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.     

The dimeric form of Ca2+-ATPase is involved in Ca2+ transport in the sarcoplasmic reticulum

To identify the functional unit of Ca(2+)-ATPase in the sarcoplasmic reticulum, we assessed Ca(2+)-transport activities occurring on sarcoplasmic reticulum membranes with different combinations of active and inactive Ca(2+)-ATPase molecules. We prepared heterodimers, consisting of a native Ca(2+)-ATPase molecule and a Ca(2+)-ATPase molecule inactivated by FITC labelling, by fusing vesicles loaded with each type of Ca(2+)-ATPase. The heterodimers exhibited neither Ca(2+) transport nor ATP hydrolysis, suggesting that Ca(2+) transport by the Ca(2+)-ATPase requires an interaction between functional Ca(2+)-ATPase monomers. This finding implies that the functional unit of the Ca(2+)-ATPase is a dimer.     

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.     

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.     

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.     

Functional coupling between ryanodine receptors, mitochondria and Ca(2+) ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.     

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.     

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.     

Developmental changes in capacitative Ca(2+) entry in mouse mammary epithelial cells

Developmental changes in capacitative Ca(2+) entry and Ca(2+) release from intracellular stores were measured using fura-2 fluorescence method during the pregnancy period (day 3-;18) in mouse mammary epithelial cells. Ca(2+) release was identified with the transient intracellular Ca(2+) ([Ca(2+)](i)) increase induced by thapsigargin addition in a Ca(2+)-free solution. Capacitative Ca(2+) entry was measured by the transient [Ca(2+)](i) increase induced by re-addition of extracellular Ca(2+) after depletion of Ca(2+) stores by thapsigargin. The capacitative Ca(2+) entry was greatest at the early stage of pregnancy (i.e. day 3 of pregnancy) and decreased as pregnancy progressed, while Ca(2+) release remained unchanged throughout the developmental stages. These findings indicate that in contrast to Ca(2+) release, a close correlation exists between capacitative Ca(2+) entry and pregnancy-induced development in mammary epithelial cells.     

Epidermal growth factor-induced depletion of the intracellular Ca2+ store fails to activate capacitative Ca2+ entry in a human salivary cell line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.     

Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca(2+) alternans appears as cytosolic Ca(2+) transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca(2+) regulation. In atrial myocytes, we tested the role of voltage-gated Ca(2+) current, sarcoplasmic reticulum (SR) Ca(2+) load, and restitution properties of SR Ca(2+) release for the occurrence of pacing-induced Ca(2+) alternans. Voltage-clamp experiments revealed that peak Ca(2+) current was not affected during alternans, and alternans of end-diastolic SR Ca(2+) load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca(2+) indicator (fluo-5N), were not a requirement for cytosolic Ca(2+) alternans. Restitution properties and kinetics of refractoriness of Ca(2+) release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca(2+) releases and 2) Ca(2+) spark frequency, both during rest after a large and small alternans Ca(2+) transient; 3) the magnitude of premature action potential-induced Ca(2+) transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca(2+) signal (Ca(2+) uncaging from DM-nitrophen) to trigger additional Ca(2+) release during alternans. The results showed that the latency of global spontaneous Ca(2+) release was prolonged and Ca(2+) spark frequency was decreased after the large Ca(2+) transient during alternans. Furthermore, the restitution curve of the Ca(2+) transient elicited by premature action potentials or by photolysis-induced Ca(2+) release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca(2+) release mechanism represents a key mechanism underlying cardiac alternans.     

An apoplastic Ca2+ sensor regulates internal Ca2+ release in aequorin-transformed tobacco cells

Removal of Ca(2+) from tobacco suspension cell medium has two immediate effects on cytosolic Ca(2+) fluxes: (i) externally derived Ca(2+) influx (occurring in response to cold shock or hypo-osmotic shock) is inhibited, and (ii) organellar Ca(2+) release (induced by a fungally derived defense elicitor, caffeine, or hypo-osmotic shock) is elevated. We show here that the enhanced release of internal Ca(2+) is likely due to increased discharge from a caffeine-sensitive store in response to a signal transduced from an extracellular Ca(2+) sensor. Thus, chelation of extracellular Ca(2+) in the absence of any other stimulus directly activates release of intracellular Ca(2+) into the cytosol. Evidence that this chelator-activated Ca(2+) flux is dependent on a signaling pathway includes its abrogation by prior treatment with caffeine, and its inhibition by protein kinase inhibitors (K252a and staurosporine) and anion channel blockers (niflumate and anthracene-9-carboxylate). An unexpected characteristic of tobacco cell adaptation to low external Ca(2+) was the emergence of a new Ca(2+) compartment that was inaccessible to external EGTA, yet responsive to the usual stimulants of extracellular Ca(2+) entry. Thus, cells that are exposed to EGTA for 20 min lose sensitivity to caffeine and defense elicitors, indicating that their intracellular Ca(2+) pools have been depleted. Surprisingly, these same cells simultaneously regain their ability to respond to stimuli that usually activate extracellular Ca(2+) influx even though all external Ca(2+) is chelated. Because this gradual restoration of Ca(2+) influx can be inhibited by the same kinase inhibitors that block EGTA-activated Ca(2+) release, we propose that chelator-activated Ca(2+) release from internal stores leads to deposition of this Ca(2+) into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca(2+) entry.     

Store-operated Ca2+ entry in porcine airway smooth muscle

Ca(2+) influx triggered by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores [mediated via store-operated Ca(2+) channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca(2+) was depleted by either 5 microM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca(2+), subsequent introduction of extracellular Ca(2+) further elevated [Ca(2+)](i). SOCC was insensitive to 1 microM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM-1 mM La(3+) or Ni(2+) inhibited SOCC. Exposure to ACh increased Ca(2+) influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca(2+) release by 20 microM xestospongin D inhibited SOCC, whereas ACh-induced IP(3) production by 5 microM U-73122 had no effect. Inhibition of Ca(2+) release through ryanodine receptors (RyR) by 100 microM ryanodine also prevented Ca(2+) influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca(2+) influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca(2+) depletion. These data demonstrate that a Ni(2+)/La(3+)-sensitive Ca(2+) influx via SOCC in porcine ASM cells involves SR Ca(2+) release through both IP(3) and RyR channels. Additional regulation of Ca(2+) influx by agonist may be related to a receptor-operated, noncapacitative mechanism.     

Novel effects of propranolol. Release of internal Ca(2+) followed by activation of capacitative Ca(2+) entry in Madin Darby canine kidney cells

The effect of propranolol on Ca(2+) signalling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Propranolol increased cytosolic free Ca(2+) levels ([Ca(2+)](i)) in a concentration-dependent manner between 0.1 and 1 mM. The response was partly inhibited by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with 0.2 mM propranolol partly inhibited the [Ca(2+)](i) rise induced by 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with thapsigargin abolished propranolol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 0.2 mM propranolol in Ca(2+)-free medium. Propranolol (0.2 mM) inhibited 25% of thapsigargin-induced capacitative Ca(2+) entry. Suppression of 1,4,5-trisphosphate (IP(3)) formation by 2 microM U73122, a phospholipase C inhibitor, did not alter 0.2 mM propranolol-induced internal Ca(2+) release. Propranolol (1 mM) also increased [Ca(2+)](i) in human neutrophils. Collectively, we have found that 0.2 mM propranolol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive Ca(2+) stores in an IP(3)-independent manner, followed by Ca(2+) influx from external space. Independently, propranolol was able to inhibit thapsigargin-induced capacitative Ca(2+) entry.     

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.     

Mechanism of intracellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured endothelial CPAE cells

Intra- and intercellular Ca(2+)-signaling during mechanical stimulation in calf pulmonary artery endothelial cells (CPAE) was investigated with digital fluorescence microscopy. Mechanical stimulation of a CPAE cell in a Ca(2+)-containing solution revealed a rise of the free intracellular Ca(2+)-concentration ([Ca(2+)](i)) in the mechanically stimulated cell (MS) proceeding to the neighboring (NB) cells as an intercellular Ca(2+)-wave. Experiments in Ca(2+)-free solution, containing 2mM EGTA, demonstrated that a detectable [Ca(2+)](i)-transient in the MS cell is not always a requisite for intercellular communication (IC). The Ca(2+)-wave propagation was not affected by changes in membrane potential and was not mediated by voltage-dependent Ca(2+)-channels. Ca(2+)-influx through the Ni(2+)-sensitive Ca(2+)-pathway occurred in the MS as could be assessed by Mn(2+)-quenching experiments. The intra- and intercellular Ca(2+)-wave was triggered by the release of thapsigargin-sensitive intracellular Ca(2+)-stores. Phospholipase C (PLC) inhibition by U73122 reduced the Ca(2+)-amplitude of the MS cell and almost completely inhibited the IC, indicating that the Ca(2+)-release in the MS and NB cells is PLC/inositol 1,4,5-trisphosphate (IP(3)) mediated.     

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.     

Discrete intracellular Ca2+ pools coupled to two distinct Ca2+ influx pathways in human platelets

Ca(2+) influx is an important event associated with platelet activation and regulated by the content of intracellular Ca(2+). Previous studies have suggested two different Ca(2+) pools and two Ca(2+) influx pathways exist in platelets. In the present study, we have investigated the regulation of thrombin- and thapsigargin-induced Ca(2+) entry into human platelets, using fluorescent indicators to monitor Ca(2+) mobilization and membrane potential. It was found that depletion of thapsigargin-sensitive Ca(2+) stores was coupled to Ca(2+) influx through a Ca(2+)-selective pathway. Additional release of Ca(2+) from the thapsigargin-insensitive pool by thrombin caused the opening of a nonselective cation channel.     

Functional characterization of P2Y1 versus P2X receptors in RBA-2 astrocytes: elucidate the roles of ATP release and protein kinase C

A physiological concentration of extracellular ATP stimulated biphasic Ca(2+) signal, and the Ca(2+) transient was decreased and the Ca(2+) sustain was eliminated immediately after removal of ATP and Ca(2+) in RBA-2 astrocytes. Reintroduction of Ca(2+) induced Ca(2+) sustain. Stimulation of P2Y(1) receptors with 2-methylthioadenosine 5'-diphosphate (2MeSADP) also induced a biphasic Ca(2+) signaling and the Ca(2+) sustains were eliminated using Ca(2+)-free buffer. The 2MeSADP-mediated biphasic Ca(2+) signals were inhibited by phospholipase C (PLC) inhibitor U73122, and completely blocked by P2Y(1) selective antagonist MRS2179 and protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) whereas enhanced by PKC inhibitors GF109203X and Go6979. Inhibition of capacitative Ca(2+) entry (CCE) decreased the Ca(2+)-induced Ca(2+) entry; nevertheless, ATP further enhanced the Ca(2+)-induced Ca(2+) entry in the intracellular Ca(2+) store-emptied and CCE-inhibited cells indicating that ATP stimulated Ca(2+) entry via CCE and ionotropic P2X receptors. Furthermore, the 2MeSADP-induced Ca(2+) sustain was eliminated by apyrase but potentiated by P2X(4) allosteric effector ivermectin (IVM). The agonist ADPbetaS stimulated a lesser P2Y(1)-mediated Ca(2+) signal and caused a two-fold increase in ATP release but that were not affected by IVM whereas inhibited by PMA, PLC inhibitor ET-18-OCH(3) and phospholipase D (PLD) inhibitor D609, and enhanced by removal of intra- or extracellular Ca(2+). Taken together, the P2Y(1)-mediated Ca(2+) sustain was at least in part via P2X receptors activated by the P2Y(1)-induced ATP release, and PKC played a pivotal role in desensitization of P2Y(1) receptors in RBA-2 astrocytes.     

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.