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-N0-nitro arginine methyl ester (L-NAME), 10 and 20 mg kg-, administered intra-arterially, decreased micturition volume and bladder capacity, and increased spontaneous bladder contractions. D-NAME (20mgkg-') had no effect. No changes in the urodynamic parameters were recorded if L-NAME (20mgkg-') was administered in combination with L-arginine (200mgkg-'). 3 Cystometries performed after intra-arterial administration of sodium nitroprusside (SNP) (3mg kg-') and 3-morpholino-sydnonimin hydrochloride (SIN-1, 2mgkg-') 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 frequencydependent 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 (10-1 10-4 M), SIN-1 (10-6-3 x l0-4 M) and NO (10-5-10-3M; 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 a,p-methylene ATP and/or atropine. Small relaxations (14-41%) of detrusor strips were evoked by SNP (10-6-10-4M), SIN-1 (10-5-3 x 10-4M) 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.

BACKGROUND AND PURPOSET16Ainh-A01, CaCCinh-A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. EXPERIMENTAL APPROACHIsolated resistance arteries were maintained in a myograph and tension recorded, in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as a charge carrier. KEY RESULTSRodent arteries preconstricted with noradrenaline or U46619 were concentration-dependently relaxed by T16Ainh-A01 (0.1-10 μM): IC50 and maximum relaxation were equivalent in ±chloride (30 min aspartate substitution) and the T16Ainh-A01-induced vasorelaxation ±chloride were accompanied by membrane hyperpolarization and lowering of intracellular calcium. However, agonist concentration-response curves ±chloride, with 10 μM T16Ainh-A01 present, achieved similar maximum constrictions although agonist-sensitivity decreased. Contractions induced by elevated extracellular potassium were concentration-dependently relaxed by T16Ainh-A01 ±chloride. Moreover, T16Ainh-A01 inhibited VDCCs in A7r5 cells in a concentration-dependent manner. CaCCinh-A01 and MONNA (0.1-10 μM) induced vasorelaxation ±chloride and both compounds lowered maximum contractility. MONNA, 10 μM, induced substantial membrane hyperpolarization under resting conditions. CONCLUSIONS AND IMPLICATIONST16Ainh-A01, CaCCinh-A01 and MONNA concentration-dependently relax rodent resistance arteries, but an equivalent vasorelaxation occurs when the transmembrane chloride gradient is abolished with an impermeant anion. These compounds therefore display poor selectivity for TMEM16A and inhibition of CaCC in vascular tissue in the concentration range that inhibits the isolated conductance.

Despite considerable advances, both the central regulation of erection with processing of various stimuli, and the different steps involved in neurotransmission, impulse propagation and intracellular transduction of neural signals in penile smooth muscles, are still incompletely known. Centrally as well as peripherally, many transmitters and transmitter systems are involved. Dopamine, nitric oxide, oxytocin and ACTH=a-MSH, seem to have a facilitatory role, whereas serotonin may be either facilitatory or inhibitory, and enkephalins are inhibitory. Peripherally, the balance between contractant (eg noradrenaline, endothelins, angiotensins) and relaxant (eg NO, VIP and related peptides, prostanoids) factors controls the degree of contraction of the smooth muscle of the corpora cavernosa, and determines the functional state of the penis. Neurogenic NO is considered the most important factor for relaxation of penile vessels and corpus cavernosum. The roles of other putative transmitters=mediators and of various intracellular mechanisms, producing relaxation of vascular and corpus cavernosum smooth muscle, have not been established. For example, recent findings have suggested a role of Rho=Rho-kinase in the regulation of cavernosal tone, and that Rho-kinase antagonism could be a new potential principle for the treatment of erectile dysfunction. Further research in this area may be rewarding.

In the cat lower esophageal sphincter (LES) and esophageal body, nitric oxide synthase (NOS) immunoreactive nerves were abundant in the circular smooth muscle layer, especially in the LES region. NADPH diaphorase staining showed an identical pattern. The ability to form L-citrulline from L-arginine corresponded roughly to the distribution of NOS. Confocal microscopic analysis indicated colocalization within neurons of vasoactive intestinal peptide (VIP) in 65% of NOS-positive nerves. In LES circular smooth muscle preparations, electrically induced relaxations (single train stimuli) were generally abolished by NG-nitro-L-arginine (L-NNA). Continuous electrical stimulation for 2 min evoked a relaxation in the presence of L-NNA. This relaxation was inhibited by VIP antiserum and followed by a decrease in guanosine 3',5'-cyclic monophosphate, but not by any consistent change in adenosine 3',5'-cyclic monophosphate levels. K+ (124 mM) induced a biphasic relaxation, with L-NNA inhibiting the first phase but not the second. We conclude that nitric oxide (NO) has a major role as the mediator responsible for relaxation in the cat esophagus. NO seems also to initiate the release and enhance the effect of another transmitter.