In gastric parietal cells, cholinergically induced increases in intracellular free calcium concentrations have been well characterized, but little is known about the signaling events beyond the initial rise in intracellular calcium. In the present study, we report the isolation of a 28-kDa protein, which is rapidly phosphorylated in intact, enriched parietal cells in response to both the cholinergic agonist, carbachol, and the calcium ionophore, ionomycin. A combination of in situ 32 P labeling and one-and two-dimensional gel electrophoresis was used to acquire sufficient quantities of protein to obtain partial amino acid sequence. Cloning of the pp28 cDNA revealed a novel protein which we have named CSPP28 based on its calcium-sensitive phosphorylation. There are three CSPP28 mRNA species (1.7, 2.2, and 3.3 kilobases) that are widely distributed throughout a variety of rabbit tissues. Recombinant CSPP28 was phosphorylated by both crude parietal cell homogenate and purified CaM kinase II in a calcium/calmodulin-dependent manner. We propose that CSPP28 may play an important and ubiquitous role in the calcium signaling pathway.Intracellular signaling is generally mediated by activation of specific receptors leading to alterations in intracellular concentrations of different second messengers, including calcium, inositol phosphates, diacylglycerol, and cyclic AMP. These second messengers modulate many physiological processes that involve the phosphorylation of enzymes, receptors, and substrates by multifunctional protein kinases, namely calcium/ calmodulin-dependent protein kinase II (CaM kinase II), calcium/phospholipid-dependent protein kinase, and cyclic AMP-dependent protein kinase (for reviews, see Refs. 1-3). Although there is an abundance of information about second messengers and second messenger-dependent protein kinases, much less is known about the specific protein kinase substrates in these signaling pathways.In many secretory cells, cholinergic stimulation of muscarinic receptors activates phospholipase C, which hydrolyzes phosphoinositol 4,5-bisphosphate to liberate inositol 1,4,5-bisphosphate and diacylglycerol (2). Similarly, in HCl-secreting gastric parietal cells, cholinergic agonists elevate inositol 1,4,5-bisphosphate concentrations which, in turn, stimulate the rapid release of calcium from internal stores (4 -8). The cascade of signaling events following the rise in intracellular free calcium concentrations in parietal cells and in other cell types remains obscure. It is clear, however, that protein phosphorylation is a critical component of second messenger-dependent cascades. In parietal cells, at least three different proteins with molecular masses of 28, 36, and 66 kDa are phosphorylated in response to cholinergic stimulation (4,8,9). These phosphorylation events appear to occur by way of different protein kinaseactivating mechanisms. Since the 36-and 66-kDa phosphoproteins are phosphorylated in isolated intact parietal cells following addition of phorbol ester under calcium-chelating cond...
The role of calcium in control of HCI secretion by the gastric parietal cell was examined using a recently available intracellular calcium-releasing agent, thapsigargin, which has been shown, in some cell types, to induce sustained elevation of intracellular calcium ([Ca2+JJ, an (28 vs 600% of basal). The protein kinase C activator, 12-0-tetradecanoyl phorbol-1 3-acetate (TPA) potentiated the secretory response to thapsigargin but this combined response also did not attain the same magnitude as the maximal cholinergic response. In the presence but not the absence of medium calcium, thapsigargin potentiated acid secretory responses to histamine, which elevate both cyclic AMP (cAMP) and [Ca2+]J in parietal cells, as well as forskolin and cAMP analogues but had no effect on submaximal and an inhibitory effect on maximal cholinergic stimulation. Further-
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