Nmol Ca2 Research Articles

Post-transcriptional regulation of the [formula omitted] exchanger in aging rat heart

Altered calcium homeostasis in the senescent heart appears to be the result, at least in part, of decreased Na + Ca 2+ exchange activity. To further investigate the basis of the decrease in Na + Ca 2+ exchange activity, Na + Ca 2+ exchanger gene expression in the heart was compared in 3 and 24 month old male Fischer 344 rats. Sarcolemmal vesicles prepared from left ventricle and septum showed reduced Na +-dependent Ca 2+ uptake in 24 month old animals when compared to 3 month old animals (0.156 ± 0.005 and 0.135 ± 0.008 nmol Ca 2+/mg/10 s; mean ± S.E. for 3 month and 24 month old animals, respectively). Western analysis showed immunodetectable Na + Ca 2+ exchanger protein levels were decreased by 19% in 24 month old animals when compared to 3 month old animals. Poly(A +) RNA was purified from left and right ventricle and left and right atria and subjected to Northern analysis using digoxin labeled cDNA probes for the Na + Ca 2+ exchanger and actin. The Na + Ca 2+ exchanger probe labeled a 7 kb message in both ventricle and atria, while the actin probe labeled both β-actin (2.2 kb) and α-actin (1.4 kb). The steady state level of expression of Na + Ca 2+ exchanger Poly(A +) RNA when normalized to β-actin, was similar when ventricle and atria were compared. There were no observable differences in Na + Ca 2+ exchanger or α-actin Poly(A +) RNA steady state levels when comparing 3 and 24 month old animals. The results suggest that reduced Na + Ca 2+ exchange activity in the left ventricle of 24 month old animals was most likely the result of post-transcriptional modification of the protein that was detectable by Western analysis.

Reciprocal changes in the postnatal expression of the sarcolemmal Na +-Ca 2+-exchanger and SERCA2 in rat heart

The aim of this study was to examine the relationship between sarcolemmal Na(+)-Ca2+ exchangers and sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2) expression and the developmental differences in cardiac Ca2+ handling. Postnatal steady-state mRNA and protein levels were analysed in rat ventricular myocardium by Northern and immunoblot analysis, respectively. This was compared to Na+ gradient-induced and SR oxalate-supported Ca2 transport in isolated membranes. Na(+)-Ca2+ exchanger mRNA declined by 75% between day 1 and 30, whereas SR Ca2+ ATPase mRNA levels increased by 97% during this period. The Na(+)-Ca2+ exchanger mRNA/Ca(2+)-ATPase mRNA ratio was found to be inversely related to post-natal age. The changes in mRNA levels were associated with corresponding developmental differences in the Ca2+ transport activities of the respective membrane proteins. In crude membranes, the Na(+)-dependent Ca2+ transport activity (at 75 microM Ca2+) declined gradually (P < 0.01; mean +/- S.E.) from 17.7 +/- 2.4 nmoles Ca2+/g wet tissue/2s at day 1-3 (n = 5) to a value of 4.2 +/- 1.1 at day 40 (n =4). Conversely, SR Ca2+ uptake increased (P < 0.01) 2.6-fold during this period. The inversely related changes in the post-natal expression and function of the Na(+)-Ca2+ exchanger and SR Ca(2+)-ATPase suggest a coordinated control at the pretranslational level of the cellular Ca2+ transport processes mediated by the two membrane proteins.

Dexamethasone inhibits inorganic phosphate stimulated Ca 2+-dependent damage of isolated rat liver and renal cortex mitochondria

The prevention of Ca 2+-induced permeabilization of rat liver and kidney cortex mitochondria by dexamethasone, a common anti-inflammatory glucocorticoid, was the subject of this study. A non-specific release of matrix Ca 2+ and membrane depolarization was observed in respiring mitochondrial suspensions subjected to a 30 nmol Ca 2+/mg protein load in the presence of 2 mM inorganic phosphate (P i), or 20 nmol Ca 2+/mg protein with 1 mM P i, for liver and renal cortex mitochondria (RCM), respectively. Additions of dexamethasone prior to Ca 2+ in mitochondrial suspensions from liver or kidney cortex (80 and 200 μM final concentrations, respectively) led to 75–80% protection from these permeabilization-associated alterations of functional integrity. In conclusion, dexamethasone appears to show great promise in blocking the opening of a Ca 2+-dependent ‘non-specific pore’ in the inner membranes of mitochondria from various sources.

Thapsigargin-resistant Intracellular Calcium Pumps: ROLE IN CALCIUM POOL FUNCTION AND GROWTH OF THAPSIGARGIN-RESISTANT CELLS

Exposure of cells to the intracellular Ca2+ pump blocker, thapsigargin (TG), results in emptying of Ca2+ pools and termination of cell proliferation (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca2+ pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 microM TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of 45Ca2+ uptake into permeabilized parent DC-3F cells revealed two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG; the IC50 values for TG were 200 pM and 4 microM, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 +/- 0.10 and 0.18 +/- 0.08 nmol of Ca2+/10(6) cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca2+ pumping; however, almost all pumping was blocked at higher TG concentrations with an IC50 of 5 microM. In both cell types, each Ca2+ pump activity (regardless of TG sensitivity) had high Ca2+ affinity (Km values congruent to 0.1 microM) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca2+ pool was releasable with inositol 1,4,5-trisphosphate (InsP3) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP3-releasable. GTP-induced Ca2+ uptake in the presence of oxalate indicated Ca2+ transfer between distinct pools in the DC-3F cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca2+ accumulation was releasable with InsP3; unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca2+ transfer between pools in the presence of oxalate. Thus, whereas InsP3 releases Ca2+ only from the high TG sensitivity Ca2+ pumping pool in parent DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant Ca2+ pumping pool now contains functional InsP3 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

An improved 45Ca protocol for investigating physiological mechanisms in coral calcification

A sensitive experimental protocol using cloned corals (hereafter “microcolonies”) of the branching scleractinian coral Stylophora pistillata and 45Ca has been developed to enable reproducible measurements of physiological and biochemical mechanisms involved in calcium transport and compartmentalization during coral calcification. Cloned S. pistillata microcolonies were propagated in the laboratory from small fragments of parent colonies collected in 1990 in the Gulf of Aqaba, Jordan. Cloned microcolonies have several intrinsic properties that help to reduce unwanted biological variability: (1) same genotype; (2) similar sizes and shapes; and (3) absence of macroscopic boring organisms. Errors specifically associated with long-standing problems to do with isotopic exchange were further reduced by producing microcolonies with no skeletal surfaces exposed to the radioisotope-labelled incubation medium. The value of the technique resides principally in its superior ability to elucidate transportation pathways and processes and not in its ability to quantitatively estimate calcium deposition by corals in nature. We describe here a rapidly exchangeable calcium pool in which up to 90% of the radioactive label taken up during incubations is located. This pool (72.9±1.4 nmol Ca mg-1 protein) is presumably located within the coelenteric cavity as suggested by the following: (1) it has 4-min half-time saturation kinetics; (2) the accumulation of calcium is linearly correlated with the calcium concentration of sea-water; and (3) its insensitivity to metabolic and ion transport inhibitors indicate that membranes do not isolate this compartment. Washout of this large extracellular pool greatly improved estimates of calcium deposition as evidenced by 10 to 40% reduction in coefficients of variation when compared with previous 45Ca2+ methods described in the literature. Comparisons of calcification measurements simultaneously carried out using the alkalinity anomaly technique and the 45Ca protocol described here show that the correlation coefficient of both techniques is close to 1. Unlike previous reports, our 45Ca2+-derived measurements are slightly lower than those computed from the alkalinity depletion technique.

Rapidly Forming Apatitic Mineral in an Osteoblastic Cell Line (UMR 106—01 BSP)

This study evaluated a rapid biomineralization phenomenon exhibited by an osteoblastic cell line, UMR 106-01 BSP, when treated with either organic phosphates [beta-glycerophosphate (beta-GP), Ser-P, or Thr-P], inorganic phosphate (P(i)), or calcium. In a dose-dependent manner, these agents (2-10 mM) stimulated confluent cultures to deposit mineral in the cell layer (ED50 of approximately 4.6 mM for beta-GP (30 +/- 2 nmol Ca2+/microgram DNA) and approximately 3.8 mM (29 +/- 2 nmol Ca2+/microgram DNA) for P(i)) with a plateau in mineral formation by 20 h (ET50 approximately 12-15 h). beta-GP or P(i) treatment yielded mineral crystals having an x-ray diffraction pattern similar to normal human bone. Alizarin red-S histology demonstrated calcium mineral deposition in the extracellular matrix and what appeared to be intracellular paranuclear staining. Electron microscopy revealed small, needle-like crystals associated with fibrillar, extracellular matrix deposits and intracellular spherical structures. Mineral formation was inhibited by levamisole (ED50 approximately 250 microM), pyrophosphate (ED50 approximately 1-10 microM), actinomycin C1 (500 ng/ml), cycloheximide (50 micrograms/ml), or brefeldin A (1 microgram/ml). These results indicate that UMR 106-01 BSP cells form a bio-apatitic mineralized matrix upon addition of supplemental phosphate. This process involves alkaline phosphatase activity, ongoing RNA and protein synthesis, as well as Golgi-mediated processing and secretion.

Pulmonary microsomes contain a Ca 2+-transport system sensitive to oxidative stress

A variety of events, including inhalation of atmospheric chemicals, trauma, and ischemia-reperfusion, may cause generation of reactive oxygen species in the lung and result in airways constriction. The specific metabolic mechanisms that translate oxygen radical production into airways constriction are yet to be identified. In the lung, calcium homeostasis is central to release of bronchoactive and vasoactive chemical mediators and to regulation of smooth muscle cell contractility, i.e. airway constriction. In the present work, we characterized Ca 2+-transport in the microsomal fraction of mouse lungs, and determined how reactive oxygen species, generated by Fe 2+/ascorbate and H 2O 2/hemoglobin, affected Ca 2+ transport. The microsomal fraction of pulmonary tissue accumulated 90 ± 5 nmol Ca 2+/mg protein by an ATP-dependent process in the presence of 15 mM oxalate, and 16 ± 2 nmol Ca 2+ in its absence. In the presence of oxalate, the rate of Ca 2+ uptake was 50 ± 5 nmol Ca 2+/min per mg protein at p Ca 5.9(37°C). The Ca 2+-ATPase activity was 50–60 nmol P i/min per mg protein (p Ca 5.9, 37°C) in the presence of alamethicin. Inhibitors of mitochondrial H +-ATPase had no effect on the Ca 2+ transport. Half-maximal activation of Ca 2+ transport was produced by 0.4–0.5 μM Ca 2+. Endoplasmic reticulum Ca 2+-pump (SERC-ATPase) was found to be predominantly responsible for the Ca 2+-accumulating capacity of the pulmonary microsomes. Incubation of the microsomes in the presence of either Fe 2+/ascorbate or H 2O 2/hemoglobin resulted in a time-dependent accumulation of peroxidation products (TBARS) and in inhibition of the Ca 2+ transport. The inhibitory effect of Fe 2+/ascorbate on Ca 2+ transport strictly correlated with the inhibition of the Ca 2+-ATPase activity. These results are the first to indicate a highly active microsomal Ca 2+ transport system in murine lungs which is sensitive to endogenous oxidation products. The importance of this system to pulmonary disorders exacerbated by oxidative chemicals remains to be studied.

T-tubule depolarization-induced SR Ca2+ release is controlled by dihydropyridine receptor- and Ca(2+)-dependent mechanisms in cell homogenates from rabbit skeletal muscle.

In vertebrate skeletal muscle, the voltage-dependent mechanism of rapid sarcoplasmic reticulum (SR) Ca2+ release, commonly referred to as excitation-contraction (EC) coupling, is believed to be mediated by physical interaction between the transverse (T)-tubule voltage-sensing dihydropyridine receptor (DHPR) and the SR ryanodine receptor (RyR)/Ca2+ release channel. In this study, differential T-tubule and SR membrane monovalent ion permeabilities were exploited with the use of an ion-replacement protocol to study T-tubule depolarization-induced SR 45Ca2+ release from rabbit skeletal muscle whole-cell homogenates. Specificity of Ca2+ release was ascertained with the use of the DHPR antagonists D888, nifedipine and PN200-110. In the presence of the "slow" complexing Ca2+ buffer EGTA, homogenates exhibited T-tubule depolarization-induced Ca2+ release comprised of an initial rapid phase followed by a slower release phase. During the rapid phase, approximately 20% of the total sequestered Ca2+ (approximately 30 nmol 45Ca2+/mg protein), corresponding to 100% of the caffeine-sensitive Ca2+ pool, was released within 50 ms. Rapid release could be inhibited fourfold by D888. Addition to release media of the "fast" complexing Ca2+ buffer BAPTA, at concentrations > or = 4 mM, nearly abolished rapid Ca2+ release, suggesting that most was Ca2+ dependent. Addition of millimolar concentrations of either Ca2+ or Mg2+ also greatly reduced rapid Ca2+ release. These results show that T-tubule depolarization-induced SR Ca2+ release from rabbit skeletal muscle homogenates is controlled by T-tubule membrane potential- and by Ca(2+)-dependent mechanisms.

Effect of R56865 on cardiac sarcoplasmic reticulum function and its role as an antagonist of digoxin at the sarcoplasmic reticulum calcium release channel.

1. The effect of R56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl]-N-methyl-2- benzothiazolamine) on cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel function was investigated. The effect of R56865 on [3H]-ryanodine and [3H]-digoxin binding to SR vesicles and its effect on the ATP-stimulated 45Ca2+ uptake into SR vesicles was also studied. 2. R56865 (0.5-50 microM) had no effect on single-channel open probability (Po) when added to native cardiac SR Ca(2+)-release channels, incorporated into planar phospholipid bilayers, that had previously been activated by 10 microM Ca2+. The single-channel conductance (93 pS) and the Ca2+/Tris+ permeability ratio (12.5) were also unaffected by R56865. 3. R56865 failed to affect the rapid Ca(2+)-induced efflux of 45Ca2+ from cardiac SR vesicles. The initial efflux rate at an extravesicular [Ca2+] of 0.1 microM was 176 +/- 33 nmol 45Ca2+ mg-1 protein s-1 (n = 5). Addition of 0.5-50 microM R56865 to the efflux solution did not affect the initial efflux rate or the total amount of 45Ca2+ released from the vesicles. 4. The specific binding of [3H]-ryanodine to SR vesicles can be viewed as a marker for SR Ca(2+)-release channel activation. R56865 (0.05-50 microM) did not change the amount of specific [3H]-ryanodine bound at 10 microM activating Ca2+. Taken together these data (points 2, 3 and 4) suggest that R56865 does not affect the Ca2+ activation of the cardiac SR Ca(2+)-release channel. 5. R56865 (0.5-50 microM) decreased the ATP-stimulated uptake of 45Ca2+ into cardiac SR vesicles. The total amount of 45Ca2+ taken up into the vesicles was decreased by 26-37% and the initial rate of uptake was also reduced.6. R56865 decreased the binding of [3H]-digoxin to cardiac SR vesicles. It inhibited binding of[3H]-digoxin to both sites on SR membranes. However, it acted as a mixed type of non-competitive antagonist, as it increased the Kd and reduced the Bmax for [3H]-digoxin. Additionally, when SRCa2+-release channels incorporated into bilayers were activated by 1 nM digoxin at 10 micro M Ca2+, 5 microMR56865 decreased single-channel Po to that seen prior to activation by digoxin, confirming that R56865is an antagonist at the cardiac glycoside receptor site on the cardiac SR Ca2+-release channel.7. The results presented here show that R56865 decreases 45Ca2+ uptake into SR vesicles and also non-competitively decreases the binding of cardiac glycosides to their activation sites on the SRCa2+-release channel. Since this compound fails to affect other aspects of the functioning of the SR, we conclude that inhibition of Ca2+-uptake into the SR and/or antagonism of digoxin binding to its high affinity sites on the cardiac SR Ca2+-release channel may contribute to the protection against glycoside induced toxicity seen with R56865. Indeed the reduction of Ca2+ uptake into the SR during Ca2+overload may contribute to the protective action of R56865 against arrhythmias induced by veratridine or by ischaemia. Notwithstanding this, it is possible that the multitude of other actions on specific ion translocation processes, as well as the possible non-specific membrane effects of R56865, may be as responsible for the protection against arrhythmias as the effects on SR function we have documented here. However, the simultaneous interference with more than one of these processes by R56865 may be required for its anti-arrhythmic action.

Enhanced calcium signalling events in neuroblastoma × glioma hybrid NG108-15 cells after treatment with dibutyryl cyclic AMP

The effects of dibutyryl cyclic AMP (dbcAMP) treatment on Ca2+ channel activities, Ca2+ accumulation by intracellular Ca2+ pools, and sizes of IP3- and GTP-releasable pools in neuroblastoma × glioma hybrid NG108-15 cells were studied. High extracellular K+ induced a greater rise in intracellular calcium concentration ([Ca2+]i) in dbcAMP-treated cells than in control cells. In dbcAMP-treated cells, the initial phase of the high K+-induced [Ca2+]i rise displayed a much higher sensitivity to ω-conotoxin than it did in control cells, whereas the plateau phase of the [Ca2+]i rise was sensitive only to nifedipine. These results indicate that predominantly L-type Ca2+ channels exist in control cells, and that N-type channels develop only after dbcAMP treatment. In dbcAMP-treated cells, mitochondria showed an increased Ca2+ uptake capacity (5.3 nmol Ca2+/mg protein) compared with that in control cells (4.2 nmol Ca2+/mg protein). However, dbcAMP treatment did not cause significant change in the affinity for Ca2+. Dibutyryl cAMP treatment enhanced the Ca2+ accumulation activity by nonmitochondrial pools (from 0.84 to 0.97 nmol Ca2+/mg protein) and increased the affinity for Ca2+ (EC50 for Ca2+ decreased from 0.146 μM to 0.063 μM). Our data also indicate that the pool that is sensitive to both IP3 and GTP was enlarged. The affinities for IP3 and GTP in causing Ca2+ release remained the same before or after dbcAMP treatment.

Identification of a 20-kDa protein with calcium uptake transport activity. Reconstitution in a membrane model

This paper presents results of experiments designed to further purify the membrane system involved in mitochondrial calcium transport. A partially purified extract, which transported calcium with a specific activity of 1194 nmol 45Ca2+/mg protein/5 min, was used to obtain mouse hyperimmune serum. This serum inhibited calcium uptake both in mitoplasts and in vesicles reconstituted with mitochondrial proteins containing cytochrome oxidase. Western blot analysis of the semipurified fraction showed that the serum recognized specifically two antigens of 75 and 20 kDa. Both antibodies were purified by elution from the nitrocellulose sheets and their inhibition capacity was analyzed. The antibody that recognized the 20-kDa protein produced a higher degree of inhibition than the other one.

Thyroid hormone-induced alterations in phospholamban protein expression. Regulatory effects on sarcoplasmic reticulum Ca2+ transport and myocardial relaxation.

The aim of the present study was to determine the changes in phospholamban protein levels and their regulatory effect on sarcoplasmic reticulum (SR) Ca2+ uptake and left ventricular function in hypothyroid and hyperthyroid rat hearts. Hypothyroidism was associated with decreases in basal left ventricular function (+dP/dt and -dP/dt), whereas in hyperthyroidism these parameters were elevated compared with values for euthyroid hearts. The maximal SR Ca2+ uptake rates were 12.8 +/- 1.1, 15.5 +/- 1.2, and 21.4 +/- 1.4 nmol Ca2+ per milligram per minute, and the EC50 values for Ca2+ were 0.76 +/- 0.09, 0.41 +/- 0.07, and 0.30 +/- 0.05 mumol/L assayed in homogenates from hypothyroid, euthyroid, and hyperthyroid hearts, respectively. The relative tissue level of phospholamban was increased (135%) in hypothyroidism and decreased (75%) in hyperthyroidism compared with euthyroidism (100%). An opposite trend was observed for the SR Ca(2+)-ATPase, which was depressed (74%) in hypothyroid hearts but increased (134%) in hyperthyroid hearts. Consequently, the relative ratio of phospholamban to Ca(2+)-ATPase was highest in hypothyroid and lowest in hyperthyroid hearts, and these changes correlated with changes in the EC50 of the SR Ca2+ uptake for Ca2+. Stimulation of hearts with 0.1 mumol/L isoproterenol revealed that the relaxant effects were lower in hyperthyroid hearts and higher in hypothyroid hearts compared with euthyroid hearts, consistent with the alterations in the phospholamban levels. The maximal increases in the speed of relaxation, elicited by isoproterenol stimulation, correlated with the changes in the relative ratio of phospholamban to Ca(2+)-ATPase in these hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cellular Ca2+ by yeast vacuoles.

The role of vacuolar Ca2+ transport systems in regulating cellular Ca2+ was investigated by measuring the vacuolar Ca2+ transport rate, the free energy available to drive vacuolar Ca2+ transport, the ability of the vacuole to buffer lumenal Ca2+, and the vacuolar Ca2+ efflux rate. The magnitude of the Ca2+ gradient generated by the vacuolar H+ gradient best supports a 1 Ca2+:2 H+ coupling ratio for the vacuolar Ca2+/H+ exchanger. This coupling ratio along with a cytosolic Ca2+ concentration of 125 nM would give a vacuolar free Ca2+ concentration of approximately 30 microM. The total vacuolar Ca2+ concentration is approximately 2 mM due to Ca2+ binding to vacuolar polyphosphate. The Ca2+ efflux rate from the vacuole is less than the growth rate indicating that the steady-state Ca2+ loading level of the vacuole is dependent mainly on the Ca2+ transport rate and the rate that vacuolar Ca2+ is diluted by growth. Based on the kinetic parameters of vacuolar Ca2+ accumulation in vitro, the maximum rate of Ca2+ accumulation in vivo is expected to be approximately 0.2 nmol of Ca2+ min-1 mg protein-1, a rate that is similar to the cellular Ca2+ accumulation rate. The cytosolic Ca2+ concentration increases from 0.1 microM to 1-2 microM as the extracellular Ca2+ concentration is raised from 0.3 mM to 50 mM. The rise in cytosolic Ca2+ concentration increases cellular Ca2+ from 10 to 300 nmol Ca2+/mg by increasing the rate of vacuolar Ca2+ accumulation but does not significantly alter the cellular growth rate.

Function of skeletal muscle sarcoplasmic reticulum vesicles with exercise.

In this study the response of sarcoplasmic reticulum (SR) to prolonged moderate-intensity exercise was examined in highly purified native vesicles isolated from rat gastrocnemius muscle. Maximal Ca(2+)-dependent ATP hydrolysis was reduced by 12.6% within 2 min after the onset of exercise. The reduction in Ca(2+)-dependent adenosinetriphosphatase activity progressed to 18% at 30 min of exercise and was maintained throughout the subsequent 90-100 min of exercise. Oxalate stimulation of unidirectional Ca2+ transport (Ca2+ loading) was unaffected by exercise. However, in the absence of anion stimulation, steady-state Ca2+ uptake (bidirectional flux) was 51.2 +/- 7.3 nmol Ca2+/mg SR after exercise compared with 36.2 +/- 2.5 nmol Ca2+/mg SR for the control period (P < 0.05). Anion-induced Ca2+ release increased from a control value of 33.9 +/- 4.3 to 55.9 +/- 9.8 nmol Ca2+/mg SR after exercise (P < 0.05). The mechanistic basis for the increase in apparent Ca(2+)-ATP coupling is unclear, although the early onset of the changes suggests the potential for functional adaptation for the SR in response to increased contractile activity.

Calcium transport across plasma membrane vesicles isolated from shoots of Stellaria media and Arena sativa

Plasma membrane vesicles (ca 40% inside‐out, after one freeze‐thaw cycle) were extracted and purified from the shoots of oat (Avena sativa L.) and chickweed (Stellaria media L.) using the two‐phase aqueous polymer technique. In the presence of ATP or GTP, a rapid uptake of 45Ca2+ occurred (0.77 and 0.62 nmol Ca2+ mg‐1 protein, for ATP and GTP, respectively, in oat, and 0.53 and 0.51 nmol Ca2+ mg‐1 protein, for ATP and GTP, respectively, in chickweed). Nucleotide‐dependent Ca2+‐transport was sensitive to 1 μM Erythrosin B (with ATP. inhibited by 52% in oat and in chickweed by 72%; with GTP, inhibition was similar in both species at ca 67%); ATP‐dependent uptake was greater in oat than in chickweed, but not stimulated by calmodulin. Addition of the calcium ionophore A‐23187 resulted in the release of label from the vesicles (41% and 63% release with ATP, and 24% and 52% release with GTP, in oat and chickweed, respectively). The results obtained suggest that Ca2+‐transport is independent of the proton pump. In oat, kinetic data indicate a discontinuity in the absorption isotherm at 10 μM free calcium.

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca2+/mg protein in an energy-dependent pool when incubated at an ambient free Ca2+ concentration of 100 nM. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca2+ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca2+ efflux and ATP-driven Ca2+ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P3, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 microM, selectively reduced the size of the Ins(1,4,5)P3-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P3. At concentrations beyond 100 microM, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca2+ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca2+ from the Ins(1,4,5)P3-sensitive store by activating a Ca2+ release channel, whereas Ca2+ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca2+ pump. The postulated ruthenium red-sensitive Ca2+ release channel might be similar to the ryanodine-receptor in muscle.

Mitochondrial Ca 2+ Uptake at Submicromolar [Ca 2+] i in Permeabilized Pancreatic Acinar Cells

We studied the uptake of Ca 2+ in different intracellular Ca 2+ pools by use of steady-state measurement of 45Ca 2+ in a preparation of digitonin-permeabilized pancreatic acinar cells. The Ca 2+ content of all intracellular pools was assessed at different free Ca 2+ concentrations between 0.1 and 1 μM. At low Ca 2+ concentrations, almost 50% of Ca 2+ was taken up, ATP-dependent, in a non-mitochondrial pool. Even at 0.1 μM free Ca 2+ the mitochondrial Ca 2+ pool contained 26% (3.4 ± 0.8 nmol Ca 2+/mg prot) of the total Ca 2+ and this percentage was increased to 49% at 1 μM Ca 2+ (16.7 ± 1.1 nmol Ca 2+/mg prot). Our data also show that the uptake into the non-mitochondrial pool can be further differentiated on the basis of ATP-dependence, and that the ATP-independent uptake has a biphasic dependency on the free Ca 2+ concentration.

Effect of perfusate [Ca2+] on cardiac sarcoplasmic reticulum Ca2+ release channel in isolated rat hearts.

The effect of perfusate [Ca2+] on the function of cardiac sarcoplasmic reticulum (CSR) was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates of isolated rat hearts maintained in a modified Langendorff preparation. The total Ca2+ pumping activity of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The homogenate Ca2+ uptake rate in the absence of ruthenium red or ryanodine decreased progressively with increasing perfusate [Ca2+] (25.7 +/- 1.2, 21.4 +/- 1.5, 17.2 +/- 1.1, and 16.3 +/- 1.2 [mean +/- SEM] nmol Ca2+.min-1.mg-1 for hearts perfused for 5 minutes with 0.2, 1.4, 2.8, and 5.6 mM Ca2+, respectively; p = 0.0001; n = 8). This depression was not observed when Ca2+ uptake was assayed in the presence of ryanodine or ruthenium red. Since the Ca2+ uptake in the presence of ryanodine or ruthenium red is determined by the Ca(2+)-ATPase, this result suggests that perfusion with varying [Ca2+] did not affect the Ca(2+)-ATPase. The observed decrease in Ca2+ uptake in the absence of ryanodine or ruthenium red is caused by an increased efflux through the ryanodine-sensitive Ca2+ release channel. When hearts perfused for 5 minutes with 0.2 or 5.6 mM Ca2+ were reperfused for 10 minutes with 1.4 mM Ca2+, homogenate Ca2+ uptake rates were restored to near control levels. These effects of perfusate Ca2+ were not direct effects, because changes in the [Ca2+] of the homogenization medium did not alter the homogenate Ca2+ uptake activity in either the presence or absence of ryanodine. The homogenate Ca2+ uptake rates were unaffected by prior active loading of the CSR with Ca2+. These results suggest a regulatory role of perfusate Ca2+ in increasing the open state of the ryanodine-sensitive Ca2+ release channel that is distinct from the beat-to-beat regulation of Ca2+ release from the CSR by Ca2+ (Ca(2+)-induced Ca2+ release).

Calcium homeostasis in procyclic and bloodstream forms of Trypanosoma brucei. Lack of inositol 1,4,5-trisphosphate-sensitive Ca2+ release.

When Trypanosoma brucei procyclic trypomastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate, and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM), a range that correlates favorably with that detected in the intact cells with fura-2. The carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive Ca2+ uptake, certainly represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. When vanadate instead of carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present, the Ca2+ set point was increased to 0.6-0.7 microM. The succinate dependence and carbonyl cyanide p-trifluoromethoxyphenylhydrazone sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. When bloodstream trypomastigotes were used, neither succinate nor alpha-glycerophosphate stimulated the mitochondrial Ca2+ uptake. The mitochondrial Ca2+ transport could be measured only in the presence of ATP and 500 microM vanadate to inhibit the endoplasmic reticulum uptake. Bloodstream trypomastigotes have a lower cytosolic Ca2+ concentration, as detected with fura-2 and a smaller extramitochondrial Ca2+ pool than procyclic trypomastigotes. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and the large extramitochondrial Ca2+ pool of procyclic trypomastigotes (61.7 nmol of Ca2+/mg of protein), no inositol 1,4,5-trisphosphate-sensitive Ca2+ release could be detected in these parasites.

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.