Depletion and restoration of the putative photosensitive materials store yielding nitric oxide in the isolated mouse gastric fundus

PubMedID: 10411590We investigated the possibility of there being any photosensitive materials stores yielding nitric oxide (NO), and combined for the first time electrical field stimulation (EFS)- and UV light-induced relaxations in mouse gastric fundus. The tissue responded with relaxation to long wave UV light (366 nm). Repeated exposure to light decreased the fundic photorelaxation in that the initial photorelaxation was 31.5 + 6.9% whereas the last (10th) photorelaxation was 2.3 ± 0.8%. There were no significant differences between EFS (30 V, 0.5 ms, 1 Hz, 15 s)-induced relaxations obtained before (39.7 ± 7.7%) and after (33.4 ± 9.1%) UV irradiation, which were completely blocked by 10-4 M L-N(G)-nitroarginine methyl ester. Treatment of the tissue with NaNO2, L-N(G)nitro-arginine, S-nitrosoglutathione, or sodium nitroprusside for 30 min followed by prolonged washout restored the photorelaxation, whereas glyceryl trinitrate or L-arginine did not produce any improvement. EFS (30 V, 0.5 ms, 3 Hz) applied for 60 min significantly recovered the reduction of the photorelaxation. L-N-S-iminoethyl-L-ornithine, which does not contain NO2 moiety, abolished electrically induced relaxation; however, it did not change photorelaxations. UV irradiation caused relaxation only when the adventitial surface of the preparation was oriented to the source of UV light. These results indicate that there could be a photosensitive relaxant materials store yielding NO in the smooth muscle layer of the gastric fundus from mouse. This putative store can be refilled by NaNO2, L-N(G-nitro-arginine), sodium nitroprusside, S-nitrosoglutathione, or long-term EFS but not glyceryl trinitrate or L-arginine. Possible candidates for NO-yielding substances might not be an organic nitrate but an intracellular nitrite, nitrosylated substances, and unknown nitro-containing compounds, which could be all sensitive to UV light

Some properties of the photorelaxation induced by UV light and possible contribution of nitric oxide in the isolated mouse gastric fundal smooth muscle

Reversible photorelaxations elicited by ultraviolet light (UV, 366 nm) of the isolated longitudinal strips of mouse gastric fundus precontracted with 1 µM methacholine (MCh) were significantly enhanced by either sodium nitrite (NaNO2, 10 µM-1 mM) or N(?)-nitro-L-arginine (L-NOARG, 1-100 µM), in concentration-dependent manner. Acidified NaNO2 but not neutral NaNO2 and L-NOARG produced relaxation similar to the photorelaxation. Hemoglobin (Hb, 1-50 µM), hydroxocobalamine (Hc, 30 and 50 µM) and ferrous sulfate (FeSO4, 10 µM-1 mM) inhibited the photorelaxation observed in the absence or presence of NaNO2 or L-NOARG (both 100 µM). Similar effects were also observed with the acidified NaNO2 induced relaxations. Methylene blue (Mb, 10 and 50 µM) did not affect photorelaxations potentiated by NaNO2 and L-NOARG but inhibited photorelaxations induced by UV light alone. Superoxide dismutase (SOD, 15-100 µM) enhanced both relaxations induced by UV light and potentiation by photoactivated nitro compounds. Sodium nitroprusside (SNP, 0.1-10 µM) also potentiated photorelaxations. On the other hand, neither L-arginine (L-ARG, 500 µM and 1 mM) nor N(G)-methyl-L-arginine (L-NMMA, 10 µM) was found to be effective on photorelaxations produced by UV light alone. 0.5 µM tetrodotoxin (TTX) did not inhibit relaxation induced by photoactivated NaNO2 (100 µM), a finding eliminating the involvement of a neuronal component. These results support the notion that the release of nitric oxide (NO) may be essential for smooth muscle relaxation by UV light in isolated mouse gastric fundus

The Functional Significance of the Rho/Rho-Kinase Pathway in Human Erythrocytes

OBJECTIVE: Erythrocyte deformability, which can be influenced by various intracellular signaling mechanisms, such as nitric oxide, cAMP, cGMP, and protein kinases, is the most important physiological factor providing the blood flow in microcirculation. However, the functional significance of the Rho/Rho-kinase pathway, which contributes cell shape changes and the reorganization of the actin cytoskeleton, has yet to be explored in erythrocytes. Therefore, we examined the influence of several activators and inhibitors of Rho/Rho-kinase signaling on human erythrocyte deformability. METHODS: RhoA and ROCK-2 proteins were studied by western blotting. Influences of 2 Rho-kinase inhibitors, fasudil and Y-27632 (both 10-7 to 10-4 M), on erythrocyte deformability was determined by ektacytometer at various shear stresses (0-30 Pa) in the presence or absence of a known Rho activator, lysophosphatidic acid (LPA, 10-5 to 5x10-5 M, 1-15 min). RESULTS: LPA incubation reduced deformability with concomitant RhoA-GTP inhibition. Y-27632 and fasudil also decreased deformability, but had no effect on LPA-induced reduction of deformability. Rho inhibitor C3 had no effect on RhoA activation. Reduction in RhoA activation was induced by sub-hemolytic mechanical stress. CONCLUSION: Our findings may indicate that the Rho/Rho-kinase pathway could contribute to the regulation of deformability of human erythrocytes

Rho-kinase expression and its contribution to the control of perfusion pressure in the isolated rat mesenteric vascular bed

PubMedID: 14757149Rho-kinase expression was investigated in the rat mesenteric artery and the effects of its inhibitors, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and fasudil (HA-1077), were examined on the increase in perfusion pressure induced by two different receptor agonists, namely the ?-adrenoceptor agonist, phenylephrine and, the endothelin ETA and ETB receptor agonist, endothelin-1. Y-27632 and fasudil produced a concentration-dependent decrease in perfusion pressure. There was no difference between the concentration-response lines of these two inhibitors. The maximum decrease in the perfusion pressure induced by 10-5 M Y-27632 was 85.8±3. 7% when the tone was increased by phenylephrine. However, it was 48.1±5.4% (P<0.001) when the perfusion pressure was elevated by endothelin-1. Saponin perfusion (100 mg l-1, for 10 min), which abolished acetylcholine-induced relaxation, did not significantly modify the Y-27632-elicited relaxation. Western blot analysis revealed that rat mesenteric artery expresses Rho-kinase protein with a molecular weight of approximately 160 kDa. These results show that Rho-kinase enzyme is expressed in rat mesenteric artery and that it contributes to the control of vascular resistance. Moreover, endothelium removal had no marked effect on the vasodilatation induced by Y-27632. In addition, the endothelin-1-induced vasoconstriction was more resistant to the Rho-kinase inhibitors than was that induced by phenylephrine, probably because excitatory endothelin receptors are associated with this signal transduction pathway at a different level from that of ?-adrenoceptors. © 2003 Elsevier B.V. All rights reserved.K.B./TÜBA-GEBİP/2002-1-5This work has been supported by the Turkish Academy of Sciences, in the framework of the Young Scientist Award Program (K.B./TÜBA-GEBİP/2002-1-5). The authors are indebted to E. Özveren, H. Kubat and B. Korkmaz for their help during Western blotting

The production of vasoconstriction-induced residual NO modulates perfusion pressure in rat mesenteric vascular bed

PubMedID: 24534887In the presence of nitric oxide synthase (NOS) inhibitors, the contribution of residual NO to endothelium-dependent relaxation induced by chemical agonists acetylcholine and bradykinin has been documented in resistance vessels. However, the contribution of residual NO to the vasodilatation in response to pressure and fluid shear stress is not well understood. In this study, to demonstrate the activity of residual NO, we applied a NO scavenger, hydroxocobalamin (HCX), on the phenylephrine-induced increase in perfusion pressure in the presence of NOS inhibitors, N?-nitro-L-arginine (L-NA) or N?-nitro-L-arginine methyl ester (L-NAME) in the rat perfused mesenteric bed. The perfusion pressure was increased by phenylephrine (1-2 µM), an ?1-adrenoceptor agonist. This increase was augmented by the addition of L-NA or L-NAME. In the presence of any NOS inhibitors, the application of hydroxocobalamin (100 µM) further increased the perfusion pressure. The removal of endothelium by saponin (50 mg/L) and the use of a non-selective protein kinase inhibitor, staurosporine (5 nM), and a tyrosine kinase inhibitor, erbstatin A (30 µM), but not a calmodulin inhibitor, calmidazolium (0.5 µM), inhibited the additional pressor responses induced by L-NA or L-NAME and a combination of either of them with hydroxocobalamine. These findings show that there could be a NOS inhibitor-resistant residual NO production in response to pressure in the rat mesenteric vascular bed. This residual NO production may be associated with the activation of tyrosine kinase and protein kinases, but not calmodulin. Finally, this pressure-induced residual NO exerts a modulatory role against vasoconstriction induced by phenylephrine. © 2014 The Author(s)

Rho-kinase inhibitor, Y-27632, has an antinociceptive effect in mice

PubMedID: 16750189The possible antinociceptive effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), was investigated in mice by using the hot-plate and abdominal constriction response (writhing) tests. In addition, the expression of Rho-kinase protein (ROCK-2) was studied in the mouse brain and spinal cord by Western blotting. Male balb/c mice (n = 8, for each group) were used in the experiment. Hot-plate latency and the number of writhes were recorded in control and in Y-27632-treated (1-5 mg/kg, i.p.) groups. Y-27632 (1 mg/kg) did not affect hot-plate latency; however, it considerably diminished the number of writhes, from 89 ± 12 in control to 30 ± 6 in the mice treated with 1 mg/kg Y-27632 (P = 0.001). At a higher dose (5 mg/kg), Y-27632 prolonged the hot-plate latency from 8.7 ± 1.0 s to 14.4 ± 1.7 s (P = 0.005) and decreased the number of writhes from 80 ± 8 to 24 ± 7 (P = 0.002). Western blot analysis revealed that mouse spinal cord and brain homogenates expressed ROCK-2 protein. These results indicate that Rho-kinase may be involved in nociception and that its inhibitors, such as Y-27632, may represent a new type of antinociceptive drug. © 2006 Elsevier B.V. All rights reserved.K.B./TÜBA-GEBİP/2002-1-5This work has been supported by the Turkish Academy of Sciences, in the framework of the Young Scientist Award Program (K.B./TÜBA-GEBİP/2002-1-5)

Protective effect of L-arginine intake on the impaired renal vascular responses in the gentamicin-treated rats

PubMedID: 15795518The purpose of this study was to investigate the effect of gentamicin (100 mg/kg/day, i.p.) treatment on endothelium-dependent and -independent vasodilation in isolated perfused rat kidney, and the effect of amino acid L-arginine (in the drinking water, 2.25 g/l) on renal dysfunction induced by gentamicin. When gentamicin-treated groups were compared with the control group, it was observed that BUN and creatinine levels increased significantly. Also, the relaxant responses induced by acetylcholine, sodium nitroprusside and pinacidil decreased. Histopathological examination indicated acute tubular necrosis in this group. In animals treated with gentamicin together with L-arginine, there was a significant amelioration in the BUN and creatinine levels. The vasodilator responses were similar to those of the control group. Histopathological examination indicated only hydropic degeneration in tubular epithelium of kidney. Co-administration of L-NG-nitroarginine methyl ester (L-NAME) (112.5 mg/l), an inhibitor of nitric oxide synthase, and L-arginine to rats treated with gentamicin did not change the protective effect of L-arginine. In rats receiving L-NAME alone, the level of BUN and creatinine and vasodilation to acetylcholine were not significantly different when compared to those of the control group, while relaxant responses to sodium nitroprusside and pinacidil were increased. These results suggest that gentamicin leads to an impairment in vascular smooth muscle relaxation in addition to acute tubular necrosis in the rat kidney. Supplementation of L-arginine has an important protective effect on gentamicin-induced nephropathy. Copyright © 2005 S. Karger AG

Effect of tempol (4-hydroxy tempo) on gentamicin-induced nephrotoxicity in rats

PubMedID: 14748758We investigated the effects of tempol (4-hydroxy tempo), a membrane-permeable radical scavenger, on gentamicin-induced renal failure in rats. The rats were given gentamicin (100 mg/kg/day, i.p., once a day); and gentamicin (100 mg/kg/day, i.p.) and tempol (3.5, 7 or 14 mg/kg/day, i.p., once a day). At the end of 7 days, the gentamicin group produced the remarkable nephrotoxicity, characterized by a significantly decreased creatinine clearance and increased serum creatinine, blood urea nitrogen (BUN) and daily urine volume when compared with controls. In control the BUN value was 21.2 ± 0.07 (mg/100 mL); in comparison, it was 96.9 ± 6.03 in gentamicin group (P < 0.05). Renal histopathologic examination confirmed acute tubular necrosis in this group. In rats treated with gentamicin and tempol a partial improvement in biochemical and histologic parameters was observed. BUN values were 96.9 ± 6.03 and 36.3 ± 2.39 in gentamicin, and gentamicin plus tempol (14 mg/kg) treated groups, respectively (P < 0.05). These results suggest that the administration of tempol may have a protective effect on gentamicin-induced nephrotoxicity in rats