Interaction of holothurian triterpene glycoside with biomembranes of mouse immune cells.
ABSTRACT The in vitro interactions between triterpene glycoside, cucumarioside A(2)-2, isolated from the Far-Eastern holothurian Cucumaria japonica, and mouse splenocyte and peritoneal macrophage biomembranes were studied. Multiple experimental approaches were employed, including determination of biomembrane microviscosity, membrane potential and Ca(2+) signaling, and radioligand binding assays. Cucumarioside A(2)-2 exhibited strong cytotoxic effect in the micromolar range of concentrations and showed pronounced immunomodulatory activity in the nanomolar concentration range. It was established that the cucumarioside A(2)-2 effectively interacted with immune cells and increased the cellular biomembrane microviscosity. This interaction led to a dose-dependent reversible shift in cellular membrane potential and temporary biomembrane depolarization; and an increase in [Ca(2+)](i) in the cytoplasm. It is suggested that there are at least two binding sites for [(3)H]-cucumarioside A(2)-2 on cellular membranes corresponding to different biomembrane components: a low affinity site match to membrane cholesterol that is responsible for the cytotoxic properties, and a high affinity site corresponding to a hypothetical receptor that is responsible for immunostimulation.
- Biochimica et Biophysica Acta 03/1974; 339(1):103-15. · 4.66 Impact Factor
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ABSTRACT: 1. Histamine-stimulated [Ca2+]i oscillations were studied in > 162 HeLa cells using the techniques of Ca2+ imaging, patch clamp and single-cell indo-1 fluorescence. 2. [Ca2+]i oscillations in HeLa cells were acutely dependent on extracellular Ca2+ and were also blocked by the extracellular addition of Cd2+ (100 microM). The Mn2+ quench technique, using fura-2 fluorescence, demonstrated that agonist-stimulated Ca2+ oscillations were associated with an increase in plasma membrane Mn2+ permeability. However, no cyclic fluctuations in Mn2+ influx were resolved over the period of [Ca2+]i spiking. 3. In whole-cell patch clamped cells an imposed potential of +80 mV was shown to block the thapsigargin-induced Ca2+ influx. Histamine and Ins(2,4,5)P3-induced [Ca2+]i oscillations were tested for the phase dependence on extracellular Ca2+ by rapidly switching the membrane potential to +80 mV, reversibly blocking Ca2+ influx. Ca2+ spikes were abolished by steps to +80 mV made at the point of spike initiation but not by steps made after development of the rapid rising phase of the spike. Steps of membrane potential to +80 mV, for increasing periods of time during the interspike period, increased the latency to the next [Ca2+]i spike by up to a maximum of approximately 150% of the control interspike interval. 4. It is concluded that the extracellular Ca2+ dependence of the histamine-induced [Ca2+]i oscillations is due to a crucial role of Ca2+ influx during spike initiation and an additional important role in setting the interspike interval. The results obtained can be interpreted in terms of a constant stimulated Ca2+ influx during [Ca2+]i spiking.The Journal of Physiology 02/1995; 482 ( Pt 2):275-81. · 4.38 Impact Factor
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ABSTRACT: The responses elicited by ATP and UTP in macrophages (measured by microfluorescence and in patch-clamp) present marked differences. The release of Ca2+ from intracellular stores induced by ATP is due to the activation of P2U receptors. These receptors can be activated by ATP4- and by MgATP2-, with apparent K0.5 values of 0.65 and 6.5 microM, respectively. The release of Ca2+ due to activation of P2U receptors by either ATP or UTP is followed by the opening of ionic channels leading to an influx of Ca2+. A second pathway for Ca2+ influx results from the opening of P2Z receptor channels triggered by adenosine-5'-O(1-thiotriphosphate) or ATP but not by UTP. The form of ATP that activates P2Z receptors is ATP4- (with a K0.5 of 0.5 microM). In voltage-clamped cells, the inward current activated by ATP4- is transient, partly because it inactivates and partly because it is rapidly masked by the development of a quinine-sensitive Ca(2+)-dependent K+ current. In current-clamp, macrophages stimulated by UTP remain normally polarized, whereas ATP depolarizes them. This P2Z-mediated depolarization results in an inhibition of the influx of Ca2+, which explains part of the difference between the time courses of the Ca2+ responses elicited by ATP and UTP.Journal of Biological Chemistry 10/1993; 268(25):18640-7. · 4.65 Impact Factor