The HIV envelope glycoprotein, GP120, increases intracellular Ca2+ concentration and induces degeneration of human and animal neurons in culture. Using patch-clamp recordings and Ca2+ imaging techniques, we have now examined the contribution of intracellular stores of calcium in the effects of GP120. We report that in rat hippocampal neuronal cultures, GP120 induces a dramatic and persistent increase in [Ca2+]i which is prevented by drugs that either deplete (caffeine, carbachol, thapsigargin) or block (dantrolene) Ca2+ release from intracellular stores. In contrast, N-methyl-d-aspartate (NMDA) receptors or voltage-dependent calcium channels do not participate in these effects, as: (i) the increase in [Ca2+]i was not affected by NMDA receptor antagonists or calcium channel blockers; and (ii) and GP120 did not generate any current in whole-cell recording. Dantrolene, a ryanodine stores inhibitor, also prevented neuronal death induced by GP120. Our results show that the GP120-induced rise in [Ca2+]i originates from intracellular calcium stores, and suggest that intracellular stores of calcium may play a determinant role in the pathological actions of GP120.
Heterotrimeric G proteins may assume modulatory roles in cellular proliferation and differentiation. The G protein ␣-subunit G ␣16 , which is specifically expressed in hematopoietic cells, is highly regulated during differentiation of normal and leukemic cells. In human erythroleukemia cells, suppression of G ␣16 inhibited cellular growth rates. A reporter gene system was established to assess the role of G ␣16 on erythroid differentiation of MB-02 erythroleukemia cells. It is based on transient transfection with a plasmid that expresses green fluorescent protein under the control of the -globin promoter. Expression of G ␣16 led to a significant increase in green fluorescent protein-positive cells, as did transfection with a G ␣16 antisense plasmid (154 and 156% of controls, respectively). The GTPase-deficient, constitutively active mutant of G ␣16 , G ␣16 R186C, further stimulated differentiation to 195% of control values. Because the effect of G ␣16 is triggered most efficiently by the GTP-bound protein, an indirect action through interference of overexpressed G ␣16 with G protein ␥-subunits can be excluded. The corresponding mutant of G ␣q (G ␣q R182C), the phylogenetically closest family member of G ␣16 , had no effect. The data define a specific role for G ␣16 -dependent signal transduction in cellular differentiation: deviations from optimal levels of G ␣16 functional activity lead to reduced growth rates and promote differentiation in hematopoietic cells.
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