Net Ca Research Articles

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.

PHe, [Ca2+]e, and cell death during metabolic inhibition: role of phospholipase A2 and sarcolemmal phospholipids.

This study measures cellular lactate dehydrogenase (LDH) release during metabolic inhibition as a monitor of sarcolemmal integrity as affected by variation of external pH (pHe) and Ca2+ concentration ([Ca2+]e). The sigmoidal relationship between pHe and LDH release and pHe and net Ca2+ uptake was essentially identical with the 50% maximal value occurring at pH 7.0 for both. This suggests that a process(es) sensitive to both pHe and [Ca2+]e plays a role in cell lysis during the course of metabolic inhibition. Variation of pHe during metabolic inhibition did not alter the decline in cellular ATP, nor did it affect changes in sarcolemmal phospholipid topology. Intracellular pH followed changes of pHe with a few minutes lag. Cell lysis increased in a graded manner as pHe and [Ca2+]e were increased, but pHe was the sole determinant of lysis, i.e., [Ca2+]e level had no effect, at the lowest (6.2) and the highest (8.0) pHe levels. pHe variation did not affect the release of radiolabeled arachidonic acid, nor did inhibitors of phospholipase A2 (PLA2) affect cell lysis at varying pHe. Therefore, cellular PLA2 activation could not be implicated for a role in cell lysis in the present model of metabolic inhibition. Alternatively, we propose that Ca2+ binding to the cytoplasmic leaflet, in combination with membrane alterations secondary to the metabolic insult, combine to destabilize the sarcolemma (20). This Ca2+ binding to the negatively charged phosphatidylserine results in the expression of the bilayer destabilizing effect of phosphatidylethanolamine. This Ca2+ binding is greatly diminished by lowered pH, resulting in an attenuation of cell lysis.

スーパーオキシドラジカルによる心筋小胞体からの特異的Ca<SUP>2+</SUP>放出促進

Because the net Ca2+ uptake in the sarcoplasmic reticulum (SR) of cardiac muscle is a result of the activity of Ca(2+)-ATPase and of the SR Ca(2+)-release channel, an abnormal Ca2+ uptake may be the result of the dysfunction of either or both structures. The site or sites of action for oxygen-derived free radicals (OFR) damage are unknown, although previous studies on the SR have focused on damage to the Ca2+ pump. Direct effects of OFR on SR Ca(2+)-release channels may be important in understanding their potential contribution to myocardial ischemia/reperfusion injury. We confirmed that superoxide anion radical (O2.-) generated from hypoxanthine-xanthine oxidase reaction decreases calmodulin content and increases 45Ca2+ efflux from the heavy fraction of canine cardiac SR vesicles. Electron spin resonance study showed that hydroxyl radicals are generated in addition to O2.- from hypoxanthine-xanthine oxidase reaction, and data indicate that O2.- is responsible for the observed effect. Current fluctuations through single Ca(2+)-release channels have been also monitored after incorporation into planar phospholipid bilayers. We directly demonstrate that activation of the channel by O2.- stimulates Ca2+ release from heavy SR vesicles and suggest the importance of accessory proteins such as calmodulin in modulating the effect of O2.-.

Acute effects of prolactin on passive calcium absorption in the small intestine by in vivo perfusion technique

The acute effect of intraperitoneally administered prolactin (0.2, 0.4, and 0.6 mg/kg body weight) on passive calcium transport in duodenum, proximal jejunum, and ileum of sexually mature female Wistar rats was investigated by using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 microCi 45CaCl2 (1 Ci = 37 GBq) was perfused through the 10-cm intestinal loop for 60 min. Results showed that 0.4 mg prolactin/kg body weight significantly increased duodenal net Ca absorption (net Ca) from 23.81 +/- 1.84 to 30.56 +/- 1.57 mmol/g dry weight (p < 0.05) by stimulating the lumen to plasma calcium flux (CaL-P). The jejunum responded to 0.2, 0.4, and 0.6 mg prolactin/kg body weight by reversing from net Ca absorption of 18.60 +/- 1.70 mmol/g dry weight to net secretion of -3.30 +/- 1.56, -10.39 +/- 2.21, and -11.79 +/- 2.04 mmol/g dry weight (p < 0.01), respectively, as a result of a dose-dependent increase in plasma to lumen calcium flux (CaP-L). Calcium fluxes in the ileum on the other hand did not respond to prolactin. There was a close correlation between net water flux and net calcium flux in all three intestinal segments under basal condition regardless of the luminal sodium concentration. However, this correlation was lost after prolactin administration, which while having no effect on net water flux, altered the duodenal and jejunal calcium fluxes. By varying the luminal concentration of sodium, it was found that the stimulatory effect of 0.4 mg prolactin/kg body weight on the duodenal CaL-P was reduced when compared with control, i.e., 17.84 +/- 0.91 vs. 26.64 +/- 1.05 mmol/g dry weight at a sodium concentration of 180 mM, and 14.48 +/- 0.99 vs. 20.12 +/- 1.34 mmol/g dry weight at a sodium concentration of 140 mM. At a sodium concentration of 80 mM, the prolactin effect was absent. Since duodenal Na(+)-K+ ATPase activity was increased by prolactin from 3.77 +/- 0.16 to 4.95 +/- 0.30 mumol Pi.mg-1 protein.h-1 (p < 0.05), sodium dependency of the prolactin-enhanced lumen to plasma calcium flux may be related to both sodium-induced water flow and calcium-sodium exchange across the basolateral membrane. Thus, it was postulated that under basal condition, net calcium transport in the small intestine occurred with the sodium-induced water transport along the paracellular pathway. However, after prolactin administration, this association was lost. Prolactin-enhanced lumen to plasma calcium flux in the duodenum was sodium dependent and involved the Na(+)-K+ ATPase activity. In the proximal jejunum, prolactin stimulated plasma to lumen calcium flux, but the mechanism was not known.

Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres.

Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.

Slowly activating vacuolar channels can not mediate Ca2+‐induced Ca2+ release

In order to test the hypothesis that slowly activating vacuolar (SV) channels mediate Ca2+‐induced Ca2+ release the voltage‐ and Ca2+‐dependence of these K+ and Ca2+‐ permeable channels were studied in a quantitative manner. The patch‐clamp technique was applied to barley (Hordeum vulgare L.) mesophyll vacuoles in the whole vacuole and vacuolar‐free patch configuration. Under symmetrical ionic conditions the current‐voltage relationship of the open SV channel was characterized by a pronounced inward rectification. The single channel current amplitude was not affected by changes in cytosolic Ca2+ whereas an increase in vacuolar Ca2+ decreased the unitary current in a voltage‐dependent manner. The SV channel open‐probability increased with positive potentials and elevated cytosolic Ca2+, but not with elevated cytosolic Mg2+. An increase of cytosolic Ca2+ shifted the half‐activation potential to more negative voltages, whereas an increase of vacuolar Ca2+ shifted the half‐activation potential to more positive voltages. At physiological vacuolar Ca2+ activities (50 μM to 2 mM) changes in cytosolic Ca2+ (5 μM to 2 mM) revealed an exponential dependence of the SV channel open‐probability on the electrochemical potential gradient for Ca2+ (ΔμCa). At the Ca2+ equilibrium potential (ΔμCa = 0) the open‐probability was as low as 0.4%. Higher open‐probabilities required net Ca2+ motive forces which would drive Ca2+ influx into the vacuole. Under conditions favouring Ca2+ release from the vacuole, however, the open‐probability further decreased. Based on quantitative analysis, it was concluded that the SV channel is not suited for Ca2+‐induced Ca2+ release from the vacuole.

The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons

After loading cultured rat hippocampal neurons with the acetoxymethyl ester of the Ca 2+ buffer BAPTA, or its dimethyl analogue DMB, the magnitudes of transient (20–25 s) depolarization- or excitatory amino acid-induced Ca 2+ responses were reduced, as were the rates of increase and recovery of [Ca 2+] i. In contrast, during prolonged (3–30 min) stimulation, the magnitudes of the Ca 2+ responses were not reduced in buffer-loaded neurons, even though the rates of increase and recovery were still much slower compared to neurons loaded with the control molecule half-BAPTA-AM. The potential consequences of this action of BAPTA and DMB were then examined in an in vitro model of excitotoxicity in which we found that, in both fetal and postnatal cultures, glutamate-induced excitotoxicity was enhanced, rather than reduced. An additional and unexpected observation was that during exposure of neurons to solutions containing BAPTA-AM, dimethyl-BAPTA-AM, or half-BAPTA-AM, we observed a rapid but reversible increase in intracellular [Ca 2+] that appeared to be mediated via an activation of voltage-operated Ca 2+ channels; most probably due to a direct depolarizing effect. We suggest that the presence of artificial Ca 2+ buffers interferes with the normal Ca 2+-dependent mechanisms for limiting Ca 2+ entry during stimulation and thereby leads to an enhanced net Ca 2+ influx. One consequence of this action is to enhance the potency of glutamate as an excitotoxic agent. These results agree with previous observations that excitotoxicity is better correlated with the total net flux of Ca 2+, rather than measurements of intracellular ionic Ca 2+. Our results do not support a potential use of artificial Ca 2+ buffers as neuroprotective agents.

Effects of lactate on the relative contribution of Ca2+ extrusion mechanisms to relaxation in guinea-pig ventricular myocytes.

1. The aim of this study was to investigate the effects of 20 mM extracellular lactate on Ca2+ regulation mechanisms in enzymatically isolated single guinea-pig cardiac myocytes. 2. The activities of the Ca2+ regulation mechanisms during application of lactate were studied using rapid cooling contractures (RCCs) and fast application of caffeine. Cytoplasmic Ca2+ was monitored using the fluorescent indicator indo-1. 3. After application of 20 mM lactate for 5 min, the diastolic level of Ca2+ was increased. The change in cytoplasmic Ca2+ elicited by stimulation (Ca2+ transient) was also changed. With lactate, the amplitude of the Ca2+ transient was smaller, and its time course was slower compared with control. 4. The recovery of cytoplasmic Ca2+ during rewarming after rapid cooling in lactate was slower than under control conditions. When the rewarming was performed either in Na(+)- and Ca(2+)-free solution or in the presence of 10 mM caffeine, the rate of recovery of cytoplasmic Ca2+ in lactate was slower than under control conditions, suggesting that the activity of both SR Ca2+ uptake and Na(+)-Ca2+ exchange is affected by lactate. 5. Cytoplasmic Ca2+ recovery during application of 10 mM caffeine in lactate was slower than in the control. The rate of recovery of the caffeine-induced transient inward current was also slower supporting the hypothesis of a slower Ca2+ extrusion brought about by Na(+)-Ca2+ exchange. 6. The relative contribution of the Ca2+ extrusion mechanisms in the presence of lactate was investigated using paired RCCs. In lactate, a second RCC (RCC2) induced immediately after recovery from the first (RCC1) was greatly reduced compared with the control. RCC2/RCC1 x 100 in lactate was 39% and RCC2/RCC1 x 100 in control conditions was 60%, suggesting that the net sarcoplasmic reticulum Ca2+ uptake is smaller in the presence of lactate. 7. When Na(+)-free Ca2+ solution was used during the paired RCCs and rewarming, RCC2/RCC1 x 100 was increased to 96 and 95% in lactate and control conditions, respectively, implying that Ca2+ efflux from the cell can be maintained by the Na(+)-Ca2+ exchanger and that other Ca2+ removal mechanisms (mitochondria and sarcolemmal Ca(2+)-ATPase) remain largely unchanged in the presence of lactate.

Cutaneous transport of Ca2+ in the frog Rana pipiens: significance and specificity.

Rana pipiens were divided into four groups: controls; hypocalcemic frogs, depleted of salts by acclimation to deionized water; hypercalcemic frogs, calcium-loaded by the introduction of 40 mumol calcium gluconate; and frogs exposed to the potential competing ions Mg2+, Sr2+, and Ba2+. All groups displayed calcium influx that was proportional to external [Ca2+]; however, the group acclimated to deionized water also displayed hypocalcemia (P < 0.025) and enhanced Ca2+ influx at higher (> 0.3 mM) external [Ca2+]. Ca2+ efflux was depressed in hypocalcemic frogs, and thus net Ca2+ flux shifted from net loss in control frogs to net uptake in hypocalcemic frogs. Hypocalcemia also resulted in increased skin Ca2+ deposits which may be related to a decreased Ca2+ (and other ions) permeability as a consequence of the acclimation to deionized water. Another group of frogs was Ca(2+)-loaded by injecting calcium gluconate: Sodium gluconate controls did not significantly alter Ca2+ fluxes. The frogs that received calcium gluconate treatments became hypercalcemic (P < 0.01) and did not display significant changes in calcium fluxes, nor did they show significant changes in skin calcium deposits. We conclude that hypocalcemia leads to regulatory responses that stimulate active Ca2+ transport in Rana pipiens skin and possibly inhibits cutaneous and renal efflux. We also conclude that hypercalcemia does not alter calcium fluxes across skin. The ions from Group IIA of the Periodic Table of Elements had little effect on Ca2+ fluxes at concentrations ranging from 0.5-4.0 mM; neither Sr2+ or Ba2+ affected Ca2+ influx. The only divalent ion tested that influenced Ca2+ was Mg2+, which significantly inhibited Ca2+ influx but only at 4.0 mM or eight times the external [Ca2+]. We conclude, therefore, that the Ca2+ transport mechanism is fairly specific for Ca2+ within Group IIA.

The Modulation of Pacing-induced Changes in Intracellular Sodium Levels by Extracellular Ca2+in Isolated Perfused Rat Hearts

This study demonstrates the inverse relationship between extracellular free calcium ([Cao]f) and intracellular sodium ([Nai]) in isolated perfused rat hearts and thus supports the role of [Nai] in the “calcium paradox”. It also shows that the extent of the increase in [Nai] (Δ[Nai]), and the extent of the decrease in left ventricular developed pressure (ΔLVDP) in isolated perfused rat hearts, induced by pacing, is modulated by [Cacf7>o]f. At low (0.24 mm) as well as normal (1.15 mm) [Cao]f, [Naiincreased with pacing, progressively and significantly (P<0.01 andP<0.05, respectively), reaching a maximum of 12.56±0.46 and 9.22±0.16 mmat 500 beats/min, respectively. At high [Cao]f(2.2 mm), however, no pacing-induced increase in [Nai] was observed. Simultaneously, within the pacing range of 250–500 beats/min, the interval–force relationship was negative for all [Cao]f. With decreasing [Cao]f, a gradually increasing Δ[Nai] was induced. We hypothesise that a [Cao]f-dependent Na–Ca exchanger activity modulates Na+uptake, and thus baseline [Nai]. During incremental pacing, the increase in pacing rate induces a [Cao]f-dependent Δ[Nai], which may interact further with the sarcolemmal Na–Ca exchanger activity. As a result, both baseline [Nai] and the pacing-induced, [Cao]f-dependent Δ[Nai] modulate the net Ca2+uptake, and thus SR Ca, in a manner that results in a modulated left ventricular force development.

Downward movements of dolomite, kieserite or a mixture of CaCO3 and kieserite through the upper layers of an acid forest soil

Liming and fertilization are important tools for improving the chemical status of acid, base poor forest soils. The downward movement of dolomite, kieserite and a mixture of CaCO3 and kieserite was investigated by monitoring the leachates and exchangeable cation composition from single and combined horizon columns, reconstructed from an acid brown forest soil profile (0-15 cm). Upon entering the soil, Mg ions from kieserite displaced base cations and acidity (H and Al ions) from exchange sites, which subsequently moved down with the mobile SO42- anions. Total leaching during the initial SO42- pulse was similar with the CaCO3 + kieserite mixture. Compared to the single kieserite treatment, the joint application of CaCO3 greatly increased the proportion of Ca in the leachates from all horizons. It also decreased the leaching of acidity from the surface Oe horizon and prevented pH from dropping under this layer. With both treatments, the redistribution of magnesium with SO42- anions resulted in a rapid increase in exchangeable Mg contents throughout the studied columns. Due to the important charge increase in the Oe horizon and to kinetic restraints imposed on dissolution, downward movement of Ca and Mg ions from dolomite was very limited. Mg was however much more mobile than Ca. In the CaCO3 + kieserite and dolomite treatments, the migration of alkalinity and base cations with time was associated with a decrease in exchangeable acidity and an increase in ECEC in the two upper soil layers. By the end of the monitoring period, overall net Mg retention in the 0-15 cm columns increased in the order kieserite < CaCO3 + kieserite << dolomite with respectively 20, 35 and 85% of cumulated inputs remaining in the columns. The corresponding net Ca retention amounted to 82 and 96% of cumulated inputs for the CaCO3 + kieserite and dolomite treatments, respectively. Results from this study complement those obtained in the field by clearly demonstrating the mechanisms involved in the downward movement of some fertilizers commonly used to increase the base saturation of acid forest soils.

In vitro studies on calcium absorption from the gastrointestinal tract in small ruminants.

Unidirectional flux rates of Ca2+ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca2+ flux rates (Jms) exceeded respective flux rates in the opposite direction (Jsm) in both species and in all segments of the intestinal tract. This resulted in net Ca2+ flux rates (Jnet = Jms-Jsm) ranging between -2 and 9 nmol.cm-2.h-1 in sheep and between 10 and 15 nmol cm-2.h-1 in goats. In sheep, only Jnet in jejunum, and in goats, Jnet in duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant Jnet of Ca2+ of around 5 nmol.cm-2.h-1 could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca2+ absorption clearly indicates the presence of active mechanisms for Ca2+ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca2+ absorption in goat rumen. Jnet of Ca2+ across goat rumen epithelia was significantly reduced by 1 mmol.l-1 verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca2+ absorption in small ruminants. Stimulation of active Ca2+ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca2+ transport as described for the upper small intestines of monogastric species.

Compensatory up-regulation of cardiac SR Ca2+-pump by heat-shock counteracts SR Ca2+-channel activation by ischemia/reperfusion.

We tested the hypothesis that heat-shock protected myocardial Ca2+-cycling by sarcoplasmic reticulum from ischemia and reperfusion (I/R) injury. Twenty-four hours after increasing body temperature to 42 degrees C for 15 min, rat hearts were isolated, Langendorff-perfused, and subjected to 30 min ischemia then 30 min reperfusion. Left ventricles were homogenized and their ionized Ca2+ concentration monitored with indo- during Ca2+-uptake in the presence and absence of the Ca2+-release channel (CRC) modulator ryanodine. Tissue content of heat-shock protein 72 (HSP 72) was analyzed. Exposure to I/R resulted in a 37% enhancement of CRC activity but no effect on Ca2+-pumping activity, resulting in 25% decreased net Ca2+-uptake activity. Pre-exposure to heat-shock resulted in a 10-fold increase in HSP 72, and a 25% enhancement of maximal Ca2+-pumping activity which counteracted the effect of I/R on CRC and net Ca2+-uptake activities. This protection of SR Ca2+-cycling was associated with partial protection of myocardial physiological performance. Net Ca2+-uptake activity was correlated with the left ventricular developed pressure and its rate of change. We conclude that one of the mechanisms by which heat-shock protects myocardium from I/R injury is to upregulate SR Ca2+-pumping activity to counteract the enhanced SR Ca2+-release produced by I/R.

Potent vasoconstrictor actions of cyclopiazonic acid and thapsigargin on femoral arteries from spontaneously hypertensive rats.

1. Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents which inhibit the Ca(2+)-ATPase of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. In resting strips preloaded with fura-PE3, the addition of CPA (10 microM) or thapsigargin (100 nM) caused an elevation of cytosolic Ca2+ level ([Ca2+]i) and a contraction. These responses were significantly greater in SHR than in WKY. 3. The additional of verapamil (3 microM) to the resting strips caused a decrease in resting [Ca2+]i, which was significantly greater in SHR than in WKY. In SHR, but not in WKY, this decrease was accompanied by a relaxation from the resting tone, suggesting the maintenance of myogenic tone in the SHR artery. 4. Verapamil (3 microM) abolished differences between SHR and WKY. The effects of verapamil were much greater on the contraction than on the [Ca2+]i. 5. The resting of Ca2+ influx in arteries measured after a 5 min incubation of the artery with 45Ca was not increased by CPA or thapsigargin in either SHR or WKY. The net Ca2+ entry measured after a 30 min incubation of the artery with 45Ca was decreased by CPA or thapsigargin in both SHR and WKY. The resting Ca2+ influx was significantly higher in SHR than in WKY, and was decreased by nifedipine (100 nM) in the SHR artery, but was unchanged in the WKY artery. 6. The resting 45Ca efflux from the artery was increased during the addition of CPA (10 microM). This increase was less in SHR than in WKY. The resting 45Ca efflux was the same in SHR and WKY. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels (VDCCs) was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional elimination of SR by CPA or thapsigargin caused a large elevation of [Ca2+]i and a potent contraction in this artery. During this process, the contraction was mainly due to the basal Ca2+ influx via L-type VDCCs. The present study also showed the existence of a relatively large compartment of [Ca2+]i which does not contribute to the contraction during the addition of CPA or thapsigargin.

Microwave Fixation and Localization of Calcium in Synaptic Terminals Using X-Ray Microanalysis and Electron Energy Loss Spectroscopy Imaging

The distribution of calcium ions is demonstrated in synaptic terminals by means of a two-step chemical precipitation of calcium ions in the rat brain. K-oxalate/K-antimonate chemical replacement with simultaneous computerized microwave irradiation was used. This precipitate in nerve cell structures was investigated by computerized electron probe x-ray microanalysis (EDX) and electron energy loss spectroscopic (EELS) imaging. The values obtained by EDX agreed with those of the standard sample and theoretical values of Ca-antimonate. Typical EELS spectra of Ca:L, O:K, and Sb:M were obtained from nerve terminals in the same tissue block as that used for EDX analysis. Excellent net Ca:L and Sb:M EELS digital images were obtained after their background images were subtracted. Calcium ions were distributed in the nerve terminals, synaptic vesicles, mitochondria, and synaptic membranes.

Quantal calcium release in electropermeabilized SH-SY5Y neuroblastoma cells perfused with myo-inositol 14,5-trisphosphate

Continuous perfusion of immobilized electropermeabilized SH-SY5Y neuroblastoma cells was utilised as a novel approach to the assessment of incremental activation and inactivation of myo-inositol 1,4,5-trisphosphate (IP 3)-induced calcium (Ca 2+) mobilisation (IICM). SH-SY5Y cells when stimulated with sub-optimal IP 3 exhibited a rapid concentration dependent activation of Ca 2+ mobilization followed by a partial inactivation. Although this partial inactivation allowed net Ca 2+ mobilized to be stringently returned to basal levels, a concentration-dependent depletion of the store was maintained while ever perfusion with the stimulating IP 3 concentration was sustained. This partial inactivation of IP 3-induced quantal Ca 2+ release (QCR) was only compromised if cells, with replete Ca 2+ stores, were perfused with supra-maximally effective concentrations of IP 3 (5–10 μM). Thus, at supra-optimal IP 3 concentrations, a reproducible plateau of Ca 2+ release lying 50–150 nM above the basal Ca 2+ concentration was observed. Feedback on IP 3R sensitivity by gross cytosolic Ca 2+ levels could be eliminated as the sustained and exclusive mediator of incremental activation/inactivation cycle of IICM in SH-SY5Y cells, since released Ca 2+ was perfused away from the immobilized cells. Thus, while ever the cells were continuously perfused with IP 3, impressive incremental inactivation was apparent. Additionally, IP 3R partial agonists were found to exhibit lower intrinsic activity for both activation and inactivation of QCR, suggesting that ligand-induced inactivation of the IP 3R was more important than inactivation mechanisms reliant on either Ca 2+ flux through the channel and/or calcium store depletion. Therefore, we suggest that, in perfused SH-SY5Y cells, the most parsimonious explanation of our data is that IP 3 binding probably activates and then partially inactivates its receptor in a concentration-dependent fashion to produce the QCR phenomenon.

Ca2+-Permeable AMPA/Kainate and NMDA Channels: High Rate of Ca2+Influx Underlies Potent Induction of Injury

Neurodegeneration may occur secondary to glutamate-triggered Ca2+ influx through any of three routes: NMDA channels, voltage-sensitive Ca2+ channels (VSCC), and Ca(2+)-permeable AMPA/kainate channels (Ca-A/K). This study aims to examine Ca2+ ion dynamics in the generation of excitotoxic injury by correlating the relative amounts of 45Ca2+ that flow into cortical neurons through each of these routes over a 10 min epoch ("10 min Ca2+ loads;" a measure of influx rate), with resultant levels of intracellular free Ca2+ ([Ca2+]) and subsequent injury. Neurons possessing Ca-A/K make up a small subset (approximately 13%) of cortical neurons in culture, which can be identified by a histochemical stain based on kainate-stimulated Co2+ uptake (Co2+ (+) neurons) and which are unusually vulnerable to AMPA/kainate receptor-mediated injury. Initial studies using brief kainate exposures (to selectively destroy Co2+ (+) neurons) along with kainate-triggered 45Ca2+ influx measurements suggested that kainate causes rapid Ca2+ influx into Co2+ (+) neurons (comparable to that caused by NMDA). Influx through both Ca-A/K and NMDA channels increased proportionately with extracellular Ca2+, suggesting that these channels have high Ca2+ permeability. When cultures were subjected to exposures that gave similar 10 min Ca2+ loads through different routes, comparable levels of injury were observed, suggesting that net intracellular Ca2+ accumulation is a critical determinant of injury. However, the relationship between [Ca2+]i and influx was less direct: although exposures that gave the lowest or highest 10 min Ca2+ loads showed correspondingly lower or higher mean [Ca2+]i responses, there appears to be a wide range of exposures over which individual neuronal differences and sequestration/buffering mechanisms obscure [Ca2+]i as a reflection of influx rate.

Ca2+ influx during the cardiac action potential in guinea pig ventricular myocytes.

The relative contributions of L-type Ca2+ current (ICa) and Na+/Ca2+ exchange to Ca2+ influx during the cardiac action potential (AP) are unknown. In this study, we have used an AP recorded under physiological conditions as the command voltage applied to voltage-clamped ventricular myocytes. ICa (measured as nifedipine-sensitive membrane current) had a complex multiphasic time course during the AP. Peak ICa was typically 4 pA/pF, after which it rapidly declined (to about 60% of peak) during the rising phase of the cell-wide Ca2+ transient before increasing to a second, more sustained component. The initial decline in ICa was sensitive to the amount of Ca2+ released by the sarcoplasmic reticulum (SR), and conditions that reduce the amplitude of the Ca2+ transient (such as rest or brief application of caffeine) increased net Ca2+ influx via ICa. Dissection of the Na+/Ca2+ exchange current at the start of the AP suggested that Ca2+ influx via Na+/Ca2+ exchange is less than 30% of that due to ICa. From these data, we suggest that ICa is the primary source of Ca2+ that triggers SR Ca2+ release, even at the highly depolarized membrane potentials associated with the AP. However, Ca2+ influx via Na+/Ca2+ exchange is not negligible and may activate some Ca2+ release from the SR, especially when ICa is reduced. We propose that SR Ca2+ release inhibits ICa within the same beat, thereby providing a negative feedback mechanism that may serve to limit Ca2+ influx as well as to regulate the amount of Ca2+ stored within the SR.

Influence of Whole-plant Net Calcium Influx and Partitioning on Calcium Concentration in Snap Bean Pods

Snap beans (Phaseolus vulgaris L.) are a food source that can contribute to dietary Ca requirements in humans. Factors which might enhance the concentration of Ca in snap bean pods have been investigated by measuring whole-plant net Ca influx, whole-plant Ca partitioning, and various growth parameters in two snap bean cultivars—Hystyle and Labrador—that differ in pod Ca concentration. Plants were grown hydroponically under controlled environmental conditions while being provided adequate quantities of Ca. The concentration of Ca in pods (dry weight basis) was 52% higher in `Hystyle', relative to `Labrador', but net Ca influx throughout crop development or total plant Ca content at three stages of development were similar in both cultivars, demonstrating that pod Ca concentration differences were not due to differences in total plant Ca influx. However, `Hystyle' partitioned more total plant Ca to pods, relative to `Labrador'. Calcium flux analysis also revealed that daily rates of whole-plant net Ca influx gradually declined throughout the period of pod growth in both cultivars; this decline was not related to whole-plant water influx. These results suggest that enhancements in whole-plant net Ca influx during pod growth and/or enhancements in the xylem transport of absorbed Ca to developing pods could increase the Ca concentration of snap bean pods.

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.