Katarina Persson

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.

Nitric oxide synthase in pig lower urinary tract : immunohistochemistry, NADPH diaphorase histochemistry and functional effects

1 The distribution and colocalization of nitric oxide synthase (NOS)‐like immunoreactivity and NADPH diaphorase activity in the pig lower urinary tract were investigated by immunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS‐immunoreactivity/NADPH diaphorase staining with smooth muscle responses involving the l‐arginine/nitric oxide (NO) pathway. 2 NOS‐immunoreactivity and NADPH diaphorase activity were expressed in nerve trunks and fine nerve fibres in and/or around muscular bundles in the detrusor, trigone and urethra. Thin nerve fibres that dispersed within the muscle bundles were mainly found in the urethral/trigonal area, whereas such fibres were less common in the detrusor. 3 Almost all neuronal structures that were NOS‐immunolabelled were also stained for NADPH diaphorase. In contrast, the urothelium, which was intensively stained by the NADPH diaphorase technique, remained unstained by immunohistochemistry. 4 Electrical field stimulation of pig isolated trigonal and urethral preparations induced relaxations, which were inhibited by tetrodotoxin (1 μm) and NG‐nitro‐l‐arginine (l‐NOARG, 10 μm). 5 l‐Arginine (1 mm), but not d‐arginine, inhibited (25–30%) electrically evoked detrusor contractions. This inhibition was reversed by l‐NOARG (0.1 mm). l‐Arginine did not inhibit detrusor contractions in the presence of scopolamine (1 μm) and had no direct smooth muscle effects per se. 6 Acetylcholine (1 nm − 10 μm) caused concentration‐dependent relaxations of noradrenaline‐induced contractions in pig vesical arteries. Removal of the endothelium practically abolished the acetylcholine‐induced relaxation. Pretreatment with l‐NOARG (0.1 mm and 0.3 mm) caused a rightward shift of the concentration‐response curves to acetylcholine, but the maximal relaxation obtained was significantly reduced (to 65 ± 12%; n = 6; P < 0.05) only at 0.3 mml‐NOARG. 7 In vessel segments contracted with K+ (60 mm), acetylcholine induced concentration‐dependent relaxations. When the vessels were incubated with 0.3 mml‐NOARG and then contracted with K+ (60 mm) all relaxant responses to acetylcholine were abolished. 8 The presence of NO synthesizing enzyme in nerve fibres and the pharmacological evidence for NO‐mediated relaxation of the trigone and urethra suggest that NO or a NO‐related substance may have a role in inhibitory neurotransmission in these regions. In the detrusor, the presence of NO‐synthesizing enzyme in nerves can be demonstrated, but its functional importance is unclear. NO, as well as other endothelium‐derived factors seem to be involved in the endothelium‐dependent acetylcholine‐induced relaxation of pig vesical arteries.

Nitric oxide and relaxation of pig lower urinary tract

1 We studied the non‐adrenergic, non‐cholinergic (NANC) nerve‐mediated relaxation induced by electrical stimulation in pig isolated lower urinary tract smooth muscle, and the possible involvement of the l‐arginine (l‐ARG)/nitric oxide (NO) pathway in this response. 2 Trigonal strips, precontracted by noradrenaline (NA), carbachol or endothelin‐1 (ET‐1), relaxed frequency‐dependently in response to electrical stimulation. Maximum relaxation was obtained at 6–8 Hz, and amounted to 56 ± 2%, 77 ± 3% and 62 ± 6% of the agonist‐induced tension in preparations contracted by NA, carbachol, or ET‐1, respectively. Exposure to NG‐nitro‐l‐arginine (l‐NOARG; 10−7−10−5 m) concentration‐dependently reduced the relaxant response in preparations contracted by NA. l‐NOARG (10−6 m) reduced the maximal response to 51 ± 8% of control. l‐NOARG (10−5 m) abolished all relaxation, and unmasked a contractile component; d‐NOARG had no effect. Also in trigonal preparations, where the tension had been raised by carbachol or ET‐1, l‐NOARG (10−5 m) markedly reduced relaxations evoked by electrical stimulation. 3 In trigonal preparations contracted by NA, maximal relaxation was increased after pretreatment with l‐ARG (10−3 m), and the inhibitory effect of l‐NOARG (10−6 m) was prevented. Incubation of the trigonal strips with methylene blue had no effect on relaxations elicited at frequencies < 6 Hz, but a small inhibition was observed at higher frequencies. 4 Administration of NO (present in acidified solution of NaNO2) induced concentration‐dependent relaxations in trigonal preparations contracted by NA, carbachol, or ET‐1. l‐NOARG (10−5 m) and l‐ARG (10−3 m) had no effect on these relaxations. However, methylene blue (10−5 m) significantly shifted the concentration‐response curve for NO to the right. NANC‐relaxation and NO‐induced relaxation of trigonal preparations were both inhibited by oxyhaemoglobin (10−5 m) and pyrogallol (10−4 m). 5 In urethral preparations precontracted by NA, electrical stimulation caused frequency‐dependent relaxations. A maximum relaxation of 73 ± 4% was obtained at 10 Hz. Also in the urethra, NANC‐relaxation was blocked by l‐NOARG (10−5 m), and a contractile response generally appeared. 6 Detrusor strips treated with α‐β methylene ATP (10−5 m) and atropine (10−6 m), and then contracted by ET‐1, showed relaxations (19 ± 3% of the induced tension) in response to electrical field stimulation (2–20 Hz) only when the tension was high. No response at all, or small contractions, were found in response to electrical stimulation in K+ (35 mm)‐contracted detrusor strips. Detrusor preparations contracted by carbachol were concentration‐dependently relaxed by exogenously administered NO, SIN‐1 (NO‐donor), and isoprenaline, whereas vasoactive intestinal polypeptide had minor effects. NO and SIN‐1 induced maximal relaxations of 63 ± 3% and 70 ± 4%, respectively, of the tension induced by carbachol. Isoprenaline produced an almost complete relaxation (96 ±4%). 7 The results suggest that NANC‐nerve mediated relaxation, involving the l‐ARG/NO pathway, can be demonstrated consistently in the pig trigonal and urethral, but not in detrusor smooth muscle. The importance of this pathway for lower urinary tract physiology and pathophysiology remains to be established.

Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats

The influence of noradrenergic mechanisms involved in micturition in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was investigated using continuous cystometry in in vivo and in vitro studies on isolated bladder and urethral tissues. Compared with WKY rats, SHR had a significantly lower bladder capacity (SHR: 0.7 +/- 0. 05 ml; WKY rats: 1.3 +/- 0.06 ml; P < 0.001), micturition volume (SHR: 0.4 +/- 0.04 ml, WKY rats: 1.2 +/- 0.05 ml; P < 0.001), and an increased amplitude of nonvoiding (unstable) bladder contractions. The effects of intrathecal and intra-arterial doxazosin on cystometric parameters were more pronounced in SHR than in WKY rats. There was a marked reduction in nonvoiding contractions after intrathecal (but not intra-arterial) doxazosin in SHR. Norepinephrine (0.1 microM-1 mM) failed to evoke contractions in bladder strips from WKY rats, in contrast to a weak contractile response in SHR. The response to electrical field stimulation was significantly less in bladder strips from SHR than from WKY rats. In WKY rats, norepinephrine produced concentration-dependent inhibition (87 +/- 5%, n = 6) of nerve-evoked bladder contractions. Almost no inhibition (11 +/- 8%, n = 6) was found in SHR. Alterations in bladder function of SHR appear to be associated with changes in the noradrenergic control of the micturition reflex, in addition to an increased smooth muscle and decreased neuronal responsiveness to norepinephrine. The marked reduction in nonvoiding contractions after intrathecal doxazosin suggests that the bladder hyperactivity in SHR has at least part of its origin in supraspinal and/or spinal structures.

The spontaneously hypertensive rat: insight into the pathogenesis of irritative symptoms in benign prostatic hyperplasia and young anxious males.

Recent epidemiological studies have shown that hypertensive men are more likely to undergo surgical intervention for irritative voiding symptoms from BPH than age‐matched controls. Indeed, noradrenergic nerves which regulate vascular tone also participate in the functional component of bladder outlet obstruction due to BPH. Newer, less invasive therapies for BPH such as thermal therapy can relieve symptoms yet do not eliminate obstruction based on urodynamic studies. Coincidentally, drugs such as α‐adrenoceptor antagonists, which have been thought to relieve obstruction due to a peripheral effect, can be given intrathecally in animals to relieve urinary frequency due to obstruction. Taken together these observations implicate both peripheral and central sympathetic pathways in the motor control of the urinary bladder especially with disease states.

Micturition in conscious rats with and without bladder outlet obstruction : role of spinal α1-adrenoceptors

1 In normal rats and rats with bladder hypertrophy secondary to outflow obstruction, undergoing continuous cystometry, we examined the responses to the selective α1‐adrenoceptor antagonist doxazosin given intrathecally (i.t.) and intra‐arterially (i.a.). In addition, we investigated the effects of the drug on L‐dopa‐induced bladder hyperactivity in normal, unobstructed rats. 2 Doxazosin 50 nmol (approximately 60 μg kg−1), given i.t., decreased micturition pressure in normal rats and in animals with post‐obstruction bladder hypertrophy. The effect was much more pronounced in the animals with hypertrophied/overactive bladders. Doxazosin did not markedly affect the frequency or amplitude of the unstable contractions observed in obstructed rats. In contrast, however, doxazosin reduced L‐dopa‐induced bladder overactivity. When tested, the enantiomers of doxazosin produced qualitatively similar effects to doxazosin, but there was no evidence of stereoselectivity. 3 The results suggest that in addition to the well documented action on prostatic and lower urinary tract smooth muscle, and an effect on the sympathetic outflow to the bladder, bladder neck, prostate, and external urethral sphincter, doxazosin may have an action at the level of the spinal cord and ganglia, thereby reducing activity in the parasympathetic nerves to the bladder. This effect is more pronounced in rats with bladder hypertrophy than in normal rats.

The l-arginine/nitric oxide pathway and non-adrenergic, non-cholinergic relaxation of the lower urinary tract

The L-arginine/nitric oxide pathway and non-adrenergic, non-cholinergic relaxation of the lower urinary tract

Carbon monoxide-induced relaxation and distribution of haem oxygenase isoenzymes in the pig urethra and lower oesophagogastric junction

The distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)‐1 and ‐2 was studied by immunohistochemistry in the pigs lower urinary tract, including bladder extramural arteries, and the oesophagogastric junction (OGJ). In isolated smooth muscle from the urethra and the OGJ, the mechanisms for CO‐induced relaxations were characterized by measurement of cyclic nucleotide levels and by responses to the guanylate cyclase inhibitor methylene blue and some K+ channel inhibitors. HO‐2 immunoreactivity was observed in coarse nerve trunks within the smooth muscle of the urethra and OGJ, and in nerve cell bodies of the enteric plexuses of the OGJ. Furthermore, the vascular endothelium of the intramural vessels of the urethra, bladder and OGJ, and the extramural vessels of the bladder, displayed HO‐2 immunoreactivity. Two different antisera against HO‐1 were used, but only one displayed immunoreactivity in neuronal structures. HO‐1 immunoreactivity, as displayed by this antiserum, was seen in nerve cells, coarse nerve trunks and varicose nerve fibres in the smooth muscle of the urethra and OGJ. Some HO‐2 and/or HO‐1 (as displayed by both HO‐1 antisera) immunoreactive cells with a non‐neuronal appearance were observed within the smooth muscle of the OGJ, bladder and urethra. In the urethral preparations, exogenously applied CO (72 μm) evoked a relaxation amounting to 76±6%. The relaxation was associated with an increase in cyclic GMP, but not cyclic AMP, content. CO‐evoked relaxations were not significantly reduced by treatment with methylene blue, or by inhibitors of voltage‐dependent (4‐aminopyridine), high (iberiotoxin, charybdotoxin) and low (apamin) conductance Ca2+‐activated, and ATP‐sensitive (glibenclamide) K+ channels. Bladder strips, and ring preparations from the extramural arteries of the bladder, did not respond to exogenously administered CO (12–72 μm). In the OGJ, exogenously applied CO evoked a relaxation of 86±6%, which was associated with an increase in cyclic GMP, but not cyclic AMP, content. Treatment with 30 μm methylene blue raised the spontaneously developed muscle tone, and reduced the maximum relaxation evoked by CO to 33±9%. Addition of 4‐aminopyridine, apamin, glibenclamide, iberiotoxin, charybdotoxin or glibenclamide had no effect on the relaxations. 4‐aminopyridine (0.1–1 mm), iberiotoxin (0.1 μm) and charybdotoxin (0.1 μm) increased the spontaneously developed tone, and a combination of charybdotoxin and apamin reduced CO‐induced (24 μm CO) relaxations. The present findings demonstrate the presence of HO in both neuronal and non‐neuronal cells in the pig OGJ and lower urinary tract. CO produces relaxation of the smooth muscle in the OGJ and urethra, associated with a small increase in cyclic GMP concentration in both regions. Relaxations evoked by CO in the urethra do not seem to involve voltage‐dependent, low and high conductance, or ATP‐dependent K+ channels. However, in the OGJ relaxations evoked by CO can be attenuated by methylene blue and a combination of charybdotoxin and apamin.

Factors involved in the relaxation of female pig urethra evoked by electrical field stimulation

1 Non‐adrenergic, non‐cholinergic (NANC) relaxations induced by electrical field stimulation (EFS) were studied in pig isolated urethra. The mechanism for relaxation was characterized by measurement of cyclic nucleotides and by study of involvement of different subsets of voltage‐operated calcium channels (VOCCs). 2 EFS evoked frequency‐dependent and tetrodotoxin‐sensitive relaxations in the presence of propranolol (1 JIM), phentolamine (1 μM) and scopolamine (1 μIM). At low frequencies (<:12Hz), relaxations were rapid, whereas at high (> 12 Hz) frequencies distinct biphasic relaxations were evoked. The latter consisted of a rapidly developing first phase followed by a more long‐lasting second phase. 3 Treatment with the NO‐synthesis inhibitor NG‐nitro‐L‐arginine (L‐NOARG; 0.3 mM) inhibited relaxations at low frequencies of stimulation. At high frequencies (>12 Hz) only the first relaxation phase was affected. 4 Measurement of cyclic nucleotides in preparations subjected to continuous nerve‐stimulation, revealed an increase in guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) levels from 1.3 ± 0.3 to 3.0±0.4 pmol mg−1 protein (P<0.01). In the presence of L‐NOARG, there was a significant decrease in cyclic GMP content to control. However, there was no increase in cyclic GMP content in response to EFS. Levels of cyclic AMP remained unchanged following EFS. 5 Treatment with the N‐type VOCC‐inhibitor, ω‐conotoxin GVIA (0.1 μMs) reduced NO‐dependent relaxations, the effect being most pronounced at low frequencies (1‐4 Hz) of stimulation. The NO‐independent second phase of the relaxation, studied in the presence of L‐NOARG (0.3 mM) at 16–30 Hz, was however markedly reduced or abolished by ω‐conotoxin GVIA. ω‐Conotoxin MVIIC (1 μM) or ω‐agatoxin IVA (30 nM) had no effect on electrically evoked relaxations. 6 These results suggest that NANC‐nerve derived urethral relaxation in the pig consists of two apparently independent components. One is mediated by NO and associated with an increase in cyclic GMP content. The other mediator is unknown and produces relaxations not associated with changes in levels of cyclic nucleotides. The release of this mediator seems to involve the N‐type VOCC, since the relaxation was markedly reduced or abolished by ω‐conotoxin GVIA.

Co-existence of nitrergic, peptidergic and acetylcholine esterase-positive nerves in the pig lower urinary tract

The distribution of NO synthase (NOS) immunoreactive nerves and the possible co-existence with other neurotransmitters were investigated in the pig lower urinary tract. NOS immunoreactive nerves were found in the muscle layer, in the lamina propria and around blood vessels. The density of NOS immunoreactive nerves was more prominent in the trigone and urethra than in the detrusor. All parts of the lower urinary tract were supplied by numerous acetylcholine esterase (AChE) positive nerves. The number of adrenergic nerves in the trigone and urethra was moderate to rich, whereas only very few adrenergic nerves were demonstrated in the detrusor. A low to moderate number of nerve fibres containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were observed in the trigone and urethra, while very few were found in the detrusor. A small number of nerves, confined to the trigone and urethra, were stained for calcitonin-gene-related peptide, somatostatin and leu-enkephalin. Nerve fibres exhibiting immunoreactivity to bombesin/gastrin releasing peptide, gastrin/cholecystokinin, substance P or neurokinin A were virtually absent. Co-localization studies revealed that some NOS-immunoreactive nerves also stained for NPY, VIP or AChE. The present study shows that nitrergic nerves are present in the pig lower urinary tract in a density lower than the cholinergic, but higher than any of the studied peptidergic nerves. Coinciding localization of NOS-positive nerves with nerves expressing AChE, VIP and NPY suggests that NO may have a role as a messenger in the lower urinary tract directly and by interaction with other transmitters.