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.