Transient ATP depletion causes apoptosis in tubular epithelial cells. Prior HS inhibits apoptosis and improves survival in these cells. Novel interactions between HSP 72 and Bcl2 may be responsible, at least in part, for the protection afforded by prior heat stress against ATP depletion injury.

The interaction of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins provides the necessary steps for vesicle docking fusion. In inner medullary collecting duct (IMCD) cells, acid secretion is regulated in part by exocytotic insertion and endocytotic retrieval of an H ؉ -ATPase to and from the apical membrane. We previously suggested a role for SNARE proteins in exocytotic insertion of proton pumps in IMCD cells. The purpose of the present study was to determine whether SNARE proteins are associated with the 31-kDa subunit of H The vacuolar H ϩ -ATPase is a ubiquitous multisubunit enzyme that participates in a wide variety of cellular functions (1). The renal collecting duct is populated by cells (␣-intercalated cells and inner medullary collecting duct (IMCD) 1 cells) that are specialized for H ϩ transport (2). The vacuolar H ϩ -ATPase in these cells resides in high density in vesicles and is polarized to the apical membrane (3). Constitutive and regulated exocytotic insertion and endocytotic retrieval of H ϩ -ATPase containing vesicles to and from the apical plasma membrane regulate, in part, not only the density of H ϩ -ATPase in the apical membrane but also the rate of proton transport by these cells (4 -7). A reduction in the intracellular pH followed by an elevation of cytosolic Ca 2ϩ are the required stimuli that induce regulated exocytic insertion of proton pumps into the apical membrane (8).It is likely that the exocytotic event that regulates translocation of the H ϩ -ATPase to the apical membrane in renal acid secretory cells utilizes a mechanism for exocytosis that is similar to that described in neuronal cells. The process in neuronal cells involves the participation of a subset of highly conserved, universally present membrane proteins (v-and t-SNAREs) and soluble factors (NSF and SNAP) (9). Consistent with this proposal is the observation that SNARE proteins are present in renal collecting duct cells (10 -13) and our recent studies document that in cultured rat IMCD cells, H ϩ transport, mediated by an H ϩ -ATPase, is inhibited by clostridial toxins (14). However, to date, no studies have described the exocytotic mechanisms regulating H ϩ -ATPase insertion into the apical plasma membrane or provided direct evidence for the participation of the SNARE proteins in this process. Furthermore, the formation of a putative docking complex similar to the 20 S complex that has been described in neuronal and neuroendocrine cells (9) has not been demonstrated in renal epithelial cells.To characterize the mechanisms involved in IMCD trafficking of H ϩ -ATPase and to characterize the role of SNAREs in this process, we determined the effect of clostridial toxins on cell acidification-induced translocation of both H ϩ -ATPase and SNAREs to the apical membrane, identified the v-SNARE proteins that participate in the translocation of H ϩ -ATPase from vesicle to apical membrane following an acid load, and isolated a complex of proteins similar to the docking complex (20 S complex...

Renal epithelial cell H+secretion is an exocytic-endocytic phenomenon. In the inner medullary collecting duct (IMCD) cell line, which we have utilized as a model of renal epithelial cell acid secretion, we found previously that acidification increased exocytosis and alkalinization increased endocytosis. It is likely, therefore, that the rate of proton secretion is regulated by the membrane insertion and retrieval of proton pumps. There is abundant evidence from studies in the nerve terminal and the chromaffin cell that vesicle docking, membrane fusion, and discharge of vesicular contents (exocytosis) involve a series of interactions among so-called trafficking proteins. The clostridial toxins, botulinum and tetanus, are proteases that specifically inactivate some of these proteins. In these experiments we demonstrated, by immunoblot and immunoprecipitation, the presence in this IMCD cell line of the specific protein targets of these toxins, synaptobrevin/vesicle-associated membrane proteins (VAMP), syntaxin, and synaptosomal-associated protein-25 (SNAP-25). Furthermore, we showed that these toxins markedly inhibit the capacity of these cells to realkalinize after an acid load. Thus these data provide new insight into the mechanism for H+ secretion in the IMCD.