Effects of γ-Aminobutyrate B Receptor Modulation on Normal Micturition and Oxyhemoglobin Induced Detrusor Overactivity in Female Rats

0022-5347/02/1686-2700/0 THE JOURNAL OF UROLOGY® Copyright © 2002 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 168, 2700 –2705, December 2002 Printed in U.S.A.

DOI: 10.1097/01.ju.0000029201.01772.2f

EFFECTS OF ␥-AMINOBUTYRATE B RECEPTOR MODULATION ON NORMAL MICTURITION AND OXYHEMOGLOBIN INDUCED DETRUSOR OVERACTIVITY IN FEMALE RATS RIKARD PEHRSON, ANDERS LEHMANN

AND

KARL-ERIK ANDERSSON

From the Department of Clinical Pharmacology, Lund University Hospital, Lund and Departments of Gastrointestinal Biology and Integrative Pharmacology, AstraZeneca R & D Mo¨lndal, Mo¨lndal, Sweden

ABSTRACT

Purpose: Using baclofen (Sigma-Aldrich, Steinheim, Germany), a ␥-aminobutyrate B (GABA(B)) receptor agonist, and CGP62349 (AstraZeneca R & D Mo¨lndal, Sweden), a GABA(B) receptor antagonist, in a rat model of conscious micturition we addressed certain questions, including whether there is a tonic GABA(B) receptor influence on normal bladder function, how baclofen affects normal and C-fiber activated micturition, and where the sites of GABA(B) receptor action are. Materials and Methods: Nonanesthetized female Sprague-Dawley rats were used. The bladder was catheterized and other catheters were placed intravenously, intrathecally or intracerebroventricularly. At 3 days the rats underwent cystometric investigation in a metabolic cage. Micturition was stimulated by infusing saline intravesically. Overactivity caused by C-fiber activation was induced by intravesical oxyhemoglobin. Drugs were given intravenously, intrathecally or intracerebroventricularly. Micturition parameters were recorded and compared before and after drug administration Results: Baclofen at doses of 0.5 ␮g. intrathecally and 0.3 ␮g. intracerebroventricularly increased bladder capacity and threshold pressure. Overflow incontinence developed in 4 of 7 rats after 0.5 ␮g. baclofen intrathecally and in 5 of 7 after 1 ␮g. baclofen intracerebroventricularly. CGP62349 at a dose of 30 ␮g. intrathecally and intracerebroventricularly had a stimulatory effect on micturition, which was attenuated by baclofen. While intravenous baclofen at 1 mg. kg.⫺1 was devoid of effects, intravenous baclofen at 4 mg. kg.⫺1 tended to decrease micturition pressure, bladder capacity and micturition volume. Infusion volume decreased significantly, demonstrating a diuretic effect, which was abolished by pretreatment with subcutaneous desmopressin at 25 ng. kg.⫺1. CGP62349 at 2 mg. kg.⫺1 intravenously had a stimulatory effect on micturition, which was inhibited by baclofen. Intravesical oxyhemoglobin at 250 ␮M. induced bladder overactivity, which was attenuated by baclofen at 4 mg. kg.⫺1 intravenously and abolished by baclofen 0.5 ␮g intrathecally. Conclusions: In the normal rat stimulation of GABA(B) receptors, mainly in the central nervous system, inhibits micturition. Antagonism of GABA(B) receptors stimulates micturition, suggesting that the receptors are under tonic GABAergic influence. Baclofen intrathecally attenuated oxyhemoglobin induced detrusor overactivity, suggesting that the inhibitory actions of GABA(B) receptor agonists in the spinal cord may be useful for controlling micturition disorders caused by C-fiber activation in the urothelium and/or suburothelium. KEY WORDS: bladder; GABA agonists; muscle, smooth; urination; rats, Sprague-Dawley

The major inhibitory neurotransmitter in the central nervous system, ␥-aminobutyrate (GABA),1 elicits its activity through at least 2 membrane receptors, namely GABA(A) and GABA(B).2 GABA is known to be important for the control of micturition acting via GABA(A) and GABA(B) receptors.3 The GABA(B) receptor agonist baclofen given intrathecally is used clinically for reducing urethral resistance and detrusor overactivity associated with spasticity. The main effect of baclofen within the central nervous system is to reduce transmitter release.4 In the spinal cord it affects the activity of motoneurons and interneurons that are important for micturition and baclofen has previously been reported to have an inhibitory action on rat micturition after intrathecal administration.3, 5, 6 However, baclofen also has supraspinal7 and peripheral3 actions. The importance of enAccepted for publication June 7, 2002. Supported by Swedish Medical Research Council Grant 6837.

dogenous GABA activity for the micturition reflex is less well studied. However, a GABA re-uptake inhibitor given intrathecally has inhibited normal rat micturition.8 Activation of afferent C-fibers in the rat bladder urothelium can induce detrusor overactivity. Previous experimental approaches have been used, for example intravesical capsaicin and resiniferatoxin to stimulate micturition. Intravesical oxyhemoglobin represents a new method of inducing detrusor overactivity.9 Baclofen, which is more effective for inhibiting C-fiber than A␦-fiber afferents in spinal cord slices,10 may have an effect on C-fiber activated detrusor overactivity. We tested if there is tonic GABAergic inhibitory control of micturition in the awake rat, and if GABA(B) receptor activation can inhibit C-fiber induced bladder overactivity. In addition, the sites of GABAergic influence on micturition were further studied. Therefore, the effects of the potent and selective GABA(B) receptor antagonist CGP6234911 given intracerebroventricularly, intrathecally and intravenously on normal rat micturition were studied. The actions of ba-

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EFFECTS OF ␥-AMINOBUTYRATE B RECEPTOR MODULATION ON MICTURITION

clofen on normal and oxyhemoglobin stimulated detrusor activity were also investigated. MATERIALS AND METHODS

Animals. Female Sprague-Dawley rats weighing 200 to 225 gm. were used. The experimental protocol was approved by the animal ethics committee, Court of the City of Lund. Catheter implantations. Rats were anesthetized with intraperitoneal ketamine (Ketalar, Park Davis, Barcelona, Spain) (75 mg. kg.⫺1) and xylazine (Rompun, Bayer AG, Leverkusen, Germany) (15 mg. kg.⫺1). Catheterization of the bladder and a femoral vein or insertion of an intrathecal or intracerebroventricular catheter was performed at the same time, as described previously,8 except for intracerebroventricular catheterization. Intracerebroventricular catheter. The rat was placed in a stereotaxic frame (Narishige, Tokyo, Japan). A skin incision was made on the skull and the bregma was located. Two small screws were attached to the skull. At 0.8 mm. posterior to and 1.4 mm. right of the bregma a small hole was drilled, reaching the dura. A catheter (Plastics One, Wallingford, Connecticut) was lowered 3.5 mm. below the dura, reaching the right lateral ventricle. The catheter was secured by dental cement (Svedia Dental, Enko¨ ping, Sweden) to the screws and skull. Cystometry. At 3 days after bladder catheterization continuous cystometry was performed, as previously described.8 Drug administration was done via tubing using a PE-50 catheter (Clay-Adams, Parsippany, New Jersey) for intravenous administration and a PEG catheter (AgnTho’s, Stockholm, Sweden) with an inner diameter of 60 ␮m. for intrathecal and intracerebroventricular administration. The tubes were attached to catheter outlets on the rat neck. This setup permitted drug administration without disturbing the animal. To define baseline values cystometry recording was evaluated for 20 minutes during regular micturition before drug administration. Before baseline values were determined in rats with an intrathecal or intracerebroventricular catheter saline was given in the same volume as drug to ensure that administration alone did not cause effects on micturition. The parameters assessed or calculated were baseline pressure (pressure during urine collection), threshold pressure (pressure immediately before bladder contraction), micturition pressure (highest pressure during micturition), micturition volume, infused volume (volume infused through tubing), postvoid residual volume (remaining urine after voiding) and bladder capacity (infused plus post-void residual volumes) or micturition volume (when larger than infusion plus post-void residual volumes). Drug effects were evaluated from cystometry results directly after drug administration for 20 minutes. Detrusor overactivity was produced by infusing 250 ␮M. oxyhemoglobin intravesically. The effects of vehicle or drug administration were evaluated. Drugs and administration. Baclofen and CGP62349 were

2701

dissolved in saline on the day of the experiment. CGP62349 was not a pure diastereomer but a 50/50 mixture of CGP62349 and its epimer CGP62349 (3-[(1S)-1-[[(2S)-2-hydroxy-3-[hydroxy[(4methoxyphenyl)methyl]phosphinyl]propyl]amino]ethyl]benzoic acid). Minirin desmopressin (Ferring, Malmo¨ , Sweden) (4 ␮g. ml.⫺1) was diluted in saline to 25 ng. ml.⫺1 on the day of the experiment. Drugs were given at body temperature to minimize the rat reaction to administration. Intravenous drugs were given in a volume of 1 ml. kg.⫺1, followed by a 0.2 ml. saline flush. For intrathecal administration 10 ␮l. were injected, followed by a 15 ␮l. flush. Intracerebroventricular administration was done in a volume of 10 ␮l. Hemoglobin (Sigma-Aldrich) was diluted in distilled water to a 1 mM. concentration. Na2S2O4 (Sigma-Aldrich) was dissolved to a 10 mM. concentration. The 2 solutions were mixed, transferred to a Spectra/Por4 dialysis membrane (Spectrum, Gardena, California) and placed in a beaker with distilled water at 4C. Dialysis was accomplished in 4 hours. Thereafter the oxyhemoglobin solution, now at 500 ␮M., was diluted in saline to 250 ␮M. and stored in a freezer at ⫺20C. The solution was used within 2 days. To verify the position of the femoral catheter after an experiment an overdose of pentobarbital was given intravenously. Instant sacrifice showed a correct position. For intrathecal and intracerebroventricular catheters 10 ␮l. dye (1% methylene blue) was administered and the rat was sacrificed 15 minutes later. The position of the catheter and extent of dye distribution were examined. If dye distribution was found in sacral and lumbar parts of the spinal cord after intrathecal administration or in the third and both lateral ventricles after intracerebroventricular administration, intervention was considered successful. Student’s paired 2-tailed t test was used with p ⬍0.05 considered significant. RESULTS

Intrathecal administrations. Intrathecal baclofen at 0.5 ␮g. induced overflow incontinence in 4 of 7 rats, including directly after administration in 1 and within 10 to 15 minutes in 3. In the 6 rats that were able to micturate directly after administration mean micturition pressure plus or minus SEM decreased (37% ⫾ 9%, p ⬍0.05) and mean threshold pressure increased (56% ⫾ 18%, p ⬍0.05.). Mean bladder capacity (64% ⫾ 13%, p ⬍0.01) and mean infused volume (45% ⫾ 16%, p ⬍0.01) increased (table 1 and fig. 1). No behavioral effects were observed during or after the experiment. CGP62349 at 3 ␮g. in 6 rats and at 10 ␮g. in 4 given intrathecally had no effects on micturition. CGP62349 at 30 ␮g. in 6 rats decreased mean micturition volume (17% ⫾ 13%, p ⬍0.05) and mean bladder capacity (14% ⫾ 12%, p ⬍0.05, table 1). No behavioral effects were observed during or after the experiment. CGP62349 at 30 ␮g. given intrathecally before 0.5 ␮g. baclofen intrathecally in 4 rats reversed the effects on micturition of intrathecal baclofen. Immediately after the ad-

TABLE 1. Effects of intrathecal 0.5 ␮g. baclofen and 30 ␮g. CGP62349 given intrathecally in rats Mean Pressure ⫾ SEM (cm. water) Micturition

Threshold

Baclofen: Before 92 ⫾ 15 16 ⫾ 1 After 58 ⫾ 9 25 ⫾ 3 p Value (paired Student’s ⬍0.05 ⬍0.05 2-tailed t test) CGP62349: Before 116 ⫾ 12 14 ⫾ 2 After 124 ⫾ 15 13 ⫾ 2 * Versus before paired Student’s 2-tailed t test p ⬍0.05.

Mean Vol. ⫾ SEM (ml.)

Baseline

Mean Bladder Capacity ⫾ SEM (ml.)

Micturition

Infused

Post-Void Residual

7⫾1 5⫾1 ⬍0.05

0.77 ⫾ 0.12 1.26 ⫾ 0.10 ⬍0.01

0.75 ⫾ 0.12 0.94 ⫾ 0.17 ⬍0.05

0.76 ⫾ 0.13 1.10 ⫾ 0.12 ⬍0.01

0.03 ⫾ 0.02 0.31 ⫾ 0.13 ⬍0.05

7⫾2 6⫾2

0.77 ⫾ 0.09 0.66 ⫾ 0.09*

0.73 ⫾ 0.09 0.61 ⫾ 0.09*

0.72 ⫾ 0.10 0.63 ⫾ 0.10*

0 0

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EFFECTS OF ␥-AMINOBUTYRATE B RECEPTOR MODULATION ON MICTURITION

FIG. 1. Effects of intrathecal (i.t.) administration of 0.5 ␮g baclofen on bladder pressure in cm. water and voided volume in ml.

ministration of the 2 drugs no behavioral effects were observed. However, when the animals were transferred to cages they were hyperexcitable and that state lasted several hours. Therefore, 6 rats were given only 30 ␮g. CGP62349 intrathecally and hyperexcitability was observed starting approximately 30 to 45 minutes after administration and lasting several hours. An additional 6 rats were given 30 ␮g. CGP62349 intrathecally with 0.5 ␮g. baclofen intrathecally. Hyperexcitability developed in 4 of the 6 rats. Intracerebroventricular administration. Baclofen at 0.3 ␮g. in 9 rats increased mean bladder capacity (27% ⫾ 17%, p ⬍0.05) and tended to increase mean micturition volume (17% ⫾ 22%, not significant), mean post-void residual volume (64% ⫾ 64%, not significant) and mean threshold pressure (64% ⫾ 36%, not significant). Mean micturition pressure tended to decrease (20% ⫾ 13%, not significant, table 2). Baclofen at 1 ␮g. given in intracerebroventricular fashion in 7 rats induced overflow incontinence in 5, including instantly in 2 and within 10 minutes in 3. In 2 rats normal micturition was maintained with increased bladder capacity. The rats became calm and reduced normal activity. CGP62349 at 30 ␮g. intracerebroventricularly in 6 rats decreased mean infusion (51% ⫾ 6%, p ⬍0.01) and micturition (55% ⫾ 7%, p ⬍0.01) volume as well as mean bladder capacity (54% ⫾ 7%, p ⬍0.01, table 2). After administration the rats were aroused and when they were transferred to their cages, they were hyperexcitable, a state that lasted several hours. The effect had greater intensity than after intrathecal administration. The simultaneous administration of 0.5 ␮g. baclofen with 30 ␮g. CGP62349 in 4 rats intracerebroventricularly attenuated bladder overactivity and its duration. No hyperexcitability was observed during or after the experiment. Intravenous administration. Baclofen at 1 mg. kg.⫺1 intravenously in 8 rats did not markedly affect micturition. At 4 mg. kg.⫺1 baclofen in 6 rats tended to decrease mean micturition pressure (30% ⫾ 15%, not significant), mean bladder capacity (16% ⫾ 16%, not significant) and mean micturition volume (14% ⫾ 16%, not significant). Mean infused volume decreased significantly (32% ⫾ 10%, p ⬍0.05, table 3).

Intravenous administration of 8 and 16 mg. kg.⫺1 baclofen in 2 rats each resulted in a reversible general muscle relaxant effect that manifested as inability to move despite consciousness plus overflow incontinence. In rats pretreated with 25 ng. kg.⫺1 desmopressin subcutaneously 4 mg. kg.-1 baclofen intravenously in 6 did not change mean bladder capacity. However, mean micturition (16% ⫾ 19%, not significant) and infused (22% ⫾ 19%, not significant) volumes tended to decrease. Mean post-void residual volume increased (1,500% ⫾ 600%, p ⬍0.05, table 3). CGP62349 at 2 mg. kg.⫺1 intravenously decreased mean bladder capacity (29% ⫾ 10%, p ⬍0.01), mean micturition volume (31% ⫾ 10%, p ⬍0.01) and mean infused volume (36 ⫾ 6%, ⬍0.01, fig. 2). There were no evident changes in behavior. These effects were abolished by pretreatment with 4 mg. kg.⫺1 baclofen intravenously (table 4). Oxyhemoglobin induced detrusor overactivity. Intravesical administration of 250 ␮M. oxyhemoglobin decreased mean bladder capacity (47% ⫾ 4%, p ⬍0.001), mean micturition volume (53% ⫾ 5%, p ⬍0.001) and mean infused volume (51% ⫾ 4%, p ⬍0.001). Mean threshold (50% ⫾ 29%, p ⬍0.05) and bladder (83% ⫾ 33%, p ⬍0.01) pressures increased. Baclofen at 1 mg. kg.⫺1 intravenously had no significantly effect but at 4 mg. kg.⫺1 intravenously it significantly attenuated oxyhemoglobin induced detrusor overactivity (table 5). Baclofen at 0.5 ␮g. intrathecally in 6 rats also attenuated detrusor overactivity induced by intravesical 250 ␮M. oxyhemoglobin (table 6 and fig. 3). DISCUSSION

GABA(B) receptors are present in the rat spinal cord and concentrated at dorsal horn laminae I to III, where they outnumber GABA(A) receptors.1, 12 These GABA(B) receptors are located on A␦ and C-fiber terminals.1 Via action on these primary afferent terminals1, 2 GABA can presynaptically attenuate neurotransmission.4 Hence, intrathecal GABA(B) receptor stimulation may inhibit afferent stimuli, which can explain the increase in bladder threshold pressure

TABLE 2. Effects of 0.3 ␮g. baclofen in 9 rats and 30 ␮g. CGP62349 in 6 given in intracerebroventricular fashion Mean Pressure ⫾ SEM (cm. water) Micturition

Threshold

Baseline

Baclofen: Before 69 ⫾ 13 11 ⫾ 2 11 ⫾ 2 After 55 ⫾ 9 18 ⫾ 4* 18 ⫾ 4 CGP62349: Before 93 ⫾ 12 15 ⫾ 4 7⫾3 After 108 ⫾ 10 19 ⫾ 6 8⫾4 * Versus before paired Student’s 2 tailed t test p ⬍0.05. † Versus before paired Student’s 2-tailed t test p ⬍0.05; ††p ⬍0.01.

Mean Vol. ⫾ SEM (ml.)

Mean Bladder Capacity ⫾ SEM (ml.)

Micturition

Infused

Post-Void Residual

0.83 ⫾ 0.10 1.05 ⫾ 0.14*

0.69 ⫾ 0.08 0.81 ⫾ 0.15

0.74 ⫾ 0.09 0.89 ⫾ 0.17

0.14 ⫾ 0.04 0.23 ⫾ 0.09

0.74 ⫾ 0.05 0.35 ⫾ 0.05†

0.73 ⫾ 0.05 0.33 ⫾ 0.05†

0.66 ⫾ 0.06 0.32 ⫾ 0.04†

0.02 ⫾ 0.01 0.02 ⫾ 0.01

EFFECTS OF ␥-AMINOBUTYRATE B RECEPTOR MODULATION ON MICTURITION

2703

TABLE 3. Effects of baclofen given intravenously with or without pretreatment with subcutaneous desmopressin Baclofen (mg. kg.⫺1) 1:

No. Rats 8

Before After

Mean Pressure ⫾ SEM (cm. water)

Mean Vol. ⫾ SEM (ml.)

Micturition

Threshold

Baseline

Mean Bladder Capacity ⫾ SEM (ml.)

62 ⫾ 10 56 ⫾ 10

16 ⫾ 3 16 ⫾ 2

5⫾2 5⫾1

0.90 ⫾ 0.07 0.84 ⫾ 0.08

0.81 ⫾ 0.09 0.83 ⫾ 0.07

0.82 ⫾ 0.07 0.70 ⫾ 0.07

0.08 ⫾ 0.03 0.04 ⫾ 0.02

18 ⫾ 3 16 ⫾ 2

5⫾2 5⫾1

0.75 ⫾ 0.08 0.63 ⫾ 0.12

0.74 ⫾ 0.08 0.64 ⫾ 0.12

0.71 ⫾ 0.08 0.48 ⫾ 0.07*

0.03 ⫾ 0.02 0.03 ⫾ 0.01

16 ⫾ 3 16 ⫾ 5

7⫾2 6⫾3

0.83 ⫾ 0.12 0.85 ⫾ 0.12

0.81 ⫾ 0.12 0.68 ⫾ 0.15

0.81 ⫾ 0.12 0.63 ⫾ 0.15

0.01 ⫾ 0.00 0.16 ⫾ 0.06*

4:

6 Before 71 ⫾ 8 After 50 ⫾ 11 6 4 ⫹ 25 ng. kg⫺1 desmopressin: Before 53 ⫾ 9 After 31 ⫾ 6* * Versus before paired Student’s 2-tailed t test p ⬍0.05.

Micturition

Infused

Post-Void Residual

FIG. 2. Effects of intravenous (i.v.) administration of 2 mg. kg.⫺1 CGP62349 on bladder pressure in cm. water and voided volume in ml. TABLE 4. Effects of 2 mg. kg⫺1 CGP62349 in the absence in 6 rats or presence in 4 of 4 mg. kg⫺1 baclofen each given intravenously Mean Pressure ⫾ SEM (cm. water) Micturition

Baseline

Micturition

Infused

Post-Void Residual

19 ⫾ 1 18 ⫾ 4

10 ⫾ 1 10 ⫾ 2

0.83 ⫾ 0.08 0.59 ⫾ 0.08*

0.81 ⫾ 0.09 0.56 ⫾ 0.08*

0.72 ⫾ 0.08 0.46 ⫾ 0.04*

0.01 ⫾ 0.01 0.02 ⫾ 0.01

17 ⫾ 3 19 ⫾ 5 p ⬍0.01.

11 ⫾ 3 10 ⫾ 3

0.74 ⫾ 0.08 0.68 ⫾ 0.08

0.69 ⫾ 0.08 0.68 ⫾ 0.08

0.68 ⫾ 0.08 0.63 ⫾ 0.06

0.05 ⫾ 0.03 0.02 ⫾ 0.01

Threshold

CGP62349: Before 65 ⫾ 12 After 71 ⫾ 13 CGP62349 ⫹ baclofen:† Before 83 ⫾ 11 After 82 ⫾ 20 * Versus before paired Student’s 2-tailed t test

Mean Vol. ⫾ SEM (ml.)

Mean Bladder Capacity ⫾ SEM (ml.)

TABLE 5. Effects of intravesical 250 ␮M. oxyhemoglobin in the presence of vehicle in 10 rats, 1 mg. kg.⫺1, baclofen in 6 or 4 mg. kg⫺1 in given intravenously Mean Pressure ⫾ SEM (cm. water) Micturition Vehicle: Before 75 ⫾ 8 After 81 ⫾ 7 p Value (paired Student’s 2-tailed test) 1 Mg. kg⫺1 baclofen: Before 73 ⫾ 12 After 87 ⫾ 15 p Value (paired Student’s 2-tailed test) 4 Mg. kg⫺1 baclofen: Before 80 ⫾ 12 After 85 ⫾ 17 * Versus before paired Student’s 2-tailed t test p ⬍0.01.

Mean Vol. ⫾ SEM (ml.)

Threshold

Baseline

Mean Bladder Capacity ⫾ SEM (ml.)

14 ⫾ 3 21 ⫾ 4 ⬍0.05

6⫾2 11 ⫾ 2 ⬍0.01

0.78 ⫾ 0.07 0.41 ⫾ 0.03 ⬍0.001

0.75 ⫾ 0.08 0.35 ⫾ 0.04 ⬍0.001

0.70 ⫾ 0.08 0.34 ⫾ 0.03 ⬍0.01

0.01 ⫾ 0.01 0.06 ⫾ 0.03

16 ⫾ 4 29 ⫾ 8

7⫾4 19 ⫾ 7 ⬍0.05

0.77 ⫾ 0.07 0.52 ⫾ 0.08 ⬍0.001

0.70 ⫾ 0.08 0.46 ⫾ 0.10 ⬍0.01

0.68 ⫾ 0.08 0.41 ⫾ 0.07 ⬍0.001

0.05 ⫾ 0.04 0.06 ⫾ 0.04

15 ⫾ 3 22 ⫾ 4

7⫾1 11 ⫾ 4

0.67 ⫾ 0.06 0.56 ⫾ 0.07

0.64 ⫾ 0.04 0.56 ⫾ 0.07

0.60 ⫾ 0.05 0.36 ⫾ 0.05*

0.02 ⫾ 0.02 0.01 ⫾ 0.01

after intrathecal baclofen. Furthermore, baclofen inhibits excitatory neurotransmission in the spinal cord.13 Since GABA mediates inhibitory inputs to the sacral parasympathetic preganglionic neurons, which control efferent impulses to the bladder and urethra,3 it may explain decreased voiding efficiency after intrathecal baclofen. The GABA(B) receptor antagonist CGP62349 given intrathecally was able to stimulate micturition and counteract the effects of intrathecal baclofen. This effect was in apparent disagreement with the finding that intrathecal saclofen, which is another GABA(B) receptor antagonist, has no effects on micturition.3 However, saclofen is a weak antagonist14 with possible agonistic properties. Therefore,

Micturition

Infused

Post-Void Residual

it seems likely that spinal GABA(B) receptors are under tonic activation and blocking these receptors may stimulate micturition. Urethral function can be influenced through pontine GABAergic neurons in direct contact with spinal motoneurons to the urethral sphincter.15 In the spinal cord by a presynaptic mechanism13 baclofen exerts a potent depressant action on motoneurons and interneurons.14 It may contribute to the decrease in micturition pressure and the dribbling incontinence produced by baclofen after intrathecal administration, as shown in the current and previous3 series. In the current study intracerebroventricular baclofen had an inhibitory effect on micturition, which was in

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EFFECTS OF ␥-AMINOBUTYRATE B RECEPTOR MODULATION ON MICTURITION

TABLE 6. Effects of intravesical 250 ␮M. oxyhemoglobin in the presence of saline or 0.5 ␮g. baclofen given intrathecally in 6 rats each Mean Pressure ⫾ SEM (cm. water) Micturition

Threshold

Saline: Before 98 ⫾ 15 20 ⫾ 3 After 108 ⫾ 13 25 ⫾ 6 Baclofen: Before 87 ⫾ 12 19 ⫾ 4 After 92 ⫾ 11 27 ⫾ 6 * Versus before paired Student’s 2-tailed t test p

Mean Vol. ⫾ SEM (ml.)

Baseline

Mean Bladder Capacity ⫾ SEM (ml.)

Micturition

Infused

Post-Void Residual

14 ⫾ 4 19 ⫾ 5

0.74 ⫾ 0.08 0.49 ⫾ 0.11*

0.71 ⫾ 0.08 0.38 ⫾ 0.09*

0.71 ⫾ 0.07 0.42 ⫾ 0.10*

0.02 ⫾ 0.02 0.13 ⫾ 0.05

13 ⫾ 4 24 ⫾ 12 ⬍0.01.

0.96 ⫾ 0.16 0.81 ⫾ 0.18

0.91 ⫾ 0.16 0.65 ⫾ 0.14

0.88 ⫾ 0.14 0.72 ⫾ 0.18

0.05 ⫾ 0.04 0.14 ⫾ 0.07

FIG. 3. Effects on bladder pressure in cm. water and voided volume in ml. in rats given 250 ␮M. oxyhemoglobin intravesically (i.ves.). Top, after intrathecal (i.t.) saline. Bottom, after 0.5 ␮g. baclofen intrathecally.

agreement with previous findings.7 Intracerebroventricular CGP62349 stimulated micturition, and intracerebroventricular and intravenous CGP62349 elicited more distinct detrusor overactivity than after intrathecal administration. Hence, the tonic GABA input that is important for micturition control may be more pronounced supraspinally than spinally. Intracerebroventricular baclofen attenuated the effects of intracerebroventricular CGP62349. In a previous report intravenous bicuculline, a GABA(A) receptor antagonist, prevented the effects of intracerebroventricular GABA on micturition,5 suggesting that GABA(A) receptor is the important GABA subtype within the brain for micturition control. Supporting this view, the GABA(B) receptor antagonist phaclofen given in intracerebroventricular fashion was unable to influence the effects of intracerebroventricular baclofen on rat bladder function in anesthetized rats.6 On the other hand, the current findings suggest that GABA(B) receptors in the brain are also involved in micturition control. Pronounced hyperexcitability was observed when the rats were transferred from metabolism cages after being given CGP62349 in intracerebroventricular fashion. This finding may be explained by increased release of excitatory amino acids due to presynaptic disinhibition, possibly combined with reduced postsynaptic GABAergic tone. An epileptogenic effect of GABA(B) receptor antagonists has previously been demonstrated.16 Hyperexcitability after intrathecal administration of the antagonist was also observed even when given with baclofen. GABA(B) receptors and corresponding mRNA have been detected in the rat bladder.17 Human and guinea pig GABA or GABA re-uptake inhibition in the rat inhibits nerve mediated bladder contractions.8 However, baclofen given intravenously at high doses caused skeletal muscle relaxation.18 Therefore, the effect on skeletal muscles observed most likely

contributed to the overflow incontinence after 8 and 16 mg. kg.⫺1 baclofen intravenously. Hence, peripheral effects of baclofen on rat micturition probably have minor importance, as previously suggested.3 The facilitation of the micturition reflex seen after intravenous CGP62349 may have a peripheral site of action. However, considering the effects of intracerebroventricular and intrathecal administration of the drug a central nervous site of action seems more likely. Via effects on the hypothalamic supraoptic nucleus, which is a major central nervous system source for the release of circulating vasopressin, baclofen can affect vasopressin release.19 It may explain the diuretic effect of intravenous baclofen in the current study. Desmopressin, which is a vasopressin analogue, has a prolonged and effective antidiuretic effect in rats.20 Pretreatment with 25 ng. kg.⫺1 desmopressin prevented the diuretic response after 4 mg. kg.⫺1 baclofen given intravenously, suggesting that intravenous baclofen reduces the release of and/or renal sensitivity to vasopressin (table 3). Intravesical oxyhemoglobin has previously been shown to induce concentration dependent detrusor overactivity in rats.9 Oxyhemoglobin probably interferes with the L-arginine/nitric oxide/cyclic guanosine monophosphate pathway by scavenging nitric oxide in the urothelium, thereby stimulating the firing of afferent C-fibers in the urothelium or suburothelium.9 Baclofen attenuated the action of intravesical oxyhemoglobin after intravenous and intrathecal administration, suggesting that detrusor overactivity produced by C-fiber activation can be attenuated by baclofen via a spinal site of action. CONCLUSIONS

In the rat the GABA(B) receptor represents an effective way to influence normal micturition. While GABA(B) receptor activation causes inhibition in the normal rat, systemic,

EFFECTS OF ␥-AMINOBUTYRATE B RECEPTOR MODULATION ON MICTURITION

spinal or supraspinal GABA(B) receptor blockade stimulates micturition. This finding suggests that the receptors are under tonic GABA influence. Intrathecal baclofen abolished oxyhemoglobin induced bladder overactivity. This observation suggests that inhibitory actions of GABA(B) agonists in the spinal cord may be useful for controlling micturition disorders caused by the activation of C-fibers in the urothelium and/or suburothelium. REFERENCES

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