Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII.

K+-dependent Na+/Ca2+-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K+-dependent Na+/Ca2+-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca2+ signal dynamics in olfactory and other neurons. Synaptic Ca2+ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca2+ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca2+ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca2+ signalling by activating a signal damping and termination pathway.

[The effect of Ca(2+)-induced opening of cyclosporine-sensitive pore on the oxygen consumption and functional state of rat liver mitochondria].

The effect of Ca(2+)-induced opening of cyclosporine-sensitive pore (mitochondrial permeability transition pore, MPTP) on the oxygen consumption and mitochondrial functional state was studied in the rat liver mitochondria. It was shown that, with the use of glutamate as oxidation substrate, in the absence of depolarization MPTP opening results in the increase of steady state respiration rate because of the activation of cyclosporine-sensitive Ca2+/H(+)-exchange and Ca2+ cycling, which was supported by the simultaneous work of MPTP and Ca(2+)-uniporter. With the aid of selective blockers, cyclosporine A and ruthenium red, it was shown that MPTP and Ca(2+)-uniporter contribute equally to the Ca(2+)-cycling and mitochondrial respiration. It was shown that bioenergetic effects of MPTP opening under steady state conditions (increase in the oxygen consumption rate under substrate oxidation without ADP, decrease in respiratory control ratio as well as the effectiveness of ATP synthesis, P/O) are close to the functional alterations, which result from the increase of endogenous proton conductance of mitochondrial membrane. Uncoupling effect of MPTP opening, by itself, had no effect on phosphorylation rate, which remains relatively stable because the fall of P/O is compensated by the activation of respiratory chain and the increase in the rate of state 3 respiration. It was concluded that under physiologically normal conditions MPTP might function as the endogenous mechanism of mild uncoupling of respiratory chain.

Involvement of AE3 isoform of Na(+)-independent Cl(-)/HCO(3)(-) exchanger in myocardial pH(i) recovery from intracellular alkalization.

Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).

Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis.

Magnesium homeostasis in HL-60 promyelocytic leukemia cells was compared to that in neutrophyl-like HL-60 cells obtained by 1.3% DMSO treatment. Magnesium homeostasis was studied by the characterization of magnesium efflux, the identification of intracellular magnesium pools, and the regulation of intracellular ionized Mg2+. In both undifferentiated and neutrophyl-like HL-60 cells, magnesium efflux occurred via the Na-Mg antiporter which was inhibited by imipramine and stimulated by db cAMP and forskolin. Receptor-mediated signals such as ATP, IFN-alpha, or PGE1, which can trigger cAMP-dependent magnesium efflux, were ineffective in undifferentiated HL-60 cells but induced 60-70% increase of magnesium efflux in neutrophyl-like HL-60 cells. Selective membrane permeabilization by the cation ionophore A23187 induced a large magnesium release when cells were treated with rotenone. In both cell populations, the addition of glucose to rotenone-treated cells restored magnesium release to the control level. Permeabilization by 0.005% digitonin provoked the release of 90% cell total magnesium in both cell types. Intracellular [Mg2+]i was 0.15 and 0.26 mM in undifferentiated and neutrophyl-like HL-60 cells, respectively. Stimuli that triggered magnesium efflux, such as db cAMP in undifferentiated and IFN-alpha in neutrophyl-like HL-60 cells, induced a slow but consistent increase of [Mg2+]i which was independent from Ca2+ movements. Overall, these data indicate that magnesium homeostasis is regulated by receptor-mediated magnesium efflux which was modified during differentiation of HL-60 cells. Stimulation of magnesium efflux is paralleled by an increase of [Mg2+]i which reflects a release of magnesium from the bound cation pool.

Induction of alkalinization in cultured renal cells (MDCK line) by prostaglandin E2.

The effect of prostaglandin E2 (PGE2), on the intracellular pH (pHi) in BCECF-loaded Madin Darby Canine Kidney (MDCK) cells was investigated. PGE2 elevated the pHi. Under resting conditions, pHi of MDCK cells suspended in PBS at pH 7.4 was 7.11 +/- 0.08; PGE2 increased pHi with an EC50 of 0.16 microM. PGF2 alpha elicited a similar response to PGE2, with an EC50 of 0.24 microM. Amiloride (0.4 mM) reversed the response to PGE2 (control 7.18 +/- 0.05; PGE2 7.26 +/- 0.05; after amiloride 7.18 +/- 0.05). In MDCK cells exposed to a Na(+)-free solution, alkalinization induced by this eicosanoid was blocked (Ringer-choline 7.16 +/- 0.03; PGE2 7.16 +/- 0.02). PGE2 increased by 100% the rate of recovery after an acidification pulse with ammonium chloride. In the presence of Ringer-HCO3- (pH 7.4), there was a delay in the maximal response to this prostaglandin (PBS 2.2 +/- 0.27, Ringer-bicarbonate 3.4 +/- 0.55 min) and the pHi increment was less marked than in PBS (0.09 pH units in HCO3- versus 0.16 pH units in PBS; P < 0.001). This effect of PGE2 was not blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1.0 mM). PMA (100 nM), activator of protein kinase C, mimicked the response to PGE2, suggesting the participation of this kinase on the effect of the prostanoid. As expected, two inhibitors of protein kinase C, staurosporine and sphingosine, abolished the response to PGE2. Staurosporine (0.10 microM), an inhibitor of protein kinase C, blocked the response to PGE2 (control 7.02 +/- 0.04; PGE2 and staurosporine 7.03 +/- 0.04, n = 9, not significant). Sphingosine, another inhibitor of protein kinase C, also blocked the response to PGE2. Two analogues of cAMP did not modify the pHi. In summary, PGE2 induced an intracellular alkalinization via stimulation of a Na+/H+ exchanger, with the participation of protein kinase C, in MDCK cells.