INJECTION OF RAT HEPATOCYTE POLY(A)(-LAEVIS OOCYTES LEADS TO EXPRESSION OF A CONSTITUTIVELY-ACTIVE DIVALENT-CATION CHANNEL DISTINGUISHABLE FROM ENDOGENOUS RECEPTOR-ACTIVATED CHANNELS() RNA TO XENOPUS)

The expression of hepatocyte plasma membrane receptor-activated divale nt cation channels in immature (stages V and VI) Xenopus laevis oocyte s and the properties which allow these channels to be distinguished fr om endogenous receptor-activated divalent cation channels were investi gated. Divalent cation inflow to oocytes housed in a multiwell plate w as measured using the fluorescent dyes Fluo-3 and Fura-2. In control o ocytes, 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 the se ions to the oocytes, lonomycin-, cholera-toxin-, thapsigargin and I nsP(3)F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inh ibition 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+ an d to SK&F 96365. These results indicate that at least three types of e ndogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca2+-sensitive fluorescent dyes:lanthanide-sensitive div alent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera to xin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocy te poly(A)(+) RNA exhibited greater rates of Ca2+ and Mn2+ inflow in t he basal (no agonist) state, greater rates of Ca2+ inflow in the prese nce 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)(+)-stimulate d Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divale nt cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide -insensitive, can be expressed and detected in Xenopus oocytes.