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

Amanda M. Auld, Michael J. Bawden, Leise A. Berven, Lyn Harland, Bernie P. Hughes, Greg J. Barritt

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    5 Citations (Scopus)

    Abstract

    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 (InsP3F) and guanosine 5′-[γ-thio]triphosphate (GTPγS) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin-and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less than 5 μM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTPγS-stimulated Ca2+ inflow was insensitive to 50 μM 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 InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with 'mock'-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-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.

    Original languageEnglish
    Pages (from-to)439-452
    Number of pages14
    JournalCell Calcium
    Volume19
    Issue number5
    DOIs
    Publication statusPublished - May 1996

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