Injection of rat hepatocyte poly(A)(+) RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP(3)F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes, lonomycin-, cholera-toxin-, thapsigargin and InsP(3)F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less than 5 mu M Gd3+ for InsP(3)F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 mu M Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca2+-sensitive fluorescent dyes:lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)(+) RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP(3)F and greater rates of Ba2+ inflow in the presence of InsP(3)F, when compared with 'mock'-injected oocytes. In poly(A)(+) RNA-injected oocytes, vasopressin- and InsP(3)(+)-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.