

BLADDER FUNCTION AND DIURESIS

0 1 I I I , I I I I

CONTROLS

0

16

14

12

10 - - m

._ S 8

F c

14 -

12 -

10 - - - m

p C

C

6 - I-"

4 -

2 -

0

0

0-0 1 Day

H 2Days

4Days ** 7Days

H 14Days

'Iy 30 Days

* 60Days

+ ControlMean

I / , , / , , ,

M 1 Day

H 2 Days

A - 4 4 Days ** 7Days

0 4 14Days

18 -

16 -

14 -

- 12 - m

2 lo

- c

8 -

6 -

4 -

2 -

0

W 30Days

++ 60 Days

M 1 Day

H 2 Days

A 4Days ** 7Days

H 14Days

'~y 30 Days

t+ 60Days

+ Control Mean

1

W 30Days

++ 60 Days - * ControlMean

/ I l I I , I I

0.5 1 2 4 8 16 32 64

Frequency (Hz)

DIABETICS

16

C

0

I I I I I I

7 6 5 4

Carbachol (-log M)

DIABETICS

20

14 - ** 7Days

H 14Days - 12 - 9 W 30 Days

+ * ControlMean

Frequency (Hz)

SUCROSE

16

C Carbachoi (-log M)

SUCROSE

20

7 6 5

Carbachol (-log M)

4

FIG. 5. Frequency-response curves of bladder body strips from control (A), diabetic (B) and sucrose-drinking rats ( C ) to field stim- ulation. Each point represents mean ? standard error of mean of 7 to 12 individual bladders. Dotted line represents pooled mean of response of all control strips (N = 55).

FIG. 6. Concentration-response curves of bladder body strips from control (A), diabetic ( B ) and sucrose-drinking rats ( C ) to carbachol. Each point represents mean ? standard error of mean of 7 to 12 individual bladders. Dotted line represents pooled mean of response of all control strips (N = 55).

2019 BLADDER FUNCTION AND DIURESIS

TABLE 3. Maximal contractile responses to ATP and KC1 with time after induction of diabetes or beginning consumption of sucrose on dav 0

Day 1 Day 2 Day 4 Day 7 Day 14 Day 30 Day 60 Overall mean ATP . . ~ ~

2.65 2 0.14 Control 2.49 t 0.26 2.61 t 0.56 2.70 t 0.42 2.65 2 0.23 2.66 t 0.39 3.00 ? 0.36 2.27 2 0.35

Sucrose 2.80 t 0.29 2.95 t 0.38 2.64 t 0.47 3.78 2 0.44 3.24 t 0.30 3.00 t 0.32 3.06? 0.48

Control 7.80 2 0.61 6.54 2 0.85 6.25 2 0.60 4.79 2 0.57 8.56 t 0.91 7.28 2 0.83 7.45 ? 0.39 STZ 8.23 2 0.83 6.77 t 0.56 7.41 ? 0.44 9.33 2 1.09* 10.91 ? 0.80* 8.29? 0.73 11.07 2 0.89: Sucrose 8.88 t 0.68 7.09 2 1.00 5.71 2 0.51 7.58 t 0.55* 9.05 t 0.29 7.48 2 0.74 9.14 t 0.93 Values represent mean response (g.) 2 standard error of the mean of 7 to 12 individual observations, except for overall control mean values, where N = 54

or 55. * significant difference compared with age-matched control rats, i significant difference compared with age-matched control and sucrose-drinking rats (p <0.05).

STZ 2.77 2 0.29 3.31 2 0.27 3.21 2 0.25 3.65 t 0.69 4.26 t 0.48* 4.33 ? 0.35t 4.31 t 0.5t

KCl 6.99 ? 0.30

bladder mass. However, all bladder function studies with sucrose-drinking rats have been done after 2 months. Our data show that increases in bladder mass and contractility occur by 7 days after induction of diabetes compared with 30 to 60 days after the beginning of sucrose treatment.

Although several studies have investigated the micturition profile in STZ-diabetic rats, there has not previously been any direct correlation between drinking and micturition pro- f i l e ~ . ~ ~ ~ Furthermore, although it has been suggested that polyuria alone may explain many of the diabetes-induced effects,4.l1 detailed studies of the effects of polyuria and diabetes on drinking and micturition have not been done. Rats given sucrose to drink had a significant increase in fluid consumption and micturition frequency on the first night. The frequency decreased the following night and remained stable thereafter. Micturition volume increased more gradu- ally. We feel that this biphasic change in frequency was probably due to the initial novelty of the new solution. The micturition changes observed in sucrose-drinking rats after the initial "excitement phase" probably represent the normal physiologic response to diuresis: increases in both frequency and micturition volume. An increase in frequency alone, even in the absence of pathologic changes, must be insufficient to efficiently empty the bladder. Therefore increases in micturi- tion volume and capacity also occur.16

Rats are nocturnal animals; thus it was not surprising that the drinking and micturition profiles were biphasic. Control rats had a greater licking frequency during the dark cycle than during the light cycle. This was followed very closely by the pattern of micturition frequency. Sucrose-drinking rats had a very similar drinking profile to the controls: few mic- turitions during the light cycle and an increased number during the dark cycle, associated with a decrease in mean micturition volume at night. However, the sucrose-drinking rats differed from the controls by virtue of the substantial increase in drinking and urination during the dark cycle. The data presented here, therefore, validate the hypothesis of Longhurst et al. that sucrose-drinking rats urinate more at night because they drink more at night.9 In contrast, STZ- diabetic rats had similar frequencies of licking and micturi- tion during the light and dark cycles, suggestive of continu- ous osmotic diuresis and dehydration.

The increases in bladder mass induced by streptozotocin treatment were almost immediate. Bladder weights were significantly greater than those of age-matched control rats within 7 days, confirming our previous observations.10 In contrast, the increases in bladder weight caused by sucrose consumption were much more variable and significantly greater than control values only at 30 and 60 days. This variability is probably related to differences between rats in Preference for sucrose-containing solutions. Sucrose intake has been shown to be related to sucrose c~ncentrationl~ as well as to exposure to mild stresses.18 It has been our expe- rience that the range of volumes of sucrose consumed, even under well-controlled conditions and with sucrose solutions of fured concentration, can be considerable. Future experi-

ments will be done to establish the relationship between different sucrose concentrations, consumption, excretion and bladder mass. An additional factor that may contribute to the more rapid increase in bladder mass in diabetic rats than in sucrose-drinking rats is their micturition pattern. Diabetic rats had increased urinary frequency and micturition vol- umes during both the light and dark cycles. Their bladders were, therefore, under constant stress. In contrast, the blad- ders of the sucrose-drinking rats were distended to a much smaller extent than those of the diabetics, and distension occurred primarily during the light cycle. One important point to be noted from these data is the great variability in the weights of the control bladders. These findings empha- size the importance of using age-matched controls at each time point examined in studies of this type.

There were considerable differences between the acute ef- fects of diabetes and sucrose-drinking on contractile re- sponses of bladder strips. Previous studies, at 1 and 2 months, found significant changes in contractile responses of bladder strips from diabetic rats to field stimulation, carba- chol, ATP and KCl.596*8,19 Similar changes have also been reported for strips from sucrose-drinking rats after 2 months' treatment.4.12,16 However, in the present study increases in contractile force were apparent by 7 days after induction of diabetes, compared with 60 days after starting sucrose treat- ment, in both cases at times when increases in bladder mass were also observed. Previous studies using diabetic and su- crose-drinking rats 2 months after the start of treatment suggested that the changes observed occurred in response to diuresis and subsequent bladder enlargement.4.11,16 Our data support this theory. Moreover, because the increased responsiveness occurred so rapidly in the bladders from the diabetics, we believe that the early changes are unlikely to result from diabetic neuropathy. Rather, we postulate that the constant overdistension of the bladders from diabetic rats causes acute changes by one or more of the following mech- anisms: a) effects on nerve endings, possibly as a result of stretch-induced nerve growth factor (NGF) production; b) effects on the contractile apparatus of the smooth muscle; or c) intracellular changes within the smooth muscle cells.

Most of the work on the mechanisms by which stretch causes bladder hypertrophy and functional changes has been done with animal models of outlet obstruction. However, a few studies have examined the long-term effects of diabetes or diuresis, and some in vitro experiments have also been done. Several recent studies have shown stretch-induced in- creases in bladder DNA synthesis, mRNA for growth factors and growth factor release.20-23 In particular, bladder NGF content and release are increased by stretch or diuresis- induced distension.20.22.23 These increases in NGF may mod- ulate the neuronal plasticity changes that occur after disten- sion.

In a recent study we compared neurogenic responses of bladder strips from 2-month diabetic and sucrose-drinking rats.12 We found that strips from diabetics were more excit- able than those from sucrose-drinking and control rats. We

2020 BLADDER FUNCTION AND DIURESIS

postulated that the increased responsiveness of bladder strips from chronically diabetic rats could result from dener- vation supersensitivity, possibly related to diabetic neuropa- thy. Changes in bladder afferent innervation and neuronal neuropeptide content have been found in long-term diabetic rats2438 and are probably responsible for some of the mic- turition disturbances associated with diabetes. The acute effects of diabetes on these pathways are not known. Studies of bladder autonomic transmission after diabetes are incon- clusive. Indirect monitoring of cholinergic function by mea- surement of acetylcholinesterase or choline acetyltransferase found increases or no change in activities.29.30 Contractile studies suggest that there is an increase in the cholinergic component of the response of bladder strips to field stimula- tion 2 months after induction of diabetes.12~31 Possible mech- anisms include increases in acetylcholine release, decreased transmitter inactivation, or degenerative nerve changes.31 Studies of synthesis or release of acetylcholine or ATP from the bladder aRer induction of diabetes or sucrose treatment have not been done, but could provide important information on the time frame and etiology of the changes in bladder contractile function.

Two studies examined bladder smooth muscle histologi- cally after induction of diabetes; this has not been done with other types of diuresis. Both Uvelius and Lincoln and cowork- ers demonstrated that the smooth muscle of bladders from diabetic rats became hypertrophied.2~32 Little has been done on the effects of diabetes or diuresis on bladder contractile proteins. Samuel and coworkers found no differences in the myosin heavy chain SM1:SMP ratio, but decreases in actin- activated ATPase activity of bladders from diabetic and su- crose-drinking rats compared with controls.33 Although this study seems to rule out diuresis-induced changes in the my- osin tail contributing to contractile differences, it does not eliminate the possibility that there may be differences in the actin- or ATP-binding sites on the myosin heads.

The effects of diabetes on glucose metabolism in the blad- der have also been examined as potential mechanisms for the changes in contractility. We showed that the contractile re- sponse of bladders from diabetic and sucrose-drinking rats to glucose removal was similar to that of controls, and not influenced by the presence of insulin in the medium.34 Blad- ders from diabetic rats have a high affinity isoform of creat- ine kinase, the enzyme that catalyzes the transfer of a phos- phate group from creatine phosphate to ATP, which is not found in control rat bladders.35 Whether this isoform could modulate intracellular energetics of bladders from diabetic rats, resulting in the observed increased contractility, is not known. Similarly, the maximal activity of lactate dehydroge- nase, the enzyme that catalyzes the formation of lactate from pyruvate, is increased in bladders from diabetic rats and is accompanied by decreases in the relative proportions of the M3H and M2H2 isoforms, but no change in lactate forma- tion.36 These changes may explain the better maintenance of force in diabetic bladders.

In conclusion, our data suggest that the acute changes in bladder function that accompany the induction of diabetes in rats are related to the diuresis-induced increases in blad- der mass.

REFERENCES

1. Longhurst, P. A. and Belis, J. A.: Abnormalities of rat bladder contractility in streptozotocin-induced diabetes mellitus. J. Pharmacol. Exp. Ther., 238 773, 1986.

2. Uvelius, B.: Detrusor smooth muscle in rats with alloxan- induced diabetes. J. Urol., 136 949, 1986.

3. 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.

4. 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. 5. Longhurst, P. A., Kauer, J. and Levin, R. M.: The ability of

insulin treatment to reverse or prevent the changes in urinary bladder function caused by streptozotocin-induced diabetes mellitus. Gen. Pharmacol., 22 305, 1991.

6. Latifpour, J., Gousse, A., Kondo, S., Morita, T. and Weiss, R. M.: Effects of experimental diabetes on biochemical and functional characteristics of bladder muscarinic receptors. J. Pharmacol. Exp. Ther., 248 81, 1989.

7. Malmgren, A, Andersson, P. 0. and Uvelius, B.: Bladder func- tion in rats with short- and long-term diabetes: effects of age and muscarinic blockade. J. Urol., 142: 1608, 1989.

8. Luheshi, G. N. and Zar, M. A.: Inhibitory effect of streptozotocin- induced diabetes on non-cholinergic motor transmission in rat detrusor and its prevention by sorbinil. Br. J. Pharmacol., 101: 411, 1990.

9. Longhurst, P. A., Wein, A. J. and Levin, R. M.: In-vivo urinary bladder function in rats following prolonged diabetic and non- diabetic diuresis. Neurourol. Urodynam., 9 171, 1990.

10. Eika, B., Levin, R. M., Monson, F. C., Murphy, M. and Longhurst, P. A.: 3H-thymidine uptake by the rat urinary bladder aft.er induction of diabetes mellitus. J. Urol., 150 1316, 1993.

11. Carpenter, F. G.: Impairment and restoration of rat urinary bladder resDonsiveness following distension. Am. J. Phvsiol..

I " . 244: R106, i983.

12. Tammela. T. L. J.. Briscoe. J. A. K.. Levin. R. M. and Lonehurst. P. A.: Factors 'mderly&g the increased sensitivity to field stimulation of urinary bladder strips from streptozotocin- diabetic rats. Br. J. Pharmacol., 113 195, 1994.

13, Bergmeyer, H. U. and Bernt, E.: D-glucose determination with glucose oxidase and peroxidase. In: Methods of Enzymatic Analysis. Edited by H. U. Bergmeyer and E. Bernt. New York: Academic Press, pp. 1205-1215, 1974.

14. Longhurst, P. A., Kang, J., Wein, A. J. and Levin, R. M.: Length- tension relationship of urinary bladder strips from streptozo- tocin-diabetic rats. Pharmacology, 40: 110, 1990.

15. Miller, R. G.: Simultaneous Statistical Inference. Montreal: McGraw-Hill, 1966.

16. Eika, B., Levin, R. M. and Longhurst, P. A.: Comparison of urinary bladder function in rats with hereditary diabetes in- sipidus, streptozotocin-induced diabetes mellitus, and nondia- betic osmotic diuresis. J. Urol., 151: 496, 1994.

17. Spector, A. C. and Smith, J. C.: A detailed analysis of sucrose drinking in the rat. Physiol. Behav., 33 127, 1984.

18. Muscat, R. and Willner, P.: Suppression of sucrose drinking by chronic mild unpredictable stress - A methodological analysis. Neurosci. Biobehav. Rev., 16 507, 1992.

19. Eika, B., Levin, R. M. and Longhurst, P. A.: Collagen and blad- der function in streptozotocin-diabetic rats: effects of insulin and aminoguanidine. J. Urol., 148: 167, 1992.

20. Persson, K, Herrell, D., Tuttle, J. B. and Steers, W. D.: Regula- tion of NGF secretion bv urinarv tract smooth muscle cells. J.

21.

22.

23.

24.

25.

26.

27

Urol., 151: 444A, 1994.- Karim, 0. M. A,, Pienta, K., Seki, N. and Mostwin, J. L.: Stretch-

mediated visceral smooth muscle growth in vitro. Am. J. Physiol., 262: R895, 1992.

Koo, H. P., Santarosa, R. P., Buttyan, R., Shabsigh, R., Olsson, C. A. and Kaplan, S. A.: Early molecular changes associated with streptozotocin-induced diabetic bladder hypertrophy in the rat. Urol. Res., 21: 375, 1993.

Steers, W. D., Kolbeck, S., Creedon, D. and Tuttle, J. B.: Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J. Clin. Invest., 8 8 1709, 1991.

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 diabe- tes. J. Auton. Nerv. Syst., 47: 83, 1994.

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.

Nadelhaft, I. and Vera, P. L.: Reduced urinary bladder afferent conduction velocities in streptozotocin diabetic rats. Neurosci. Lett., 135: 276, 1992.

Andersson, P. O., Fahrenkrug, J., Malmgren, A. and Uvelius, B.: Effects of age and streptozotocin-induced diabetes on contents and effects of substance P and vasoactive intestinal polypep- tide in the lower urinary tract of the rat. Acta Physiol. Scand.,

BLADDER FUNCTION AND DIURESIS 2021

1 4 4 361, 1992. 28. 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-Schmied. Arch. Pharmacol., 3 3 5 580, 1987.

29. Lincoln, J., Crockett, M., Haven, A. J. and Burnstock, G.: Rat bladder in the early stages of streptozotocin-induced diabetes: adrenergic and cholinergic innervation. Diabetologia, 2 6 81, 1984.

30. Kudlacz, E. M., Gerald, M. C. and Wallace, L. J.: Effects of diabetes and diuresis on contraction and relaxation mecha- nisms in rat urinary bladder. Diabetes, 3 8 278, 1989.

31. Luheshi, G. N. and Zar, M. A,: The effect of streptozotocin- induced diabetes on cholinergic motor transmission in the rat urinary bladder. Br. J. Pharmacol., 103: 1657, 1991.

32. Lincoln, J., Haven, A. J., Sawyer, M. and Burnstock, G.: The

smooth muscle of rat bladder in the early stages of streptozo- tocin-induced diabetes. Br. J. Urol., 56.24, 1984.

33. Samuel, M., Longhurst, P. A., Levin, R. M. and Chacko, S.: Characteristics of urinary bladder myosin isolated from dia- betic rats. J. Urol., 145 356A, 1991.

34. Longhurst, P. A., Briscoe, J. A. K, Leggett, R. E., Samadzadeh, S. and Levin, R. M.: The influence of diabetes-mellitus on glucose utilization by the rat urinary bladder. Metabolism, 42. 749, 1993.

35. Levin, R. M., Haugaard, N., Levin, S. S. and Wein, A. J.: Crea- tine kinase activity in normal and hypertrophied rabbit uri- nary bladder tissue (following partial outlet obstruction). Mol. Cell. Biochem., 106: 143, 1991.

36. Amer, A., Malmqvist, U., Osterman, A. and Uvelius, B.: Energy turnover and lactate dehydrogenase activity in detrusor smooth muscle from rats with streptozotocin-induced diabetes. Acta Physiol. Scand., 147: 375, 1993.