The effects of cyanide on intracellular ionic exchange in ferret and rat ventricular myocardium

The effects of cyanide on Ca2+ exchange in isolated ventricular myocytes and on the intracellular concentrations of Ca2+, Na+ and H+ have been investigated to assess the contribution that mitochondria might play in cellular Ca2+ metabolism. Ionic levels were measured with ion-selective electrodes. KCN (2.5 mM) inhibited a component of Ca2+ exchange in myocytes that could be attributed to mitochondrial exchange, but was without effect on non-mitochondrial Ca2+ exchange. NaCN (2.5 mM) caused a transient reduction of [H+]i, [Na+]i and [Ca2+]i when applied to the superfusate bathing ventricular trabeculae or papillary muscles. The transient changes of [Na+]i were accentuated when the preparation was exposed to a solution which would be expected to increase the cellular calcium content. The reduction of [Na+]i which accompanies a reduction of the extracellular sodium concentration, [Na]o, was attenuated in the presence of NaCN, but the intracellular acidosis resulting from a reduction of [Na]o was unaffected by NaCN. A small, but significant, rise of [Ca2+]i accompanied a reduction of [Na]o but only when NaCN was present in the superfusate. It is concluded that cyanide ions have a reasonably specific action on cardiac cellular ionic metabolism. Its primary action is to prevent mitochondrial Ca2+ sequestration. It is postulated that a Na+/H+ exchange, possibly at the sarcolemma, could account for some of the changes to sarcoplasmic ionic levels observed. In a solution of low [Na]o, it is concluded that mitochondria could sequester at least 30% of the calcium accumulated by the cell even though the sarcoplasmic [Ca2+] does not exceed 0.3 microM.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotrophs.

In rat pituitary somatotrophs, the stimulation of growth hormone secretion by growth hormone-releasing hormone (GHRH) is a Ca(2+)-dependent event involving Ca2+ influx. The presence of calcium-induced calcium release (CICR) Ca2+ stores has been suggested in these cells. The aim of our study was to demonstrate the presence of CICR stores in rat somatotrophs and to determine their function in GHRH Ca2+ signalling. To this end we measured cytosolic free Ca2+ concentration ([Ca2+]i), using indo-1 in purified rat somatotrophs in primary culture, while altering intracellular Ca2+ stores. Ionomycin (10 ttM) or 4-bromo-A23187 (10 ItM), used to mobilise organelle-bound Ca2+, raised [Ca2+]i in the absence of extracellular Ca2+. Caffeine (5 to 50 mM), used to mobilise Ca2+ from CICR stores, transiently raised [Ca2+]i in 65% of cells tested. The response to 40 mM caffeine was abolished when Ca2+ stores were depleted, with 1 microM thapsigargin or with 10 microM ryanodine. All cells that responded to 40 mM caffeine responded to 10 nM GHRH. The [Ca2+]i response to 10 nM GHRH was reversible and repeatable. However, the second response was 38% smaller than the first. Ryanodine treatment abolished the reduction in the second [Ca2+]i response, while thapsigargin increased the reduction by 67%. We conclude that rat somatotrophs possess CICR Ca2+ stores and that they account for 30-35% of the GHRH-induced increase in [Ca2+]i, and that their partial depletion is involved in somatotroph desensitization.     

Mechanism of Inhibition of N-Methyl-d-Aspartate-Stimulated Increases in Free Intracellular Ca2+ Concentration by Ethanol

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. These cells were sensitive to low N-methyl-D-aspartate (NMDA) concentrations with EC50 values for NMDA-induced intracellular Ca2+ concentration ([Ca2+]i) increases of approximately 7-16 microM measured in the absence of Mg2+. NMDA-stimulated [Ca2+]i increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mM KCl prior to NMDA addition. Under these predepolarized conditions, 100 mM ethanol inhibited 25 microM NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA-induced increases in [Ca2+]i in a concentration-dependent manner at 4, 16, 64, and 256 microM NMDA. Whereas NMDA-induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 microM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 microM) enhanced 25 microM NMDA-induced increases in [Ca2+]i by approximately 50%. Glycine (1-100 microM) prevented the 100 mM ethanol inhibition of NMDA-stimulated [Ca2+]i observed in the absence of exogenous glycine. MK-801 (25-400 nM) inhibited 25 microM NMDA-stimulated rises in [Ca2+]i in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretory responses of rat peritoneal mast cells to high intracellular calcium

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.     

Maintenance of intracellular calcium in Escherichia coli

Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells. Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli. Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid. The concentration of free [Ca2+]i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium. Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium. In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration. In starved cells [Ca2+]i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM. Addition of glucose lowered the Ca2+ levels to 90 nM. Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux. Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool. These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+]i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.     

Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N',-tetraacetic acid

The calcium ion dependence of calcium transport by isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been investigated by means of the Calcium-stat method, in which transport may be measured in the micromolar free calcium ion concentration range, in the absence of calcium buffers. At pH 7.2 and 20 degrees C, ATP, in the range 1 to 10 mM, decreased [Ca2+]0.5 from 2.0 microM to 0.3 microM and decreased Vmax of oxalate-supported transport from 0.5 to 1.3 mumol min-1 mg-1. Simultaneous measurements of transport and of ATPase activity in the range 0.8 to 10 microM free Ca2+ showed a ratio of 2.1 calcium ions translocated/molecule of ATP hydrolyzed. Transport, in the presence of 5 mM ATP, ceased when calcium ion concentration fell to 0.6 to 1.2 microM, whilst ATPase activity of 90 nmol of ATP hydrolyzed min-1 mg-1 persisted. The data obtained by the Calcium-stat method differed from those described previously using calcium buffers, in that they showed lower apparent affinities of the transport site for calcium ions, more marked sigmoidal behavior, an effect of ATP concentration on Ca2+ concentration dependence and lower ATPase activity in the absence of transport. The calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) had no effect when transport was stimulated maximally at saturating free Ca2+ concentrations. However, at calcium ion levels below [Ca2+]0.5, 70 microM CaEGTA stimulated transport to rates of 20 to 45% of Vmax. Half-maximal stimulation of transport occurred at 19 microM CaEGTA. CaEGTA, 50 microM, decreased [Ca2+]0.5, determined at 5 mM ATP, from 1.3 microM to 0.45 microM. It is proposed that a ternary complex, E . Ca2+ . EGTA4-, is formed as an intermediate species during CaEGTA-stimulated calcium transport by sarcoplasmic reticulum membranes and stimulates the calcium pump at limiting free Ca2+ ion concentration.     

Clearance of large Ca2+ loads in a single smooth muscle cell: examination of the role of mitochondrial Ca2+ uptake and intracellular pH

Redistribution of cytosolic free Ca2+ following Ca2+ influx into the cytoplasm was studied in single smooth muscle cells isolated from guinea-pig urinary bladder. Voltage-clamped cells were loaded with a low-affinity fluorophore Indo-1FF. A decay of free intracellular Ca2+ ([Ca2+]i) after the termination of the depolarizing pulse (1 s from -50 mV to +20 mV) was fitted with a single exponential and the effect of various substances on the time constant was compared. At a holding potential of +80 mV the [Ca2+]i decay was 1.56 times slower compared to that at -50 mV suggesting the presence of a voltage-dependent process redistributing Ca2+. In the presence of cyclopiazonic acid (CPA, 10 microM), an inhibitor of sarco(endo)plasmatic Ca2+ pump (SERCa), the [Ca2+]i decay was 3.93 times slower than that in the absence of the inhibitor. Introduction of a polycation Ruthenium Red (RR) (20 microM), an inhibitor of the mitochondrial Ca2+ uniporter, into a cell or collapsing a transmitochondrial H+ gradient with the protonophore CCCP (2 microM) slowed down the [Ca2+]i decay 6.05-fold and 9.78-fold, respectively. The apparent amplitude of [Ca2+]i increments was also increased by CCCP. Increasing H+ buffering power in the intracellular solution from 10 mM to 40 mM of HEPES greatly reduced the effect of CCCP on [Ca2+]i decay. A further increase in HEPES concentration to 100 mM eliminated the effects of CCCP both on the time course of [Ca2+]i decay and on the amplitude of [Ca2+]i increment. Perfusion of RR together with 100 mM HEPES into the cytoplasm was without effect on the decay time course of [Ca2+]i. The effect of CPA on [Ca2+]i decay was also reduced in cells loaded with 100 mM HEPES; the time constant in the presence of CPA was slowed down by a factor of 2.18. Application of 10 mM Na(+)-butyrate to the cells loaded with 10 mM HEPES resulted in a slowing down of [Ca2+]i decay: the time constant was increased by a factor of 5.84. Measurement of intracellular pH with SNARF-1 confirmed cytoplasmic acidification during application of Na(+)-butyrate and CCCP. It is concluded that the contribution of mitochondrial Ca2+ uptake to the rapid [Ca2+]i decay is much less than could be extrapolated from action of protonophores in these smooth muscle cells. The results also demonstrate the importance of intracellular pH for Ca2+ handling in the cytoplasm of smooth muscle cells.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

Regulation of Calcium Concentrations in Synaptosomes: α2-Adrenoceptors Reduce Free Ca2+ by Closure of N-Type Ca2+ Channels

The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K(+)-depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT (10.7 mM) was approximately 10,000 times higher than [Ca2+]i (118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaT was freely exchangeable. This pool was much larger than the free Ca2+ pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+ concentration in the buffer, suggesting that [Ca2+]i is not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time- and dose-dependent manner. Maximum inhibition (40% at 100 microM) occurred at 2 min and the IC50 was 80 nM. The reduction caused by clonidine (1 microM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+ pool size. The effects of clonidine (1 microM) on [Ca2+]i (26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+ concentration (0.1-1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i.     

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.     

Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

Calcium stimulates ATP-Mg/Pi carrier activity in rat liver mitochondria

Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.