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 increases 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 [14C]TC and the fluorescent dye quin-2, respectively. Bt2cAMP, forskolin, and 8-bromo-cAMP stimulated Na(+)-dependent, but not Na(+)-independent TC uptake. Bt2cAMP increased the maximal rate of Na+/TC cotransport without affecting the apparent Km. Increases in TC uptake and [Ca2+]i by Bt2cAMP were inhibited in hepatocytes preloaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA) or preincubated with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate (TMB8). Calmodulin antagonists inhibited Bt2cAMP-induced increases in TC uptake, but not [Ca2+]i. Other Ca(2+)-mobilizing agents (thapsigargin, vasopressin, phenylephrine, and ionomycin) increased [Ca2+]i but failed to stimulate TC uptake, indicating that an increase in [Ca2+]i alone is not a sufficient stimulus for TC uptake. However, increases in TC uptake by 1 and 10 microM Bt2cAMP were further increased by thapsigargin, indicating a permissive role for Ca2+/calmodulin. Bt2cAMP-induced increases in TC uptake and [Ca2+]i were inhibited by known inhibitors of PKA and by an activator of PKC, but they remained unaffected by a specific inhibitor of PKC. Unlike thapsigargin, vasopressin inhibited Bt2cAMP-induced increases in TC uptake. Taken 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.
Grüne
S
S
Department of Medicine, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536.
Engelking
L R
LR
Anwer
M S
MS
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D013656:Taurocholic Acid / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D014667:Vasopressins / Q000494:pharmacology
D047428:Protein Kinase Inhibitors; D013729:Terpenes; D014667:Vasopressins; D005576:Colforsin; D010656:Phenylephrine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015759:Ionomycin; D013656:Taurocholic Acid; D003994:Bucladesine; D019284:Thapsigargin; D012964:Sodium; D000242:Cyclic AMP; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium