Abstract
Nonhydrolyzable guanine nucleotide analogues were used to evaluate the role of guanine nucleotide binding (G) proteins in regulating pepsinogen secretion from streptolysin O-permeabilized chief cells from guinea pig stomach. In the presence of 100 nM calcium, 100 microM guanosine 5'-(beta,gamma-imido)triphosphate or guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) caused a 2- to 4-fold increase in pepsinogen secretion. GTP gamma S stimulated secretion in the absence of calcium (up to 10 mM EGTA). With or without added calcium, GTP analogues caused a 2- to 3-fold increase in cAMP, whereas guanosine 5'-O-2-(thio)diphosphate and calcium alone had no effect on cAMP levels. GTP analogue-induced activation of phospholipase C was evidenced by a calcium-independent increase in cytidine diphospho-1,2-diacylglycerol levels (50% above basal). Phorbol ester- and GTP gamma S-stimulated phosphorylation of a 72-kDa acidic protein was abolished by an inhibitor of protein kinase C (CGP 41251). However, GTP gamma S-induced pepsinogen secretion was only partially inhibited by adding CGP 41251 or a protein kinase C inhibitor peptide. These results indicate that guanine nucleotides activate major signaling pathways in gastric chief cells. Nevertheless, GTP gamma S can induce pepsinogen secretion independently of changes in calcium, cAMP, or activation of protein kinase C.
Highlights
From the Division of Digestive Diseases, Department of Medicine, State University of New York, Health Science Center, Brooklyn, New York 11203-2098
Nonhydrolyzable guanine nucleotide analoguwesere (PKC)’ [5,6].Increases in cellular levels of CAMPand calcium used to evaluate the role of guanine nucleotide bindirnesgult in activation of protein kinases and/or phosphatases (G) proteins in regulating pepsinogen secretion from that, by currently unknown mechanisms, mediate the final streptolysin 0-permeabilized chief cells from guinea steps that result in pepsinogen secretion [1].The possibility pig stomach
With or without added calcium, GTP analoguTeshe introduction of nonhydrolyzable guanine nucleotide caused a2- to %fold increase in CAMP, whereas guaanna-logues into permeabilized secretory cells, thereby moduosine 5’-0-2-(thio)diphosphateand calcium alone had lating the activity of G proteins, has been used by several no effect on CAMPlevels
Summary
Inc.; protein kinase C inhibitor peptide (PKC[19-31]) from Upstate Biotechnology, Inc. (Lake Placid, NY); Iso-Lytes and 129-albumin from ICN; and [32P]ATPfrom Du Pont-New England Nuclear. Tissue Preparationand Permeabilization-Dispersed chief cells from guinea pig stomach (>go% chief cells) were prepared as described previously [13] and were kept in standard incubation solution containing Ca2+and M%+ for 10 min before proceeding. Cellular CAMP-Chief cell cAMP was determined by radioimmunoassay using the procedure described previously [2]. In these experiments, 100 p~ IBMX was included in the incubation medium. The cells were washed with permeabilization buffer without SLO and incubated in the presence of 10 mM LiCl for an additional 10 min. Following a 3-min incubation at 37 "C,the cell suspension was placed on ice and adjusted to 2% Nonidet P-40, 9.5 M urea, 5% P-mercaptoethanol, and 2% IsoLytes (consisting of 0.8% 5-8, 0.8% 6-8, and 0.4% 3-10 Iso-Lyte).
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