Characterization of the inositol 1,4,5-trisphosphate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate.
The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.
Föhr
K J
KJ
Abteilung Anatomie und Zellbiologie der Universität Ulm, Oberer Eselsberg, Federal Republic of Germany.
Scott
J
J
Ahnert-Hilger
G
G
Gratzl
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000310:Adrenal Gland Neoplasms / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002452:Cell Count; D002463:Cell Membrane Permeability / Q000187:drug effects; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006902:Hydroxymercuribenzoates / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007340:Insulinoma / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010673:Pheochromocytoma / Q000378:metabolism; D051381:Rats; D014638:Vanadates / Q000494:pharmacology*
D006902:Hydroxymercuribenzoates; C030601:4-hydroxymercuribenzoate; D014638:Vanadates; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D002118:Calcium; D004229:Dithiothreitol