Ergin Şingirik

An in vitro study of nonadrenergic-noncholinergic activity on the cavernous tissue of mouse

The relaxant effects of electrical field stimulation (EFS) and exogenously applied acetylcholine (ACh) or acidified NaNO2 (a-NaNO2) were investigated in the isolated mouse corpus cavernosum precontracted with phenylephrine hydrochloride (PE). Tetrodotoxin (TTX) blocked the relaxant effects of EFS completely, whereas it had no effect on the responses to ACh or a-NaNO2. Guanethidine and indomethacin failed to affect the electrically or ACh-induced relaxations. Atropine completely blocked the effect of ACh; however, it caused a slight reduction in the relaxation evoked by EFS.NG- Nitro-l-arginine (l-NOARG) reduced the effects of EFS and ACh significantly, but it was ineffective on the relaxations induced by a-NaNO2. The inhibitory action ofl-NOARG was partly restored byl-arginine, but not byd-arginine. Methylene blue (MB) and hydroxocobalamin (HC) exhibited significant inhibition on the relaxations evoked by EFS, ACh and a-NaNO2. Hydroquinone (HQ) reduced relaxation due to a-NaNO2, but did not affect that of EFS and ACh. Our findings suggest that EFS-induced relaxations of mouse cavernosal tissue are mediated by a transmitter which probably resembles an organic nitrate.

Protective Effect of L-Arginine Intake on the Impaired Renal Vascular Responses in the Gentamicin-Treated Rats

The 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.

Protective effect of quercetin, a polyphenolic compound, on mouse corpus cavernosum.

Flavonoids are plant‐based phenolic compounds, and quercetin is the most abundant dietary member of this family. One of the most important characteristics of quercetin is its antioxidant property. The aim of this study was to investigate antioxidant effects of quercetin on corpora cavernosa of mice. Corpora cavernosa were isolated in organ baths, precontracted with phenylephrine (0.5 μm) and relaxant responses were mediated by acetylcholine (0.1–1 μm), electrical field stimulation (EFS, 1–16 Hz, 0.5 ms, 30 V) or acidified sodium nitrite (a NaNO2, 0.5 mm). Superoxide anion generators; pyrogallol (50 μm), hydroquinone (100 μm), LY 83583 (6‐Anilinoquinolin‐5,8‐quinone, 10 μm) and superoxide dismutase (SOD) inhibitor; diethyldithiocarbamic acid (DETCA, 8 mm) were used in order to expose corpus cavernosa to oxidant stress. Acetylcholine (0.1–1 μm) induced relaxant responses were significantly inhibited in LY 83583 (10 μm) and DETCA + LY 83583 applicated trials. EFS‐induced relaxant responses were significantly inhibited in DETCA (8 mm) and DETCA + LY 83583 administrated trials. On the other hand, acidified sodium nitrite‐induced responses were inhibited by all of the superoxide anion generators tested. Quercetin (10 μm) failed to improve the inhibitions on endothelium and electrically stimulated responses. Acidified sodium nitrite (0.5 mm) mediated relaxant responses were significantly restored by quercetin except the groups in which LY 83583 were used. The data suggest that quercetin acts as a protective agent in mouse corpus cavernosum, increasing the bioavailability of exogenous nitric oxide by protecting it from superoxide anion (O2–).

Molecular mechanism of vasorelaxant and antiatherogenic effects of the statins in the human saphenous vein graft

In this study we aimed to investigate the vasorelaxant and antiatherogenic effects of the statins (fluvastatin and pravastatin) in the human saphenous vein grafts at the molecular level by using histopathologic, pharmacological and immunochemical techniques. The saphenous vein grafts evaluated histopathologically displayed a loss in their endothelium up to a ratio of 30% and set forth indications of functional deterioration. The pharmacological evaluations proved that the relaxation responses induced by fluvastatin and pravastatin were significantly inhibited by nitric oxide synthase inhibitor, N(G)-nitro-l-arginine, and cyclooxygenase inhibitor, indomethacin, while these responses were significantly increased by angiotensin converting enzyme inhibitors, captopril and enalapril, and rho kinase inhibitor, Y27632. The results of immunochemical studies are in accordance with the results of the pharmacological studies that the related statins increased the levels of nitric oxide, phospholipase A(2) and they decreased the levels of angiotensin II and active rho kinase. On the other hand mevalonolactone, a substrate of lipid metabolism, failed to change the effects of fluvastatin and pravastatin in the related tissue. The experimental results indicate that activation of nitric oxide synthase and phospholipase A(2)-cyclooxygenase pathway and inhibition of angiotensin converting enzyme and rho kinase may have a role on the effects of fluvastatin and pravastatin in the human saphenous vein grafts. It seems that the vasorelaxant and antiatherogenic effects of the related statins are independent of their lipid lowering mechanism.

The impact of extracellular and intracellular Ca2+ on ethanol-induced smooth muscle contraction

AbstractAim:To evaluate the impact of extracellular and intracellular Ca2+ on contractions induced by ethanol in smooth muscle.Methods:Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer.Results:Ethanol (164 mmol/L) produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine (50 and 100 μmol/L), a local anesthetic agent, and hexamethonium (100 and 500 μmol/L), a ganglionic blocking agent, failed to affect these contractions, verapamil (1–50 μmol/L) and nifedipine (1–50 μmol/L), selective blockers of L-type Ca2+ channels, significantly inhibited the contractile responses of ethanol. Using a Ca2+-free medium nearly eliminated these contractions in the same tissue. Ryanodine (1–50 μmol/L) and ruthenium red (10–100 μmol/L), selective blockers of intracellular Ca2+ channels/ryanodine receptors; cyclopiazonic acid (CPA; 1–10 μmol/L), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase; and caffeine (0.5–5 mmol/L), a depleting agent of intracellular Ca2+ stores, significantly inhibited the contractile responses induced by ethanol. In addition, the combination of caffeine (5 mmol/L) plus CPA (10 μmol/L), and ryanodine (10 μmol/L) plus CPA (10 μmol/L), caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine (5 mmol/L), ryanodine (10 μmol/L) and CPA(10 μmol/L) eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice.Conclusion:Both extracellular and intracellular Ca2+ may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.

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.

Pharmacological profile of a nitric oxide donor spermine NONOate in the mouse corpus cavernosum.

BACKGROUND/AIM To investigate the effects of spermine NONOate in the cavernous tissue obtained from mice treated or untreated with sildenafil. MATERIALS AND METHODS We studied the effects of spermine NONOate on the tone and nitrergic relaxation responses of isolated mouse corpus cavernosum and compared them with sodium nitroprusside in the absence or presence of L-nitroarginine, hydroxocobalamin, pyrogallol, diethyldithiocarbamate (DETCA), or 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The neurogenic contractions and relaxations of the tissues were induced by electrical field stimulation. Some mice received a single oral dose of sildenafil and after 1 h the effects of spermine NONOate were evaluated by in vitro studies. RESULTS Spermine NONOate relaxed mouse corpus cavernosum in a concentration-dependent manner. Spermine-NONOate-induced relaxation was relatively slow to develop and it was reversible and reproducible. These relaxations were significantly suppressed by hydroxocobalamin, diethyldithiocarbamate, or ODQ, but not by L-nitroarginine or pyrogallol. Spermine NONOate potentiated the nitrergic relaxations to electrical field stimulation (EFS), whereas it significantly reduced EFS-induced contractions. Sildenafil treatment can enhance the relaxant responses to spermine NONOate and EFS. CONCLUSION These findings suggest that spermine NONOate has a potent relaxant action in cavernous tissue and this effect can be potentiated by oral sildenafil treatments. Spermine NONOate may be considered an attractive treatment for erectile dysfunction in pathologic disorders with a lack of endogenous NO production.

Protective effect of alpha-linolenic acid on gentamicin-induced ototoxicity in mice

Abstract Alpha-linolenic acid is one of the fatty acids known as omega 3. Previous studies have shown the antioxidant and anti-inflammatory effects of alpha-linolenic acid, which prevented cell damage by inhibiting apoptotic pathway. Also, it is known that gentamicin activates apoptotic mediators and causes necrosis in the kidney. Due to this reason, we planned a study to evaluate the protective effects of alpha-linolenic acid on gentamicin induced ototoxicity by evaluating inflammation and apoptotic mediators. For this purpose, 100 mg/kg gentamicin (i.p; intraperitoneally) and 200 mg/kg alpha-linolenic acid (gavage) are administered to mice for 9 days. On 9th and 10th days, rotarod performance was assessed to test the effect of gentamicin and alpha-linolenic acid treatment on the motor coordination of mice. Gentamicin treatment decreased fall latency of mice and gentamicin treatment together with alpha-linolenic acid increased fall latency of mice. Gentamicin treatment also increased expression of phospholipase A2(plA2), cyclooxygenase-2(COX-2) and inducible nitric oxide syntheses (iNOS). Furthermore, it increased Bax and caspase-3, which are proapoptotic proteins and decreased bcl-2 that is an antiapoptotic protein. Gentamicin treatment together alpha-linolenic acid recovered the change of expression of these enzymes. In conclusion, this study showed that alpha-linolenic acid will be useful to prevent gentamicin-induced ototoxicity by inhibiting apoptosis and inflammation.

Ethanol-induced relaxation of mouse esophagus: subcellular mechanisms

Ethanol (164 mm) produced reproducible relaxations in isolated mouse esophageal strips. Hexamethonium (10–500 μm), a ganglionic blocking agent, and lidocaine (10–100 μm), a local anesthetic agent, failed to affect the relaxations induced by ethanol in the mouse esophagus. Although verapamil (10–500 μm), a selective blocker of L‐type Ca2+ channels, failed to affect the relaxations to ethanol, ruthenium red (10–100 μm), a selective blocker of ryanodine receptors (intracellular Ca2+ channels), and cyclopiazonic acid (1–10 μm), a selective blocker of sarcoplasmic reticulum Ca2+ ATPase (SERCA), significantly inhibited these relaxations. In addition, tetraethylammonium (10–100 μm), a potassium‐selective ion channel blocker and Nω‐nitro‐l‐arginine (l‐NOARG; 10–500 μm), a specific inhibitor of nitric oxide synthase (NOS), neomycin (10–500 μm), a phospholipase C inhibitor and indomethacine (1–10 μm), a non‐selective COX inhibitor, significantly inhibited the relaxations induced by ethanol. In contrast ouabain (10–100 μm), an inhibitor of Na+–K+‐ATPase, failed to cause significant alteration on these relaxations in the same tissue. The results of the present study suggest that the inhibitory effect of ethanol on the mouse esophagus may be direct effect of ethanol on the muscle tissue rather than neuronal effect. In addition, intracellular but not extracellular Ca2+ may have a role on ethanol‐induced relaxations in isolated mouse esophageal strips. Potassium channels and nitric oxide may also have a role on these relaxations. Similarly, phospholypase C and arachidonic acid pathways may contribute the relaxations to ethanol. However Na+–K+‐ATPase may not have a role on relaxations induced by ethanol in the mouse esophagus.

Molecular mechanism of KCl-induced relaxation of the esophagus

KCl (40 mM) caused reproducible relaxations in frog esophagus. N(G)-nitro-L-arginine (L-NOARG; 1-100 microM), a steriospecific inhibitor of nitric oxide synthase (NOS), completely inhibited the relaxations induced by KCl but not those induced by vasoactive intestinal polypeptide (VIP) antagonist. The inhibitory effect of L-NOARG was prevented by L-arginine (L-ARG; 0.1-1 mM), the precursor of nitric oxide (NO) biosynthesis, but not by D-arginine (D-ARG; 0.1-0.5 mM), the enantiomer of L-arginine. L-ARG or D-ARG alone did not significantly modify the effect of KCl. The relaxations to KCl were significantly inhibited by omega conotoxin (omega-conotoxin; 0.1 microM), a selective blocker of N-type calcium channels. Propranolol (0.1-1 microM), a nonselective blocker of beta-adrenergic receptors, prazosine (0.01-0.1 microM), a selective blocker of alpha(1)-adrenergic receptors, phentolamine (0.1-1 microM), a nonselective blocker of adrenergic receptors, atropine, a selective blocker of muscarinic cholinergic receptors, and lidocaine (1-10 microM), a blocker of sodium channels, had no effect on KCl-evoked relaxations. Caffeine (500 microM), an intracellular calcium releasing agent, did not significantly modify the effect of KCl. In contrast, ruthenium red (100 microM), a selective blocker of ryanodine receptors (intracellular Ca(2+) channels), significantly inhibited these relaxations. Similarly, potassium channel blockers such as 4-aminopyridine (4-AP; 100 microM) and tetraethylammonium (TEA; 100 microM) caused a significant inhibition on relaxations to KCl. In addition, ouabain (100 microM), a specific blocker of Na(+)-K(+)-ATPase, also caused a significant inhibition on these relaxations. The results suggest that NO, Na(+)-K(+)-ATPase and potassium channels may have a role on relaxations induced by 40 mM KCl in the frog esophagus.