Domingo Triguero

Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications.

1 To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO‐mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L‐[14C]‐arginine to L‐[14C]‐citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2 NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg−1 protein min−1): urethra (33 ± 3.3), detrusor (13.1 ± 1.1) and ureter (1.5 ± 0.2). No activity was detected in the particulate fraction of any region. 3 NOS activity was dependent on Ca2+‐calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4 NOS activity was inhibited to a greater extent by NG‐nitro‐l‐arginine (l‐NOARG) and its methyl ester (l‐NAME) than by NG‐monomethyl‐l‐arginine (l‐NMMA), while 7‐nitroindazole (7‐NI) was a weak inhibitor and L‐cannavine had no effect. 5 Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin‐sensitive manner, suggesting feedback inhibition of NOS by NO. 6 EFS induced prominent NO‐mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L‐NOARG = L‐NAME > 7‐NI > L‐NMMA. 7 In conclusion, we have demonstrated the presence of NO‐synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO‐mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.

NADPH-diaphorase and NANC relaxations are correlated in the sheep urinary tract

The present investigation was performed with the purpose of revealing by histochemical examination of NADPH-diaphorase activity and electrical field stimulation (EFS) of isolated preparations in vitro, whether a nitrergic innervation is present in the lower urinary tract of the sheep. NADPH-diaphorase positive fibers were found in the trigone and urethra, but not in detrusor and ureter. EFS elicited L-nitroarginine-sensitive relaxations of precontracted preparations from the trigone and urethra while it did not relax detrusor and ureteral preparations. The present results show a direct regional correlation between the NADPH-diaphorase activity and EFS-induced relaxations, and suggest the presence of an inhibitory nitrergic innervation, which might be of importance for relaxation of the bladder neck and urethra during voiding.

Interstitial cells of Cajal in the urethra are cGMP-mediated targets of nitrergic neurotransmission

While interstitial cells of Cajal (ICC) in the urethra respond to nitric oxide (NO) donors by increasing cGMP, it remains unclear whether urethral ICC are functionally innervated by nitrergic nerves. We have addressed this issue in the rat and sheep urethra, where cGMP production and relaxation were compared in preparations subjected to electrical field stimulation (EFS; 2 Hz, 4 min) of nitrergic nerves or to exogenous S-nitroso-L-cysteine (SNC; 0.1 mM, 4 min). Upon EFS, cGMP immunoreactivity (cGMP-ir) was observed in both smooth muscle cells (SMC) and in spindle-shaped cells that contained c-kit and vimentin, features of ICC. Similarly, cGMP-ir was preferentially, but inconsistently, found in ICC of the outer muscle layer on exposure to SNC. We found separate functional groups of ICC within the urethra. Thus only ICC present in the muscle layers (ICC-M) but not those in the serosa (ICC-SR) and lamina propia (ICC-LP) seem to be specifically influenced by activation of neuronal NO synthase (nNOS). Thus the increase in cGMP-ir in the ICC-M induced by EFS was prevented by Nomega-nitro-L-arginine and ODQ. Urethral ICC did not express nNOS, although they were closely associated with nitrergic nerves. cGMP-ir was also present in the urothelium (in the rat but not in the sheep) and the vascular endothelium but not in neural structures, such as the nerve trunks and nerve terminals. Together, these results suggest a model of parallel innervation in which both SMC and ICC-M are effectors of nerve-released NO in the urethra.

Local regulation of oviductal blood flow.

1. Blood flow to the oviduct is implicated in the genesis and maintenance of oviductal fluid, in this way contributing to the creation of an adequate medium for ovum/embryo physiology. Therefore, factors controlling the tone of the vessels supplying the oviduct would be expected to affect its luminal environment. In addition, cyclic changes in oviductal blood flow have been suggested to have mechanical functions in the transport of the ovum/embryo. 2. The vascular supply to the oviduct has a prominent adrenergic vasomotor control. A dense adrenergic innervation, together with the presence of a predominant population of alpha(1)-adrenoceptors, provides a contractile regulatory mechanism of oviductal blood flow. No evidence is available on the presence of beta-adrenoceptors. The scanty cholinergic innervation of mammalian oviduct is mainly confined to the vessels, where acetylcholine (ACh) has a vasodilatory effect by releasing endothelium-derived relaxing factors. 3. The presence of nerves containing neuropeptides has been shown in the oviduct. Specifically, a high density of neuropeptide Y- and vasointestinal peptide-containing nerve fibers has been found in relation to blood vessels, but their role in the neutral control of the oviduct blood flow remains to be established. To date, it is not known whether or not oviductal blood vessels receive perivascular nitrergic nerves. 4. Relaxing and contracting factors derived from endothelium also seem to have a modulatory role on oviductal vascular tone. Neurotransmitters or autacoids, such as ACh and histamine, acting on endothelial receptors, release nitric oxide (NO), which relaxes oviductal arteries through guanylyl cyclase activation and accumulation of cyclic GMP. In addition, the release of an endothelium-derived hyperpolarizing factor (EDHF), distinct from NO, by ACh has been shown in oviductal arteries. It acts through the opening of low-conductance Ca(2+)-activated K+ channels leading to hyperpolarization and relaxation. Furthermore, potent and long-lasting contractions induced by the endothelium-derived contractile factor, endothelin (ET), points to its role in the long-term regulation of oviductal vascular tone. 5. A particularly high density of 5-hydroxytryptamine (5-HT) and histamine, present in mast cells clustered in the vicinity of blood vessels, has been described in the oviduct. It is known that histamine elicits a relaxation of oviductal arteries that is partially endothelium-dependent and mediated by the activation of H1-receptors. The implication of histamine in both the increase in blood flow and edema around ovulation, as well as the existence of a functional antagonism between histamine and 5-HT in the regulation of oviductal blood flow, await further investigation. 6. Other factors, such as relaxing and contracting cyclooxygenase-derived products, may also participate in the modulation of blood flow to the oviduct. 7. An overall endocrine regulation of the oviductal vascular supply exists, acting by both direct effects on smooth muscle and modulation of neural and autocrine factors. This control enables cyclic changes in blood flow to the oviduct that are tightly coupled to the reproductive functions of the tube.

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoine‐1‐oxyl 3‐oxide (carboxy‐PTIO), the specific guanylate cyclase inhibitor 1H‐[1,2,4]‐oxadiazole‐[4,3‐a]‐quinoxalin‐1‐one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 μM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml−1) inhibited NO‐mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy‐PTIO (0.1–0.5 mM) inhibited NO‐mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml−1). Relaxations to NO were reduced, but not abolished, by ODQ (10 μM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5′‐dithio‐bis (2‐nitrobenzoic acid) (DTNB, 0.5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N‐ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L‐cysteine (L‐cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD‐independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open.

Presence of the Ca2+-activated chloride channel anoctamin 1 in the urethra and its role in excitatory neurotransmission

We investigated the cellular distribution of the calcium-activated chloride channel (CaCC), anoctamin 1, in the urethra of mice, rats, and sheep by both immunofluorescence and PCR. We studied its role in urethral contractility by examining the effects of chloride-free medium and of several CaCC inhibitors on noradrenergic and cholinergic excitatory responses, and on nitrergic relaxations in urethral preparations. In all species analyzed, CaCC played a key role in urethral contractions, influencing smooth muscle cells activated by increases in intracellular calcium, probably due to calcium influx but with a minor contribution by IP(3)-mediated calcium release. The participation of CaCC in relaxant responses was negligible. Strong anoctamin 1 immunoreactivity was detected in the smooth muscle cells and urothelia of sheep, rat, and mouse urethra, but not in the interstitial cells of Cajal (ICC) in any of these species. RT-PCR confirmed the expression of anoctamin 1 mRNA in the rat urethra. This anoctamin 1 in urethral smooth muscle probably mediates the activity of chloride in contractile responses in different species, However, the lack of anoctamin 1 in ICCs challenges its proposed role in regulating urethral contractility in a manner similar to that observed in the gut.

CHOLINERGIC MODULATION OF NON-ADRENERGIC, NON-CHOLINERGIC RELAXATION IN ISOLATED, SMALL CORONARY ARTERIES FROM LAMBS

Abstract The presence of cholinergic innervation of small coronary arteries in the lamb was investigated by measuring choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities and by performing in vitro experiments in a microvascular myograph to establish whether or not there is a cholinergic component in the response to electrical field stimulation (EFS). ChAT-specific activity was present in proximal coronary segments, but was significantly higher in small coronary arteries. AChE-positive ganglia and fibres were distributed within the adventitia and outer third of the media in proximal coronary segments, and dense perivascular nerve plexuses were observed in small coronary arteries. Acetylcholine induced contractions in all preparations examined and relaxations in 20% of the segments contracted with the thromboxane analogue U46619. EFS did not induce neurogenic contractions in lamb small coronary arteries. In the presence of the α-adrenoceptor antagonist, phentolamine, EFS caused frequency-dependent reproducible relaxations that were enhanced by the blocker of cholinergic transmission, botulinum neurotoxin. An inhibitor of AChE, physostigmine, had no significant effect on the relaxations caused by EFS, while both the muscarinic receptor antagonist, atropine, and the muscarinic M2-receptor antagonist, AFDX 116, enhanced these responses. Blockade of sympathetic neurotransmission with guanethidine or incubation with the P2-receptor antagonist, suramin, abolished the relaxations induced by EFS, whereas propranolol was without effect. Low-frequency EFS caused less relaxation in preparations activated by acetylcholine than in those contracted with U46619, while sensitivity and maximal relaxation induced by adenosine 5′-triphosphate (ATP) were not different in U46619- and acetylcholine-contracted arteries. The presence of the enzymes necessary for both biosynthesis and degradation of acetylcholine and the finding that blockers of cholinergic neurotransmission enhance EFS-induced relaxations suggest that small coronary arteries are cholinergically innervated.

Spontaneous photo-relaxation of urethral smooth muscle from sheep, pig and rat and its relationship with nitrergic neurotransmission

In the present work we have characterized the relaxant response induced by light stimulation (LS) in the lower urinary tract from sheep, pig and rat, establishing its relationship with nitrergic neurotransmission. Urethral, but not detrusor, preparations showed pronounced photo‐relaxation (PR) which declined progressively following repetitive LS. Sheep urethral PR was again restored either spontaneously or (to a greater extent) by exogenous nitric oxide (NO) addition and by electrical field stimulation (EFS) of intrinsic nitrergic nerves. Greater NO generation was detected from sheep urethral than from detrusor homogenates following illumination. Sheep urethral PR was inhibited by oxyhaemoglobin, but not by methaemoglobin, carboxy‐PTIO, extracellular superoxide anion generators or superoxide dismutase. Guanylyl cyclase but not adenylyl cyclase activation mediates urethral relaxation to LS. Urethral PR was more resistant to inhibition by L‐thiocitrulline than EFS‐induced responses, although this agent prevented PR restoration by high‐frequency EFS. Urethral PR was TTX insensitive and partially modified in high‐K+ solutions. Cold storage for 24 h greatly impaired urethral PR, although it was restored by high‐frequency EFS. Repetitive exposure to LS, EFS or exogenous NO induced changes in the shape of the EFS‐induced nitrergic relaxation, possibly by pre‐synaptic mechanisms. In conclusion, we suggest the presence of an endogenous, photo‐labile, nitro‐compound store in the urethra, which seems to be replenished by neural nitric oxide synthase activity, indicating a close functional relationship with the nitrergic neurotransmitter.

Presence of cyclic nucleotide-gated channels in the rat urethra and their involvement in nerve-mediated nitrergic relaxation

We have addressed the distribution of cGMP-gated channels (CNG) in the rat urethra for the first time, as well as their putative role in mediating of the relaxation elicited by electrical field stimulation of nitrergic nerves. Functional studies have shown that specifically blocking CNG with L-cis-diltiazem leads to the rapid inhibition of urethral relaxation induced either by nitric oxide (NO) released by the nerves or by soluble guanylate cyclase activated with YC-1. By contrast, nerve-mediated noradrenergic contractions were only slowly and mildly reduced by L-cis-diltiazem. This effect was mimicked by lower concentrations of the D-diltiazem isomer, probably due to the nonspecific inhibition of voltage-dependent calcium channels. However, D-diltiazem did not affect relaxation responses. The expression of heteromeric retinal-like CNGA1 channels was demonstrated by conventional PCR on mRNA from the rat urethra. These channels were located in a subpopulation of intramuscular interstitial cells of Cajal (ICC) as well as in smooth muscle cells, although they were less abundant in the latter. CNG channels could not be visualized in any nervous structure within the urethral wall, in agreement with the emerging view that a subset of ICC serves as a target for NO. These channels could provide a suitable ionic mechanism to associate the changes in cytosolic calcium with the activation of the nitric NO-cGMP pathway and relaxation although the precise mechanisms involved remain to be elucidated.

Endothelin receptor-mediated Ca2+ mobilization and contraction in bovine oviductal arteries : Comparison with noradrenaline and potassium

1. The effects of endothelin-1 (ET-1) were studied in bovine oviductal arteries and compared to those of noradrenaline (NA) and high K+ (K+). The influence of endothelium, the receptor subtypes involved, and the mechanisms of Ca2+ mobilization were assessed. 2. ET-1 (0.1-300 nM) induced concentration-dependent contractions with a potency of 10(3) and 10(2) times higher than NA (0.1 microM-0.1 mM) and K+ (9.5-119 mM), respectively. Removal of endothelium or NG-nitro-L-arginine (L-NOARG, 0.1 mM) pretreatment did not affect responses to either ET-1 or K+, whereas the NA response was significantly increased. Indomethacin (1 microM) had no effect on either of these agonists. 3. The rank order of potency for the ET isopeptides was: ET-1 = ET-2 > ET-3. The ETA receptor-selective agonist, sarafotoxin 6c (S6c), had no effect. The ETA receptor-selective antagonist, BQ-123, showed a competitive antagonism on the ET-1 response (pA2 value of 6.58 +/- 0.01), whereas contractions to ET-3 were completely abolished by BQ-123 at 0.1 microM. 4. Concentration-response curves to both ET-1 and NA were shifted to the right and their maximum response reduced to approximately 56% and 65% of controls, respectively, under 30 min of incubation in Ca(2+)-free solution, whereas responses to K+ were almost abolished by this treatment. Contractions to both NA (30 microM) and ET-1 (30 nM) were maximally inhibited after 10 min of extracellular Ca2+ deprivation. 5. Contractions to ET-1 were more potently inhibited by nickel (Ni2+, 0.3 mM), whereas nifedipine (1 microM) and cadmium (Cd2+, 0.1 mM) induced only a slight effect. In contrast, opposite effects were found for both NA and K+. 6. Treatment with ryanodine (100 microM) and caffeine (10 mM) in Ca(2+)-free solution reduced the tension measured 5 min after NA (30 microM) and ET-1 (30 nM) addition, but the sustained response (tension at 25 min) remained unaffected. 7. Calphostin C (1 microM), a specific protein kinase C (PKC) inhibitor, reduced the maximum contractile response to ET-1 by about 50% without significantly affecting its pD2 value. 8. These results suggest that ET-1 acts in bovine oviductal arteries by directly activating a homogenous population of ETA receptors in smooth muscle, without endothelial modulation. Several Ca2+ activation mechanisms seem to be involved in the contractile action of the peptide, including: (1) extracellular Ca2+ entrance through Ni(2+)-sensitive and L-type Ca2+ channels; (2) intracellular Ca2+ release from a ryanodine-sensitive Ca2+ store; and (3) sensitization of the contractile machinery to Ca2+ via PKC.