Transmembrane signaling by the B subunit of cholera toxin: increased cytoplasmic free calcium in rat lymphocytes

It has previously been shown that the B subunit of cholera toxin, which binds solely to the plasma membrane ganglioside GM1, stimulates the proliferation of rat thymic lymphocytes (Spiegel, S., P. H. Fishman, and R. J. Weber, 1985, Science [Wash. DC], 230:1285-1287). The purpose of this study was to identify which transmembrane signaling system(s) are activated by the B subunit of cholera toxin. We compared the effects of B subunit and concanavalin A (Con A), a potent mitogenic lectin, on a number of second messenger systems that are putative mediators of T cell activation. Changes in the fluorescence of quin2- loaded cells revealed that mitogenic doses of either B subunit or Con A induced rapid and sustained increases in cytoplasmic free Ca2+ ([Ca2+]i). Within 5 min, [Ca2+]i increased from a basal level of 69 +/- 4 to 136 +/- 17 and 185 +/- 24 nM, respectively. The effects of B subunit and Con A were additive and largely dependent on the presence of extracellular Ca2+, though release of Ca2+ from intracellular stores could be detected for Con A, but not B subunit, using indo-1. The B subunit had no effect on either inositol phosphate levels or on the distribution of protein kinase C, indicating that, unlike Con A, the B subunit does not activate phosphoinositide hydrolysis. Fluorimetric measurements on cells loaded with bis(carboxyethyl)-5,6- carboxyfluorescein revealed that Con A induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas B subunit had no effect on intracellular pH. Finally, by monitoring bis-oxonol fluorescence, we found that Con A induced a small hyperpolarization of the membrane potential, whereas B subunit had no acute effect. These data suggest that the biological effects of B subunit are mediated by an increase in [Ca2+]i resulting from a net influx of extracellular Ca2+.