RESUMEN
Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca(2+) measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mosmol/kgH2O), although the Ca(2+) response was unchanged. The lowering of the external Cl(-) by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring the NKCC1 bumetanide-dependent NH4 (+) transport; Na(+)-K(+)-2Cl(-) transport was upregulated >40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed, to a large extent, to the specific activation of the NKCC1.
Asunto(s)
Líquidos Corporales/metabolismo , Presión Osmótica/fisiología , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Glándula Submandibular/metabolismo , Animales , Bumetanida/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Carbacol/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Concentración Osmolar , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 2 de la Familia de Transportadores de Soluto 12/efectos de los fármacosRESUMEN
Cevimeline and pilocarpine are muscarinic agonists used clinically to treat dry mouth. In this study, we explored fluid secretion from mouse submandibular glands to determine the mechanism of cevimeline, pilocarpine, and an experimentally used agent carbachol. Cevimeline evoked almost the same amount of secretion at concentrations from 30 µM to 1 mM. Pilocarpine also induced secretion at a concentration as low as 1 µM and was the most powerful secretagogue at 10 µM. Secretion was induced by carbachol at 0.1 µM, with maximum secretion at 1.0 µM. Cevimeline induced monophasic secretion at all concentrations tested, whereas higher concentrations of pilocarpine and carbachol induced secretion with variable kinetics, i.e., an initial transient high flow rate, followed by decreased secretion after 2 to 3 min. In the presence of an epithelial Na(+) channel blocker, amiloride, neither carbachol nor pilocarpine affected the Na(+) level of secreted saliva; however, it significantly increased the Na(+) content of cevimeline-induced saliva. The intracellular Ca(2+) response of acinar cells was almost identical among all three agents, although recovery after drug removal was slower for cevimeline and pilocarpine. A profound decrease in intracellular pH was observed during pilocarpine and carbachol treatment, whereas intracellular acidification induced by cevimeline was only seen in the presence of a Na(+)/H(+) exchange inhibitor. When external HCO(3)(-) was removed, cevimeline-induced saliva significantly decreased. These findings suggest that cevimeline specifically activates Na(+)/H(+) exchange and may promote Na(+) reabsorption by stabilizing epithelial sodium channel activity.