CALCIUM INFLUX MEDIATED BY THE ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN-B (ST(B))

The heat-stable enterotoxin B (ST(B)) of Escherichia coli is a 48-amino acid extracellular peptide that induces rapid fluid accumulation in animal intestinal models. Unlike other E. coli enterotoxins that elicit cAMP or cGMP responses in the gut [heat-labile toxin (LT) and heat-stable toxin A (ST(A)), respectively], ST(B) induces fluid loss by an undefined mechanism that is independent of cyclic nucleotide elevation. Here we studied the effects of ST(B) on intracellular calcium concentration ([Ca2+]i), another known mediator of intestinal ion and fluid movement. Ca2+ and pH measurements were performed on different cell types including Madin-Darby canine kidney (MDCK), HT-29/C1 intestinal epithelial cells, and primary rat pituitary cells, Ca2+ and pH determinations were performed by simultaneous real-time fluorescence imaging at four emission wavelengths. This allowed dual imaging of the Ca2+- and pH-specific ratio dyes (indo-1 and SNARF-1, respectively). ST(B) treatment induced a dose-dependent increase in [Ca2+]i with virtually no effect on internal pH in all of the cell types tested. ST(B)-mediated [Ca2+]i elevation was not inhibited by drugs that block voltage-gated Ca2+ channels including nitrendipine, verapamil (L-type), omega-conotoxin (N-type), and Ni2+ (T-type). The increase in [Ca2+]i was dependent on a source of extracellular Ca2+ and was not affected by prior treatment of MDCK cells with thapsigargin or cyclopiazonic acid, agents that deplete and block internal Ca2+ stores. In contrast to these results, somatostatin and pertussis toxin pretreatment of MDCK cells completely blocked the ST(B)-induced increase in [Ca2+]i. Taken together, these data suggest that ST(B) opens a GTP-binding regulatory protein-linked receptor-operated Ca2+ channel in the plasma membrane. The nature of the ST(B)-sensitive Ca2+ channel is presently under investigation.