Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations

Although many cell functions are regulated by Ca(2+) oscillations induced by a cyclic release of Ca(2+) from intracellular Ca(2+) stores, the pacemaker mechanism of Ca(2+) oscillations remains to be explained. Using green fluorescent protein-based Ca(2+) indicators that are targeted to intracellular Ca(2+) stores, the endoplasmic reticulum (ER) and mitochondria, we found that Ca(2+) shuttles between the ER and mitochondria in phase with Ca(2+) oscillations. Following agonist stimulation, Ca(2+) release from the ER generated the first Ca(2+) oscillation and loaded mitochondria with Ca(2+). Before the second Ca(2+) oscillation, Ca(2+) release from the mitochondria by means of the Na(+)/Ca(2+) exchanger caused a gradual increase in cytoplasmic Ca(2+) concentration, inducing a regenerative ER Ca(2+) release, which generated the peak of Ca(2+) oscillation and partially reloaded the mitochondria. This sequence of events was repeated until mitochondrial Ca(2+) was depleted. Thus, Ca(2+) shuttling between the ER and mitochondria may have a pacemaker role in the generation of Ca(2+) oscillations.     

Analysis of Mn(2+)/Ca(2+) influx and release during Ca(2+) oscillations in mouse eggs injected with sperm extract

Repetitive Ca(2+) release from the endoplasmic reticulum (ER) is necessary for activation of mammalian eggs. Influx and release of Mn(2+) and Ca(2+) during Ca(2+) oscillations induced by injection of sperm extract (SE) into mouse eggs were investigated by Mn(2+)-quenching of intracellular Fura-2 after adding Mn(2+) to external medium. Mn(2+)/Ca(2+) influx was detected at the resting state. A marked Mn(2+)/Ca(2+) influx occurred during the first Ca(2+) release upon SE injection, and persistently facilitated Mn(2+)/Ca(2+) influx was observed during steady Ca(2+) oscillations. As intracellular Mn(2+) concentration ([Mn(2+)](i)) increased progressively, periodic [Mn(2+)](i) rises appeared, corresponding to each Ca(2+)transient but taking a slower time course. A numerical simulation based on continuous Mn(2+)/Ca(2+) influx-extrusion across the plasma membrane and release-uptake across the ER membrane in a competitive manner mimicked well the Mn(2+) oscillations calculated from experimental data, strongly suggesting that repetitive Mn(2+) release develops after Mn(2+) entry and uptake into the ER. In other experiments, a marked Mn(2+) influx occurred upon Mn(2+) addition to Ca(2+)-free medium after depletion of the ER using an ER Ca(2+) pump inhibitor plus repeated injection of inositol 1,4,5-trisphosphate (InsP(3)). No significant increase in Mn(2+) influx was induced by injection of SE, InsP(3), or Ca(2+), when Ca(2+) release was prevented by pre-injection of an antibody against the InsP(3) receptor. We concluded that Ca(2+) influx is activated during the initial large Ca(2+)release possibly by a capacitative mechanism and kept facilitated during steady Ca(2+) oscillations. The finding that repetitive Mn(2+) release is caused by continuous Mn(2+) entry suggests that continuous Ca(2+) influx may play a critical role in refilling the ER and, thereby, maintaining Ca(2+)oscillations in mammalian fertilization.     

Mitochondrial Ca(2+) uptake depends on the spatial and temporal profile of cytosolic Ca(2+) signals

Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.     

A novel role for Bcl-2 in regulation of cellular calcium extrusion

The antiapoptotic protein Bcl-2 plays important roles in Ca(2+) signaling by influencing inositol triphosphate receptors and regulating Ca(2+)-induced Ca(2+) release. Here we investigated whether Bcl-2 affects Ca(2+) extrusion in pancreatic acinar cells. We specifically blocked the Ca(2+) pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca(2+) concentration after a period of enhanced Ca(2+) entry. Because external Ca(2+) was removed and endoplasmic reticulum Ca(2+) pumps were blocked, Ca(2+) extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca(2+) level much faster than control cells. The enhanced Ca(2+) extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na(+)/Ca(2+) exchange activity, because removal of external Na(+) did not influence the Ca(2+) extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca(2+) extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca(2+) extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca(2+) pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca(2+) extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.     

Relationship between Ca2+ sparklets and sarcoplasmic reticulum Ca2+ load and release in rat cerebral arterial smooth muscle

Ca(+) sparklets are subcellular Ca(2+) signals produced by the opening of sarcolemmal L-type Ca(2+) channels. Ca(2+) sparklet activity varies within the sarcolemma of arterial myocytes. In this study, we examined the relationship between Ca(2+) sparklet activity and sarcoplasmic reticulum (SR) Ca(2+) accumulation and release in cerebral arterial myocytes. Our data indicate that the SR is a vast organelle with multiple regions near the sarcolemma of these cells. Ca(2+) sparklet sites were located at or <0.2 μm from SR-sarcolemmal junctions. We found that while Ca(2+) sparklets increase the rate of SR Ca(2+) refilling in arterial myocytes, their activity did not induce regional variations in SR Ca(2+) content or Ca(2+) spark activity. In arterial myocytes, L-type Ca(2+) channel activity was independent of SR Ca(2+) load. This ruled out a potential feedback mechanism whereby SR Ca(2+) load regulates the activity of these channels. Together, our data suggest a model in which Ca(2+) sparklets contribute Ca(2+) influx into a cytosolic Ca(2+) pool from which sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps Ca(2+) into the SR, indirectly regulating SR function.     

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca(2+) channels was assessed by whole-cell patch-clamp recordings of Ca(2+) current and complementary experiments in which internal [Na(+)] was reduced. However, Ca(2+) entry via Na(+)-Ca(2+) exchange measured in the absence of L-type Ca(2+) current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca(2+) channels increase [Ca(2+)] near Na(+)-Ca(2+) exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca(2+) release in heart cells under various conditions.     

粘盖牛肝菌不同菌株对Zn2+、Cd2+的吸附及其对油松Zn2+、Cd2+的耐受性

为了探讨同一菌根真菌不同菌株对重金属的耐受性,选用采集于内蒙古阴山山脉不同地区的粘盖牛肝菌(Suillus bovinus)的不同菌株进行研究。首先,在不同浓度Zn^2 、Cd^2 液体培养基中培养菌丝体,以了解各菌株菌丝体对重金属的耐受性及吸附能力,采用烘干法和原予吸收法分别测定菌丝体的生物量和菌丝体、培养液中Zn^2 、Cd^2 含量,结果表明：劈柴沟粘盖牛肝菌在Zn^2 、Cd^2 胁迫条件下,生物量、吸附能力约为其余各菌株的1．5～2倍。其次,为了探明油松(Pinus tabulaeformis)形成菌根后对Zn^2 、Cd^2 胁迫的耐受性及其耐受机理,采用一次性定量浇灌不同浓度Zn^2 、Cd^2 溶液的方法,测定了菌根化油松苗地上、地下生物量及Zn^2 、Cd^2 含量的分配,结果表明：菌根形成后能显著促进油松的生长及对Zn^2 、Cd^2 胁迫的耐受性,并且菌根能够帮助油松吸收基质中大量的Zn^2 、Cd^2 ,根中重金属的含量是茎叶中的2～3倍以上,非菌根苗在重金属浓度稍高(Zn^2 400mg／kg;Cd^2 40mg／kg)时就会死亡。经方差分析及多重比较证实,劈柴沟粘盖牛肝菌对Zn^2 、Cd^2 的耐受性及对油松的促生效果与其它各菌株存在显著的差异,这可能是它通过把吸收的Zn^2 、Cd^2 最大限度地输送到根中的同时,也输送到了茎叶中,使重金属在体内得到一定程度的稀释,使自身免受毒害。     

Factors that determine Ca2+ sensitivity of photoreceptor guanylyl cyclase. Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1)

We explored the possibility that, in the regulation of an effector enzyme by a Ca(2+)-sensor protein, the actual Ca(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca(2+)-sensor protein for Ca(2+) but also by the relative affinities of its Ca(2+)-bound versus Ca(2+)-free form for the effector enzyme. As a model, we used Ca(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca(2+) range. We find that at physiological concentrations of Mg(2+) this mutation increases the free Ca(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 microM. Similar to rod outer segment cyclase, Ca(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg(2+), but the shift in Ca(2+) sensitivity for the R838S mutant relative to the wild type is Mg(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca(2+)-bound and Ca(2+)-free GCAP-1 and found that the net shift in Ca(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca(2+)-free versus Ca(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca(2+)/GCAP is determined by both the affinity of GCAP for Ca(2+) and relative affinities of the effector enzyme for the Ca(2+)-free versus Ca(2+)-loaded GCAP.     

Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.     

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.     

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.     

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.     

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.     

Differential tyrosine phosphorylation of plasma membrane Ca2+-ATPase and regulation of calcium pump activity by carbachol and bradykinin

We investigated the effects of thapsigargin (TG), bradykinin (BK), and carbachol (CCh) on Ca(2+) entry via endogenous channels in human embryonic kidney BKR21 cells. After depletion of Ca(2+) stores by either TG, BK, or CCh, the addition of Ca(2+) gave a much larger rise in Ca(2+) levels in CCh-treated and TG-treated cells than in cells treated with BK. However, in experiments performed with Ba(2+), a cation not pumped by Ca(2+)-ATPases, only a modest difference between CCh- and BK-stimulated Ba(2+) entry levels was observed, suggesting that the large difference in the Ca(2+) response is mediated by a differential regulation of Ca(2+) pump activity by CCh and BK. This hypothesis is supported by the finding that when Ca(2+) is removed during the stable, CCh-induced Ca(2+) plateau phase, the decline of cytosolic Ca(2+) is much faster in the absence of CCh than in its presence. In addition, if Ca(2+) is released from a caged Ca(2+) compound after a UV pulse, the resulting Ca(2+) peak is much larger in the presence of CCh than in its absence. Thus, the large increase in Ca(2+) levels observed with CCh results from both the activation of Ca(2+) entry pathways and the inhibition of Ca(2+) pump activity. In contrast, BK has the opposite effect on Ca(2+) pump activity. If Ca(2+) is released from a caged Ca(2+) compound, the resulting Ca(2+) peak is much smaller in the presence of BK than in its absence. An investigation of tyrosine phosphorylation levels of the plasma membrane Ca(2+)-ATPase (PMCA) demonstrated that CCh stimulates an increase in tyrosine phosphorylation levels, which has been reported to inhibit Ca(2+) pump activity, whereas in contrast, BK stimulates a reduction of PMCA tyrosine phosphorylation levels. Thus, BK and CCh have a differential effect both on Ca(2+) pump activity and on tyrosine phosphorylation levels of the PMCA.     

Calcium spark properties in ventricular myocytes are altered in aged mice

This study determined whether whole cell Ca(2+) transients and unitary sarcoplasmic reticulum (SR) Ca(2+) release events are constant throughout adult life or whether Ca(2+) release is altered in aging ventricular myocytes. Myocytes were isolated from young adult (approximately 5 mo old) and aged (approximately 24 mo old) mice. Spontaneous Ca(2+) sparks and Ca(2+) transients initiated by field stimulation were detected with fluo-4. All experiments were conducted at 37 degrees C. Ca(2+) transient amplitudes were reduced, and Ca(2+) transient rise times were abbreviated in aged cells stimulated at 8 Hz compared with young adult myocytes. Furthermore, the incidence and frequency of spontaneous Ca(2+) sparks were markedly higher in aged myocytes compared with young adult cells. Spark amplitudes and spatial widths were similar in young adult and aged myocytes. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Resting cytosolic Ca(2+) levels and SR Ca(2+) stores were assessed by rapid application of caffeine in fura-2-loaded cells. Neither resting Ca(2+) levels nor SR Ca(2+) content differed between young adult and aged cells. Thus increased spark frequency in aging cells was not attributable to increased SR Ca(2+) stores. Furthermore, the decrease in Ca(2+) transient amplitude was not due to a decrease in SR Ca(2+) load. These results demonstrate that alterations in fundamental SR Ca(2+) release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca(2+) release may reflect age-related changes in fundamental release events rather than changes in SR Ca(2+) stores and diastolic Ca(2+) levels.     

Mechanisms of vasopressin-induced intracellular Ca2+ oscillations in rat inner medullary collecting duct

Arginine vasopressin (AVP) causes increase in intracellular Ca(2+) concentration with an oscillatory pattern. Ca(2+) mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca(2+) oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca(2+) oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca(2+) oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca(2+) oscillations and endogenous cADPR plays a major role in the AVP-induced Ca(2+) oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP(3)) induced Ca(2+) release but did not maintain sustained Ca(2+) oscillations. Removal of extracellular Ca(2+) halted ongoing AVP-mediated Ca(2+) oscillation, suggesting that it requires extracellular Ca(2+) entry. AVP-induced Ca(2+) oscillation was unaffected by nifedipine. Intracellular Ca(2+) store depletion induced by 20 μM thapsigargin in Ca(2+)-free medium triggered store-operated Ca(2+) entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl(3) and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca(2+)-free medium, application of extracellular Ca(2+) also triggered Ca(2+) influx, which was sensitive to GdCl(3) and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca(2+) oscillations in IMCD are mediated by the interplay of Ca(2+) release from RyRs and a Ca(2+) influx mechanism involving nonselective cation channels that resembles SOCE.     

跨膜Ca~(2+)梯度对肌质网Ca~3+-ATP酶调节的特异性

我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。     

Inactivation of L-type calcium channels in cardiomyocytes. Experimental and theoretical approaches

The L-type calcium current (ICa) plays an important role in excitation-contraction coupling of heart cells. It is critical for forming the major trigger for Ca(2+)-induced Ca(2+) release from the sarcoplasmic reticulum and hence its feedback regulation is of fundamental biological significance. The channel inactivation sharpens the kinetics and temporal precision of the Ca(2+) signals so that it prevents longer-term increases in free intracellular Ca(2+) concentration. Cardiac L-type Ca(2+) channels are known to inactivate through voltage- and Ca(2+)-dependent mechanisms. Pure voltage-dependent inactivation has a much slower time course of development than Ca(2+)-dependent inactivation and plays minor role in inhibition of Ca(2+) influx into the cell. The major determinant of the inactivation kinetics of Ca(2+) current during depolarization is Ca(2+)-dependent mechanisms. Furthermore, it is possible to distinguish two phases in Ca(2+)-dependent inactivation of calcium current: a slow phase that depends on Ca(2+) flow through the channels (Ca(2+) current-dependent inactivation) and a fast one that depends on Ca(2+) released from the sarcoplasmic reticulum (Ca(2+) release-dependent inactivation). Although both Ca(2+) released from the SR and Ca(2+) permeating channels play a role, SR-released Ca(2+) is the most effective inactivation mechanism in inhibition of Ca(2+) entry through the channel.     

Na+-Ca2+ exchange in mouse oocytes: modifications in the regulation of intracellular free Ca2+ during oocyte maturation

Increases in intracellular free Ca(2+)+ concentration (Ca(2+)+ oscillations) occur during meiotic maturation and fertilization of mammalian oocytes but little is known about the mechanisms of Ca(2+) homeostasis in these cells. Cells extrude Ca(2+) from the cytosol using two main transport processes, the Ca(2+)-ATPase and the Na(+)-Ca(2+) exchanger. The aim of this study was to determine whether Na(+)-Ca(2+) exchange activity is present in immature and mature mouse oocytes. Na(+)-Ca(2+) exchange can be revealed by altering the Na(+) concentration gradient across the plasma membrane and recording intracellular free Ca(2+) concentrations using Ca(2+)-sensitive fluorescent dyes. Depletion of extracellular Na(+) caused an immediate increase in Ca(2+) concentration in immature oocytes and a delayed increase in mature oocytes. The Na(+) ionophore, monensin, caused an increase in intracellular Ca(2+) in immature oocytes similar to that induced by Na(+)-depleted medium. In mature oocytes, monensin had no effect on intracellular Ca(2+) but the time taken for Ca(2+) to reach a peak value on removal of extracellular Na(+) was significantly decreased. Finally, addition of Ca(2+) to immature oocytes incubated in Ca(2+)-free medium caused an increase in the concentration of intracellular Ca(2+) that was dependent upon the presence of extracellular Na(+). This effect was not seen in mature oocytes. The data show that Na(+)-Ca(2+) exchange occurs in immature and mature mouse oocytes and that Ca(2+) homeostasis in immature oocytes is more sensitive to manipulations that activate Na(+)-Ca(2+) exchange.