Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

Abstract

The mechanism of regulation of intracellular pH (pHi) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2',7'-bis(2-carboxyethyl)-5(and -6)-carboxyfluorescein. The pHi in the TRIS/HEPES-buffered standard solution was 7.29 +/- 0.01. Addition of 1 mumol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pHi. These agents decreased pHi in the absence of external Na+ or in the presence of amiloride. The rate of pHi recovery from an acid load after NH+4 prepulse was a linear function of pHi and increased as pHi became more acidic. Addition of ACh shifted the relationship towards a more alkaline pHi range. The increase in pHi induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mumol/l). Addition of 0.1-1 mumol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pHi within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pHi. In Ca(2+)-free solution with 50 mumol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pHi and cytosolic Ca2+ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mumol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na+/H+ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na+/H+ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na+/H+ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca2+ concentration. The implication of the cytosolic acidification and cell volume change in pHi regulation is discussed.

Targeted Disruption of the Nhe1 Gene Prevents Muscarinic Agonist-induced Up-regulation of Na+/H+ Exchange in Mouse Parotid Acinar Cells

“…4B). The muscarinic-induced alkaline shift generated a subsequent intracellular alkalinization (0.13-pH unit increase) relative to the initial resting intracellular pH and is consistent with previous observations for rat submandibular (28) and parotid (23) acini. However, both of these up-regulatory events were completely abolished in acini…”

“…Available evidence suggests that a combination of elevated [Ca 2ϩ ] i , cell shrinkage, and/or Cl Ϫ loss plays a central role and that this increased activity is independent of protein kinase C, calmodulin, and phosphorylation of NHE1 (8,23,26,28,29). A null background affords, by expression of a mutated form of the Nhe1 gene, a unique opportunity to perform in vivo structure/function analysis to determine the exact mechanism for activation.…”

Modulation of Na(+)‐H+ exchange by altered cell volume in perfused rat mandibular salivary gland.

“…Therefore, the shrinkage of salivary acinar cells that follows muscarinic stimulation could, in principle, be the main trigger for the upregulation of the exchanger. Some preliminary data support this hypothesis (Manganel & 1J T Seo and others Turner, 1991;Okada et al 1991) while other data do not (Robertson & Foskett, 1992Foskett, Wong, Sue-aQuan & Robertson, 1994). This study examines the role of cell shrinkage in the upregulation of the Na+-H+ exchanger following stimulation of the rat mandibular salivary gland with acetylcholine (ACh).…”

“…The shrinkage of the cells is due to the efflux of K+ and Cl-ions through channels that are activated by an elevation of cytosolic Ca2+ ([Ca2+]i) (Nauntofte, 1992). Also associated with muscarinic stimulation is an upregulation of the basolateral Na+-H+ exchanger (Manganel & Turner, 1989;Steward, Seo & Case, 1989;Dissing & Nauntofte, 1990;Okada, Saito, Sawada & Nishiyama, 1991) which serves both to counteract the increased metabolic activity during fluid secretion, and also to compensate the secretion of HC03-7 It may also contribute, in parallel with Cl--HC03-exchange, to the accumulation of intracellular Cl that provides the main driving force for fluid secretion (Case, Hunter, Novak & Young, 1984;Novak & Young, 1986;Pirani, Evans, Cook & Young, 1987).…”

“…Increased protein kinase C activity has been excluded in studies of isolated acini from the rat parotid (Manganel & Turner, 1990 and rat mandibular gland (Okada et al 1991). On the other hand, elevation of [Ca2+]i increases the activity of the exchanger, and the upregulation observed during muscarinic stimulation is dependent on the rise in [Ca2+]i (Manganel & Turner, 1990Okada et al 1991). WVhat remains to be established is whether the exchanger is activated directly by Ca2P or by other cellular changes resulting from the increase in [Ca2+]i, such as cell shrinkage.…”