Urocortin, a member of corticotropin releasing factor (CRF) peptide family, has positive chronotropic and inotropic effects on heart and also shows a vasodilatory effect. However, the mechanism underlying its vasodilatory effect has yet to be elucidated. Endothelium-dependent relaxation of resistance arteries is mainly achieved by activation of K+ channels. Therefore, we investigated possible role of K+ channels and hyperpolarization for the vasodilatory effect of urocortin using the isolated perfused rat mesenteric arteries. Urocortin (0.2 nM) produced a slow-onset decrease in the perfusion pressure of the mesenteric vascular bed, which was elevated by an alpha1-adrenoceptor agonist, phenylephrine (2-4 microM). Urocortin also hyperpolarized the main mesenteric artery. Removal of endothelium with saponin treatment considerably inhibited the relaxation and hyperpolarization induced by urocortin. In contrast, the hyperpolarization was not significantly changed by cyclooxygenase inhibitor, indomethacin (1 microM) and/or nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (100 microM). Urocortin-induced relaxation was not affected by the combination of a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), indomethacin and N(omega)-nitro-L-arginine. However, the relaxation and hyperpolarization were abolished by high extracellular potassium concentration (40 mM) or by a large conductance Ca(2+)-activated K+ channel blocker, charybdotoxin (1 nM). Glibenclamide (1 microM), an ATP-dependent K+ channel inhibitor, did not affect the relaxation and hyperpolarization. These results suggest that urocortin causes endothelium-dependent relaxation and hyperpolarization of rat mesenteric arteries, probably through the activation of charybdotoxin sensitive Ca2+-activated K+ channels. These findings also indicate an essential role of the endothelium for the urocortin-elicited vascular relaxation and hyperpolarization.

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.

Gentamicin is an aminoglycoside antibiotic and has therapeutic importance in the treatment of Gram-negative aerobic bacterial infections. However, its clinical use is limited by nephrotoxicity, in which oxidative stress plays an important role. In this study, we investigated the protective effects of Cyclotrichium niveum, an endemic plant with antioxidant properties, against gentamicin-induced nephrotoxicity in rats. Renal injury and protection were assessed using blood urea nitrogen (BUN) and serum creatinine as markers of renal function, malondialdehyde (MDA) as a marker of oxidative stress, and histopathological evaluation of renal cortical injury. We first characterized the chemical composition of C. niveum essential oil (EO) by GC-MS, identifying pulegone as its major constituent (79.6%). Next, we evaluated the in vitro antioxidant activity of EO and isolated pulegone using the DPPH assay. Their protective effects were then examined in vivo at two dose levels each (EO, 200 and 400 mg/kg/day; pulegone, 50 and 100 mg/kg/day, p.o.) in rats receiving gentamicin (100 mg/kg/day, i.m.) for 8 days. Both EO and pulegone exhibited DPPH radical-scavenging activity. Gentamicin induced pronounced renal injury, whereas co-administration of either EO or pulegone provided protection, with more pronounced effects at the higher doses. In particular, gentamicin increased BUN and creatinine 2.74- and 2.37-fold, respectively (p < 0.001 for both), and produced grade 4–5 cortical injury in all histologically evaluated animals. At the higher doses, EO and pulegone reduced BUN by 56% and 64%, respectively (p = 0.042 and p < 0.001), and MDA by 57% and 55%, respectively (p = 0.006 for both). Pulegone additionally reduced creatinine by 39% (p = 0.041). Both treatments significantly improved histopathological injury grade (p = 0.002 and p = 0.008 for EO and pulegone, respectively). Together, these findings demonstrate that C. niveum EO and pulegone attenuate gentamicin-induced renal injury across functional, oxidative, and structural measures, with the strongest protection observed at the higher doses. Their in vitro antioxidant activity and attenuation of lipid peroxidation in vivo support an antioxidant contribution to the nephroprotective response, while the activity of isolated pulegone identifies it as an important bioactive contributor to the protective effects of C. niveum EO.