Isolated secretion granules from parotid glands of chronically stimulated rats possess an alkaline internal pH and inward-directed H+ pump activity.

Abstract

Abstract. Secretion granules have been isolated from the parotid glands of rats that have been chronically stimulated with the fi-adrenergic agonist, isoproterenol. These granules are of interest because they package a quantitatively different set of secretory proteins in comparison with granules from the normal gland. Polypeptides enriched in proline, glycine, and glutamine, which are known to have pI's >10, replace a-amylase (prs = 6.8) as the principal content species. The internal pH of granules from the treated rats ranges from 7.8 in a potassium sulfate medium to 6.9 in a choline chloride medium. The increased pH over that of normal parotid granules (,x,6.8) appears to reflect the change in composition of the secretory content. Whereas normal mature parotid granules have practically negligible levels of H ÷ pumping ATPase activity (Aryan, P., G. Rudnick, and J. D. Castle, 1985, J. Biol. Chem., 260, 14945-14952) the isolated granules from isoproterenol-treated rats undergo a timedependent internal acidification (,,00.2 pH uni0 that requires the presence of ATP and is abolished by an H + ionophore. Additionally, an inside-positive granule transmembrane potential develops after ATP addition that depends upon ATP hydrolysis. Two independent methods have been used that exclude the possibility that contaminating organelles are the source of the H ÷-ATPase activity. Together these data provide clear evidence for the presence of an H ÷ pump in the membranes of parotid granules from chronically stimulated rats. However, despite the presence of H+-pump activity, fluorescence microscopy with the weak base, acridine orange, reveals that the intragranular pH in live cells is greater than that of the cytoplasm. IN contrast to the peptide-containing secretory vesicles of neural and endocrine cells (29,39), mature exocrine granules of the rat parotid gland do not have an acidic internal pH (7) and exhibit almost no inward-directed H + pump activity (8). However, both exocrine and endocrine granules originate within compartments located at the trans aspect of the Golgi complex (15, 23), and several recent studies provide reason to suspect that this stage of the secretory pathway (Golgi/post-Golgi) may include a compartment that possesses H+-ATPase activity at levels sufficient to cause internal acidification. First, membrane vesicles derived from the secretion grannie-like fractions of hepatocyte Golgi complexes undergo an ATP-dependent internal acidification that is inhibitable by the sulflaydryl-reactive reagents N-ethyl maleimide and 7-chloro-4-nitrobenz-2-oxa-l,3-diazole (Nbd-C1; I reference 22). Second, the addition of chloroquine (a membrane-permeating weak base that elevates the 1. Abbreviations used in this paper: A~, transmembrane electrical potential; ApH, pH difference across the granule membrane (pHi.-pHo~t); AMP-PNP, Mg 2+-I$, -imidoadenosine-5'-triphosphate; AO, acridine orange; CCCP, earbonyl cyanide m-chloromethoxyphenyl hydrazone; MOPS, morpholino propane sulfonic acid; Nbd-C1, 7-chioro-4-nitrobenz-2-oxa...

Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

“…This is supported by measurements of the mean internal space in isolated parotid secretory granules. Thus, granules from isoproterenol-treated rats exhibit twice the internal space (expressed as μL/mg protein) as granules from untreated rats (Arvan et al, 1984;Arvan and Castle, 1986). Isoproterenol treatment induces the expression of basic and acidic prolinerich proteins (PRPs) (Robinovitch et al, 1977), including sulfated proteoglycans (Blair et al, 1991).…”

Thea3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells

“…The secretory granules have a luminal pH of 5.0-5.5 (Arvan and Castle, 1986;Orci et al, 1986;Urbe et al, 1997), and their acidification has important roles in the condensation and maturation of their contents (Hutton, 1994). The electrochemical proton gradient across the membrane provides the energy for accumulation of neurotransmitters into synaptic vesicles (Moriyama and Futai, 1990;Nelson, 1993).…”

XBP1 and MIST1 are not sufficient to induce the regulated secretory pathway in parotid cell lines.

“…These specific vesicles are called dense-core secretory granules (DCSG). Until stimulated, these DCSGs are stored and continue to remain in the apical end of the cell, as seen in Figure 1A ( Arvan & Castle, 1986;Borgonovo, Ouwendijk, & Solimena, 2006). The…”