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.     

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.