ROLE OF INTRACELLULAR CALCIUM AND PROTEIN-KINASES IN THE ACTIVATION OF HEPATIC NA+ TAUROCHOLATE COTRANSPORT BY CYCLIC-AMP/

Glucagon and dibutyryl cyclic AMP (Bt2cAMP) stimulate Na+/taurocholate (TC) cotransport and increase the intracellular Ca2+ concentration ([ Ca2+]i) of hepatocytes. Whether the effect of cAMP is mediated via inc reases in [Ca2+]i, cAMP-dependent protein kinase (PKA), and/or protein kinase C (PKC) was investigated in this study. TC uptake and [Ca2+]i were determined in isolated rat hepatocytes using [C-14]TC and the flu orescent dye quin-2, respectively. Bt2cAMP, forskolin, and 8-bromo-cAM P stimulated Na+-dependent, but not Na+-independent TC uptake. Bt2cAMP increased the maximal rate of Na+/TC cotransport without affecting th e apparent K(m). Increases in TC uptake and [Ca2+]i by Bt2cAMP were in hibited in hepatocytes preloaded with mino-5-methylphenoxy)-ethane-N,N ,N',N'-tetraacetic acid (MAPTA) or preincubated with 8-diethylaminooct yl 3,4,5-trimethoxybenzoate (TMB8). Calmodulin antagonists inhibited B t2cAMP-induced increases in TC uptake, but not [Ca2+]i. Other Ca2+-mob ilizing agents (thapsigargin, vasopressin, phenylephrine, and ionomyci n) increased [Ca2+]i but failed to stimulate TC uptake, indicating tha t an increase in [Ca2+]i alone is not a sufficient stimulus for TC upt ake. However, increases in TC uptake by 1 and 10 muM Bt2cAMP were furt her increased by thapsigargin, indicating a permissive role for Ca2+/c almodulin. Bt2cAMP-induced increases in TC uptake and [Ca2+]i, were in hibited by known inhibitors of PKA and by an activator of PKC, but the y remained unaffected by a specific inhibitor of PKC. Unlike thapsigar gin, vasopressin inhibited Bt2cAMP-induced increases in TC uptake. Tak en together these results indicate that stimulation of hepatic Na+/TC cotransport by cAMP 1) is mediated via PKA; 2) is potentiated, but not mediated, by Ca2+/calmodulin-dependent processes; and 3) may be down- regulated by PKC.