The involvement of prostaglandins on the effects

The involvement of prostaglandins on the effects induced by 1-nitro-2-phenylethane is similarly unlikely because vasorelaxation was not changed by indomethacin [20]. The inhibition of adenylyl cyclase with MDL-12330A [21] or cAMP-dependent protein kinase A with KT-5720 [22] also did not interfere with its relaxant actions. Similarly, neither endothelium removal nor L-NAME-induced nitric oxide (NO) synthase inhibition influenced vasodilation in aortic rings, suggesting that the endothelial release of NO is not involved. Considering that the experiments that used antagonists were performed in preparations that were separate from those with 1-nitro-2-phenylethane alone and that only some concentration-effect curves were significantly shifted to the right, the putative occurrence of tolerance can be discarded. Indeed, experiments in endothelium-intact aortic rings under repeated (i.e., five repetitions separated by 15min intervals) exposure to 1-nitro-2-phenylethane (200μM) resulted in relaxant responses of a similar order of magnitude (data not shown). In vivo studies in either normotensive [7] or hypertensive [8] rats also indicate that the occurrence of tolerance in the cardiovascular effects of 1-nitro-2-phenylethane is unlikely. In addition to vascular relaxation, 1-nitro-2-phenylethane also increased cGMP levels in aortic rings. Notably, these effects were significantly inhibited by the guanylate cyclase inhibitor ODQ [23]. Methylene blue, another guanylate cyclase inhibitor [24], also reduced the vasodilator effect of 1-nitro-2-phenylethane in aortic rings. Corroborating the effects of Hyp9, 1-nitro-2-phenylethane decreased the transient contraction induced by capacitative Ca2+ entry triggered by Ca2+ store depletion following repeated stimulation with Gemfibrozil and subsequent Ca2+ restoration [25], an effect blocked by ODQ. Similarly, the supposedly inositol triphosphate-induced transient contraction caused by stimulation with phenylephrine under Ca2+-free conditions [26] was inhibited by 1-nitro-2-phenylethane in an ODQ-preventable manner. Some of these effects of 1-nitro-2-phenylethane resembled the effects induced by sodium nitroprusside, in which this NO donor was able to decrease the transient contraction induced by phenylephrine in a Ca2+-free medium in an ODQ-preventable manner. Similar to 1-nitro-2-phenylethane, sodium nitroprusside increased cGMP levels in aortic tissues. Conversely, the phasic contractile response induced by caffeine under Ca2+-free conditions, which results in activation of a Ca2+-induced Ca2+ release mechanism mediated by the ryanodine receptor-elicited opening of Ca2+ channels in the sarcoplasmic reticulum [27], was the only contractile response not presently influenced by 1-nitro-2-phenylethane. Other authors reported similar ineffectiveness of sodium nitroprusside against caffeine-induced contractions under Ca2+-free conditions [28], [29], [30]. Therefore, the present findings suggest that the vasorelaxant effects of 1-nitro-2-phenylethane involve stimulation of the sGC pathway. One might consider the possibility that 1-nitro-2-phenylethane is a NO donor. The spontaneous release of NO from 1-nitro-2-phenylethane, however, is unlikely because the –NO2 group is disposed in a small aliphatic chain attached to an aromatic ring with resonance properties that are able to delocalize electrons of the symmetric π-orbital system in the aromatic ring to satisfy the nitrogen's positive charge imposed by the electronegative influence of oxygen atoms (Fig. 1) [31]. Additionally, its chemical structure differs from classical NO donors that generally possess nitrate or S-nitroso groups, which are the source for NO [32]. In vascular smooth muscle cells, the guanylate cyclase's heme moiety is the major endogenous intracellular site that binds NO, augmenting the catalytic activity of the enzyme several hundred-fold and resulting in cGMP from guanosine triphosphate. Notwithstanding, 1-nitro-2-phenylethane unlikely displaces the original histidine (HIS105) by directly binding to the five-coordinate ferrous heme (Fe[II]), which occurs with NO [33]. Although not presently addressed, a putative hypothesis is that P450 (CYP)-catalyzed conversion is able to release NO from 1-nitro-2-phenylethane, which has already been shown for glyceryl trinitrate [34].