Intracellular Na+ inhibits voltage-dependent N-type Ca2+ channels by a G protein βγ subunit-dependent mechanism

N-type  voltage-dependent  Ca²⁺ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na⁺ concentration inhibits N-VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba²⁺ current via N-VDCCs was inhibited by Na⁺ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba²⁺ currents were inhibited by Na⁺ influx and enhanced by depletion of Na⁺, after incubation in a Na⁺-free extracellular solution. The Na⁺-induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ-dependent process. Na⁺-induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na⁺ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na⁺-induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na⁺ion, regulated by the Na⁺ concentration via Gβγ.     

Inhibition of T3 Mediated T-Cell Proliferation by Ca2+-Channel Blockers and Inhibitors of Ca2+/Phospholipid-Dependent Kinase

The potential roles of Ca2+ ions in the response of T lymphocytes to stimulation with monoclonal antisera to the T3 antigen were investigated by means of pharmacological agents that predominantly inhibit the flux of Ca2+ ions into cells (verapamil, nifedipine) or the activity of Ca2+-dependent kinases (trifluoperazine, polymyxin B). As assessed by uptake of [3H]thymidine, proliferation induced with anti-T3 +/- recombinant IL-2 at 72 h was inhibited by greater than 80% in the presence of nifedipine at 50 microM, and almost completely arrested (greater than 95% inhibition) with the other agents at the same concentration. Further quantitative assays of the effects of polymyxin B and trifluoperazine on C-kinase labelling of exogenous substrate showed a major reduction with both agents, but inhibition was substantially greater with polymyxin B that with trifluoperazine (IC50 = 14 and 70 microM respectively). These results were confirmed by qualitative assessment of Ca2+/phospholipid-dependent phosphorylation of endogenous substrates, which demonstrated major phosphoproteins of MW 56,000, 52,000, 43,000, and 20,000, and dose-dependent reduction in labelling in the presence of polymyxin B. Similar results were obtained under more physiological conditions in intact cells labelled with 32P orthophosphate. These findings indicate several possible roles for Ca2+ in T-cell activation, and several possible levels of activity, including modulation of calmodulin-dependent kinases and effects on Ca2+/phospholipid-dependent kinases and Ca2+ channels.     

Role of Ca2+ mobilization and Ca2+ sensitization in 8-iso-PGF2α-induced contraction in airway smooth muscle

Background Isoprostanes are prostaglandin (PG)-like compounds synthesized by oxidative stress, not by cyclooxygenase, and increase in bronchoalveolar lavage fluid of patients with asthma. The airway inflammation implicated in this disease may be amplified by oxidants. Although isoprostanes are useful biomarkers for oxidative stress, the action of these agents on airways has not been fully elucidated. Objective This study was designed to determine the intracellular mechanisms underlying the effects of oxidative stress on airway smooth muscle, focused on Ca²⁺ signalling pathways involved in the effect of 8-iso-PGF_2α. Methods Using simultaneous recording of isometric tension and F₃₄₀/F₃₈₀ (an indicator of intracellular concentrations of Ca²⁺, [Ca²⁺]_i), we examined the correlation between tension and [Ca²⁺]_i in response to 8-iso-PGF_2α in the fura-2 loaded tracheal smooth muscle. Results Augmented tension and F₃₄₀/F₃₈₀ by 8-iso-PGF_2α were attenuated by ICI-192605, an antagonist of thromboxane A₂ receptors (TP receptors). Moreover, D609, an antagonist of phosphatidylcholine-specific phospholipase C, markedly reduced both the tension and F₃₄₀/F₃₈₀ induced by 8-iso-PGF_2α, whereas U73122, an antagonist of phosphatidylinositol-specific phospholipase C, modestly inhibited them by 8-iso-PGF_2α. SKF96365, a non-selective antagonist of Ca²⁺ channels, markedly reduced both tension and F₃₄₀/F₃₈₀ by 8-iso-PGF_2α. However, diltiazem and verapamil, voltage-dependent Ca²⁺ channel inhibitors, modestly attenuated tension although their reduction of F₃₄₀/F₃₈₀ was not different from that by SKF96365. Y-27632, an inhibitor of Rho-kinase, significantly attenuated contraction induced by 8-iso-PGF_2α without reducing F₃₄₀/F₃₈₀, whereas GF109203X and Go6983, protein kinase C inhibitors, did not markedly antagonize them although reducing F₃₄₀/F₃₈₀ with a potency similar to Y-27632. Conclusion 8-iso-PGF_2α causes airway smooth muscle contraction via activation of TP receptors. Ca²⁺ mobilization by SKF96365- and D609-sensitive Ca²⁺ influx and Ca²⁺ sensitization by Rho-kinase contribute to the intracellular mechanisms underlying the action of 8-iso-PGF_2α. Rho-kinase may be a therapeutic target for the physiologic abnormalities induced by oxidative stress in airways.     

Amplification of exocytosis by Ca2+-induced Ca2+ release in INS-1 pancreatic β cells

Modulation of spontaneous electrical activities (slow waves, pacemaker potentials and follower potentials) in response to hyperpolarization produced by the ATP-sensitive K⁺ channel openers (KCOs) pinacidil or nicorandil was investigated in smooth muscle tissues of the guinea-pig stomach antrum. With hyperpolarization, the amplitude of slow waves and follower potentials was reduced and that of pacemaker potentials was increased, with a minor modulation of their frequency. The attenuation of slow waves was associated with an inhibition of the 1st component and abolition of the 2nd component. All these actions of KCOs were antagonized by glibenclamide. An increase in the extracellular K⁺ concentration prevented the KCO-induced hyperpolarization with partial restoration of slow waves, suggesting that the inhibition was produced mainly by a decrease in membrane resistance. Exposure of tissues to KCOs for a long period of time (> 20 min) resulted in the reappearance of slow waves displaying both 1st and 2nd components. The 2nd component of the slow wave, which displayed a slower recovery, was inhibited again by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-sensitive K⁺ channels. Noradrenaline hyperpolarized the membrane by activating apamin-sensitive K⁺ channels and increased the amplitude and frequency of slow waves through activation of α₁-adrenoceptors, actions different from those of KCOs. Thus, inhibition of slow waves by KCOs may be primarily related to the decrease in amplitude of a passive electrotonic component, possibly due to a reduction of the input resistance. The hyperpolarization shifted the threshold potential for generation of the 2nd component of slow waves to negative levels, presumably due to modulation of mitochondrial functions.     

Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.