Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus

We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked. NO donors reduced [Ca2+]i and tension in a concentration-dependent manner. A close relationship was noted between these parameters. In terms of the [Ca2+]vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO. When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension. Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked. Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension. In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non-electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles. Muscles of the fundus generate tonic contraction or relaxation and contribute to the regulation of gastric volume. Upon ingestion of food, the proximal stomach relaxes to accommodate the increase in volume without large changes in pressure (Paton & Vane, 1963). This accommodation is known as adaptive relaxation (Meyer, 1987), and it depends upon the synthesis and release of nitric oxide (NO), and possibly other inhibitory transmitters released from non-adrenergic, non-cholinergic (NANC) enteric inhibitory motoneurons (Desai, Sessa & Vane, 1991). The muscles of the fundus share many similarities with phasic muscles of the gastrointestinal tract, but the mechanisms by which fundus muscles deliver Ca2+ to the contractile apparatus and handle Ca2+ appear to be somewhat unique. Several mechanisms contribute to the regulation of tone in fundus muscles. (i) There appears to be a relationship between membrane potential and force. Morgan and co-workers (Morgan, Muir & Szurszewski, 1981) depolarized muscles with elevated external K+ while simultaneously recording membrane potential and contractile force. These authors noted that a steep relationship exists between voltage and contraction. The membrane potentials of fundus cells typically lay positive to the voltage at which mechanical force could be detected, so small changes in membrane potential yielded large effects on tone. Inhibitory and excitatory junction potentials elicited by transmural stimulation of intrinsic nerves were thus thought to mediate changes in tone (see review and Fig. 15 in Szurszewski, 1987). (ii) Part of the membrane potential regulation of tone is thought to depend upon Ca2+ entry via dihydropyridine-sensitive channels (i.e. L-type Ca2+ channels; Petkov & Boev, 1996). However, another, possibly more significant, portion of the voltage-dependent regulation of tone is achieved via a dihydropyridine-insensitive pathway, because these compounds block only a fraction of the tone in the canine fundus (Boev, Golenhofen & Lukanov, 1976). The conductance responsible for this alternative, voltage-dependent Ca2+ entry pathway is unknown. (iii) A significant pharmaco-mechanical mechanism (i.e. release and reuptake of Ca2+ by internal stores) is apparent because large tonic contractions can be elicited repeatedly in the absence of external Ca2+ (Filipponi, Golenhofen, Hofstetter, Hohnsbein, Lammel & Lukanow, 1987). In addition to these mechanisms for Ca2+ delivery, there may be means of regulating the sensitivity of the contractile apparatus to Ca2+, as observed in other smooth muscles (Nishimura & van Breemen, 1989) and in other regions of the canine stomach (Ozaki, Blondfield, Hori, Sanders & Publicover, 1992b). At present, the post-junctional mechanisms utilized by enteric inhibitory transmitters to elicit relaxation in the proximal stomach are not clearly understood. Although NO or NO donors elicit hyperpolarization of fundus muscles (Burns, Lomax, Torihashi, Sanders & Ward, 1996), it is unclear whether the mechanical effects of NO are linked to changes in membrane potential or to other mechanisms. In the present study we have characterized changes in membrane potential, [Ca2+]i and force in response to stimulation of intrinsic enteric neurons and exogenous NO donors. After observing a temporal dissociation between hyperpolarization responses and changes in [Ca2+]i and force, we also investigated whether electrical changes could be disassociated from changes in [Ca2+]i and force. Our findings suggest that membrane potential changes are largely unnecessary for mechanical responses to enteric inhibitory stimuli in the canine fundus.