Effects of ethanol withdrawal on the activity of rho-kinase in rat brain.

Besides its effect regarding addiction, ethanol also damages the central nervous system when it is used at high doses for a long time. The increase in the activity of Rho/Rho kinase pathway leads to central nervous system pathologies such as cerebral injury and epileptogenesis. The aim of this study was to investigate the contribution of Rho/Rho Kinase pathway to the degenerative and addictive effects of ethanol. For this purpose, we determined the Rho-kinase activity in striatum and hippocampus of rat brain. Wistar rats were treated with ethanol in a special liquid diet for 21 days. An isocaloric liquid diet without ethanol was given to the rats in the control group during the study. At the end of the 21 day ethanol exposure, one group was kept on taking ethanol and another group was withdrawn from ethanol. The rats were decapitated and their brains were taken out. Striatum and hippocampus were isolated. Phospho-moesin protein levels were measured in striatum and hippocampus homogenates using by Western blot analysis. The Rho-kinase (ROCK) activity in the striatum was found to be significantly decreased in ethanol exposed rats. In the hippocampus, there was a significant increase in the ROCK activity in the ethanol group. Our results indicated that ethanol caused some significant changes in Rho/Rho Kinase pathway in rat brain (Fig. 2, Ref. 25).

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

In 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, Nω-nitro-L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in the rat perfused mesenteric bed. The perfusion pressure was increased by phenylephrine (1-2 µM), an α1-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.

G protein-coupled estrogen receptor1 (GPER1) may mediate Rho-kinase (ROCK-2) up-regulation in coronary endothelial cells.

OBJECTIVE: Effect of estrogenic compounds and 17ß-estradiol (E2), which induces endothelial cell motility, was investigated on ROCK-2 expression in rat coronary vascular endothelial cells (CVEC). METHODS: The CVEC were isolated from the heart of Wistar rats by collagenase (0.04%) and incubated with E2 (1-100 nM), estrogen receptor α (ERα) agonist: propyl pyrazole triol (PPT, 10 nM); ERß agonists: (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN, 10 nM) and E2-conjugate with bovine serum albumin (E2-BSA, 1 nM); and GPER1 agonist: G1 (100 nM). Furthermore, the effect of combination of E2 with estrogen receptors (ERs) antagonist and GPER1 agonist, ICI-182780 (10 µM), physiological estrogen antagonists: progesterone (P4, 10-100 nM) and testosterone (T, 10-100 nM); transcription inhibitor: actinomycin-D (1 µg/ml); GPER1 antagonist: G-15 (100 nM), superoxide dismutase, (SOD, 500 U/ml); Gi/o protein inhibitor: pertussis toxin (PTX, 100 µg/ml); and epidermal growth factor receptor (EGFR) blocker: AG-1478 (10 µM) was tested. After 24h incubation, ROCK-2 and GPER1 protein expressions were detected in the CVEC by Western-blotting. RESULTS: E2, ICI-182780, and G1 but not E2-BSA significantly up-regulated ROCK-2 expression, which was suppressed by actinomycin-D, PTX, AG-1478, and G-15. However, PPT and DPN had no effects on the ROCK-2 expression. ICI-182780, P4, T or SOD did not antagonize the E2 action. GPER1 expression was demonstrated in the CVEC. CONCLUSIONS: Estrogens could up-regulate ROCK-2 in the rat CVEC through GPER1 and EGFR transactivation.

Antiepileptic effects of two Rho-kinase inhibitors, Y-27632 and fasudil, in mice.

BACKGROUND AND PURPOSE: Rho/Rho-kinase signalling is involved in many cellular events, including some in the CNS. However, the role of this pathway in epilepsy has not yet been assessed. Therefore, we determined the effects of two Rho-kinase inhibitors, Y-27632 and fasudil, on seizures induced by pentylenetetrazole (PTZ) or maximal electroconvulsive shock (MES). EXPERIMENTAL APPROACH: Effects of Y-27632 (5-10 mg kg(-1)) and fasudil (5-25 mg kg(-1)) on duration of myoclonic jerks, clonic and tonic convulsions, tonic hindlimb extensions and percentage of tonic convulsion index, as well as recovery latency for righting reflex were investigated in mice stimulated with PTZ (65 mg kg(-1)) or MES (50 Hz, 50 mA and 0.4 s). These inhibitors were also tested on a model of kindling induced by PTZ (35 mg kg(-1), for 11 days). Membrane and cytosolic levels of RhoA protein were measured in brain homogenates from kindled mice. KEY RESULTS: Y-27632 and fasudil diminished onset of myoclonic jerks, clonic convulsions and tonic hindlimb extensions in mice given PTZ. These inhibitors suppressed the percentage of tonic convulsion index and recovery latency for righting reflex in the mice excited with MES. Western blotting demonstrated that Rho translocation to plasma membrane increased in the brain homogenates obtained from PTZ-kindled mice. However, the Rho-kinase inhibitors at the given doses did not change motor coordination of the mice. CONCLUSIONS AND IMPLICATIONS: Rho/Rho-kinase signalling may play a role in epilepsy induced by PTZ and MES. Furthermore, Rho-kinase inhibitors could be novel important antiepileptic agents.

Effect of trapidil, an antiplatelet and vasodilator agent on gentamicin-induced nephrotoxicity in rats.

This study was carried out to evaluate the effect of trapidil, an antiplatelet and vasodilator drug, on the nephrotoxicity by an aminoglycoside, gentamicin, in rats. Forty female Wistar rats were divided into six different groups. One group served as a control group and the other groups were treated as follows: gentamicin (50 mg kg(-1) twice daily)-treated, gentamicin plus trapidil (4 or 20 mg kg(-1) daily)-treated and only trapidil-treated (4 or 20 mg kg(-1) daily) groups. Serum urea, creatinine and nitrite/nitrate levels were measured. Moreover, histopathological as well as electron microscopic examinations were performed. At a lower dose (4 mg kg(-1)) trapidil did not prevent the development of renal tubular damage by gentamicin. However, a higher dose of trapidil (20 mg kg(-1)) inhibited the ability of gentamicin to increase the levels of creatinine and urea. Furthermore, both light and electron microscopic evaluation confirmed the nephroprotective effect of the higher dose of trapidil. The level of the stable nitric oxide (NO) metabolite, nitrite, was also increased by trapidil. In conclusion, trapidil at a higher dose may protect against gentamicin nephrotoxicity. The mechanism underlying trapidil nephroprotection is not known, but may result from the antagonism of platelet-derived growth factor (PDGF), vasodilatation, inhibition of trombosit aggregation, and/or NO release.

Simultaneous determination of codeine and ethyl morphine HCL in tablet formulations using LC.

A reverse phase high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of codeine (methyl morphine) and dionin (ethyl morphine hydrochloride) in antitussive analgesic tablet formulations. A C(18) column and methanol-water (1:2) mixture mobile phase (pH 3.0) were used. Spectrophotometric detection was carried out at 210 nm. The total elution time was shorter than 7 min. This method was found to be quite precise and reproducible. This proposed method was successfully applied to the determination of codeine and ethyl morphine hydrochloride in tablets produced by the Turkish Army Drug Factory.

Formation of nitric oxide from nitroxyl anion: role of quinones and ferricytochrome c.

1. Our previous finding that copper ions oxidize nitroxyl anion released from Angeli's salt to nitric oxide prompted us to examine if copper-containing enzymes shared this property. 2. The copper-containing enzyme, tyrosinase, which catalyses the hydroxylation of monophenols to diphenols and the subsequent oxidation of these to the respective unstable quinone, failed to generate nitric oxide from Angeli's salt by itself, but did so in the presence of tyrosine. 3. L-DOPA, the initial product of the reaction of tyrosinase with tyrosine, was not the active species, since it failed to generate nitric oxide from Angeli's salt. Nevertheless, L-DOPA and two other substrates, namely, catechol and tyramine did produce nitric oxide from Angeli's salt in the presence of tyrosinase, suggesting involvement of the respective unstable quinones. In support, we found that 1,4-benzoquinone produced a powerful nitric oxide signal from Angeli's salt. 4. Coenzyme Q(o), an analogue of ubiquinone, failed to generate nitric oxide from Angeli's salt by itself, but produced a powerful signal in the presence of its mitochondrial complex III cofactor, ferricytochrome c. 5. Experiments conducted on rat aortic rings with the mitochondrial complex III inhibitor, myxothiazol, to determine if this pathway was responsible for the vascular conversion of nitroxyl to nitric oxide were equivocal: relaxation to Angeli's salt was inhibited but so too was that to unrelated relaxants. 6. Thus, certain quinones oxidize nitroxyl to nitric oxide. Further work is required to determine if endogenous quinones contribute to the relaxant actions of nitroxyl donors such as Angeli's salt.

Effects of some divalent cations on nitrergic relaxations in the mouse corpus cavernosum.

Acute effects of some divalent cations (Cd2+, Ni2+, Co2+, Zn2+, Mn2+ and Sn2+) were investigated on neurogenic and endothelium-dependent relaxations in the isolated mouse corpus cavernosum. Neither neurogenic nor endothelium-dependent relaxation was affected by cations at the concentrations used (up to 100 microM), except Cd2+. Although Cd2+ (20 and 40 microM) did not cause any significant alteration in the acetylcholine- (ACh) or sodium nitroprusside- (SNP) induced relaxation, it inhibited electrical field stimulation- (EFS) produced relaxation significantly. Zn2+ and selenium could not reverse this inhibitory action. Cd2+ did block the EFS-evoked guanethidine-sensitive contraction in the presence of N(G)-nitro-L-arginine. Elevation of external Ca2+ content significantly reduced the inhibitions due to Cd2+ on the EFS-induced relaxation and on the EFS-evoked guanethidine-sensitive contraction. In the Ca2+-omitted medium, EFS-induced relaxation disappeared, while acetylcholine-elicited relaxation resisted. Verapamil was ineffective on the relaxation produced by EFS or acetylcholine. However, it significantly diminished phenylephrine-induced contractions. These findings suggest that unlike other cations at the concentrations used in the present study, Cd2+ may have an effect on an external Ca2+-dependent mechanism at the neuronal level, and this effect may be responsible for its acute inhibitory action on the neurogenic relaxation in the mouse corpus cavernosum.

Oxidation of nitroxyl anion to nitric oxide by copper ions.

1. This study made use of a nitric oxide-sensitive electrode to examine possible means of generating nitric oxide from nitroxyl anion (NO(-)) released upon the decomposition of Angeli's salt. 2. Our results show that copper ions (from CuSO(4)) catalyze the rapid and efficient oxidation of nitroxyl to nitric oxide. Indeed, the concentrations of copper required to do so (0.1 - 100 microM) are roughly 100-times lower than those required to generate equivalent amounts of nitric oxide from S-nitroso-N-acetyl-D,L-penicillamine (SNAP). 3. Experiments with ascorbate (1 mM), which reduces Cu(2+) ions to Cu(+), and with the Cu(2+) chelators, EDTA and cuprizone, and the Cu(+) chelator, neocuproine, each at 1 mM, suggest that the oxidation is catalyzed by copper ions in both valency states. 4. Some compounds containing other transition metals, i.e. methaemoglobin, ferricytochrome c and Mn(III)TMPyP, were much less efficient than CuSO(4) in catalyzing the formation of nitric oxide from nitroxyl, while FeSO(4), FeCl(3), MnCl(2), and ZnSO(4) were inactive. 5. Of the copper containing enzymes examined, Cu-Zn superoxide dismutase and ceruloplasmin were weak generators of nitric oxide from nitroxyl, even at concentrations (2500 and 30 u ml(-1), respectively) vastly greater than are present endogenously. Two others, ascorbate oxidase (10 u ml(-1)) and tyrosinase (250 u ml(-1)) were inactive. 6. Our findings suggest that a copper-containing enzyme may be responsible for the rapid oxidation of nitroxyl to nitric oxide by cells, but the identity of such an enzyme remains elusive.

Evidence that the nitrergic neurotransmitter and endothelium-derived relaxing factor might be S-nitrosothiols in the mouse corpus cavernosum.

The effects of thimerosal, a sulfhydryl oxidizing agent on nitrergic, endothelium-dependent and -independent relaxations were investigated to examine the possibility that the nitrergic neurotransmitter and endothelium-derived relaxing factor (EDRF) could be S-nitrosothiol or free nitric oxide (NO) in the isolated mouse corpus cavernosum. Thimerosal (5 x 10(-6)-2 x 10(-5) M) inhibited or almost abolished electrical field stimulation--(EFS, 30V, 0.5 ms, 15 sec, 1, 2, 4, 8, 16 Hz), acetylcholine--(ACh, 5 x 10(-8)-1.25 x 10(-6) M), glyceryl trinitrate--(GTN, 3 x 10(-7)-3 x 10(-6) M), and S-nitrosoglutathione--(GSNO, 5 x 10(-6)-1.25 x 10(-4) M) induced relaxations. Thiomerosal inhibition seems to be specific to L-arginine NO pathways since it had no effect on acidified sodium nitrite--(10(-4)-5 x 10(-4) M), photoactivated sodium nitrite--(2 x 10(-4) M), isoprenaline--(10(-6) M), or papaverine--(10(-4) M) elicited relaxations. Moreover, the inhibitory effect of thimerosal on the nitrergic, ACh- or GTN-induced relaxations were partly reversed by sulfhydryl-containing compounds, L-cysteine (10(-3) M), dithiothreitol (10(-3) M), or glutathione (10(-3) M). However L-methionine (10(-3) M), which contains a methyl group on the sulphur atom, failed to restore the thimerosal inhibition. Thimerosal did not change the contraction produced by 10(-4) M NG-nitro-L-arginine methyl ester. These findings indicate that the nitrergic neurotransmitter as well as EDRF may not be free NO but NO-transferring molecules, probably S-nitrosothiols, in the mouse corpus cavernosum.

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

We 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)-nitro-arginine methyl ester. Treatment of the tissue with NaNO(2), 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-(delta)iminoethyl-L-ornithine, which does not contain NO(2) 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 NaNO(2), 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.