Dissociation of store release from transmembrane influx of calcium in human neutrophils

Abstract

Release of C,t:" from intracc!lular stores wa~ wsuahsed in individual neutrophih m the pre~ence of the Mn '* or SKF 96365. influx of Mw'" quenched rata-2 close to the plabma membrane but did not quench fnra-2 at the s~le of stole release The size and location of the 'cloud' of elewtted Ca:" was unaffected by the channel blocker SKF 96365. Furthe]morc, the ~ze and Ioeatton was unaffected by the ple~ence of extracellular Ca"'. This dhsoe;.'ttion of transmembrane reflux from ~tore release delnonstrates that the entry of Ca "-~ into the cyto~ol of neutrophils oceur~ directly into the cylosol and not vta the sto~¢ ~ite.

Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane

“…The data also further support the hypothesis that cations do not first enter endothelial cells via the internal stores, but by a direct route into the cytoplasm (Putney, 1986(Putney, , 1990. Although our evidence strongly suggests a selectivity of ionomycin for endomembranes [as has previously been only hinted at (Stauderman and Pruss, 1989; Albert and Tashjian, 1986;Neylon and Irvine, 1991)], there are reports of ionophore being unaffected by conditions which can nevertheless blunt the agonist-stimulated Ca2+ entry (McCarthy et al, 1989;Davies et al, 1992;Graier et al, 1992). We cannot fully explain such discrepancies in the light of our data, but suggest that there may be differences in experimental protocols (e.g.…”

“…Furthermore, ionomycin-induced cation entry in human umbilical-vein endothelial cells (HUVEC) may share a common route of entry with histamine-induced entry, since both responses were inhibited by thrombin (Neylon and Irvine, 1991). On the other hand, others have shown that inhibitors of Ca2+ entry such as SK&F 96365 (Davies et al, 1992;Graier et al, 1992) or phorbol ester (McCarthy et al, 1989) had no effect on ionophore responses, while decreasing agonist-driven effects, which is more consistent with the ionophores directly translocating bivalent cations across the plasma membrane.…”

“…Given that both CPA and ionomycin could discharge the agonist-sensitive pool, we next examined whether they could, in turn, trigger cation influx in a manner analogous to histamine (Jacob, 1990). The unidirectional entry of Mn2 , monitored by the quenching of fura-2, has frequently been used as an index for stimulated cation entry (Merritt et al, 1989;Jacob, 1990;Montero et al, 1990;Neylon and Irvine, 1991;Davies et al, 1992). Figure 4 indicates the ability of histamine, ionomycin and CPA to stimulate Mn2+ influx by depletion of internal stores in Ca2+-free solution.…”

“…Although transition-metal ions are commonly employed as non-specific blockers of Ca2+ entry (Rink, 1990), their potential for competing with Ca2+ for the co-ordination sites in the ionomycin molecule (Stiles et al, 1991) rendered them unsuitable agents. Instead, the store-regulated Ca2+-entry blocker SK&F 96365 was used (Merritt et al, 1990;Davies et al, 1992;Schilling et al, 1992;Graier et al, 1992) and, consistent with its blockade of influx, was found to inhibit the plateau, but not the initial mobilization, of intracellular stores by histamine, CPA or ionomycin. Curiously, with histamine, inhibition was only significant after a preincubation with the blocker.…”

Characterization of a complement-fragment-C5a-stimulated calcium-influx mechanism in U937 monocytic cells

“…Taken together these results indicate that [Ca2+]i is controlled in U937 monocytes by mechanisms distinct from those which appear to operate in other myeloid cells, such as neutrophils, stimulated with C5a and formylpeptide. be dependent on the emptying of internal Ca2+ stores (Putney, 1990;Alonso-Torre et al, 1993), although influx does not appear to occur via the organelle which contains the Ca2+ store (Davies et al, 1992). The elevation of [Ca2+]1 in fMLP-stimulated neutrophils appears to be entirely dependent on pertussis-toxinsensitive G-proteins (Goldman et al, 1985).…”