Initial Rate Of Ca2 Research Articles

Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ± 0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period.

Acute Tobacco Smoke Exposure Promotes Mitochondrial Permeability Transition in Rat Heart

Chronic exposure to tobacco smoke is known to impair mitochondrial function. However, the effect of acute tobacco smoke exposure (ATSE) in vivo, as might occur in social settings, on mitochondrial function and calcium handling of cardiac cells has not been examined. It was hypothesized that ATSE might adversely modify mitochondrial function as reflected in mitochondrial energetics, membrane potential, and calcium transport. Mitochondria were isolated from the hearts of adult rats either exposed to 6 h of environmental tobacco smoke (∼60 mg/mm3 tobacco smoke particles) or sham exposure. To model a calcium stress similar to ischemia/reperfusion, mitochondria were exposed to a Ca2+ bolus with measurement of membrane potential, energetics, Ca2+uptake and release, and redox state. ATSE mitochondria were characterized by significantly higher ADP-stimulated ATP production and a more reduced redox state (NADH ratio) under basal conditions without observed changes in resting Ψm. Exposure of ATSE mitochondria to Ca2+stress resulted in significantly more rapid depolarization of Ψm. The initial rate of Ca2+uptake was not altered in ATSE mitochondria, but CsA-sensitive Ca2+ release was significantly increased. ATSE does not significantly alter resting mitochondrial function. However, ATSE modifies the response of cardiac mitochondria to calcium stress, resulting in a more rapid depolarization and subsequent release of Ca2+ via the mitochondrial permeability transition (MPT). This research was supported by a Philip Morris External Research program grant. We thank Mike Goldsmith and Dale Uyeminami at the Center for Health and Environment for invaluable assistance in the smoke exposure experiments.

Calcium Signals Are Affected by Ciprofloxacin as a Consequence of Reduction of Mitochondrial DNA Content in Jurkat Cells

The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular ATP formation, and capacitative Ca(2+) entry into Jurkat cells were investigated. In cells incubated for several days with 25 mug/ml ciprofloxacin, a 60% reduction of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a gradual inhibition of the capacitative Ca(2+) entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca(2+) entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin at a concentration of 25 mug/ml seriously affects cellular energy metabolism and calcium homeostasis.

Functional Coupling between TRPC3 and RyR1 Regulates the Expressions of Key Triadic Proteins

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.

Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-dependent refilling of Ca2+ stores.

Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca2+ concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca2+/Mn2+ influx in intact platelets and to monitor Ca2+ uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca2+ and Mn2+ influx developed rapidly (within 30 s) after fast release of Ca2+ from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca2+ leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) with 30 micromol/L 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). NO (1 micromol/L) inhibited capacitative Ca2+ and Mn2+ influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca2+ and Mn2+ influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca2+-free medium, NO reduced the peak Ca2+ rise caused by thrombin and significantly promoted Ca2+ back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 micromol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca2+ concentration. Inhibition of Ca2+ uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca2+ into the stores after inositol-1,4,5-trisphosphate-induced Ca2+ release or after addition of Ca2+ (1 micromol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca2+ uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca2+ stores.

Effects of La3+ and Gd3+ on Ca2+ influx in rat hepatoma H-35 cells

Effects of La3+ and Gd3+ on Ca2+ influx were investigated in rat hepatoma H-35 cells by measuring the initial rate of Ca-45(2+) uptake. It was found that the maximum initial rate of Ca2+ uptake was increased six- to ten-fold at low concentrations of La3+ and Gd3+. Kinetic analyses by measuring the initial rate of Ca2+ influx at different external Ca2+ concentrations indicated the existence of two intracellular exchangeable components in the basal Ca2+ system, with low and high affinities for Ca2+, and only one class of Ca2+ binding sites was observed in the La3+- or Gd3+-treated cells. For high affinity, La3+ and Gc(3+) increased both kinetic parameters K-m and V-max of basal Ca2+ influx. La3+ and Gd3+ compete directly with Ca2+ for Ca2+ binding site for low affinity. The kinetics is competitive.

ERcalcistorin/Protein-disulfide Isomerase Acts as a Calcium Storage Protein in the Endoplasmic Reticulum of a Living Cell: COMPARISON WITH CALRETICULIN AND CALSEQUESTRIN

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI), a high capacity low affinity Ca2+-binding protein in the endoplasmic reticulum of sea urchin eggs (Lebeche, D., and Kaminer, B. (1992) Biochem. J. 287, 741-747), shares 55% sequence identity with mammalian PDI and has PDI activity (Lucero, H., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112-23119). We report on ECaSt/PDI functioning as a Ca2+ storage protein in the endoplasmic reticulum (ER) of a living cell and compare it with calsequestrin and calreticulin, high capacity low affinity Ca2+-binding proteins in the sarcoplasmic reticulum and ER, respectively. Stably transfected Chinese hamster ovary cell clones expressed these proteins, which were localized in the ER of the cell. Microsomes from cells expressing ECaSt/PDI, calreticulin, and calsequestrin accumulated 17.2 +/- 0.27, 20.0 +/- 0.82, and 38.0 +/- 0.28 nmol of Ca2+/mg of protein, respectively; control microsomes accumulated from 2.6 +/- 0.17 to 2.9 +/- 0.14 nmol of Ca2+/mg of protein. The initial rate of Ca2+ uptake was similar in microsomes from transfected and control cells. Microsomes containing an ECaSt/PDI mutant in which 45% of the acidic residue pairs in the C terminus were truncated had a reduced Ca2+ storage capacity. This supports our previous hypothesis that the degree of low affinity Ca2+ binding is dependent on the number of pairs of carboxyl groups in the molecule. The maximal Ca2+ accumulation by microsomes containing the expressed ECaSt/PDI, C-terminally truncated ECaSt/PDI, calreticulin, or calsequestrin correlates approximately with the Ca2+ binding capacity of the respective proteins.

Voltage-dependent Ca 2+ uptake by right-side-out plasma membrane vesicles derived from maize shoots

Cellular responses to a variety of environmental, developmental and pathological stimuli are initiated by an increase in cytoplasmic Ca2+. This may be effected by the opening of Ca2+ channels in the plasma membrane, activated by membrane depolarisation. The presence of such Ca2+ channels in plasma membranes from shoots (coleoptile plus rolled leaves) of maize (Zea mays L. var. Clipper) is demonstrated. Voltage-dependent Ca2+ uptake by right-side-out plasma membrane vesicles derived from shoots of maize was characterised using a valinomycin/K+ gradient voltage-clamp. When assayed at −125 mV, Ca2+ accumulation by vesicles increased linearly over an initial period of 10 min. Thereafter, the rate of Ca2+ uptake declined, and vesicle content of Ca2+ reached a constant value after about 1 hour. Addition of A23187 after uptake released Ca2+ from vesicles, and the intravesicular Ca2+ concentration after 1 hour suggested voltage-driven Ca2+ uptake. The initial rate of Ca2+ uptake (Ca2+ influx, measured over a 5 min period) increased as the voltage was clamped to more negative values between zero and −125 to −150 mV, then declined at extreme negative voltages. These are characteristics of a channel activated by depolarisation. When assayed at −125 mV, Ca2+ influx increased with increasing extravesicular concentration to a maximum at between 25 to 100 μmol/L CaCl2. As extravesicular CaCl2 concentration was increased further, Ca2+ influx progressively declined. Ca2+ influx was inhibited by micromolar concentrations of divalent cations (Ba2+ > Sr2+ > Mg2+), trivalent cations (Nd3+ > Al3+ > Gd3+ > La3+) and ruthenium red. Ca2+ influx was not affected by micromolar concentrations of Cs+, nifedipine, verapamil or diltiazem. Tetra-ethylammonium blocked Ca2+ influx when applied extravesicularly at millimolar concentrations. The characteristics and physiological role of the putative Ca2+ channel described here are discussed with reference to previously characterised Ca2+ channels.

Effect of emergent and long-term adaptation to physical strength on the resistance of Ca-transporting system of myocardial sarcoplasmic reticulum

Experiments were carried out on rats adapted to physical exercise (on the next day after completion of 4, 11, 15, and 30 swimming sessions). Catalase and superoxide dismutase activities were similar in all rats. The resistance of Ca transport into sarcoplasmic reticulum to high Ca2+ concentrations and autooxidaton increased starting from 4 swimming sessions, and to thermal inactivation from 11 sessions; the maximum resistance was attained after 15 sessions 1.5- to 2-fold surpassing the initial level. Maximum initial rate of Ca2+-transport (155% of the control) was observed after 30 swimming sessions. In acute physical strength and at the initial stages of adaptation (4 swimming sessions) functional properties of myocardial Ca-transporting system were preserved under optimal conditions, but can be readily disturbed by adverse factors.

Role of Platelet-Activating Factor in Hepatocellular Ca2+Alterations during Hemorrhagic Shock

A role of the potent proinflammatory Ca2+agonist platelet-activating factor (PAF) on hepatocellular Ca2+homeostasis and oxidant injury was investigated, since Ca2+dysregulation has been demonstrated as a pivotal pathomechanism causing hepatic dysfunction during hemorrhagic or septic shock. In anesthetized male Sprague-Dawley rats, blood was withdrawn to a mean arterial blood pressure of 40 mm Hg for 60 min. Rats were resuscitated with 60% of shed blood and threefold the shed blood volume of lactated Ringers' (shock group). After 60 min of resuscitation, hepatocytes were isolated by portal collagenase perfusion. Hepatocellular Ca2+uptake (Caup2+), initial rate of Ca2+influx (Cain2+), and membrane Ca2+flux (Caflux2+) were determined using45Ca2+incubation techniques. Hepatocyte oxidant injury was fluorometrically determined by thiobarbituric acid reactive substances. Caup2+(3.37 ± 0.15 vs. 2.53 ± 0.08 nmole Ca2+/mg protein), Cain2+(0.42 ± 0.1 vs. 0.27 ± 0.02 nmole Ca2+/mg protein/min), and Caflux2+(31.3 ± 4.3 vs. 16.9 ± 2.4 pmole Ca2+/mg/min) significantly increased in the untreated shock group compared to untreated sham-operated rats (P< 0.05). The specific PAF receptor antagonist BN52021 given 5 min before (5 mg/kg b.w.) and continuously during resuscitation (5 mg/kg/hr) significantly reduced Caup2+in the shock group (2.73 ± 0.2;P< 0.01) and prevented hepatocyte lipid peroxidation (shock: 91.9 ± 1; shockBN52021: 66.7 ± 2 nmole/mg wet weight;P< 0.01). These data suggest that platelet-activating factor plays a pivotal role in promoting hepatocyte Ca2+overload during hemorrhagic shock by releasing Ca2+agonistic mediators and inducing oxidative membrane alterations both of which are capable of enhancing cellular Ca2+influx.

Inositol 1,4,5-Trisphosphate and Calcium Regulate the Calcium Channel Function of the Hepatic Inositol 1,4,5-Trisphosphate Receptor

The regulation of the inositol 1,4,5-trisphosphate (IP3) receptor in liver was analyzed using a novel superfusion method. Hepatic microsomes were loaded with 45Ca2+, and superfused at high flow rates to provide precise control over IP3 and Ca2+ concentrations ([Ca2+]) and to isolate 45Ca2+ release from reuptake. 45Ca2+ release was dependent on both [Ca2+] and IP3. The initial rate of 45Ca2+ release was a biphasic function of [Ca2+], increasing as [Ca2+] approached 3 microM but decreasing at higher concentrations, suggesting that the hepatic IP3 receptor is regulated by [Ca2+] at two sites, a high affinity potentiation site and a low affinity inhibitory site. The relationship between initial rates and IP3 concentration was steep (Hill coefficient of 3.4), suggesting that activation of the calcium channel requires binding of at least 3 IP3 molecules. IP3 concentrations above 10 microM produced rapid decay of release rates, suggesting receptor inactivation. Superfusion with 10 microM IP3 under conditions that minimize calcium release ([Ca2+] < 1 nM) inhibited 45Ca2+ release in response to subsequent stimulation (400 nM Ca2+). These data suggest sequential positive and negative regulation of the hepatic IP3 receptor by cytosolic calcium and by IP3, which may underlie hepatocellular propagation of regenerative, oscillatory calcium signals.

Coordinate regulation of SR Ca(2+)-ATPase and phospholamban expression in developing murine heart.

Phospholamban, the regulator of Ca(2+)-adenosinetriphosphatase (ATPase) activity in cardiac sarcoplasmic reticulum (SR), is an important determinant of basal myocardial performance. To determine whether phospholamban expression is developmentally regulated in the mouse and whether such regulation reflects alterations in Ca2+ pump activity, hearts from different stages of development were processed for molecular biological and biochemical studies. Both phospholamban and Ca(2+)-ATPase mRNAs were approximately 40% of adult (100%) levels at birth and gradually increased to approach adult levels by day 15 of development. These changes in transcript levels were indicative of changes at the protein level for both phospholamban and Ca(2+)-ATPase. Analysis of the initial rates of Ca2+ uptake demonstrated that over the course of development the upregulation of Ca(2+)-ATPase correlated with increases in the maximal rates of Ca2+ uptake and the constant apparent stoichiometric ratio of phospholamban to Ca(2+)-ATPase correlated with maintenance of a constant affinity of this enzyme for Ca2+ (0.25 +/- 0.03 microM Ca2+). Furthermore, targeted ablation of phospholamban in the mouse resulted in a much higher affinity of Ca2+ uptake for Ca2+ (0.10 +/- 0.02 microM Ca2+) than that observed in wild-type hearts, and this increased affinity was also maintained across different stages of postnatal development. These findings suggest that phospholamban is a major regulator of the affinity of Ca(2+)-ATPase for Ca2+, and coordinate regulation of the expression levels of these two SR proteins may be necessary for maintaining Ca2+ homeostasis in the developing mammalian heart.

Opposite effect of adaptation to physical exercise on myocardium and skeletal muscle. Ca-transporting system of the sarcoplasmic reticulum and antioxidant defense enzymes

Adaptation to physical exercise was achieved via 60-min sessions of swimming at 32°C for 45 days, the duration of swimming being increased from 15 to 60 min during the first 14 days. Under these conditions, against the background of reduced catalase and superoxide dismutase activity the Ca-transporting system of the sarcoplasmic reticulum in the heart is shown to work more effectively: Ca2+ transport is characterized by a higher initial rate and is inactivated 1.5 times more slowly byin vitro-induced lipid peroxidation and not inhibited by high concentrations of free Ca2+. In the skeletal muscle, on the other hand, catalase and superoxide dismutase activity rise, but this does not improve the functioning of the Ca pump: the initial rate of Ca2+ transport drops, its resistance to autooxidation is not increased in comparison with the control, and the resistance of the Ca2+-transporting system to the inhibiting influence of free Ca2+ is lowered.

Highly Supralinear Feedback Inhibition of Ca2+ Uptake by the Ca2+ Load of Intracellular Stores

Net Ca2+ uptake into intracellular Ca2+ stores of homogenized cells is transient, even when the extravesicular Ca2+ concentration is kept constant. To study the mechanism underlying the phenomenon, we have investigated 45Ca2+ uptake by HL-60 cell homogenates. The initial rate of Ca2+ uptake as well as the final amount of stored Ca2+ were a function of the extravesicular Ca2+ concentration. However, Ca2+ uptake stopped independently of the extravesicular Ca2+ concentration after approximately 10 min. Studies using Ca2+-ATPase inhibitors demonstrated that the transient nature of the net uptake was not due to Ca2+ efflux. Monovalent cation ionophores did not influence the Ca2+ uptake curves, excluding a relevant involvement of pH and membrane potential. Together with the observation of a continued Ca2+ uptake in the presence of the intralumenal Ca2+ chelator oxalate, these results strongly suggest a feedback inhibition of Ca2+ uptake by the Ca2+ load of intracellular stores. The concentration-inhibition relationship between the Ca2+ load and the rate of Ca2+ uptake was highly supralinear (slope factor >/= 4). IC50 and maximum of the dose-inhibition curve, but not the slope factor were a function of the extravesicular free Ca2+ concentration. A series of three logistic equations derived from our data allowed an appropriate description of the behavior of Ca2+ uptake. Our results suggest, in addition to its well known activation by cytosolic Ca2+ concentration, a highly supralinear feedback inhibition of Ca2+ uptake by the Ca2+ load of intracellular stores. The steepness of the feedback inhibition might have a profound effect on spatial and temporal behavior of the Ca2+ signal.

Selective inhibition of oxalate-stimulated Ca2+transport by cyclopiazonic acid and thapsigargin in smooth muscle microsomes

45Ca2+ uptake and efflux studies were performed on membranes prepared from dog mesenteric artery and rat vas deferens. Oxalate-stimulated, ATP-dependent Ca2+ uptake in microsomal vesicles, a property characteristic of sarcoplasmic reticulum, was completely inhibited in a concentration-dependent manner by cyclopiazonic acid (0.1-30 microM) and thapsigargin (10 nM-10 microM). Using discontinuous sucrose gradient centrifugation, rat vas deferens microsomes were separated into two fractions, one enriched in plasma membrane (F2), the other enriched in sarcoplasmic reticulum (F3). The F3 fraction had a major increase in Ca2+ uptake in the presence of oxalate, which was completely inhibited by either cyclopiazonic acid or thapsigargin. In the F2 fraction Ca2+ uptake in the presence of oxalate was lower than in F3 and was not completely inhibited by thapsigargin and cyclopiazonic acid. Instead, the F2 fraction had a thapsigargin-insensitive and cyclopiazonic acid insensitive, saponin-sensitive component of uptake, which probably represents Ca2+ uptake by plasma membrane. In the absence of oxalate, the inhibition of Ca2+ uptake by saponin and cyclopiazonic acid or thapsigargin was additive in the F2 and F3 fractions, suggesting that cyclopiazonic acid and thapsigargin selectively inhibited sarcoplasmic reticulum derived Ca2+ uptake and did not affect plasma membrane derived Ca2+ uptake. Measurement of the initial rate of Ca2+ uptake in the presence and absence of oxalate by rat vas deferens microsomes demonstrated selective inhibition of oxalate-stimulated Ca2+ uptake by cyclopiazonic acid and thapsigargin. Ca2+ efflux from rat vas deferens microsomes actively loaded with 45Ca2+ either in presence or the absence of oxalate was not increased by cyclopiazonic acid or thapsigargin, showing that the inhibition of Ca2+ accumulation was not due to an increase in Ca2+ efflux. In both rat vas deferens and dog mesenteric artery, the maximal inhibitory effects of cyclopiazonic acid developed rapidly, whereas for maximal inhibition thapsigargin required pretreatment of microsomes prior to measurement of Ca2+ uptake. In rat vas deferens microsomes the inhibitory effects of cyclopiazonic acid could be quickly and completely reversed, whereas the effects of thapsigargin were not easily reversed. Collectively, these results suggest selectivity of cyclopiazonic acid and thapsigargin for the sarcoplasmic reticulum Ca2+ pump. Their selective inhibitory properties and differences in onset and offset of inhibition make cyclopiazonic acid thapsigargin useful pharmacological tools in the study of the physiological and pathophysiological roles of the sarcoplasmic reticulum Ca2+ pump in regulating smooth muscle Ca2+.

Alterations of cytosolic calcium in platelets and erythrocytes of lyon hypertensive rats

Platelet cytosolic free calcium concentration ([Ca2+]i) and intracellular pH (pHi) (including their responses to thrombin), as well as erythrocyte [Ca2+]i and 45Ca2+ influx, were studied in Lyon hypertensive (LH) and normotensive (LN) rats aged 3 months. Platelets of LH rats were characterized by substantially elevated basal [Ca2+]i values, higher [Ca2+]i levels after thrombin stimulation, and enhanced initial rate of thrombin-induced Mn2+ entry through receptor-operated Ca2+ channels. Basal platelet pHi values were not significantly different in LH and LN animals but thrombin elicited a significant alkalinization only in LH platelets. Erythrocytes of LH rats had an enhanced initial rate of 45Ca2+ and tended to elevated [Ca2+]i levels. Our data indicate profound alterations in cell Ca2+ handling in platelets and erythrocytes of LH rats, similar to those previously described in spontaneously hypertensive rats of the Okamoto-Aoki strain. The analysis of the relations between blood pressure, plasma lipids, and cell Ca2+ handling suggested that triglycerides, but not cholesterol, might be involved in altered platelet Ca2+ handling in LH rats.

Cryopreservation extenders affect calcium flux in bovine spermatozoa during a temperature challenge.

Bovine spermatozoa are commercially cryopreserved by diluting the cells in media, known as extenders, followed by slow cooling and freezing. Previous work has shown that this process of cryopreservation alters the cells' ability to control divalent calcium (Ca2+) movement. This study evaluated the effect of a brief exposure to common extenders on bovine spermatozoa during subsequent cooling and rewarming. Three fresh ejaculates from each of three bulls were each split and incubated for 30 minutes at 25 degrees C in milk extender or phosphate-buffered saline (PBS) (control); three other fresh ejaculates from each of three bulls were similarly incubated in egg yolk-Tris extender (EYT) or PBS. Spermatozoa were washed and the fluorescent Ca2+ indicator, indo-1 acetoxymethyl ester, was used to monitor the internal Ca2+ in the spermatozoa in Ca(2+)-free PBS over a continuous temperature gradient of 25 degrees C (15 minutes), cooling to 5 degrees C (32 minutes), at 5 degrees C (15 minutes), rewarming to 25 degrees C (25 minutes), and at 25 degrees C (15 minutes). Milk exposure reduced the initial percentage of missing acrosomes and EYT exposure improved the initial viability and acrosome morphology compared to the controls; only milk immediatetly increased internal Ca2+. The initial rate of Ca2+ uptake at 25 degrees C was greater for milk or EYT-exposed spermatozoa than controls (P < 0.05). During cooling, the rate of Ca2+ uptake in all spermatozoa increased (P < 0.01), and it continued to increase during the 15 minutes at 5 degrees C. During rewarming to 25 degrees C, the internal Ca2+ in all spermatozoa declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid filtration studies of the effect of cytosolic Ca2+ on inositol 1,4,5-trisphosphate-induced 45Ca2+ release from cerebellar microsomes

Using microsomal membrane vesicles derived from sheep cerebellum, we measured the rate of inositol 1,4,5-trisphosphate (InsP3)-dependent 45Ca2+ efflux from 45Ca(2+)-loaded compartments during rapid perfusion with a medium containing InsP3 and various concentrations of free 40Ca2+ on the cytosolic side (pH 7.1, 5 mM Mg2+, in the absence of ATP at 20 degrees C). At 0.15 microM InsP3 and pCa 6.5, half-45Ca2+ release was attained within less than 200 ms. At low Ca2+ concentrations, the initial rate of 45Ca2+ release depended smoothly on InsP3 concentration, and InsP3 activated release with moderate positive cooperativity. Preliminary experiments performed at various free 40Ca2+ concentrations were consistent with a bell-shaped 40Ca2+ dependence of 45Ca2+ release. In the range of micromolar or higher free 40Ca2+ concentrations, the apparent inhibition of 45Ca2+ release was dependent on InsP3 concentration, and 45Ca2+ release for intermediate InsP3 concentrations was transient; under selected conditions, a second perfusion period, identical to the first one but separated from it by a short recovery period, was found to allow renewed 45Ca2+ efflux. At high Ca2+ concentration, fast reversible Ca(2+)-dependent desensitization of the channel, and not heterogeneity, was therefore responsible for the termination of InsP3-triggered 45Ca2+ efflux at submaximal concentrations of InsP3. At lower Ca2+ concentrations, a large fraction of the apparent activating effect of submicromolar 40Ca2+ concentrations on 45Ca2+ efflux that we had observed in the preliminary experiments proved to be the artifactual consequence of an inhibitory effect exerted by metal-free Ca2+ chelators on InsP3-dependent efflux at nanomolar 40Ca2+ concentrations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3 were all effective inhibitors. When this inhibition was taken into account, a rise in free 40Ca2+ concentration from 30 to 300 nM only weakly enhanced 45Ca2+ fluxes in the presence of a low concentration of InsP3. As a result, submicromolar free 40Ca2+ appears to be only a poor activator of InsP3-induced Ca2+ release under these experimental conditions.

Kinetics of calcium transport across the lymphocyte plasma membrane

We have investigated plasma membrane Ca2+ transport by monitoring the fluorescence of human peripheral T-lymphocytes loaded with fura 2. Thapsigargin (TG) was utilized the block the Ca(2+)-ATPase of the endoplasmic reticulum and elevate the cytosolic Ca2+ (Ca2+i). Ca2+ influx was inhibited by chelating extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The rate of decline in the Ca2+i signal of TG-treated lymphocytes after exposure to EGTA was used to assess Ca2+ extrusion across the plasma membrane. Initial rates of Ca2+i decline were examined in cells suspended in Na(+)-containing and Na(+)-free solutions; initial rates were linearly related to the [Ca2+]i at the onset of the Ca2+i decline and were unaffected by varying the extracellular Ca2+. Extracellular Na+ increased the rate of Ca2+ extrusion and decreased the threshold [Ca2+]i for extrusion, indicating a substantial role for the Na(+)-Ca2+ exchange in Ca2+i homeostasis. Both decreased temperature and calmodulin inhibition significantly slowed the Ca2+i decline in Na(+)-free HEPES-buffered solution, suggesting Ca2+ extrusion under these conditions was mediated by the Ca2+ pump. Protein kinase C (PKC) activation or inhibition did not affect the Ca2+i decline parameters. However, Ca2+ accumulation and Mn2+ (a Ca2+ surrogate) uptake were significantly and Mn2+ (a Ca2+ surrogate) uptake were significantly inhibited by activators of PKC. Cyclic nucleotides altered neither the parameters of the Ca2+i decline nor Mn2+ uptake. Thus human T-lymphocytes exhibit Na(+)- and Ca(2+)-dependent transporters characterized as the Na(+)-Ca2+ exchanger and Ca2+ pump. The main effect of PKC in these cells is the modulation of Ca2+ entry across the lymphocyte plasma membrane.