Yasuhiko Igawa

Effects of inhibition of the L-arginine/nitric oxide pathway in the rat lower urinary tract in vivo and in vitro

1 The present study was performed to investigate how blockade of the l‐arginine/nitric oxide (NO) pathway influences the function of the lower urinary tract in vivo, as studied by cystometry in conscious rats and in vitro, in isolated muscle preparations from the rat detrusor and urethra. 2 l‐NG‐nitro arginine methyl ester (l‐NAME), 10 and 20 mg kg−1, administered intra‐arterially, decreased micturition volume and bladder capacity, and increased spontaneous bladder contractions. d‐NAME (20 mg kg−1) had no effect. No changes in the urodynamic parameters were recorded if l‐NAME (20 mg kg−1) was administered in combination with l‐arginine (200 mg kg−1). 3 Cystometries performed after intra‐arterial administration of sodium nitroprusside (SNP) (3 mg kg−1) and 3‐morpholino‐sydnonimin hydrochloride (SIN‐1, 2 mg kg−1) showed a decrease in bladder capacity, micturition volume and threshold pressure. SIN‐1, but not SNP, induced spontaneous bladder contractions. 4 Isolated precontracted urethral preparations responded to electrical stimulation with a frequency‐dependent tetrodotoxin‐sensitive relaxation. l‐NAME (10−4 m), but not d‐NAME, reduced the maximal relaxation to 31 ± 8% (n = 8) of the response prior to drug administration. The inhibition induced by l‐NAME was completely reversed by l‐arginine (10−3 m). SNP (10−8−10−4 m), SIN‐1 (10−6−3 × 10−4 m) and NO (10−5−10−3 m; present in acidified solution of NaNO2), caused relaxation (93–100%) of urethral preparations. l‐NAME did not affect these relaxations. 5 Detrusor strips contracted by carbachol or K+ showed contractions in response to electrical stimulation, even when pretreated with α,β‐methylene ATP and/or atropine. Small relaxations (14–41%) of detrusor strips were evoked by SNP (10−6−10−4 m), SIN‐1 (10−5−3 × 10−4 m) and NO (10−5−10−3 m). Electrically (20 Hz) induced contractions of the detrusor muscle were unaffected by addition of l‐NAME (10−6−10−4 m) or l‐arginine (10−3 m). 6 The present results suggest that the l‐arginine/NO pathway is of functional importance for the bladder outlet region, but that its role in the detrusor is questionable. They also suggest that the site of action of l‐NAME for inducing bladder hyperactivity in the rat is the outlet region rather than the detrusor muscle.

Effects of GABA-Receptor Stimulation and Blockade on Micturition in Normal Rats and Rats with Bladder Outflow Obstruction☆

The effects on micturition of the GABA receptor agonists muscimol (selective for GABAA-receptors) and baclofen (selective for GABAB-receptors), given intrathecally and intra-arterially to unanesthetized rats with and without bladder hypertrophy secondary to outflow obstruction, were studied by continuous cystometry. When given intrathecally in increasing doses, both muscimol (1 to 10 micrograms) and baclofen (0.1 to 5 micrograms) produced a dose-dependent inhibition of micturition with progressive increases in bladder capacity and residual volume, and a decrease in micturition pressure ending with urinary retention and dribbling incontinence. Generally, the effects were similar in normal rats and rats with bladder hypertrophy. In normal rats, bicuculline (blocking GABAA-receptors) decreased bladder capacity and micturition volume, and increased micturition pressure. In both types of rat, the effects of muscimol and baclofen were partly counteracted by bicuculline and baclofen, respectively. In rats with bladder hypertrophy, the amplitude of the spontaneous bladder contractions during filling was significantly increased after administration of muscimol and baclofen. When given intra-arterially in high doses, muscimol (2 to 16 mg./kg.) and baclofen (8 to 16 mg./kg.) produced effects on cystometric parameters and spontaneous bladder contractions similar to those obtained with intrathecal administration. These results suggest that muscimol and baclofen have insignificant peripheral effects on the lower urinary tract, but depress micturition by an effect on the central nervous system. They also suggest that the inhibitory GABA-ergic system does not play a role in the genesis of bladder hyperactivity in rats with outflow obstruction.

Functional importance of cholinergic and purinergic neurotransmission for micturition contraction in the normal, unanaesthetized rat

1 The cholinergic and purinergic neurotransmission involved in micturition in the normal, unanaesthetized rat was investigated by means of continuous cystometry. 2 ATP (1 and 5 mg kg−1), administered intra‐arterially (i.a.) close to the bladder, produced rapid, phasic, dose‐dependent increases in bladder pressure with micturition immediately after injection. The micturition pressure of the following spontaneous voidings increased, and bladder capacity, micturition volume, and residual volume decreased. Pretreatment with α,β‐methylene ATP (1 mg kg−1, i.a.) blocked the effects of ATP (5 mg kg−1). 3 α,β‐Methylene ATP (0.25, 0.5 and 1 mg kg−1, i.a.) produced rapid, phasic, increases in bladder pressure with micturition immediately after injection. The effects of α,β‐methylene ATP (0.25 mg kg−1, i.a.) were not affected by pretreatment with indomethacin (0.5–2 mg kg−1, i.a.). The micturition pressure of the subsequent spontaneous voidings decreased, and bladder capacity and residual volume increased. 4 Carbachol (5–50 μg kg−1, i.a.) produced rapid, sustained, dose‐dependent increases in bladder pressure with micturition, and then increased basal pressure, threshold pressure, and micturition pressure, and decreased bladder capacity and micturition volume during the following spontaneous voidings. 5 Atropine (1 mg kg−1, i.a.) decreased micturition pressure and micturition volume, but did not induce dribbling incontinence. Micturition contractions still occurred after the injection, but changed in appearance and were of shorter duration than before. In the presence of atropine (1mg kg−1, i.a.), α,β‐methylene ATP (1 mg kg−1, i.a.) produced initially a phasic increase in bladder pressure with micturition and then dribbling incontinence in all animals tested. 6 After blockade of the micturition reflex with morphine (10 μg intrathecally), ATP (5 mg kg−1, i.a.), α,β‐methylene ATP (0.25–1 mg kg−1, i.a.), and carbachol (5–500 μg kg−1, i.a.) were unable to empty the bladder. 7 The results suggest that drug‐induced bladder emptying in the normal, unanaesthetized rat requires an intact micturition reflex and they support the view that the two physiologically important transmitters involved in micturition are acetylcholine and ATP.

Capsaicin-induced bladder hyperactivity in normal conscious rats.

Capsaicin, instilled intravesically in normal, unanesthetized rats induced a concentration-dependent bladder hyperactivity, which could be abolished by hexamethonium, given intra-arterially near the bladder, or by morphine administered intrathecally. The effect was reversible and could be repeated. The NK-2 receptor selective antagonist SR 48,968 and the nonselective NK receptor antagonist spantide, given intra-arterially near the bladder, which by themselves, in the concentrations used, did not affect cystometric parameters, both counteracted the capsaicin-induced hyperactivity, whereas the NK-1 receptor selective antagonist RP 67,580 failed to do so. Blockade of tachykinin receptors in the urinary bladder does not seem to produce changes of the micturition reflex associated with bladder filling in the conscious rat. However, tachykinins released from capsaicin-sensitive nerves by various stimuli may, through stimulation of NK-2 receptors, lower the threshold for initiation of the micturition reflex. In the rat, intravesical capsaicin may be a suitable model for studies of afferent activity caused by stimuli releasing peptides from sensory nerves in the bladder, thereby provoking bladder hyperactivity.

Role of intrathecal tachykinins for micturition in unanaesthetized rats with and without bladder outlet obstruction

1 The effects on micturition of RP 67,580, a selective NK1 receptor antagonist, and SR 48,968, a highly potent antagonist at NK2 receptor sites, given intrathecally (i.t.) or intra‐arterially (i.a.) near the bladder, were investigated in unanaesthetized rats with and without bladder outlet obstruction. 2 In normal rats, RP 67,580, given i.t. in doses of 2 and 20 nmol per rat, decreased micturition pressure, but did not change other cystometric parameters. After 20 nmol of RP 67,580, dribbling incontinence due to retention was observed in 1 out of 7 animals. This effect was reversible. I.t. RP 67,580 in a dose of 2 nmol, had no effect on hyperactivity induced by intravesically instilled capsaicin. 3 In animals with bladder hypertrophy secondary to outflow obstruction, RP 67,580, given i.t. in a dose of 2 nmol per rat, decreased the micturiton pressure, but had no effect on other cystometric parameters. After 20 nmol, dribbling incontinence due to retention was observed in 5 out of 7 animals. 4 RP 67,580, given i.a. in a dose of 4 nmol, had little effect on the cystometric parameters investigated, both in normal animals and rats with bladder hypertrophy. 5 SR 48,968, given i.t. in doses of 2 and 20 nmol per rat, had no clear‐cut effects on the micturition pattern in normal rats, or rats with bladder hypertrophy. However, the drug reduced capsaicin‐induced bladder hyperactivity. When given i.a. in a dose of 4 nmol, SR 48,968 had no effect on cystometric parameters in normal rats or rats with bladder hypertrophy. 6 The effects of both RP 67,580 and SR 48,968 were stereoselective, their enantiomers (RP 68,651 and SR 48,965) being inactive. 7 These results thus suggest that at the spinal level there is a tachykinin involvement (via NK1 receptors) in the micturition reflex induced by bladder filling, both in normal rats, and, more clearly, in animals with bladder hypertrophy secondary to outflow obstruction. The bladder response to filling was not influenced by blockade of vesical NK1 and NK2 receptors. On the other hand, the bladder hyperactivity evoked by intravesical capsaicin seems to involve NK2 receptors both at the bladder and spinal levels.

A rat model for investigation of spinal mechanisms in detrusor instability associated with infravesical outflow obstruction

SummaryA rat model of infravesical outflow obstruction was modified to allow cystometric investigation in conscious, free-moving animals after intrathecal drug administration. The catheter position and extent of drug distribution were controlled by injection of dye and dissection of the spinal canal. Continuous cystometries were performed in awake normal rats as well as rats with bladder hypertrophy and hyperactivity following infravesical outflow obstruction. In some animals of each group, cystometry was performed with simultaneous recording of intra-abdominal pressure. The possible effects of the presence of the intrathecal catheter were studied, as well as the effects of saline, local anesthetics, morphine and naloxone administered through the catheter. Neither the presence of the intrathecal catheter nor injection of saline affected the cystometric pattern. Bupivacaine (50 μg) produced paralysis of both lower extremities and a complete, though reversible, suppression of micturition in normal rats. In rats with hypertrophy, intrathecal bupivacaine in doses of 50 μg and 100 μg produced decreases in micturition pressure, increases in bladder capacity and dribbling incontinence. However, the amplitude of spontaneous contractile activity increased after the administration. The inhibitory effects of morphine (0.5–10 μg) on micturition in normal rats, which were rapidly reversed by naloxone, were in accordance with results obtained in previous studies in anesthetized animals. Rats with bladder hypertrophy showed a similar response to morphine and naloxone. However, the bladder hyperactivity was not inhibited by morphine. We conclude that the present model seems reliable for the study of spinal mechanisms in the development of detrusor instability associated with infravesical outflow obstruction.

Facilitatory effect of vasoactive intestinal polypeptide on spinal and peripheral micturition reflex pathways in conscious rats with and without detrusor instability.

In unanesthetized, normal rats, and rats with bladder hypertrophy following infravesical outflow obstruction, cystometry was performed to investigate the effects of spinal and peripheral administration of vasoactive intestinal polypeptide (VIP) on micturition. In addition, the direct effects of the peptide on isolated smooth muscle preparations of detrusor and urethra were studied. In normal animals, 10 micrograms. of VIP administered intrathecally as well as intra-arterially close to the bladder, but not intravenously, decreased micturition volume and bladder capacity, and facilitated spontaneous bladder contractions. In animals with bladder hypertrophy, the same dose of VIP intrathecally had similar effects on these three parameters, but the effects of VIP given intra-arterially were less pronounced. VIP given intravenously was ineffective. Hexamethonium 5 mg. x kg.-1 given intraarterially did not block the stimulatory effect of VIP 10 micrograms. given intra-arterially to normal animals. VIP had negligible effects on isolated detrusor muscle contracted by carbachol or electrical stimulation, or on urethral preparations contracted by noradrenaline. These results suggest that VIP has a facilitatory action on the micturition reflex at the spinal cord and ganglionic levels.

Effects of morphine metabolites on micturition in normal, unanaesthetized rats.

1 By means of continuous cystometry in normal, unanaesthetized rats, the effects on micturition of intrathecally (i.t.) administered morphine‐3‐glucuronide (M3G) and morphine‐6‐glucuronide (M6G), the two main metabolites of morphine, were studied and compared with those of i.t. morphine. 2 Both M6G (0.01, 0.1, and 0.5 μg) and M3G (5 μg) were found to have significant effects on micturition. Like morphine (0.1, 0.5, and 10 μg), M6G was able to inhibit the micturition reflex, and produce urinary retention and dribbling incontinence in a dose‐dependent manner. The potency of M6G for inhibiting micturition was approximately 10 times higher than that of morphine, and the duration of its effect was longer. All effects of M6G could be reversed by naloxone. 3 M3G (5 μg) facilitated the micturition reflex, resulting in decreases in bladder capacity and micturition volume, and an increase in spontaneous contractile activity. Pretreatment with naloxone (10 μg), which by itself had no effect on micturition, enhanced the facilitatory effects of M3G. In addition, M3G tended to counteract the inhibitory effects of both morphine and M6G on micturition. M3G (5 μg) also produced an excitatory behavioural syndrome. 4 It is concluded that in rats, i.t. M3G has excitatory effects on micturition and behaviour, probably not mediated via opioid receptors. I.t M6G has a potent inhibitory effect on micturition mediated by stimulation of opioid receptors. It may have effects on somatosensory afferent input in lower doses than those required for effects on micturition.