Protein kinase A regulates the disposition of Ca²⁺ which enters the cytoplasmic space through store-activated Ca²⁺ channels in rat hepatocytes by diverting inflowing Ca²⁺ to mitochondria

The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca²⁺ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca²⁺ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca²⁺ (Ca^2t₀) to cells incubated in the absence of added Ca^2t₀. Guanosine 5′-[γ-thio]-triphosphate (GTP[S]) and AlF₄^- inhibited the thapsigargin-stimulated Ca²⁺₀ induced increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3′,5′-(cyclic)-phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca²⁺₀-induced increase in [Ca²⁺]_c by GTP[S], but had little effect on thapsigargin-induced release of Ca²⁺ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca²⁺-induced increase in [Ca²⁺]_c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulatcd Ca²⁺₀-induced increase in [Ca²⁺]_c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca²⁺, which enters the cytoplasmic space through store-activated Ca²⁺ channels, by directing some of this Ca²⁺ to the mitochondria. The idea that caution should be exercised in using observed values of Ca²⁺₀-induced increase in [Ca²⁺]_c as estimates of rates of agonist-stimulated Ca²⁺ inflow is briefly discussed.