Increase In Cellular Ca2 Research Articles

Sarcolemmal phospholipid asymmetry and Ca fluxes on metabolic inhibition of neonatal rat heart cells.

The present study examines the hypothesis that during depletion of high-energy phosphates a change will occur in the phospholipid topology and in Ca fluxes in cultured neonatal cells and that these two events may be causally related. A combination of 2-deoxyglucose and iodoacetic acid was used to produce graded changes in the adenine nucleotides in the cells. An on-line technique for 45Ca measurement was used to follow Ca uptake and compartmentation by the cells, and chemical and enzymatic probes were used to study sarcolemmal phospholipid topology. After 15 min of metabolic inhibition (ATP = 10% of control) an increase in cellular Ca occurs, which progresses with time. Over 70% of this Ca accumulates in the mitochondria. After 30 min of metabolic inhibition (ATP < 10% of control) a change in the phospholipid topology is observed, and an increased amount (two times control) of sarcolemmal phosphatidylethanolamine is present in the outer monolayer of the sarcolemma. This change in phospholipid topology was independent of the extracellular Ca concentration. The sequence of altered Ca fluxes and distribution followed by the altered phospholipid topology is discussed in terms of its possible role in the pathogenesis of sarcolemmal disruption.

Free radical theory of aging: A hypothesis on pathogenesis of senile dementia of the Alzheimer’s type

Senile dementia of the Alzheimer’s type (SDAT) is the major cause of dementia. It is a spontaneous, i.e. sporadic, systemic disorder whose major manifestations are in the brain. It is hypothesized that SDAT may be the result of one of a number of potential mutations in a mitochondrial DNA molecule, early in development and after germ cell segregation, that impairs oxidative phosphorylation and increases production of $$O\mathop \cdot \limits_2^ -$$ and H2O2. Replicative segregation distributes the mutated mtDNA to the cells of the developing organism in such a manner that with advancing age cellular dysfunction occurs first in areas of the brain associated with Alzheimer’s disease. Cell damage and death is attributed to random free radical damage secondary to falling ATP production and increasing formation of $$O\mathop \cdot \limits_2^ -$$ and H2O2 due to aging of the normal and defective mitochondria. The increasing oxidative stress contributes to cell damage and eventual death in part by impairing cellular control of Ca2+ concentration; sustained increases in cellular Ca2+ disrupts the cytoskeleton and activates calcium dependent catabolic enzymes. SDAT is essentially due to acceleration of normal neuronal aging. The amyloid associated with SDAT is apparently a consequence of normal neuronal metabolism and of the changes associated with normal neuronal death. The incidence of SDAT may be decreased by efforts to prevent mutation of a mtDNA molecule early in development, for example, possibly by decreasing maternal deleterious free radical reactions by dietary modulation and/or antioxidant supplements. Similar efforts by the general population may also decrease the incidence by disproportionately slowing the rate of production of free radical damage in those individuals destined to develop Alzheimer’s disease owing to their higher rate of initiation of deleterious free radical reactions. The function of SDAT patients may be temporarily improved by “ rescuing” some impaired neurons, and the underlying mitochondrial decline slowed, by measures similar to those employed with other mitochondrial disorders.

Characterization of the energy-dependent, mating factor-activated Ca 2+ influx in Saccharomyces cerevisiae

The yeast mating pheromones, a and α factors, bind to specific G protein-coupled receptors in haploid cells and bring about both growth arrest in the early G 1 phase of the cell cycle and differentiation into mating capable cells. This induces an increase in Ca 2+ influx leading to elevated intracellular calcium concentrations, which has been shown to be essential for subsequent downstream events and the mating process itself [1]. We have characterized the α factor induced increase in cellular Ca 2+ in wild type S. cerevisiae and in the temperature-sensitive cell division cycle mutants cdc7 and cdc28 which are growth-arrested at the G 0–G 1 border at the nonpermissive temperature. We observed a 2–4 fold increase in the initial velocity of Ca 2+ influx in α factor-treated wild-type cells and in cdc7 and cdc28 cells grown at the nonpermissive temperature. Calcium influx was energy dependent, inhibited by membrane depolarization and slightly increased by hyperpolarization. Furthermore, Ca 2+ influx was sensitive to both divalent and trivalent cations, but was unaffected by nifedipine and verapamil. These data demonstrate that budding yeast possesses a regulated Ca 2+ transport mechanism, the activation of which is dependent upon exit out of the cell cycle and growth cessation. This transport mechanism has many similarities to that observed in mitogen-stimulated mammalian cells.

The rapid nongenomic actions of 1 alpha,25-dihydroxyvitamin D3 modulate the hormone-induced increments in osteocalcin gene transcription in osteoblast-like cells.

We have previously shown that one of the rapid nongenomic actions of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), the increase in intracellular calcium (Ca2+), accompanies the increased osteocalcin (OC) mRNA steady-state levels in rat osteosarcoma cells. To determine the functional significance of the nongenomic actions, we have measured changes in intracellular Ca2+ as an indicator of the rapid effects and have assessed the effect of inhibition of the rapid increase in cellular Ca2+ by the inactive epimer, 1 beta, 25-dihydroxyvitamin D3 (1 beta,25-(OH)2D3), on OC mRNA steady-state levels and transcription. 1 beta,25-dihydroxyvitamin D3 inhibited 1 alpha,25-(OH)2D3 induced increases in intracellular Ca2+ and OC mRNA transcription at 1 hr and OC mRNA steady state levels at 3 hr. 1 beta,25-Dihydroxyvitamin D3 did not alter the binding of the vitamin D receptor complex to the vitamin D responsive element of the OC gene. The results demonstrate the functional importance of the rapid, nongenomic actions of 1 alpha,25-(OH)2D3 in the genomic activation of the OC gene by the hormone in rat osteoblast-like cells, perhaps by modifying subtle structural and/or functional properties of the vitamin D-receptor DNA complex or by affecting other protein DNA interactions that support OC gene transcription.

Removal of sarcolemmal sialic acid residues results in a loss of sarcolemmal functioning and integrity.

Treatment of neonatal rat heart cells with neuraminidase results in a large increase of cellular-associated Ca2+. The study described below was designed to test the hypothesis that neuraminidase produces its effects by increasing the transient Ca2+ channel current, as proposed by Yee et al. (24). This ICa,T can be inactivated by dodecylsulfate (DDS) (17). The experimental data show that 1) the increase in cellular Ca2+ during neuraminidase treatment cannot be explained by an increased ICa,T; 2) neuraminidase treatment has a much more profound effect on sarcolemmal permeability than has been recognized previously; and 3) the effect of neuraminidase treatment can be prevented by 50 microM DDS. The study indicates that glycocalyx-lipid bilayer interactions are important in maintenance of selective permeability of the sarcolemma. The protective effect of 50 microM DDS is probably mediated by insertion of the negatively charged amphiphilic molecule in the sarcolemma, although the exact mechanism remains to be elucidated.

Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin-3-dependent macrophage-like cell line.

An interleukin 3 (IL-3)-dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)-prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca(2+)-dependent and alkaline pH-optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5-10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane-associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL-3-dependent M phi-like cells.

Mechanisms of vasopressin-induced increase in intracellular Na+ in vascular smooth muscle cells.

The present study was undertaken to examine the interrelationship between the arginine vasopressin (AVP)-induced dynamic changes in intracellular Ca2+ and Na+ concentrations in cultured rat aortic vascular smooth muscle cells (VSMC) by the direct measurements of intracellular Na+ concentration [( Na+]i), cytosolic free calcium [( Ca2+]i), and intracellular pH (pHi) using fluorescence dyes. AVP increased [Ca2+]i and [Na+]i in a dose-dependent manner; the rise in [Ca2+]i preceded the rise in [Na+]i. Pretreatment with the V1 antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid) 2-(O-methyl)-tyrosine] AVP [( d(CH2)5Tyr(Me)]AVP) completely blocked the effects of AVP on [Ca2+]i and [Na+]i. The V2 agonist 1-desamino-8-D-AVP, DDAVP, did not affect basal [Na+]i or the AVP-induced increase in [Na+]i. Also, Ca(2+)-free solution completely blocked the AVP-induced increase in [Na+]i. Moreover, Ca(2+)-free solution decreased the AVP-induced intracellular acidification and blunted the later AVP-induced intracellular alkalinization. These results therefore indicate that after binding to the V1 receptor AVP increases [Na+]i mediated by the activation of Na(+)-Ca2+ and Na(+)-H+ exchanges in VSMC. All of these cellular events are completely dependent on an increase in cellular Ca2+ uptake produced by AVP.

Effects of neuraminidase on cellular calcium and contraction in cultured cardiac myocytes

Mammalian plasma membranes, including the myocardial sarcolemma, are abundantly glycosylated. Sialic acid is a ubiquitous anionic sugar found at the periphery of sarcolemmal glycoconjugates. The physiological role of this sugar is not clear, but neuraminidase, which specifically hydrolyzes sialic acid from the sarcolemma, has been found to increase calcium exchange, cause electrophysiological abnormalities, and enhance the transient (T) calcium current in cardiac myocytes. The purpose of this study was to better characterize the effect of neuraminidase on cellular calcium (Ca) and contractile function. Neuraminidase removed up to 57% of total sialic acid from the cells. 45Ca exchange was measured and neuraminidase was found to increase cell calcium proportional to the amount of sialic acid removed (18·6 ± 0·8 mmol/kg dry w, maximally). Over 80% of the increment in calcium remained rapidly exchangeable ( t 1 2 < 15 s ) under non-perfusion limited conditions and was inhibited by cations (La > Cd > Mn > Mg) and nifedipine. Using a video-monitoring system, neuraminidase was observed to transiently increase cell shortening during contraction (30 ± 9%), with progression to arrhythmias followed by cessation of contraction. These results indicate that neuraminidase, probably by removing sarcolemmal sialic acid residues, greatly augments cellular calcium in cultured cardiac myocytes. Most of the increment in Ca induced by neuraminidase was very rapidly exchangeable and most likely mediated by a Ca specific mechanism. Additionally, neuraminidase treatment altered contractile function in a manner consistent with elevated cellular Ca. Despite the many-fold increase in cellular Ca induced by sialic acid removal, cells recovered and demonstrated rhythmic contractions upon return to control incubation conditions.

Role of Calcium-Channel Bockers in Preventing Acute and Chronic Renal Injury

Calcium-channel blockers (CCBs) have been shown to afford protection against acute (ARF) and chronic renal failure (CRF). The effects of CCBs against acute renal injury occur at both the vascular and tubular epithelial level. At the vascular level, experimental ARF-associated loss of renal autoregulation and hypersensitivity to renal nerve stimulation has been shown to be reversed by CCBs. These beneficial vascular effects of CCBs occur on the background of the finding that renal ischemic injury is associated with an increase in cellular Ca2+ concentration. A rise in tubular epithelial Ca2+ concentration also occurs very early after a renal ischemic insult. This effect of ischemia is associated with evidence of membrane depolarization, opening of slow calcium channels, increased cellular Ca2+ uptake, and reversal by CCBs. There is evidence that the increased cellular Ca2+ uptake activates phospholipases, which prolong and increase membrane damage. Experimental CRF is also associated with increased renal cellular Ca2+ concentration, an effect that can be attenuated by CCBs. The CCBs probably slow progression of CRF by both cytoprotective and antihypertensive effects. These findings of vascular and tubular effects of CCBs in experimental ARF and CRF have led to their clinical use to prevent initial dysfunction of cadaveric kidney transplants, cyclosporine nephrotoxicity, radiocontrast-induced ARF, and progression of CRF. Randomized clinical studies are necessary to further examine the efficacy of CCBs in ARF and CRF.

Effect of vasopressin on Na + kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.

Hypoxia-reoxygenation induced increase in cellular Ca 2+ in myocytes and perfused hearts: the role of mitochondria

Reoxygenation of isolated rat cardiac myocytes following a period of hypoxia and substrate deprivation resulted in a 1.5–2-fold increase in the total Ca 2+ content which could be inhibited by 1 μ m antimycin A or ruthenium red (50% inhibition at 2.5 μ m). This increase in Ca 2+ content was not accompanied by any release of creatine kinase into the medium. Treatment of reoxygenated cells with digitonin also resulted in an antimycin A-sensitive increase in Ca 2+ but this was inhibited by a lower concentration of ruthenium red (50% inhibition at 0.25 μ m) and was associated with a substantial release of creatine kinase from the cells. It is concluded that the reoxygenation-stimulated increase in Ca 2+ is dependent on functioning mitochondria and does not occur as a result of physical damage to the sarcolemma. In a parallel series of experiments, the effects of antimycin A and ruthenium red on the reoxygenation-induced increase in Ca 2+ and release of cytosolic contents in the perfused heart (the oxygen paradox) were also investigated. As was observed with the isolated myocytes, each of the compounds significantly reduced the magnitude of the Ca 2+ increase that occurred on reoxygenation: the compounds also reduced the extent of release of cell contents in the perfused heart. The implications of these results for the series of events occurring on reoxygenation of the hypoxic myocardium are discussed.

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the 45Ca-exchange method to determine free cellular Ca++, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca++). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca++] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca++ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca++] results in a release of the fusion-block and an increase in cellular Ca++ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca++] must exist. The latter suggests further that cell surface Ca++ was also critical.

Ouabain treatment of cardiac cells induces enhanced Na+-Ca2+ exchange activity

Inhibition of the cardiac Na+-K+-ATPase with cardiac glycosides causes a rise in internal Na+ and a subsequent increase in cellular Ca2+ due to the sarcolemmal Na+-Ca2+ exchange mechanism. We investigated the adaptation of cultured cardiac cells to prolonged elevation of internal Ca2+ after exposure to ouabain. Cultured neonatal rat heart cells were treated with 100 microM ouabain for 4-48 h. This ouabain concentration inhibited Na+-K+-ATPase activity by approximately 45% and caused modest cellular Ca2+ loading. We found that cells adapted to ouabain treatment by increasing the amount of sarcolemmal Na+-Ca2+ exchange activity by 50-90% over a 24-h period. Kinetic and immunological data indicate that the increase was due to increased numbers of functional exchangers. Neither total cellular nor total sarcolemmal protein content was affected by the ouabain treatment. These results may be relevant toward understanding the effects of therapeutic use of cardiac glycosides.

Kinetics of long-term (72 hr) calcium content during mitogen activation of cultured human T lymphocytes.

In vitro stimulation of human blood lymphocytes with mitogen resulted in an increased intracellular content of Ca2+ per unit cell volume. This increase in Ca2+ content of lectin-activated cells reached a maximum after 24 hr of culture and thereafter slowly declined. Brief treatment of cells at 24 hr of culture with the Ca2+ ionophore A23187 in combination with EGTA resulted in a larger release of Ca2+ from cells in mitogen-stimulated cultures than from cells in control cultures. This indicates that the Ca2+ is accumulated intracellularly but is readily exchangeable. At 24 hr of culture the increase in cellular Ca2+ correlated well with the proliferative response as measured by 3H-thymidine incorporation. Ca2+ influx at 24 and 48 hr of culture was markedly enhanced in the mitogenically stimulated cells as compared either to cells cultured for 1 and 72 hr or cells cultured without mitogen.

An immediate increase in calcium accumulation by rat thymocytes induced by triiodothyronine: its role in the subsequent metabolic responses.

In previous studies we have shown that the thyroid hormone T3 induces a prompt increase in the cAMP concentration of rat thymocytes in vitro. This is followed by and very likely leads to an increase in cellular uptake of the glucose analog 2-deoxyglucose (2-DG). Since these effects of T3 were shown to require the presence of Ca2+ in the suspending medium, it seemed reasonable to determine whether T3 would influence the metabolism of calcium itself. In standard medium containing 1 mM Ca2+ and 45Ca as a tracer, T3 induced a very prompt, dose-related, but transient, increase in thymocyte calcium accumulation. This effect was evident within the first minute after the addition of T3 (including processing time) and is to our knowledge the most rapid effect of T3 yet demonstrated. The effect reached a maximum very shortly thereafter and then abated within a few minutes. Uptake of 45Ca from Ca2+-free medium (5 microM Ca2+ as a contaminant) was also increased by T3, but under these conditions, the increase was sustained for the entire 120-min period of study. At a concentration of 25 microM, lanthanum (La3+) unexpectedly produced a rapid and marked (4- to 6-fold) increase in 45Ca accumulation in the thymocytes. The increase in cellular calcium accumulation produced by La3+ was not associated with any effect on thymocyte cAMP concentration or 2-deoxyglucose (2-DG) uptake. However, La3+ potentiated the response of the cell to T3 in respect to calcium accumulation, cAMP concentration, and 2-DG uptake, shifting the dose-response curve of the latter two effects to the left. In view of the calcium dependence of the T3 effect on thymocyte cAMP concentration and 2-DG uptake, and the effect of La3+ to concomitantly enhance the sensitivity of the cell to the effects of T3 on calcium accumulation and cAMP and 2-DG metabolism, we suggest that the prompt increase in cellular Ca2+ uptake induced by T3 that we demonstrated is causally related to its subsequent effect on cellular cAMP concentration and 2-DG uptake.

Calcium messenger system: an integrated view.

Calcium messenger system: an integrated view.

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange.

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.

Regulation of abalone sperm cyclic AMP concentrations and the acrosome reaction by calcium and methylxanthines

When Ca 2+ is added to abalone sperm ( Haliotis rufescens) in Ca 2+-free artificial seawater (CaFASW) to a final concentration of 9.6 m M a 4-fold elevation in sperm cAMP occurs within 15–30 sec. The methylxanthines, theophylline and 1-methyl-3-isobutylxanthine (MIX), also elevate sperm cAMP concentrations. In CaFASW, either compound causes up to a 3-fold increase in cAMP within 15–30 sec. MIX (150 μ M), added to sperm in the presence of 9.6 m M Ca 2+, elevates sperm cAMP 100-fold within 15–30 sec and also triggers the acrosome reaction (AR) in the same period. Under identical conditions theophylline (1.67 m M) is much less potent at elevating cAMP and inducing the AR. The effects of methylxanthines on cAMP of sperm incubated in the presence of Ca 2+ appear to respresent a potentiation by these compounds of the action of Ca 2+. Neither compound induces the AR in the absence of Ca 2+. All of the observed effects on sperm cAMP and the AR are dependent on Ca 2+ and methylxanthine concentrations. Added cyclic nucleotides or their derivatives do not induce the AR in either the absence or presence of Ca 2+. Experiments with isolated sperm heads and flagella indicate that the dramatic stimulatory response of sperm cAMP to Ca 2+ plus MIX is present in the head region (acrosome, nucleus, midpiece) of the cell. The data suggest that the dramatic elevation of cAMP by MIX in the presence of Ca 2+ may occur directly by an inhibition of phosphodiesterase activity and indirectly by an increase in cellular Ca 2+. A strong temporal correlation between the cAMP elevation and the abalone AR exists, although cAMP elevation by itself does not act as the primary mediator of this exocytotic event.

Effects of inorganic phosphate on ion exchange, energy state, and contraction in mammalian heart.

Effects of inorganic phosphate (Pi) on contractile function, ionic exchange, and cellular energetic state were investigated in isolated, arterially perfused, interventricular rabbit septa. The addition of 10 or 20 mM Pi to a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered perfusate showed 1) a decrease in developed tension due to a decrease in free Ca concentration; 2) an increase in diastolic tension; 3) a decrease in 42K uptake at the same time that 42K efflux was depressed; 4) an increase of 24Na activity; 5) an increase in tissue 47Ca activity; and 6) a decrease in tissue adenosine 5'-triphosphate (ATP) levels. If a 10-mM Pi intervention was applied without changing the free Ca concentration of the perfusate, the mechanical record showed a transient positive inotropic effect without changes in K efflux. These results are consistent with Pi-induced stimulated mitochondrial Ca uptake and Na-K pump inhibition that combine to increase cellular Ca uptake. The increase in cellular Ca and the decrease in cellular ATP would both tend to increase diastolic tension. In the presence of antimycin A, respiration-dependent Ca uptake is inhibited, and mitochondrial Ca uptake is then maintained at the expense of cellular ATP. This process of mitochondrial Ca uptake could account for the deficiency of ATP and the rigor type contracture that occurs with antimycin A. This energy-deficient state is modified if mitochondrial ATP consumption is blocked by oligomycin, and such treatment prevents the ATP-deficient rigor. Under these conditions, although mitochondrial Ca uptake is blocked by the presence of antimycin A and oligomycin, Ca uptake remains augmented secondary to Na-K pump inhibition by Pi.

The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN'N'-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.