Carbachol-induced contractions of rat stomach fundus strips, obtained in a nutrient solution containing 1.8 mM Ca2+, were resistant to Ca2+ withdrawal, even after 1 h of bathing the tissues in a nominal 0 Ca2+ solution. This was not observed when K+ was used to evoke contractions, which were rapidly inhibited after Ca2+ removal (t1/2=2 min). The effect of carbachol in 0 Ca2+ solution was reduced by using drugs that reduce intracellular pools of Ca2+, such as caffeine (1-3 mM), ryanodine (30 microM) or thapsigargin (1 microM), corroborating the involvement of intracellular Ca2+ stores. On the other hand, when the 0 Ca2+ solution contained EGTA, a complete decline of carbachol effects was observed within about 8 min, indicating the involvement of extracellular Ca2+. Atomic absorption spectrometry showed that our 0 Ca2+ solution still contained 45 microM Ca2+, which was drastically reduced to 5.9 nM in the presence of EGTA. Taken together, our results indicate that the effects of carbachol are due to the mobilization of caffeine-, ryanodine- and thapsigargin-sensitive intracellular Ca2+ stores, and that these stores are not inactivated or depleted if micromolar concentrations (45 microM), but not nanomolar concentrations (5.9 nM) of Ca2+ are maintained in the extracellular milieu.
Relaxation induced by NANC-nerve stimulation is reduced by nitric oxide synthase (NOS) inhibitors but not by superoxide generators or NO scavengers, casting doubts on the precise nature of the neurotransmitter being released by these nerves. The lack of effect of superoxide anion generators to inhibit nitrergic nerve-mediated relaxations has been attributed to the protective action of high tissue levels of superoxide dismutase (SOD). The effects of hydroquinone, hydroxocobalamin and carboxy-PTIO, three NO inactivators which do not depend on superoxide anion generation, upon nitrergic nerve-mediated relaxations of the rat proximal duodenum were determined in order to elucidate whether they are mediated by free NO. GABA and nicotine caused relaxations of isolated segments of the rat proximal duodenum in a concentration-dependent manner that were abolished by tetrodotoxin (TTX). Similarly, transmural electrical stimulation (TES) caused frequency-dependent relaxations that were also abolished by TTX. The NOS inhibitors L-NAME and L-NOARG reduced in a concentration-dependent manner nerve-mediated relaxations elicited by TES, nicotine and GABA. The effect of NOS inhibitors was prevented by L-arginine but not D-arginine. NO caused concentration-dependent relaxations that were not affected by TTX or L-NOARG but were abolished by hydroquinone, hydroxocobalamin and carboxy-PTIO. In contrast, these compounds failed to affect TES-, nicotine- and GABA-induced relaxations. The lack of effect of hydroquinone, hydroxocobalamin and carboxy-PTIO upon nerve-mediated relaxations was unaltered by pretreatment with the SOD irreversible inhibitor DETCA. The present findings show that nitrergic nerve-mediated relaxations of the rat duodenum are unaffected by NO inactivators that do not generate superoxide anion. It is suggested that either a NO-containing molecule that is unreactive with the inactivators tested is the inhibitory neurotransmitter released by nitrergic nerves or that NOS activity fulfills another role in nitrergic nerves which could be related to the release of an still unidentified transmitter.
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