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Pulmonary arterial hypertension (PAH) is a syndrome caused by restricted blood flow in the pulmonary circulation, which results in a poor patient prognosis. The serotonin (5-HT), TRPC1 (Transient receptor potencial channel 1), TRPC6 (Transient receptor potencial channel 6), calcineurin A, and NFATc3 (an isoform of nuclear factor of activated T-cells family) are involved in cell proliferation and hypertrophy and the crosstalk between these molecules may play an essential role in the pathogenesis of pulmonary arterial hypertension. We hypothesized that 5-HT promotes PAH by affecting TRPC channels. We investigated the effects of sarpogrelate, a 5-HT 2A receptor antagonist, on pulmonary arterial pressure, cardiac remodeling, pulmonary artery remodeling, and TRPC1, TRPC6, calcineurin A, and NFATc3 expression in pulmonary arteries from rats with PAH. The results showed that sarpogrelate reduced pulmonary arterial pressure, cardiac remodeling, pulmonary artery remodeling, and expression of TRPC1, TRPC6, calcineurin A, and NFATc3 in pulmonary arteries. In conclusion, Sarpogrelate reduced the severity of PAH in rat model and decreased the expression of TRPC1, TRPC6, calcineurin A, and NFATc3 in pulmonary arteries.

Pancreatic β-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca 2+ entry. To elucidate how mitochondrial activation in β-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca 2+ ] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca 2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca 2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca 2+ signals. We propose that the combined effects of Ca 2+ and redox reactions can trigger insulin secretion by these two mechanisms., Competing Interests: Competing interestsThe authors are employees of Nestlé Institute of Health Sciences, which is part of the Nestlé Group., (© 2017. Published by The Company of Biologists Ltd.)

Ethylenediaminetetraacetic acid, ethylenediamine-N,N'-disuccinic acid and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid are polyaminocarboxylic acids that are able to sequester metal ions. Calcium is implicated in maintenance of intercellular matrix, zonula occludens (tight junctions) and zonula adherens of epithelium and endothelium cells. Corneal epithelium is impervious to many aqueous formulations due to it being lipophilic, whereby transcellular drug transit is resisted, whilst tight junctions restrict access via the paracellular route. Research has shown that integrity of tight junctions breaks down through loss of Ca(2+) for endothelial and epithelial cells. This study investigates different Ca(2+) sequestering compounds and their effect on corneal permeability of riboflavin at physiological pH. Riboflavin is a topically administered ocular drug applied during UV-induced corneal cross-linking for the treatment of keratoconus., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles.

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulation.

Although serotonin (5-HT) induced proliferation of vascular smooth muscle cells is considered to involve changes in intracellular Ca2+ ([Ca2+]i), the mechanism of Ca2+ mobilization by 5-HT is not well defined. In this study, we examined the effect of 5-HT on rat aortic smooth muscle cells (RASMCs) by Fura-2 microfluorometry for [Ca2+]i measurements. 5-HT was observed to increase the [Ca2+]i in a concentration- and time-dependent manner. This action of 5-HT was dependent upon the extracellular concentration of Ca2+ ([Ca2+]e) and was inhibited by both Ca2+ channel antagonists (verapamil and diltiazem) and inhibitors of sarcoplasmic reticular Ca2+ pumps (thapsigargin and cyclopia zonic acid). The 5-HT-induced increase in [Ca2+]i was blocked by sarpogrelate, a 5-HT2A-receptor antagonist, but not by different agents known to block other receptor sites. 5-HT-receptor antagonists such as ketanserin, cinanserin, and mianserin, unlike methysergide, were also found to inhibit the 5-HT-induced Ca2+ mobilization, but these agents were less effective in comparison to sarpogrelate. On the other hand, the increase in [Ca2+]i in RASMCs by ATP, angiotensin II, endothelin-1, or phorbol ester was not affected by sarpogrelate. These results indicate that Ca2+ mobilization in RASMCs by 5-HT is mediated through the activation of 5-HT2A receptors and support the view that the 5-HT-induced increase in [Ca2+]i involves both the extracellular and intracellular sources of Ca2+.

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca2+ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca2+ release from mitochondria when respiration is supported either by the NAD(+)-linked substrate beta-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca2+ much better with succinate than with beta-hydroxybutyrate as respiratory substrate. When Ca2+ release is induced by t-butylhydroperoxide succinate-supported Ca2+ retention is impeded by rotenone. Ca2+ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca2+ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca2+ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.

In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD). Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(1,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.

Rotenone-poisoned rat liver mitochondria energized by succinate addition, after a 5-min period of preincubation in presence of 10 microM Ca2+, produce H2O2 at much faster rates, undergo extensive swelling, and are not able to retain the membrane potential and accumulated Ca2+. Similar results were obtained when a suspension of rat liver mitochondria preincubated in anaerobic medium for 5 min was reoxygenated. The addition of either ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ruthenium red, catalase, or dithiothreitol, just before succinate or O2 addition, prevented mitochondrial swelling, indicating the involvement of Ca2+, reactive oxygen species, and oxidation of membrane protein thiols in this process of membrane permeabilization. Inhibition of mitochondrial swelling by cyclosporin A suggests that the membrane alterations observed under these experimental conditions are related to opening of the permeability transition pore. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which prevents Ca2+ cycling across the membrane, did not inhibit mitochondrial swelling when Ca2+ influx into the mitochondrial matrix was driven by a high Ca2+ gradient. When rotenone plus antimycin A-poisoned mitochondria were energized by N,N,N',N'-tetramethyl-p-phenylenediamine, which reduces respiratory chain complex IV, mitochondrial swelling did not occur, unless succinate, which reduces coenzyme Q, was also added. It is concluded that reduced coenzyme Q is the electron source for oxygen radical production during Ca(2+)-stimulated oxidative damage of mitochondria.

Mitochondria from bovine frozen semen are capable of preserving oxidative energy. The purpose of this work was to study calcium uptake by the inner mitochondrial membrane which, together with the plasmatic membrane, is involved in spermatic motility and viability regulation. Mitochondrial coupling was confirmed. In the active state, there was a drop in oxygen uptake rate using calcium concentrations ranging from 20 to 100 microM. In the resting state, 75 microM calcium concentration mimicked the active respiration state. In both states, concentrations greater than 100 microM induced mitochondrial uncoupling. Maximal mitochondrial uptake was recorded in the active state with 10 microM lactate. At calcium concentrations greater than 100 microM, there was a drop in mitochondrial calcium uptake for both respiration states. The ability to preserve oxidative energy allows the spermatozoon to take up mitochondrial calcium, thus regulating its functional activity.

The role of coenzyme Q on the process of mitochondrial membrane damage associated with oxidative stress was studied in a suspension of uncoupled mitochondria exposed to Ca2+ in the presence of Fe(II)citrate or t-butyl hydroperoxide. Reduction of coenzyme Q by succinate was shown to inhibit both inner membrane lipid peroxidation and permeabilization induced by Fe(II)citrate. In contrast, the inner membrane permeabilization induced by Ca2+ alone or Ca2+ plus t-butyl hydroperoxide was potentiated by the presence of succinate. These results support our previous proposition that the mitochondrial damage associated with oxidative stress generated by these pro-oxidants in the presence of Ca2+ is mediated by different mechanisms.

The effect of Ca2+ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (delta psi) and alkalinization of the medium. This typical behaviour is drastically changed if Ca2+ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of delta psi, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 microM Ca2+ and a preincubation for 2 min with 0.4-1.0 microM Ca2+. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca2+ concentrations as low as 0.3 microM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca2+] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyz A. Evtodienko YuV. Duszyński J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148-1154; Czyz A. Teplova VV. Sabala P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539-544], the present results suggest that cytoplasmic Ca2+ plays a crucial role in the Crabtree effect.

Fuel metabolism generates multiple signals that interact to stimulate insulin secretion. These studies explored the mechanism by which fuels activate phosphoinositide (PI) hydrolysis and the role of this signal transduction pathway in fuel-stimulated insulin secretion. High potassium (30 mM), which depolarizes the membrane and increases Ca2+ influx, caused only a transient monophasic release of insulin. In contrast, glucose (20 mM) or monomethylsuccinate (MMSucc; 10 mM) markedly stimulated a sustained insulin secretory response, indicating that fuel metabolism generates a signal(s) in addition to Ca2+ influx that is required for a sustained secretory response. On the other hand, diazoxide, an ATP-sensitive K+ channel activator that prevents membrane depolarization and Ca2+ influx in response to fuel metabolism, reduced the secretory responses to glucose and MMSucc to baseline levels, demonstrating that Ca2+ influx was essential to fuel-stimulated insulin secretion. The further addition of high K+ bypassed the diazoxide block and restored insulin secretory rates. The insulin secretory response to glucose or MMSucc in the presence of diazoxide and K+ was inhibited by the Ca2+ channel antagonist nitrendipine and the protein kinase-C inhibitor staurosporine. Changes in PI hydrolysis paralleled those in insulin secretion. High potassium alone induced only a modest 2.5-fold increase in inositol phosphate accumulation. This response was significantly less than that to glucose or MMSucc, which increased inositol phosphate accumulation by 6.8- or 5.2-fold, respectively. Like its effect on secretion, diazoxide markedly reduced glucose- or MMSucc-stimulated PI hydrolysis, and this inhibition was reversed with high K+. In contrast, diazoxide had no effect on receptor-activated PI hydrolysis stimulated by 100 nM cholecystokinin (CCK), and the effects of CCK were not dependent on added fuel, indicating that fuel and CCK activate PI hydrolysis by distinct pathways. These findings demonstrate that mitochondrial metabolism of glucose or MMSucc generates a signal(s) that interacts with Ca2+ influx to stimulate PI hydrolysis and sustained insulin secretion. This pathway of fuel-activated PI hydrolysis is distinct from that of CCK receptor-activated PI hydrolysis. These studies suggest that fuel-activated PI hydrolysis plays an important role in fuel-stimulated insulin secretion.

The effects of the new 5-HT2A receptor antagonist sarpogrelate on the cellular action of serotonin were examined in cultured rat mesangial cells by measuring cytosolic free calcium concentration ([Ca2+]i). Sarpogrelate inhibited serotonin-induced increases in [Ca2+]i in a concentration-dependent manner. M1, a major metabolite of sarpogrelate, also exhibited an inhibitory effect exceeding that of sarpogrelate. The inhibitory effects of sarpogrelate and M1 were abolished by washing out these compounds. In contrast, the increase in [Ca2+]i induced by angiotensin II or arginine vasopressin was not affected by pretreatment of the cells with sarpogrelate or M1. These results suggest that sarpogrelate and its major metabolite (M1) act as reversible and specific 5-HT2A receptor antagonists against the contractile action of platelet-derived serotonin in mesangial cells.

In previous work, calcium ions, bound at the lumenal side of the CF0H+ channel, were suggested to keep a H+ flux gating site closed, favoring sequestered domain H+ ions flowing directly into the CF0-CF1 and driving ATP formation by a localized delta approximately mu H+ gradient. Treatments expected to displace Ca++ from binding sites had the effect of allowing H+ ions in the sequestered domains to equilibrate with the lumen, and energy coupling showed delocalized characteristics. The existence of such a gating function implies that a closed-gate configuration would block lumenal H+ ions from entering the CF0-CF1 complex. In this work that prediction was tested using as an assay the dark, acid-base jump ATP formation phenomenon driven by H+ ions derived from succinic acid loaded into the lumen. Chlorpromazine, a photoaffinity probe for many proteins having high-affinity Ca(++)-binding sites, covalently binds to the 8-kDa CF0 subunit in the largest amounts when there is sufficient Ca++ to favor the localized energy coupling mode, i.e., the "gate closed" configuration. Photoaffinity-bound chlorpromazine blocked 50% or more of the succinate-dependent acid-base jump ATP formation, provided that the ionic conditions during the UV photoaffinity treatment were those which favor a localized energy coupling pattern and a higher level of chlorpromazine labeling of the 8-kDa CF0 subunit. Thylakoids held under conditions favoring a delocalized energy coupling mode and less chlorpromazine labeling of the CF0 subunit did not show any inhibition of acid-base jump ATP formation. Chlorpromazine and calmidazolium, another Ca(++)-binding site probe, were also shown to block redox-derived H+ initially released into sequestered domains from entering the lumen, at low levels of domain H+ accumulation, but not at higher H+ uptake levels; ie., the closed gate state can be overcome by sufficiently acidic conditions. That is consistent with the observation that the inhibition of lumenal succinate-dependent ATP formation by photoaffinity-attached chlorpromazine can be reversed by lowering the pH of the acid stage from 5.5 to 4.5. The evidence is consistent with the concept that Ca++ bound at the lumenal side of the CF0 H+ channel can block H+ flux from either direction, consistent with the existence of a molecular structure in the CF0 complex having the properties of a gate for H+ flux across the inner boundary of the CF0. Such a gate could control the expression of localized or delocalized delta approximately mu H+ energy coupling gradients.

Isolated rat liver mitochondria undergo extensive swelling and disruption of membrane potential when they accumulate Ca2+ in the presence of a prooxidant such as diamide or t-butylhydroperoxide. The phenothiazinic drug trifluoperazine, at concentrations (15-35 microM) which do not inhibit respiration or the influx of Ca2+ into mitochondria, significantly protected mitochondria against the deleterious effects of Ca2+ plus a prooxidant. In contrast, at concentrations higher than 100 microM the drug potentiated these deleterious effects of Ca2+ and prooxidants and had a damaging effect per se on the inner mitochondrial membrane. It is proposed that the protection conferred by the drug is mediated by changes in membrane protein structure that decrease the production of protein thiol cross-linkings which occur when mitochondria accumulate calcium under oxidant stress conditions.

In this paper the authors studied the effects of thyroid hormones and their structural analogues on the mitochondrial calcium transport activities. The thyroid hormones, 3,5,3' L-triiodothyronine (LT3) and 3,5,3'5' L-tetraiodothyronine (LT4) at physiological intracellular concentrations between 7.2 and 9 nM, decouple total Ca++ transport, as well as inhibit the passive transport of Ca++, either due to oxidation of pyruvate, malate or succinate or after inhibition with rotenone. The optical isomers 3,5,3' D-triiodothyronine (DT3) and 3,5,3',5' D-tetraiodothyronine (DT4) are less effective at all the used concentrations. Furthermore the structural analogues 3,3',5' L-triiodothyronine (LrT3), 3,5-dicloro, 3',5' L-diiodothyronine (LDiClT2) and 3,5 L-diiodothyronine (LT2) furnished even less effects on the same activities. The effect of the thyroid hormones and of their structural analogues has revealed that the mitochondrial calcium transport may be influenced both by a stereospecific interaction between hormones and protein ligands and by a lipophilic chaotropic action on the mitochondrial membranes lipids. In this context it is interesting to consider that both thyroid hormones and Ca++ transport activity are interacting with the energetic metabolism by means of phosphorylation and substrate oxidation mechanism.

Calcium uptake into filipin-treated bovine spermatozoa is completely inhibited by the uncoupler CCCP or by ruthenium red. Both Pi and mitochondrial substrates are required to obtain the maximal rate of calcium uptake into the sperm mitochondria. Bicarbonate and other anions such as lactate, acetate or beta-hydroxybutyrate do not support a high rate of calcium uptake. There are significant differences among various mitochondrial substrates in supporting calcium uptake. The best substrates are durohydroquinone, alpha-glycerophosphate and lactate. Pyruvate is a relatively poor substrate, and its rate can be greatly enhanced by malate or succinate but not by oxalacetate or lactate. This stimulation is blocked by the dicarboxylate translocase inhibitor, butylmalonate and can be mimiced by the non-metabolized substrate D-malate. The Ka for pyruvate was found to be 17 microM and 67 microM in the presence and absence of L-malate, respectively. The Ka for L-malate is 0.12 mM. It is suggested that in addition to the known pyruvate/lactate translocase there is a second translocase for pyruvate which is malate/succinate-dependent and does not transport lactate. In the presence of succinate, glutamate stimulates calcium uptake 3-fold, and this effect is not inhibited by rotenone. In the presence of glutamate plus malate or oxalacetate there is only an additive effect. It is suggested that glutamate stimulates succinate transport and/or oxidation in bovine sperm mitochondria. The alpha-hydroxybutyrate is almost as good as lactate in supporting calcium uptake. Since the alpha-keto product is not further metabolized in the citric acid cycle, it is suggested that lactate can supply the mitochondrial needs for NADH from its oxidation to pyruvate by the sperm lactate dehydrogenase x. Thus, when there is sufficient lactate in the sperm mitochondria, pyruvate need not be further metabolized in the citric acid cycle in order to supply more NADH.

The mechanism by which palmitoyl-CoA inhibits Ca2+ uptake in liver and heart mitochondria was examined. At a given concentration of palmitoyl-CoA, the extent of inhibition is inversely related to the concentration of the respiratory substrate succinate. Palmitoyl-CoA inhibition of uncoupler-stimulated respiration and respiration stimulated by ionophore-A23187-induced Ca2+ cycling is also relieved by high succinate concentrations. These effects of palmitoyl-CoA and succinate concentration are distinct from the increase in inner-membrane permeability, which can be produced by palmitoyl-CoA and Ca2+ [Beatrice, Palmer & Pfeiffer (1980) J. Biol. Chem. 255, 8663-8671]. The apparent K0.5 of the mitochondrial Ca2+ pump is not altered by palmitoyl-CoA. No or negligible effects of palmitoyl-CoA on the Ca2+-uptake rate are observed when ascorbate replaces succinate as an energy source. These findings, together with the known activity of palmitoyl-CoA as a competitive inhibitor of the dicarboxylate carrier [Morel, Lauquin, Lunardi, Duszynski & Vignais (1974) FEBS Lett. 39, 133-138], indicate that palmitoyl-CoA inhibits energy-linked Ca2+ transport by limiting the rate of electron transport through limitation of succinate entry into the mitochondria rather than by directly inhibiting the Ca2+ carrier.

In a study of the Ca2+ kinetics of mitochondria of chick epiphyseal chondrocytes, the rate of Ca2+ uptake was linear up to a medium Ca2+ concentration of 30 mum. The half maximal transport rate occurred at 34 mum Ca2+. The Ca2+ uptake rate, expressed as a function of time, was 35 nmoles/mg protein/min; the presence of Mg2+ had little effect on Ca2+ accumulation. While these kinetic parameters did not differ significantly from mitochondria of cells of nonmineralizing tissues, the respiratory characteristics of the chondrocyte organelles exhibited functional differences. Thus, up to 350 nmoles Ca2+/mg protein, chondrocyte mitochondria performed coupled oxidative phosphorylation. Calcium uptake was energy supported, while Ca2+ binding was low. Addition of respiratory inhibitors and uncouplers to these mitochondria resulted in a rapid loss of more than 80% of the total Ca2+. The Ca/Pi ratio of the extrudate was very similar to the ratio of the ions in cartilage septum fluid. In the most mineralized zones of the epiphyseal plate, there was little change in the state 4 respiratory rate, but nonspecific Ca2+ binding was elevated and a high percentage of the total Ca2+ was in a nonextrudable form. The results indicate that in cells preparing for mineralization, much of the total mitochondrial Ca2+ is in a form that can be transported to the calcification front. In cells close to the calcification front, nonextrudable Ca2+ may form calcium phosphate granules described by other investigators.

The addition of chlortetracycline (CTC) to succinate-energized rat liver mitochondria resulted in depolarization of the membrane potential and decreased respiratory control. CTC inhibited both processes at concentrations that were half maximally effective at approximately 13 and 16 microM, respectively. These inhibitory effects were prevented by either the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or the inhibitor of mitochondrial Ca2+ influx, ruthenium red. These findings are consistent with the formation of a membrane associated calcium-CTC complex and suggest that CTC can alter mitochondrial energy metabolism during transmembrane Ca2+ cycling.

It has been found that addition of iron(III)-gluconate complex to rat liver mitochondria disturbed the mitochondrial Ca2+ transport. Indirect evidence when the changes in the membrane potential during the transport of Ca2+ were followed, as well as direct evidence, when the fluxes of Ca2+ were monitored by a Ca2+-selective electrode, indicated that this iron complex induced an efflux of Ca2+ from liver mitochondria. The mechanisms by which iron induced Ca2+ release appeared to be linked to the induction of lipoperoxidation of mitochondrial membrane. The mitochondrial membrane, however, did not become irreversibly damaged under these conditions, as indicated by its complete repolarization. It was also shown that the induction by iron of lipoperoxidation brought about an efflux of K+ from mitochondria.

Cholesterol side-chain-cleavage enzyme activity in mitochondria isolated from heavily luteinized rat ovaries was determined using isocitrate as an electron donor and [4-14C]cholesterol to start the reaction. The activity of the enzyme was reduced when more than 10 microM calcium was added to the mitochondrial preparations. When 100 microM EGTA or 2 mM ATP was added to the reaction an increase in enzyme activity was observed and ATP was able to partially overcome the calcium-induced inhibition.

The association of Mg2+ ions with mitochondria isolated from guinea pig cerebral cortex is investigated and resolved into two components, that bound to the surface of both the outer and the inner membranes and that transported into the mitochondrial matrix. When rotenone-treated mitochondria are preincubated in a Mg2+-containing medium, Mg2+ binding can be measured and actual Mg2+ transport determined after the addition of succinate. Mg2+ uptake as well as retention within mitochondria is an energy-dependent process linked to substrate oxidation. EGTA completely prevents Mg2+ uptake, while the Ca2+ uniporter inhibitor Ruthenium Red, along with prevention of Mg2+ uptake, induces a slow efflux of accumulated Mg2+ ions. These findings suggest that both inward and outward Mg2+ movements follow Ca2+ fluxes across the mitochondrial membrane. Modulation of Mg2+ movements by mitochondria is therefore suggested to occur within nerve terminals.

The use of a fast-responding Ca2+-selective electrode has allowed the simultaneous measurement of the true Ca2+-activity in the medium and of the Ca2+-transport rate, eliminating the uncertainties connected with the use of indirect methods of estimating Ca2+ transport rates (e.g. O2-stimulation, K+-efflux). The results on the kinetics of Ca2+ uptake by rat liver mitochondria obtained in this study are qualitatively similar to those obtained by Bragadin et al. [Bragadin, M., Pozzan, T. & Azzone, G. F. (1979) Biochemistry, 18, 5973--5978] by monitoring the K+-efflux from mitochondria. In the valinomycin-driven Ca2+ uptake, the Ca2+ translocase is the limiting factor, whereas in the system driven by succinate extraneous limiting factors play a role. Thus, liver mitochondria, when studied under appropriate non-limiting conditions, exhibit a hyperbolic dependence on the Ca2+ uptake rate on Ca2+-activity analogous to that of mitochondria from other tissues. The sigmoidal dependence of the uptake rate on Ca2+-activity in liver mitochondria, observed under conditions which produce hyperbolic kinetics in mitochondria from other tissues, can thus be attributed to factors which are extraneous to the Ca2+ transport system per se.

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.

The ability of mouse mastocytoma cells to take up 45Ca2+ was measured in normal growth medium. As previously observed in physiological buffers with succinate and Pi, cells grown for 18h with N6,O2'-dibutyryladenosine 3',5' cyclic monophosphate (DB cyclic AMP) to inhibit growth took up more 45Ca2+ than untreated cells. However 45Ca2+ uptake by cells in growth medium was less sensitive to respiratory inhibitors or uncouplers than 45Ca2+ uptake in physiological buffer. Increased 45Ca2+ uptake by 18h cyclic nucleotide-treated cells was not a result of tighter mitochondrial coupling since mitochondria prepared from cyclic nucleotide-treated cells were less coupled than those from untreated cells. Nevertheless studies with uncouplers suggested that the bulk of the intracellular Ca2+ was associated with mitochondria. DB cyclic AMP-treated cells contained less total Ca2+ than untreated cells indicating that net Ca2+ efflux occurred during the 18h period of drug treatment. These observations suggest that Ca2+ fluxes increase in DB cyclic AMP-treated PY815 cells and that a net efflux of Ca2+ occurs during growth inhibition by the cyclic nucleotide.

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252-257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.

Calcium uptake was studied on thymocytes from rabbits with 45Ca loading followed by rapid filtration. Datta shown that the substrates of the mitochondrial electron transport chain did not allowed calcium uptake. Exogenous ATP was required to observe a large influx of calcium in thymocytes. Succinate, magnesium and phosphate ions increased this ATP induced influx of calcium, which was not completely inhibited with atractyloside or ruthenium red.

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.

1. Axoplasm isolated from giant axons of the squid Loligo and of the polychaete worm Myxicola continues to bind Ca and maintain an ionized Ca concentration close to 0.1 microgram which is similar to that seen in intact axons. 2. Injection of Ca into isolated axoplasm only produces a transient rise in ionized Ca showing that axoplasm can buffer a Ca challenge. 3. In order to characterize the Ca-binding systems isolated axoplasm was placed in small dialysis tubes and exposed to a variety of artificial axoplasms containing 45Ca. 4. In the presence of ATP, orthophosphate and succinate, Ca uptake appreciable and after 4 hr exposure of Loligo axoplasm to 0.1 microgram-Ca, approximately 100 mumole Ca/kg axoplasm was bound. Binding could be divided operationally into two distinct processes, one that requires ATP or succinate togeth with orthophosphate and is blocked by cyanide and oligomyocin, and one that is unaffected by these reagents. 5. Energy-dependent binding has a large capacity, but a rather low affinity for Ca, being half-maximal between 20 and 60 microgram-Ca. In Loligo, its properties closely parallel those of a crude mitochondrial preparation isolated from axoplasm; but there are some interesting differences in Myxicola. Energy-independent binding is half-maximal at ionized Ca concentrations between 80 and 160 nM but is readily saturated and has a capacity of 6-60 mumole/kg axoplasm. 6. Ca binding by Loligo is greatest in media containing roughly physiological concentrations of K and is reduced by isosmotic replacement of K by Na. This effect seems to be confined to the energy-dependent, presumed mitochondrial, component of binding. 7. Ca binding by Loligo axoplasm is reduced by both La and Mn ions.

The association between reported intestinal fluid secretory activity of bile acids and Ca2+ ionophore properties was investigated in a pig jejunal brush border vesicle system. Secretory and nonsecretory bile acids and hydroxy-fatty acids were tested to see if Ca2+ ionophore activity is a general property of all bile acids and hydroxy-fatty acids, or if it is confined to compounds with recognized fluid secretory activity. Ionophore activity was attributed to compounds which could increase rates of Ca2+ influx and efflux from brush border vesicles under conditions where nonspecific permeability to sorbitol was not affected. The recognized secretory agents chenodeoxycholate and dioctyl sulfosuccinate had Ca2+ ionophore activity in the test system. The nonsecretory agents cholate, hyodeoxycholate and 4-hydroxybutyrate had no detectable activity, while ursodeoxycholate showed minor ionophore activity. Due to complications resulting from Ca2+ sequestration it was impossible to determine the Ca2+ ionophore activity of 12-hydroxystearate in this system. The detergent properties of all these agents are known to increase intestinal permeability, but detergent strength, as measured by concentration required to increase mannitol exchange across vesicle membranes, did not correlate well with secretory activity. We conclude that intestinal fluid secretion caused by bile acids and hydroxy-fatty acids could be controlled partially by Ca2+ ion interactions which could include intracellular signal effects of Ca2+ on anion permeability of the brush border membrane as well as possible increases in permeability of the tight junctions, and local hypertensive effects.

The calcium-complexing abilities of acetate, bicarbonate, lactate, succinate, and citrate were quantified, and were found to differ substantially. Acetate was a more potent vasodilator than lactate, and the vasorelaxant potencies of acetate and lactate failed to correlate with their calcium-complexing ability. The vasodilator action of acetate was not abolished after correction for complexing of ionized calcium. Strips constricted with vasopressin were especially sensitive to the vasodilator effects of acetate.

The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenal glands. In the presence of succinate, addition of calcium chloride in the concentration range 20-150 muM caused an inhibition of pregnenolone formation in both decapsulated and capsular adrenal mitochondria. 11beta-hydroxylation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP+. Under these conditions, calcium chloride in the concentration range 120-875 muM caused a 2-4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone. The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium by adrenal mitochondria can stimulate pregnenolone formation by increasing the interaction of mitochondrial cytochrome P-450 with endogenous substrate.

Sodium-dependent citrate uptake (v) by rabbit renal brush border vesicles was studied as a function of pH and Ca2+, citrate, and succinate concentrations to evaluate the hypothesis that factors which alter renal citrate reabsorption also alter v. At pH 8 a 10-fold increment in citrate concentration (up to 3.3 mM) was required to achieve the same v observed at pH 7. This provides proof that the protonated citrates are the effective substrates for v. At higher citrate concentrations (5-10 mM) and pH 8, substrate inhibition of v occurred. Studies of the substrate concentration dependency of v at various pH levels and calcium concentrations indicated that v was competitively inhibited by citrate3-. Changes in calcium concentrations altered v by altering the protonated citrate and citrate3- concentrations; v showed countertransport with succinate. Sodium-dependent succinate uptake (vs) was not pH dependent but was more inhibited by 1 mM citrate at pH 7 than at pH 8, indicating that the protonated citrates are inhibitors of vs. However, citrate3- was also found to inhibit vs. Thus, changes in v at the brush border membrane due to the effects of pH or calcium concentration on the protonated citrate concentration and to competition between polycarboxylates may be relevant to changes in renal citrate reabsorption in vivo.

Microliter samples of cytoplasm containing mitochondria were aspirated from giant axons of the marine annelid Myxicola infundibulum into polyethylene tubes. The small molecular constituents within these cytoplasmic samples were controlled by a dialysis capillary with a 6,000 molecular weight cut off. The negative log of the calcium ion activity (pCa) (6.72 +/- 0.03, n = 40) and, in some cases, the pH (7.51 +/- 0.01, n = 7) of the samples were monitored with ion-sensitive microelectrodes. Adding 5 mM succinate or 5 mM ATP at pH 7.5 caused the Ca activity in the cytoplasm to drop from an experimentally elevated value of approximately 10 microM to below 1 microM. This decrease could be inhibited with ruthenium red, suggesting a mitochondrial mechanism. Ca uptake, following the addition of either succinate or ATP, was reversibly slowed when the cytoplasmic pH was elevated to approximately 8.3. When ruthenium red was added after mitochondria had taken up Ca, the Ca activity in the extramitochondrial cytoplasm gradually increased suggesting ongoing release of Ca from storage sites. Increasing the cytoplasmic pH to approximately 8.5 in the presence of ruthenium red did not increase the ongoing release over that found with ruthenium red alone. The apparent washout of Ca from the energy-independent, nonmitochondrial Ca buffers was only slightly affected by pH (pH 7.5-8.5). It is concluded that elevating intracellular pH to 8.3 slows the Ca uptake by mitochondria. Thus cytoplasmic pH may have a function in regulating mitochondrial Ca metabolism and/or extramitochondrial calcium activity.

The conditions of potassium transport were investigated in isolated rabbit hear mitochondria. In the presence of 5 to 40 mM potassium chloride there is a rapid initial phase of potassium influx of less than 2 min duration in which 65-75% of the total K+ uptake occurs. This is followed by a slow phase of K+ uptake of about 45 min during which 25 to 35% of the total potassium was taken up. While the rapid phase is due to passive diffusion, the slow phase is an active process which requires either ATP or respiratory substrates as an energy source. It can be totally abolished by 0.5 mM antimycin A. An increase of the calcium concentration in the incubation medium to more than 5 X 10(-5)M resulted in an inhibition of the energy-dependent uptake of potassium which was directly proportional to the calcium level. k-Strophanthin induced a distinct inhibition only at very high concentration (greater than 10(-4)M). The inhibition of potassium uptake by 1. 3 or 5 mM calcium chloride was gradually cancelled by increasing k-strophanthin concentrations above 10(-8)M. Maximal cancellation was found with 10(-5)M of k-strophanthin. This concentration almost completely prevented the inhibition by calcium ions. On the other hand, 10(-5)M k-strophanthin had no inhibitory effect on the energy-dependent uptake of 0.5 mumoles of calcium chloride or on the oxidative phosphorylation of 0.5 mumoles of ADP added to the incubation medium.

The effect of aging on Ca2+ -transport in synaptosomal preparations from rat brains was assessed by measuring the accumulation of radioactive 45Ca within these particles. Four groups of rats at 6, 12 24 and 30 months of age were used for this study. Synaptosomal particles were isolated from the cerebral cortex of each animal and the radioactive 45Ca inside the particles were measured after incubating the particles with media containing an energy source and 45Ca Cl2. Results indicated that the transport of 45Ca was lower in the younger rats than the older groups. A 20% increase was consistent with the old rats (30 mo) as compared with the young ones (6 mo). The increase in Ca2+ -transport across synaptic plasma membranes may be related to the transmitter release and behavioral activity after senescence.