SULFHYDRYL-REAGENTS AND CAMP-DEPENDENT KINASE INCREASE THE SENSITIVITY OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN HEPATOCYTES

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepato cytes in a way that resembles responses to Ca2+-mobilizing hormones (S aikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266 , 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P . (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount o f Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from perm eable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Ta ylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D . C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512) . The effects of sulfhydryl reagents were studied in fura-2-injected r at and guinea pig hepatocytes and compared with the actions of cAMP (B urgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1 991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinje ction of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous leve ls of (1,4,5)IP3 or were first injected with a sub-threshold concentra tion of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TB HP in (2,4,5)IP3-injected guinea pig cells were also blocked by hepari n. In many respects, the actions of TBHP appeared to be similar to tho se of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op . cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca 2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP 3-injected guinea pig hepatocytes differed, however, in that the incre ase in [Ca2+]i evoked by elevating intracellular cAMP was greatly redu ced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on th e Ca2+ transients induced by TBHP. This provides evidence that the sen sitizing effect of TBHP is not mediated by PKA and is more likely to b e a direct effect on the inositol trisphosphate receptor. It is possib le, however, that the sulfhydryl reagents and PKA act on a common regu latory site on the receptor protein.