Field-stimulated Relaxation Research Articles

The Novel Imidazopyridine 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) Is a Highly Selective Inhibitor of the Inducible Nitric-Oxide Synthase

We have identified imidazopyridine derivatives as a novel class of NO synthase inhibitors with high selectivity for the inducible isoform. 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) showed half-maximal inhibition of crudely purified human inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NO synthases at 86 nM, 17 microM, and 162 microM, respectively. Inhibition of inducible NO synthase was competitive with l-arginine, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. In radioligand and surface plasmon resonance experiments, BYK191023 exhibited an affinity for iNOS, nNOS, and eNOS of 450 nM, 30 microM, and >500 microM, respectively. Inhibition of cellular nitrate/nitrite synthesis in RAW, rat mesangium, and human embryonic kidney 293 cells after iNOS induction showed 40- to 100-fold higher IC(50) values than at the isolated enzyme, in agreement with the much higher l-arginine concentrations in cell culture media and inside intact cells. BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations. The inhibitory potency of BYK191023 was tested in isolated organ models of iNOS (lipopolysaccharide-treated and phenylephrine-precontracted rat aorta; IC(50) = 7 microM), eNOS (arecaidine propargyl ester-induced relaxation of phenylephrine-precontracted rat aorta; IC(50) > 100 microM), and nNOS (field-stimulated relaxation of phenylephrine-precontracted rabbit corpus cavernosum; IC(50) > 100 microM). These data confirm the high selectivity of BYK191023 for iNOS over eNOS and nNOS found at isolated enzymes. In summary, we have identified a new highly selective iNOS inhibitor structurally unrelated to known compounds and l-arginine. BYK191023 is a valuable tool for the investigation of iNOS-mediated effects in vitro and in vivo.

Does potassium induce the release of nitric oxide in the rabbit corpus cavernosum?

We investigated the effects of increases in the extracellular potassium concentration on the function of the rabbit corpus cavernosum. The resting tissue tension increased as the potassium concentration was increased from 4.7 mM to 20 mM or 30 mM. The maximum contraction induced by 200 microM phenylephrine was significantly decreased in the presence of 30 mM potassium compared with 4.7 mM potassium. After precontraction was induced with 200 microM phenylephrine, the magnitude of field-stimulated relaxation increased significantly as the potassium concentration was increased from 4.7 mM to 10 or 20 mM, but was almost completely abolished at 30 mM potassium. There was no difference in the suppressive effect of L-NAME on field-stimulated relaxation between specimens treated with 4.7 mM or 20 mM potassium. ATP- and bethanechol-induced relaxation was not affected by increases in the extracellular potassium concentration. A high-dose potassium solution (124 mM) induced contraction of the corpus cavernosum. In tissue precontracted with phenylephrine, a high-dose potassium solution that contained phenylephrine induced relaxation of corpus cavernosum; this relaxation was completely suppressed by L-NAME. These findings suggest that small increases in the extracellular potassium concentration increase field-stimulated relaxation of the corpus cavernosum and that this relaxation is not related to the effects of nitric oxide. Relaxation induced by high-dose potassium in tissue precontracted with phenylephrine is probably the result of release of nitric oxide.

Evidence for a role of intracellular-calcium release in nitric oxide-stimulated relaxation of the rabbit corpus cavernosum.

Erection is mediated by relaxation of the smooth muscle elements within the sinusoids of the corpus cavernosum. Although cavernosal relaxation can be mediated by a variety of mechanisms including purinergic stimulation, prostoglandins, and beta-adrenergic stimulation the major mechanism involves the stimulated release of nitric oxide (NO) and subsequent relaxation of the corporal smooth muscle. Experimentally, NO can be released both by direct stimulation of NO-containing nerves (using field stimulation) and indirectly via cholinergic stimulation of NO release from the endothelium (using bethanechol). Preliminary studies have indicated that NO release and/or NO-stimulated relaxation of corporal smooth muscle is an active process involving both an increase in cytosolic calcium and an increase in metabolic energy utilization. Ryanodine is a pharmacological agent that can inhibit calcium-stimulated calcium release from the sarcoplasmic reticulum. The results of the current study demonstrated that ryanodine inhibited both field-stimulated relaxation and bethanechol-stimulated relaxation but did not affect relaxation induced by adenosine triphosphate (ATP) or nitroprusside. These studies strongly support the hypothesis that NO-stimulated relaxation is mediated, in part, by calcium release from the sarcoplasmic reticulum through ryanodine-sensitive channels.

Effect of alteration in the extracellular potassium and calcium on field-stimulated relaxation of the rabbit corpus cavernosum

Changes in extracellular potassium or calcium concentration can alter the calcium equilibrium between intracellular and extracellular calcium pools in smooth muscle. The current study was designed to investigate the effect of alterations in the extracellular potassium and calcium concentrations, primarily on field-stimulated relaxation of the corpus cavernosum. The results can be summarized as follows: 1. 1. Increasing extracellular calcium at constant potassium mediated a moderate increase in the contractile response to phenylephrine; 2. 2. Increasing extracellular potassium at constant calcium did not affect the contractile response to phenylephrine; 3. 3. Increasing the concentration of extracellular calcium at constant potassium resulted in an increase in field-stimulated relaxation only at low frequency stimulation; 4. 4. Increasing the concentration of extracellular potassium at constant calcium resulted in a marked increase in field-stimulated relaxation, especially prominent at high-frequency stimulation; 5. 5. Increasing the extracellular potassium concentration at constant calcium, or increasing the calcium at constant potassium, had no significant effects on the relaxant effects of ATP and bethanechol; 6. 6. Our findings suggest that, within the corpus cavernosum, neuronal release or synthesis of nitric oxide depends, at least in part, on intracellular bioavailability of calcium.

Comparative Studies on the Ontogeny and Autonomic Responses of the Fetal Calf Bladder at Different Stages of Development : Involvement of the Nitric Oxide on Field Stimulated Relaxation

Comparative Studies on the Ontogeny and Autonomic Responses of the Fetal Calf Bladder at Different Stages of Development : Involvement of the Nitric Oxide on Field Stimulated Relaxation

Comparative Studies on the Ontogeny and Autonomic Responses of the Fetal Calf Bladder at Different Stages of Development: Involvement of Nitric Oxide on Field Stimulated Relaxation

This initial study correlates the passive length-tension relationship, contractile and relaxant responses to field stimulation and contractile responses to specific autonomic agonists and antagonists with gestational age. Fetal bovine bladders were separated into three groups based on the head-rump length (FL): 30 to 45cm. (early gestation), 50 to 65cm. (middle gestation) and 70 to 85cm. (late gestation). Each bladder was separated into upper and lower bladder segments; longitudinal strips of smooth muscle were isolated and placed in individual muscle baths.Passive length-tension studies demonstrated that compliance was greatest in the bladder of late gestation and lowest in the bladder of early gestation period. Field stimulation (FS) elicited frequency-dependent contractile responses in all strips. In the upper bladder, the maximal response and maximal rate of tension generation to FS was lowest in the youngest fetuses and increased in proportion to the gestational age. In the lower bladders, there were no gestational age-related differences in the maximal response or maximal rate of tension generation in response to field stimulation.The maximal response of the upper bladder to bethanechol increased significantly from the youngest gestational age to mid-gestation, with no further changes between mid- and late gestation. The maximal response to field stimulation and bethanechol were equal between upper and lower bladder segments for the youngest gestational bladders, whereas for the oldest gestational ages, the maximal response of the upper bladder to FS and bethanechol were significantly greater than the responses of the lower bladder.In the presence of maximal precontraction with bethanechol, FS induced a rapid and marked decrease in tension. The magnitude of the relaxation was substantially greater for the strips of lower bladder than for the strips of upper bladder at late gestation. In lower bladders, the magnitude of the field stimulated relaxation was greater in the strips from the older fetuses than in the strips from younger fetuses. In all strips, field stimulated relaxations were completely inhibited by pretreatment with L-NAME (an inhibitor of nitric oxide synthesis), indicating that the FS-induced relaxation was due to nitric oxide. In addition to nitric oxide-induced relaxation, beta adrenergic stimulation also induced a significant relaxation of the isolated strips.In summary, these data suggest that, in the tubular shaped fetal bovine bladder, there were distinct differences in the autonomic responses between the upper bladder segment and the lower bladder segment in the late gestation period.

Comparative studies on rabbit corpus cavernosal contraction and relaxation. An in vitro study.

Erectile function (erection and detumescence) involves the complex interaction of direct neuronal stimulation of corporal smooth muscle, neurohumoral release of specific endothelial contractile and relaxant factors, and secondary modulation by a variety of putative neuropeptides and vasoactive modulators including nitric oxide. The specific aim of the current study was to determine the relative contribution of nitric oxide, adrenergic, purinergic, and cholinergic stimulation in the relaxant response to field stimulation. The results demonstrate that virtually all of the inhibitory effects of field-stimulated relaxation could be explained by the release of nitric oxide. L-NAME (L-NG-nitro arginine methyl ester, a competitive inhibitor of NO synthase) reduced field-stimulated relaxation by over 95% at all frequencies. Neither atropine nor propranolol (or the combination of the two) had any significant effect on field-stimulated relaxation. L-NAME blocked both field-stimulated relaxation and bethanechol-stimulated relaxation. However, methylene blue (a guanyl cyclase inhibitor) was significantly more potent at blocking bethanechol-stimulated relaxation than field-stimulated relaxation. Neither L-NAME nor methylene blue had any effect on nitroprusside (a direct liberator of NO) nor ATP-stimulated relaxation. Isoproterenol had only a minor inhibitory effect on phenylephrine-contracted tissue. These data suggest that 1) Methylene blue, which inhibits guanyl cyclase, is a relatively poor inhibitor of field-stimulated relaxation. 2) L-NAME is a potent inhibitor of NO synthesis and can in a dose-dependent fashion inhibit over 95% of field-stimulated relaxation. 3) Equipotent relaxation of corporal smooth muscle can be effected through pharmacologic stimulation with ATP (2 mM), nitroprusside (200 microM), and field stimulation (32 Hz).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cold storage on relaxation responses in the rat oesophageal tunica muscularis mucosae.

Tetrodotoxin-resistant relaxations produced by electrical field stimulation, 5-hydroxytryptamine (5-HT), and A23187 (calimycin) were investigated in the rat isolated oesophageal tunica muscularis mucosae subjected to cold storage of varied duration. Cold storage for 2 days abolished cholinergic nerve mediated contractions to field stimulation; however, field stimulation evoked relaxations and muscarinic agonist evoked contractions persisted undiminished. After 5 days of cold storage, field-stimulated relaxations, as well as tension generating capacity of the tissue, were significantly reduced. Proximodistal differences were observed in tunica muscularis mucosae sensitivity to the relaxant effects of serotonin and A23187 and these were exaggerated after cold storage. In the distal segment, cold storage for 2 days unmasked ketanserin-sensitive 5-HT receptors mediating contractions. Similarly, A23187 induced contractions rather than relaxation in cold-stored distal tunica muscularis mucosae; however, this effect was resistant to ketanserin. Immunohistochemical staining by means of the peroxidase-antiperoxidase technique revealed 5-HT-like immunoreactive "mast cells" within the tunica muscularis mucosae. These cells appeared to be associated with the smooth muscle rather than the vasculature. It is concluded that field-stimulated relaxation is not dependent on intramural nerves, and it is unlikely that the release of 5-HT from mast cells or other cells mediates field-stimulated relaxation. However, confirmation awaits the provision of an antagonist against the novel 5-HT receptor that mediates 5-HT-induced relaxations.

Tetrodotoxin-sensitive and -insensitive relaxations in the rat oesophageal tunica muscularis mucosae.

1. Relaxation responses were produced by vagal and field stimulation, respectively, of the whole oesophagus preparation from the rat and of the isolated tunica muscularis mucosae (t.m.m.) preparation from the rat. These relaxation responses persisted in the presence of antagonists of histamine, serotonin, noradrenaline and acetylcholine. 2. Unlike vagally evoked relaxation, that evoked by low-frequency field stimulation, i.e. field-stimulated relaxation (f.s.r.) was generally resistant to tetrodotoxin (TTX). 3. Both types of relaxations exhibited remarkable temperature sensitivity and were abolished by lowering the bath temperature from 37 to 28 degrees C. 4. TTX-resistant relaxations were also produced by scorpion (Leiurus quinquestriatus) venom, the calcium ionophore, A23187 (calimycin) and by increasing the extracellular potassium by 2 mM. The failure of these agents to inhibit f.s.r. is inconsistent with a releasing and/or depleting action on any endogenous mediator. 5. Relaxations produced by vasoactive intestinal peptide (VIP) could be blocked by alpha-chymotrypsin which, however, failed to abolish f.s.r., suggesting that VIP is not the mediator of f.s.r. 6. F.s.r. was completely blocked by the calcium channel antagonists, verapamil (10(-6) M), nifedipine (10(-7) M), and by magnesium (20 mM). 7. Our results indicate that TTX-insensitive relaxations in the isolated t.m.m. are dependent upon extracellular calcium, are due to activation of potential-operated calcium channels and are not mediated by VIP.