Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.
The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.
Bànhegyi
G
G
Istituto di Patologia Generale, Università di Siena, Italy.
Fulceri
R
R
Bellomo
G
G
Romani
A
A
Pompella
A
A
Benedetti
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004357:Drug Synergism; D005215:Fasting; D005502:Food; D005943:Gluconeogenesis; D005947:Glucose / Q000096:biosynthesis; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000494:pharmacology*
D005958:Glucosephosphates; D014667:Vasopressins; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000001:Calcimycin; D019298:Glucose-6-Phosphate; D003994:Bucladesine; D000255:Adenosine Triphosphate; D005947:Glucose; D002118:Calcium