Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes.

In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NO(i)) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency-dependent increases in NO(i) that were blocked by the calmodulin (CaM) inhibitor 10 microM W-7 and partially inhibited by the phosphatidylinositol 3'-kinase (PI-(3)K) inhibitor 10 microMm LY294002. Increasing extracellular [Ca(2+)] caused a concentration-dependent increase in FS-induced NO(i) that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 microM) decreased cell shortening and NO(i) production without concomitant changes in L-type Ca(2+) current (I(Ca,L)) or [Ca(2+)](i) transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 microM) increased cell shortening and NO(i) production without concomitant changes in I(Ca,L) or [Ca(2+)](i) transients. FS-induced NO(i) production was decreased in myocytes infected (100 multiplicity of viral infection (MOI); 24 h) with a replication-deficient adenovirus expressing a dominant-negative mutant of protein kinase B (Akt) compared with cells infected with a control adenovirus expressing beta-galactosidase. FS-induced NO(i) was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS-induced [Ca(2+)](i) transients were increased by the nNOS inhibitor nNOS-I (0.24 microM), decreased by the eNOS inhibitor L-NIO (1 microM) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS-induced NO(i) production requires both Ca(2+)-dependent CaM signalling and Ca(2+)-independent PI-(3)K-Akt signalling activated by contractile activity. FS activates NO(i) production from both eNOS and nNOS, and each source of NO(i) exerts opposing effects on [Ca(2+)](i) transient amplitude. These findings are important for understanding the regulation of NO(i) signalling in the normal and mechanically failing heart.

Phenylephrine acts via IP3-dependent intracellular NO release to stimulate L-type Ca2+ current in cat atrial myocytes.

This study determined the effects of alpha1-adrenergic receptor (alpha1-AR) stimulation by phenylephrine (PE) on L-type Ca2+ current (I(Ca,L)) in cat atrial myocytes. PE (10 microm) reversibly increased I(Ca,L) (51.3%; n = 40) and shifted peak I(Ca,L) activation voltage by -10 mV. PE-induced stimulation of I(Ca,L) was blocked by each of 1 microm prazocin, 10 microm L-NIO, 10 microm W-7, 10 microm ODQ, 2 microm H-89 or 10 microm LY294002, and was unaffected by 10 microm chelerythrine or incubating cells in pertussis toxin (PTX). PE-induced stimulation of I(Ca,L) also was inhibited by each of 10 microm ryanodine or 5 microm thapsigargin, by blocking IP3 receptors with 2 microm 2-APB or 10 microm xestospongin C or by intracellular dialysis of heparin. In field-stimulated cells, PE increased intracellular NO (NOi) production. PE-induced NOi release was inhibited by each of 1 microm prazocin, 10 microm L-NIO, 10 microm W-7, 10 microm LY294002, 2 microm H-89, 10 microm ryanodine, 5 microm thapsigargin, 2 microm 2-APB or 10 microm xestospongin C, and unchanged by PTX. PE (10 microm) increased phosphorylation of Akt, which was inhibited by LY294002. Confocal microscopy showed that PE stimulated NOi release from subsarcolemmal sites and this was prevented by 2 mm methyl-beta-cyclodextrin, an agent that disrupts caveolae formation. PE also increased local, subsarcolemmal SR Ca2+ release via IP3-dependent signalling. Electron micrographs of atrial myocytes show peripheral SR cisternae in close proximity to clusters of caveolae. We conclude that in cat atrial myocytes PE acts via alpha1-ARs coupled to PTX-insensitive G-protein to release NOi, which in turn stimulates I(Ca,L). PE-induced NOi release requires stimulation of both PI-3K/Akt and IP3-dependent Ca2+ signalling. NO stimulates I(Ca,L) via cGMP-mediated cAMP-dependent PKA signalling. IP3-dependent Ca2+ signalling may enhance local SR Ca2+ release required to activate Ca2+-dependent eNOS/NOi production from subsarcolemmal caveolae sites.

Reduction of Ca2+-transporting systems in memory T cells.

Antigen-specific B and T lymphocytes make up the material grounds of immune memory, their main functional distinction from the so-called "naive" cells is due to the rapid and enhanced response to the antigen-pathogen. An essential distinction between the memory and naive T cells is different sensitivity of these two subpopulations of T lymphocytes to Ca2+-ionophores. Comparative analysis of Ca2+ responses of the immune memory T lymphocytes and naive T cells of mouse CBA/J line to the addition of Ca2+-mobilizing agents concanavalin A, thapsigargin, and ionomycin was carried out. These compounds in concentrations increasing [Ca2+]i in naive cells had no effect on [Ca2+]i in memory cells. Thus, the Ca2+ entrance into memory cells was not activated by exhaustion of intracellular resources. Estimation of intracellular resources of Ca2+, mobilized by ionomycin and thapsigargin in Ca2+ free medium has shown the absence in memory T cells of the intracellular Ca2+ pool, which may be one of factors of their resistance to ionophores. Reduction of the system of Ca2+ influx into memory T cells was shown using the SH-reagent thimerosal. Memory T cells appear to be resistant to "Ca2+ -paradox." Their incubation with 0.5 mM EDTA in the presence or absence of Ca2+ -mobilizing compounds followed by addition of 2 mM CaCl2 did not result in induction of Ca2+ influx into these cells.

Mechanism of action of calcium ionophores on intact cells: ionophore-resistant cells.

Calcium ionophores are generally assumed to directly facilitate the transport of Ca2+ across the plasma membrane. The ability of Ca2+ ionophores ionomycin and A23187 to increase Ca2+ concentration in the cytosol ([Ca2+]i) in different cells was analyzed in detail using fluorescent Ca2+ probes. In fura-2-loaded cells, the dependence of the level of [Ca2+]i on ionomycin and A23187 concentrations had a complex character and could not be explained by ionophoric properties only. The Ca2+ signal induced by the Ca2+ ionophores consisted of three components. The first component was due to the activation of Ca2+ influx through native Ca2+ channels and was sensitive to drugs which inhibited the receptor-operated Ca2+ influx. The second component originated from phospholipase C-dependent mobilization of Ca2+ from intracellular stores. An additional influx of Ca2+ into the cells was activated in this case by a store-regulated mechanism. The third ionophoric component was very small at low concentrations of the ionophores. The effect of the ionophores on Ca2+ influx and Ca2+ mobilization was demonstrated on different cells such as Ehrlich ascites tumour cells, murine peritoneal neutrophils, macrophages, and T-lymphocytes. Thymocytes, neutrophils, and Ehrlich ascites tumour cells were more sensitive to the Ca2+ ionophores. Memory T-cells and brown preadipocytes were ionophore-resistant. The insensitivity to Ca2+ ionophores correlated with the absence of Ca2+ in the intracellular Ca2+ stores and the low activity of plasma membrane store-regulated Ca2+ channels.

Priming effect of calcium ionophores on phorbol ester-induced respiratory burst in mouse peritoneal neutrophils.

The abilities of three calcium ionophores (A23187, 4-bromo-A23187, and ionomycin) to modulate the respiratory burst of neutrophils induced by phorbol ester and to increase the concentration of free intracellular Ca2+ ([Ca2+]i) were compared. The production of reactive oxygen species (ROS) was determined by luminol-dependent chemiluminescence and [Ca2+]i was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 microM) and ionomycin (0.001-0.5 microM) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burst induced by phorbol ester. The integral response (total production of ROS over 6 min) had a bell-shaped dependence on the concentration of ionomycin and A23187 with increase and decrease at low and high concentrations of the ionophores, respectively. The maximal effect was found at 0.5 microM ionomycin and 2 microM A23187, these concentrations resulting in transient increases in [Ca2+]i to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophores had no effect in calcium-free media, though they increased [Ca2+]i to approximately 400 nM through the mobilization of intracellular Ca2+. In cells with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with phorbol ester amplified twofold the production of ROS. The inhibition of phospholipase A2 with 4-bromophenacyl bromide significantly decreased the production of ROS. Thus, the entrance of Ca2+ and generation of arachidonic acid under the influence of phospholipase A2 are necessary for the ionophore-induced priming of production of ROS during cell activation with phorbol esters.

[Effect of structural analogs of platelet activating factor on the intracellular signal transduction in murine peritoneal neutrophils and macrophages of P388D1 line]. / Deistvie strukturnykh analogov factora aktivatsii trombotsitov na peredachu vnutrikletochnykh signalov v peritoneal'nykh neitrofilakh myshi i makrofagakh linii P388D1.

Direct and modulate effects of platelet activating factor (PAF), its structural analogues and ATP on primary and second processes at peritoneal neutrophils and P388D1 cells activation has been studied. The effect of compounds was evaluated on changes in Ca2+ transport and generation of reactive oxygen species. It was shown, that the synthetic analogues of MS series interact with PAF receptor, mobilize Ca2+ from thapsigargin-dependent intracellular stores and inhibit Ca2+ response on PAF in both types of cells. Unlike PAF the analogues do not induce the formations of reactive oxygen species in neutrophils and inhibit the PMA-induced respiratory burst. The activation of pyrinoreceptor of P388D1 cells by exogenous ATP does not inhibit PAF induced Ca2+ rise in cytoplasm, though partly releases Ca2+ from the same store.

The effect of total saponins from Panax Ginseng C.A. Meyer on the intracellular signalling system in Ehrlich ascites tumor cells.

The mechanisms of action of total saponins from Panax Ginseng C.A. Meyer on the elements of intracellular signalling system in Ehrlich ascites tumor cells were studied. The action of total saponins was compared with the effect of ATP, a classical activator of these cells. Saponins at concentrations of 10(-6)-10(-3)% increased [Ca2+]i, mobilized Ca2+ ions from the endoplasmic reticulum (ER) and activated the influx of Ca2+ to cells. Like ATP, saponins activated the Na+/H+ exchange and Ca(2+)-dependent K+ channels. Of all the parameters, only the activation of Ca2+ influx in cell is directly affected by saponins. The changes in other parameters are connected with nonspecific activation of purinoreceptors. The analysis of the kinetic data suggests that, as distinct from ATP-dependent activation of purinoreceptor, saponins first activate the Ca2+ influx to cells and only then induce the mobilization of Ca2+ from ER.

[Mechanism of activation of Ehrlich ascites carcinoma cells using the total fraction of saponins from Korean ginseng]. / Mekhanizm aktivatsii kletok astsitnoi kartsinomy Erlikha obshchei fraktsiei saponinov iz koreiskogo zhen'sheniia.

Molecular mechanisms of action of the total fraction of saponins from Red Korean Ginseng on the elements of the intracellular signalling system of the cells of ascitic Ehrlich carcinoma (AEC) were studied. The action of the total fraction of the saponins on the AEC cells was compared with that of the classic activator of such cells i.e. ATP. It was shown that the action of the total fraction of the saponins was similar to that of ATP. In concentrations of 10(-6) to 10(-3) per cent saponin induced an increase of [Ca2+]j mobilizing Ca2+ from the endoplasmic reticulum (ER) and activating the Ca2+ inlet to the cells. The same as in case with the use of ATP the Ca2+ mobilization from ER was reversible. In comparison to ATP saponin induced higher activation of the Ca2+ inlet to ER and the cells. The same as ATP saponin activated the Na+/H+ exchange and the Ca2+ - dependent K+-channels. Out of all the mention-ed parameters only the activation of the Ca2+ inlet to the cells was probably the direct result of the saponin action. The changes in the other parameters were mediated by nonspecific activation of the purine receptor. The analysis of the kinetic data demonstrated that unlike the ATP-dependent activation of the purine receptor the saponins first of all activated the inlet of Ca2+ to the cells and only after that the mobilization of the latter from ER.