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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.

ABSTRACT: 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+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5'-[gamma-thio]-triphosphate (GTP[S]) and AlF4(-) inhibited the thapsigargin-stimulated Ca2+o-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+o-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+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-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+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.

SUBMITTER: Fernando KC

PROVIDER: S-EPMC1219259 | biostudies-other | 1998 Mar

REPOSITORIES: biostudies-other

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Project description:The roles of the filamentous actin (F-actin) cytoskeleton and the endoplasmic reticulum (ER) in the mechanism by which store-operated Ca(2+) channels (SOCs) and other plasma-membrane Ca(2+) channels are activated in rat hepatocytes in primary culture were investigated using cytochalasin D as a probe. Inhibition of thapsigargin-induced Ca(2+) inflow by cytochalasin D depended on the concentration and time of treatment, with maximum inhibition observed with 0.1 microM cytochalasin D for 3 h. Cytochalasin D (0.1 microM for 3 h) did not inhibit the total amount of Ca(2+) released from the ER in response to thapsigargin but did alter the kinetics of Ca(2+) release. The effects of cytochalasin D (0.1 microM) on vasopressin-induced Ca(2+) inflow were similar to those on thapsigargin-induced Ca(2+) inflow, except that cytochalasin D did inhibit vasopressin-induced release of Ca(2+) from the ER. Cytochalasin D (0.1 microM) inhibited vasopressin-induced Mn(2+) inflow (predominantly through intracellular messenger-activated non-selective cation channels), but the degree of inhibition was less than that of vasopressin-induced Ca(2+) inflow (predominantly through Ca(2+)-selective SOCs). Maitotoxin- and hypotonic shock-induced Ca(2+) inflow were enhanced rather than inhibited by 0.1 microM cytochalasin D. Treatment with 0.1 microM cytochalasin D substantially reduced the amount of F-actin at the cell cortex, whereas 5 microM cytochalasin D increased the total amount of F-actin and caused an irregular distribution of F-actin at the cell cortex. Cytochalasin D (0.1 microM) caused no significant change in the overall arrangement of the ER [monitored using 3',3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] in fixed cells] but disrupted the fine structure of the smooth ER and reduced the diffusion of DiOC(6)(3) in the ER in live hepatocytes after photobleaching. It is concluded that (i) the concentration of cytochalasin D is a critical factor in the use of this agent as a probe to disrupt the cortical F-actin cytoskeleton in rat hepatocytes, (ii) a reduction in the amount of cortical F-actin inhibits SOCs but not intracellular messenger-activated non-selective cation channels, and (iii) inhibition of the activation of SOCs and reduction in the amount of cortical F-actin is associated with disruption of the organization of the ER.

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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.

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