Passive Ca2 Research Articles

Effects of thyroid hormone on Ca 2+ efflux and Ca 2+ transport capacity in rat skeletal muscle

The present study was undertaken to investigate the effects of 3,5,3′-triiodothyronine (T 3) treatment on passive Ca 2+ efflux, Ca 2+-dependent Mg 2+-ATPase (Ca 2+-ATPase) concentration and active Ca 2+ transport in isolated rat skeletal muscle. In addition, the question was examined whether changes in Ca 2+ efflux at rest and during electrical stimulation in the hyperthyroid state were accompanied by parallel changes in 3-O-methylglucose efflux. The resting Ca 2+ efflux from rat soleus muscle was increased by 25% after 8 days of treatment with T 3 ( 20 μg 100 g body weight). This was associated with a 78% increase in the basal efflux of 3-O-methylglucose. Electrical stimulation resulted in a rapid stimulation of Ca 2+ efflux and 3-O-methylglucose efflux in the two groups of rats, and the levels obtained were significantly higher in the T 3-treated group. The stimulating effect of the alkaloid veratridine on Ca 2+ efflux was 60% larger in 8-day hyperthyroid rats. Within 24 h after the start of T 3 treatment, a significant (21%) increase in Ca 2+-ATPase concentration was detected. Significant increases in active Ca 2+ uptake and passive Ca 2+ efflux were not observed until after 2 and 3 days of T 3 treatment, respectively. It is concluded that T 3 stimulates the synthesis of Ca 2+ ATPase and augments the intracellular Ca 2+ pools (sarcoplasmic reticulum and mitochondria). The latter results in enhancement of the passive Ca 2+ leak, which in turn, may lead to activation of substrate transport systems. The suggested increase in intracellular Ca 2+ cycling after T 3 treatment may, at least partly, explain the T 3-induced stimulation of energy metabolism.

Stimulation of ATP-driven Ca 2+ pump in the basal-lateral plasma membranes of kidney cortex during compensatory renal growth

During compensatory renal growth 45Ca 2+ transport in basal-lateral plasma membrane vesicles isolated from the rat renal cortex have been investigated. Stimulation of Ca 2+-ATPase activity was observed, without an effect of compensatory renal growth on Na +/Ca 2+ exchanger activity and on passive Ca 2+ permeability of the vesicles. Twelve hours following unilateral nephrectomy about 40% increase of Ca 2+-ATPase activity above control value was observed and this effect was present until the end of the experimental period (7 days). When kinetic parameters for Ca 2+-ATPase were studied in native membranes, an increase V max was observed, whereas the K m for Ca 2+ was similar in control vesicles and vesicles isolated from the remnant kidney. Depletion of endogenous calmodulin resulted in a decrease of V max and an increase of K m(Ca 2+, while its addition reversed these parameters and increased the Hill coefficient from about 1 to about 2. Once again, only a significant increase of V max in vesicles isolated from the remnant kidney above the control value was observed. Finally, increase of Ca 2+-ATPase activity during compensatory renal growth could be abolished by actinomycin D, indicating that its stimulation is due to protein synthesis.

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

The effects of platelet-activating factor (PAF) on Na(+)-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 microM) significantly inhibited Na(+)-Ca2+ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na(+)-Ca2+ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na(+)-Ca2+ exchange, suggesting that the effect of PAF on Na(+)-Ca2+ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca2+ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na(+)-Ca2+ exchange. Interestingly, passive Ca2+ binding to cardiac sarcolemma was increased by 40 microM PAF. This study indicates that a depression of Na(+)-Ca2+ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na(+)-Ca2+ exchange is discussed.

Effects of active oxygen generated by [formula omitted] on cardiac [formula omitted] exchange and membrane permeability to Ca 2+

Sarcolemmal vesicles isolated from bovine heart were preincubated at 37°C with an oxygen radical generating system consisting of 1 m m dithiothreitol (DTT) and 50 μ m FeSO 4. Exposure of the vesicles for 1 to 40 mins stimulated Na + Ca 2+ exchange about 2.5-fold. The DTT Fe 2+ treatment decreased the apparent K m for Ca 2+ of Na i +-dependent Ca 2+ uptake by 80% (from 63 to 13 μ m). The effect on V max was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (95%) or catalase (60%). In contrast, superoxide dismutase and sodium formate did not prevent the effects of DTT Fe 2+ on the exchanger. Neither Zn 2+ nor Ga 3+ could replace Fe 2+ in the stimulation of Na + Ca 2+ exchange. Passive Ca 2+ efflux was determined by first allowing Na + Ca 2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. Ca 2+ leakage from the vesicles was slightly but significantly ( P < 0.05) increased by the action of DTT Fe 2+ , the rate constants for the passive Ca 2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytes in ischemia/reperfusion injury suggests that the stimulation of Na + Ca 2+ exchange by active oxygen may moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as the stimulation depends in part on the K m for Ca 2+ prior to treatment.

Ca 2+ transients and Mn 2+ entry in human neutrophils induced by thapsigargin

Human neutrophils, preloaded with the fluorescent probe, Fura-2, were exposed to Ca 2+-releasing agents. The monitored traces of fluorescence were transformed by computer to cytosolic Ca 2+ concentration ([Ca 2+] i). Due to quenching of Fura-2, the addition of Mn 2+ enabled us to compute the cytosolic concentration of total manganese ([Mn] i). The agents used were the novel Ca 2+-mobilizing agent, thapsigargin (Tg), the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), and the divalent cation lonophore, A23187. The agents caused translent rises of [Ca 2+] i and monotonous rises of [Mn] i, suggesting influx but no efflux of Mn 2+. The rise time of [Ca 2+] i and the time constants and magnitude of the apparent Mn 2+ influx were strongly dependent on the sequence of addition of the agonist and Ca 2+. Contrary to FMLP, Tg needed several minutes to exert its full effect on the rise of [Ca 2+] i and on the influx of Mn 2+, the latter being dependent on two phases, activation and partial inactivation. Pretreatment with phorbol 12-myristate 13-acetate (PMA) inhibited the responses of Tg, FMLP and A23187. For comparison, human red blood cells were tested. Contrary to A23187, Tg did not Induced Ca 2+ uptake in ATP-depleted red cells but increased the Ca 2+ pump flux in intact red cells by 10%. The experimental data and computer simulations of the granulocyte data suggest that time-dependent changes of both passive Ca 2+ flux into the cytosol and Ca 2+ flux of the plasma membrane pump are involved in the translent [Ca 2+] i response.

Evidence for the involvement of carboxyl groups in passive calcium uptake by liver plasma membrane vesicles and in agonist-induced calcium uptake by hepatocytes

The hydrophobic reagents DCCD and EEDQ, each of which reacts with protein carboxyl groups, were found to inhibit both passive Ca 2+ uptake by plasma membrane vesicles isolated from rat liver and agonist-induced Ca 2+ uptake by hepatocytes. The data raise the possibility that the Ca 2+ inflow pathway(s) in liver has a specific requirement for a reactive carboxyl group or groups.

Role of phosphatidylinositol in cardiac sarcolemmal membrane sodium-calcium exchange

The purpose of this investigation was to study the effects of a distinct type of phospholipase C on sarcolemmal Na+-Ca2+ exchange. With this phospholipase C (Staphylococcus aureus), treatment of cardiac sarcolemmal vesicles resulted in a specific hydrolysis of membrane phosphatidylinositol. This hydrolysis of phosphatidylinositol also released two proteins (110 and 36 kDa) from the sarcolemmal membrane. Phospholipase C pretreatment of the sarcolemma resulted in an unexpected stimulation of Na+-Ca2+ exchange. The Vmax of Na+-Ca2+ exchange was increased but the Km for Ca2+ was not altered. This stimulation was specific to the Na+-Ca2+ exchange pathway. ATP-dependent Ca2+ uptake was depressed after phospholipase C treatment, but passive membrane permeability to Ca2+ was unaffected. Sarcolemmal Na+,K+-ATPase activity was not altered, whereas passive Ca2+ binding was modestly decreased after phospholipase C pretreatment. The stimulation of Na+-Ca2+ exchange after phosphatidylinositol hydrolysis was greater in inside-out vesicles than in a total population of vesicles of mixed orientation. This finding suggests that the cardiac sarcolemmal Na+-Ca2+ exchanger is functionally asymmetrical. The results also suggest that membrane phosphatidylinositol is inhibitory to the Na+-Ca2+ exchanger or, alternatively, this phospholipid may anchor an endogenous inhibitory protein in the sarcolemmal membrane. The observation that a transsarcolemmal Ca2+ flux pathway may be stimulated solely by phosphatidylinositol hydrolysis independently of phosphoinositide metabolic products like inositol triphosphate is novel.

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum.

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplasmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (less than 100 micrograms/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28-50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (greater than 100 micrograms/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20-200 micrograms/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca 2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.

Heterogeneity of the effects of monensin on two types of contractions evoked by inhibition of Na +-K + ATPase in the guinea-pig vas deferens

The interactions between ouabain or K +-free medium and monensin on noradrenaline (NA) release and Ca 2+-evoked contractions were asseseed in guinea-pig vas deferens. Ouabain (10 μM) produced large, sustained contractions and momensin (1 μM) small, transient contractions. The ouabain-evoked contraction tended to be potentiated in the presence of monensin. The marked contraction induced by ouabain plus monensin was nearly abolished by phentolamine or by treatment with 6-hydroxydopamine. Ouabain caused the release of NA from the tissue, as determined in an HPCL-ECD study. This ouabain-evoked release of NA was enhanced by the simultaneous administration of monensin. High Ca 2+ (10 mM) per se, ouabain, monensin or K +-free medium did not elicit contraction of the tissue in the presence of phentolamine. However, 10 mM Ca 2+ caused a large contraction 45–65 min after exposure to ouabain or K +-free medium and this contraction was suppressed by monensin. The contraction evoked by Ca 2+ in the presence of ouabain was further inhibited by amiloride, an inhibitor of Na +-Ca 2+ or Na +-H + exchange transport, but not by nifedipine, a voltage-dependent Ca 2+ antagonist. In contrast, the high KCl-evoked contraction was notably reduced by nifedipine, but was virtually unaffected by monensin or amiloride. These results suggest that the contraction evoked by Ca 2+ in the presence of ouabain could be mediated by an influx of Ca 2+ via a passive Ca 2+ transport system, but not through voltage-dependent Ca 2+ channels. In contrast to the contractile response to ouabain alone, the reason why the contractile response to high Ca 2+ in the presence of the cardiac glycoside was markedly inhibited by monensin is discussed in relation to the ion transport system.

Cytokine-Induced Peritoneal Fibrosis in CAPD Effects of Ca++ and 1,25(OH)2D3

We have demonstrated in some continuous ambulatory peritoneal dialysis (CAPD) patients with ultrafiltration (UF) loss, the role of increased peritoneal lymphocyte (PLy) and macrophage (PMO) Ca++ concentrations in the release of large amounts of gamma-interferon (gamma-IFN) and Interleukin-1 (IL-1), which stimulate peritoneal fibroblast proliferation. We have also shown in vitro and in vivo that the calcium channel blocker verapamil (VPM) is able to normalize the previously high PLy and PM0 Ca++ concentrations, and cytokine release; to decrease fibroblast proliferation; and to increase UF in 60% of the CAPD patients with UF loss due to a cytokine-mediated hyperproliferation of peritoneal fibroblasts, while in the remaining 40% there is little improvement (VPM responders and low-responders, respectively). To evaluate which mechanisms, in addition to passive Ca++ influx, can play a role in the Ca++-dependent activation of peritoneal immune cells, we evaluated the effects in vitro of different doses of Ca++ alone; with VPM; and with 1,25(OH)2D3 on: 1) PLy and PM0 cytoplasmic Ca++ levels; 2) gamma-IFN and IL-1 release by PLy and PM0; and 3) peritoneal fibroblast proliferation in six CAPD VPM low-responder patients. Results showed a direct correlation between Ca++ levels in the medium and PLy and PM0 Ca++ concentrations; IL-1 and gamma-IFN release; and peritoneal fibroblast proliferation. These effects were enhanced by the addition of low doses of 1,25(OH)2D3 to the medium, while both high 1,25(OH)2D3 doses and verapamil abrogated the Ca++-induced PLy and PM0 activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of intracellular free Ca and rate of Ca influx on the Ca pump

The activity of the Ca pump of human red blood cells was studied in resealed ghosts as a function of intracellular free Ca (fCai). Resealed ghosts were made by the agarose column method to contain, in addition to other constituents, less than 0.1 microM fCai, 100 microM arsenazo III, and either 1 mM ATP plus an ATP regenerating system (active ghosts) or no added ATP and no regenerating system (passive ghosts). The rate of Ca influx into these ghosts was manipulated by suspending them in solutions containing various combinations of free Ca (1-30 microM) and the Ca ionophore A23187 (0.1-0.7 microM). Entering Ca increased the fCai and stimulated the pump in active ghosts. In passive ghosts, all the Ca movement could be described by a single rate constant. The activity of the Ca pump was calculated from the rate of net Ca uptake in the active ghosts, using the rate constant for passive Ca movement as determined in the passive ghosts. fCai and the rates of Ca transport in both active and passive ghosts were calculated from the absorbance of entrapped arsenazo III. In general, increasing fCai from 1 to 10 microM activated the pump. Higher fCai caused an inhibition compared with peak activity. The maximum rate of pumping was 80 microM/min. The major new finding is that the rate of active transport at a given fCai appeared to vary with the rate of fCai accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinction of two components of passive Ca 2+ transport into human erythrocytes by Ca 2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.

Passive calcium influx by plasma membrane vesicles isolated from rat liver

Passive Ca 2+ influx independent of ATP addition to the incubation medium, took place in plasma membrane vesicles isolated from rat liver. The rate of Ca 2+ influx was found to depend on the concentration of added Ca 2+, and on the incubation temperature, and was inhibited by La 3+, Hg 2+ and by p-chloromercuribenzoate. Influx was not blocked by calcium channel blockers, or affected by a range of uncouplers. Addition of the Ca 2+ ionophore A23187 to vesicles that had taken up the ion induced a rapid efflux of Ca 2+ especially when EGTA also was added to the incubation medium. A number of divalent cations inhibited Ca 2+ influx. The vesicles could be frozen and stored overnight with little loss in activity. The kinetics of Ca 2+ influx could be related to that which occurs in the unstimulated perfused rat liver. The data suggest that the plasma membrane vesicle preparation may be useful for further studies on the basal liver cell Ca 2+ influx system in vitro.

Regulation of the ATP dependent calcium uptake activity of heart sarcolemmal vesicles by endogenous cytosolic proteins

In a previous study we described the inhibitory action of a cytosolic protein fraction from heart muscle on ATP-dependent Ca2+ uptake by sarcoplasmic reticulum; further, this inhibition was shown to be blocked by an inhibitor antagonist, also derived from the cytosol (Narayanan et al. Biochim Biophys Acta 735: 53-66, 1983). The present study examined the effects of the endogenous cytosolic Ca2+ transport inhibitor and its antagonist on ATP-dependent Ca2+ uptake by sarcolemmal vesicles isolated from rat and canine heart. The cytosolic inhibitor caused strong inhibition (up to 97%) of Ca2+ uptake by sarcolemma (SL); this inhibition could be reversed by the cytosolic inhibitor antagonist. Studies on the characteristics of inhibition revealed the following: a) Inhibition was dependent on the concentration of the inhibitor (50% inhibition with approximately 80 micrograms inhibitor protein). b) The inhibitor reduced the velocity of Ca2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca2+ but caused greater than 2-fold decrease in its apparent affinity for ATP. c) The rates of unidirectional passive Ca2+ release from actively Ca2+ loaded SL vesicles were not altered by low concentrations of the inhibitor (less than 100 micrograms/ml) which were effective in producing marked inhibition of Ca2+ uptake; at higher concentrations (greater than 100 micrograms/ml), the inhibitor caused increase in the rates of passive Ca2+ release. These findings demonstrate that the activity of the ATP-driven Ca2+ pump of cardiac SL can be regulated in vitro by endogenous cytosolic proteins.

Effects of chronic swimming training on cardiac sarcolemmal function and composition.

Cardiac contractile function is dependent on the integrity and function of the sarcolemmal membrane. Swimming exercise training is known to increase cardiac contractile performance. The purpose of the present study was to examine whether a swimming exercise program would alter sarcolemmal enzyme activity, ion flux, and composition in rat hearts. After approximately 11 wk of exercise training, cardiac myosin and actomyosin Ca2+-adenosinetriphosphatase (ATPase) activity was significantly higher in exercised rat hearts than in sedentary control rat hearts. Glycogen content was increased in plantaris and gastrocnemius muscles from exercised animals as was succinic dehydrogenase activity in gastrocnemius muscle of exercised rats in comparison to sedentary rat preparations. Sarcolemmal vesicles were isolated from hearts of exercise-trained and control rats. Sarcolemmal Na+-K+-ATPase and K+-p-nitrophenylphosphatase activities, Na+-Ca2+ exchange, and passive Ca2+ binding did not differ between the two groups. ATP-dependent Ca2+ uptake and 5'-nucleotidase activity were elevated in the cardiac sarcolemmal vesicles isolated from exercised animals compared with sedentary control rats. Sarcolemmal phospholipid composition was not altered by the exercise training. Our results demonstrate that swimming training in rats does not affect most parameters of cardiac sarcolemmal function or composition. However, the elevated sarcolemmal Ca2+ pump activity in exercised rats may help to reduce intracellular Ca2+ and augment cardiac relaxation rates. The enhanced 5'-nucleotidase activity may stimulate adenosine production, which could affect myocardial blood flow. The present results further our knowledge on the subcellular response of the heart to swimming training in the rat.

Na+-Ca2+ exchange in cardiac sarcolemma: modulation of Ca2+ affinity by exercise.

The high activity of the cardiac Na+-Ca2+ exchanger has led to the suggestion that it plays an important role in the regulation of myocardial contractility. We have proposed that exercise training increases stroke volume as a consequence of an enhanced contractility caused by an adaptation in Ca2+ transport across the cardiac plasma membrane (sarcolemma). The present study examined the possibility that the Na+-Ca2+ exchanger in heart muscle is modified in response to training. Sprague-Dawley rats (female, n = 72) were randomly divided into exercise-trained (T) and sedentary control (C) groups. As a result of the 11-wk treadmill-training paradigm, group T had a 7.6% higher (P less than 0.005) heart-to-body weight ratio and a 36% increase (P less than 0.01) in gastrocnemius mitochondrial enzyme activity. Na+-Ca2+ exchange was studied in highly purified sarcolemmal vesicles using rapid-quenching techniques. The absolute initial rate of uptake was significantly higher in T vs. C at calcium concentrations [( Ca2+]) ranging from 10 to 80 microM. This increased uptake appears to be due solely to the fact that the apparent Km of the myocardial Na+-Ca2+ exchanger for Ca2+ was significantly lower in T vs. C (15.7 +/- 1.1 vs. 36.1 +/- 2.6 microM), since the maximum velocity was unchanged. The observed increase in the affinity of the exchanger for Ca2+ is not attributable to group differences in vesicular purity, cross-contamination, or passive Ca2+ efflux. This observation is consistent with observed alterations in sarcolemmal composition in response to exercise training. We propose that the modification of the Na+-Ca2+ exchanger may play an important role in the adaptation of the heart to exercise.

Possible mechanism responsible for mechanical dysfunction of ischemic myocardium: A role of oxygen free radicals.

It has been proposed that a major target organelles damaged by the ischemic process, probably by the oxygen free radicals generated, is the portion of the excitation-contraction coupling system that regulates Ca2+ delivery (the sarcoplasmic reticulum and sarcolemma) to the contractile proteins. We tested this hypothesis by studying the effect of in vitro generation of oxygen free radicals from xanthine-xanthine oxidase system or dihydroxyfumarate (DHF)/Fe3+-ADP system on Ca2+ flux behavior of canine cardiac sarcoplasmic reticulum (SR); sarcolemmal (Na+, K+)-ATPase and Na+-Ca2+ exchange activities; and myofibrillar (Ca2+, Mg2+)-ATPase activity. Generation of oxygen free radicals by xanthine oxidase acting on xanthine as a substrate increased the passive Ca2+ efflux and decreased intravesicular Ca2+ with no effect on active Ca2+ influx (Ca2+-ATPase) of SR vesicles. Similar exposure of sarcolemmal vesicles to xanthine plus xanthine oxidase stimulated Na+-Ca2+ exchange activity. When sarcolemmal vesicles were incubated with DHF plus Fe3+-ADP, (Na+, K+)-ATPase activity was decreased. It is postulated that the SR Ca2+ efflux pathways but not catalytic activity of the Ca2+ pump and sarcolemmal (Na+, K+)-ATPase involving Na+-Ca2+ exchange activity are altered by oxygen free radicals, and such changes may partly account for the occurrence of intracellular Ca2+ overload during the course of myocardial ischemia. Interestingly, oxygen free radicals from xanthine-xanthine oxidase system had no effect on myofibrillar pCa-ATPase curve. From this set of observations we would hypothesize that the SR and sarcolemma may be the principal target organelles of oxygen free radicals attack in the ischemic injury and not the contractile proteins per se.

Transport Properties of the Tomato Fruit Tonoplast

Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca(2+) transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca(2+). A low affinity Ca(2+) uptake system (K(m) > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H(+)/Ca(2+) antiport. A high affinity Ca(2+) uptake system (K(m) = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca(2+) transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12 degrees C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca(2+)/H(+) antiport activity could only by partially ascribed to an effect of low temperature on H(+)-ATPase activity, ATP-dependent H(+) transport, passive H(+) fluxes, or passive Ca(2+) fluxes. These results suggest that low temperature directly affects Ca(2+)/H(+) exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca(2+)/H(+) exchange protein or by an indirect effect of temperature on lipid interactions with the Ca(2+)/H(+) exchange protein.

Na +[sbnd]Ca 2+ exchange and calcium permeability in canine basolateral membrane vesicles: The effects of dibutyryl cAMP and specific inhibitors

The role of dibutyryl 3′,5′-cyclic adenosine monophosphate (dibutyryl cAMP) as putative second messenger for parathyroid hormone (PTH) in regulating canine proximal tubular basolateral membrane Na + Ca 2+ exchange and passive calcium permeability was assessed, as was the nature of this passive calcium permeability. Dibutyryl cAMP (50 mg) infused in vivo over 30 min increased fractional phosphate excretion from 4.9 ± 1.8%to20.5 ± 4.6%, P < 0.05, n = 6, but had no effect on either passive Ca 2+ efflux or sodium-stimulated Ca 2+ efflux from Ca 2+-preloaded basolateral membrane vesicles (BLMV). Both of these mechanisms have been previously shown to be stimulated by PTH. Further studies were performed to investigate the mechanism of the passive calcium flux. Calcium uptake by BLMV was blocked by lanthanum (La 3+) but not by the calcium-channel blocker verapamil. La 3+ blocked efflux of Ca 2+ from preloaded vesicles when it was placed in the external solution. This La 3+-blockable efflux was larger in potassium equivalent BLMV prepared from normal dogs than in the BLMV prepared from thyroparathyroidectomized dogs. Benzamil produced 50% inhibition of sodium-stimulated Ca 2+ uptake at 250 μM whereas neither amiloride nor diltiazem achieved 50% inhibition at the maximal doses studied. Benzamil, 1 mM, had no effect on passive calcium efflux and neither did the substitution of sucrose for potassium, which has been shown to affect Ca 2+ Ca 2+ exchange by the Na 2+ Ca 2+ exchanger. This suggests that the calcium flux under potassium equivalent conditions was not mediated by Ca 2+ Ca 2+ exchange by the Na 2+ Ca 2+ exchanger. These results demonstrate that the basolateral membrane of proximal tubular cells possesses both a Na 2+ Ca 2+ exchanger inhibitable by benzamil and a passive calcium permeability not inhibited by benzamil nor by verapamil but by La 3+. Neither of these two mechanisms of calcium flux was affected by dibutyryl cAMP whereas both have been shown to be stimulated by PTH.