Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.     

Activation of the oxidative burst in aequorin-transformed Nicotiana tabacum cells is mediated by protein kinase- and anion channel-dependent release of Ca2+ from internal stores

The source of Ca2+ involved in transducing an oxidative-burst defense signal was examined in aequorin-transformed tobacco (Nicotiana tabacum L.) cells using modulators of Ca2+ entry. Treatments that either increased or decreased the influx of Ca2+ from external stores were found to have little effect on the magnitude or kinetics of an osmotically stimulated oxidative burst. In contrast, treatments that reduced the discharge of Ca2+ from internal stores inhibited dilution-activated H2O2 production. Curiously, most of the modulators commonly employed in animal studies as internal Ca(2+)-release inhibitors were neither effective in blocking discharge of intracellular Ca2+ nor in preventing the oxidative burst. When three different biochemical elicitors of the oxidative burst were similarly examined, both the H2O2 production and Ca2+ fluxes stimulated were found to be sensitive to modulators of internal Ca2+ release, but neither was impacted by alterations in externally derived Ca2+ influx. We hypothesize, therefore, that the oxidative burst does not depend on the influx of external Ca2+, but instead may generally be mediated by the release of internal Ca2+ in a manner that depends on the proper function of kinases and anion channels. These Ca2+ pulses trigger downstream signaling events that include the activation of Ca(2+)-regulated protein kinases, which are required for stimulation of the oxidative burst.     

Activation of 45Ca2+ influx and 22Na+/H+ exchange by epidermal growth factor and vanadate in A431 cells is independent of phosphatidylinositol turnover and is inhibited by phorbol ester and diacylglycerol

Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2-3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF-stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2-acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor.     

The relationships between early ionic events, the pattern of protein synthesis, and oocyte activation in the surf clam, Spisula solidissima

The ionic events linked to activation of surf clam (Spisula solidissima) oocytes include a transient increased Ca2+ influx and an acid release. The aim of the present work was to further elucidate the respective roles of these two ionic events and to clarify the possible role of protein kinase C in the sequence of events leading to oocyte activation. K+-enriched seawater, ammonium chloride, and the phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, were tested for their ability to promote germinal vesicle breakdown (GVBD), an acid release, increased 45Ca2+ uptake, and a shift in the pattern of protein synthesis. Oocytes activated by addition of K+ ions release an amount of H+ similar to that induced by fertilization, with the same time course, show an increased, verapamil-sensitive, 45Ca2+ uptake that is proportional to the amount of added K+, and undergo a shift in their pattern of protein synthesis, which requires the presence of external Ca2+. Ammonium chloride, at concentrations causing a higher production of acid than that induced by K+ ions or fertilization, does not trigger GVBD nor any increased 45Ca2+ uptake or any detectable shift in the pattern of protein synthesis. Combined additions of ammonium chloride with subthreshold concentrations of K+ ions allow GVBD to occur, thus revealing a synergistic effect of ammonia and K+ ions. TPA slowly induces GVBD, an Na+-dependent acid release, and a shift in the pattern of protein synthesis, in the absence of increased 45Ca2+ uptake. Our results lead us to propose the following sequence of events for the activation of Spisula oocytes: an increased Ca2+ influx contributes to activate protein kinase C which causes a Na+-dependent acid release leading to a rise of pHi. This rise of pHi, although insufficient by itself, may set the pHi in a permissive range for activation to occur through the action of other protein kinase C-sensitive events leading to the production of meiosis-inducing proteins.     

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Augmented platelet calcium uptake in response to serotonin stimulation in patients with major depression measured using Mn2+ influx and 45Ca2+ uptake

There is an augmented platelet intracellular calcium response to serotonin stimulation in major depression. The role that calcium influx has in this process is not known. The objective of this study was to determine platelet calcium influx in response to serotonin by two methods, Mn2+ influx and 45Ca2+ uptake, in order to observe if the uptake response to serotonin was augmented in major depression by comparing the response to normal controls. The use of the two methods of calcium influx showed that serotonin stimulates calcium uptake into platelets. Furthermore, patients with major depression have significantly augmented platelet calcium uptake in response to serotonin. The interesting finding was that calcium uptake into platelets is biphasic, occurring immediately and after five minutes. These results may support the two pool model for calcium oscillations within cells whereby extracellular calcium is needed for intracellular calcium release, and for replenishment of depleted stores once intracellular calcium is released.     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovlent cation ionophroes

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K+ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K+ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux wre inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100,000 dalton ATPase to 50,000 dalton fragments, which was not associated with changes in Ca2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca2+-activated ATPase protein.     

Dopamine inhibits calcium flux in the 7315a prolactin-secreting pituitary tumour

Cells of the 7315a prolactin-secreting tumour express biochemically normal cell-surface receptors for dopamine. However, dopamine inhibits prolactin release from these cells only when the basal rate of prolactin release is augmented by increasing the intracellular and/or extracellular calcium concentration of the tumour cells. This suggests that dopaminergic modulation of calcium ion flux could have a central physiological role in these neoplastic cells. In 7315a cells we examined the ability of dopamine to regulate 45Ca2+ influx and fractional 45Ca2+ efflux under conditions of enhanced calcium flux using the calcium channel activator, maitotoxin. It was observed that unidirectional calcium influx stimulated by maitotoxin was significantly inhibited by dopamine. Maitotoxin stimulated fractional efflux and prolactin release from the tumour cells and dopamine simultaneously inhibited both processes by a haloperidol-reversible mechanism. Therefore, in 7315a cells dopamine receptor activation is coupled to inhibition of calcium flux as at least one component in the regulation of prolactin release. These cells may provide further opportunity to study intracellular signalling mechanisms that are modulated by dopamine receptor activity.     

The role of calcium in the mechanism of corticotropin releasing factor mediated ACTH release

To investigate the role of calcium (Ca+2) in CRF stimulated ACTH release, we studied the effect of the following conditions on CRF (10 nM) mediated ACTH release in primary pituitary monolayer culture: different concentrations of Ca+2; EGTA; lanthanum (La+3) and nifedipine, blockers of calcium cell influx and penfluridol, trifluoperazine, and pimozide, inhibitors of calmodulin activation. Higher concentrations of Ca+2 in the culture medium led to greater amounts of CRF induced ACTH release. EGTA at 3 mM decreased the amount of CRF stimulated ACTH release by 60% but did not alter the spontaneous release of ACTH. At 0.5 mM and 1.0 mM La+3, ACTH release induced by CRF was inhibited by 23% and 35% respectively (p less than 0.01). Nifedipine (both 10(-5) and 10(-4) M) inhibited CRF stimulated ACTH release but only to a maximum of 30%. This inhibition was completely overcome by the addition of 12 mM calcium. Penfluridol, pimozide, and trifluoperazine blocked the release of ACTH induced by CRF by 63%, 26%, and 0% respectively. In conclusion, extracellular Ca+2, Ca+2 influx, and calmodulin play a role in the mechanism of CRF stimulated ACTH in vitro.     

Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis

We have investigated the unusual observation that depolarization of rat basophilic leukemia cells in high potassium not only fails to induce secretion, but also inhibits the secretion induced when receptors for IgE are aggregated by antigen. Antigen-stimulated 45Ca uptake and the rise in cytoplasmic free ionized calcium measured with the fluorescent indicator quin2 were both inhibited in depolarized cells. 45Ca efflux, on the other hand, was unaffected, which confirms that IgE receptor activation was not impaired in high potassium. Unlike the large increase in total cell calcium seen when cells in normal saline solution were stimulated with antigen, there was a decrease in total cell calcium when depolarized cells were stimulated. This is consistent with our finding that 45Ca uptake was inhibited while 45Ca efflux was unaffected. Inhibition of 45Ca uptake and secretion closely paralleled the decrease in membrane potential, and could be overcome by increasing the extracellular calcium concentration. We conclude that changes in the electrochemical gradient for calcium are important in determining calcium influx and the magnitude of antigen-stimulated secretion from rat basophilic leukemia cells, while the release of calcium from intracellular stores is unaffected.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Thrombin-induced calcium movements in platelet activation

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.     

Lysophosphatidic acid is a mediator of Trp-Lys-Tyr-Met-Val-d-Met-induced calcium influx

Intracellular calcium (Ca(2+)) homeostasis is very strictly regulated, and the activation of G-protein-coupled receptor (GPCR) can cause two different calcium changes, intracellular calcium release, and calcium influx. In this study, we investigated the possible role of lysophosphatidic acid (LPA) on GPCR-induced Ca(2+) signaling. The addition of exogenous LPA induced dramatic Ca(2+) influx but not intracellular Ca(2+) release in U937 cells. LPA-induced Ca(2+) influx was not affected by pertussis toxin and phospholipase C inhibitor (U73122), ruling out the involvement of pertussis toxin-sensitive G-proteins, and phospholipase C. Stimulation of U937 cells with Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), which binds to formyl peptide receptor like 1, enhanced phospholipase A(2) and phospholipase D activation, indicating LPA formation. The inhibition of LPA synthesis by phospholipase A(2)-specific inhibitor (MAFP) or n-butanol significantly inhibited WKYMVm-induced Ca(2+) influx, suggesting a crucial role for LPA in the process. Taken together, we suggest that LPA mediates WKYMVm-induced Ca(2+) influx.     

Amylase release from rat parotid glands. II. Calcium kinetics.

The kinetics of 45Ca2+ uptake, efflux, and calcium potentiation of amylase release by slices of rat parotid glands were examined. Pretreatment of the tissue with 11.25 mM 45Ca2+ medium increased the total tissue 45calcium content. Lanthanum (1 mM) decreased tissue uptake, blocked the slow components of exchange and appeared to inhibit transcellular calcium movement. Neither dibutyryl cyclic AMP nor caffeine caused consistently significant effects on 45Ca2+ kinetics, or total 45calcium content. Carbamylcholine increased the initial rate of 45Ca2+ uptake, but had no effect on total uptake. Elevation of the extracellular Ca2+ concentration to 11.25 mM during stimulation of amylase release resulted in an initial decrease in the rate of amylase release followed by a potentiation of release which developed slowly, requiring 40--50 min to reach the maximal response. The inability to detect release-related changes in either calcium influx or mobilization, and the lengthy times and high Ca2+ concentrations required to achieve calcium potentiation suggests that calcium does not couple amylase release.     

Schistosoma mansoni: silver ion (Ag +) stimulates and reversibly inhibits lipid-induced cercarial penetration.

Certain long-chain polyunsaturated fatty acids (FA) found on mammalian skin trigger cercariae to penetrate and transform into schistosomules; however, the mechanism by which FAs stimulate cercariae is unknown. In order to determine whether argentophilic papillae concentrated at the apical region of the cercariae are the chemoreceptors that may mediate cercarial response to FAs, an assay assessed the proportion of cercariae that penetrated a 0.25% agar matrix in the presence (61%) and the absence (2.3%) of linolenic acid at 0.22 mM. Silver nitrate (Ag+) which selectively binds to cercarial papillae (Short and Cartrett, J. Parasitol. 59, 1041, 1973) is nontoxic (at 0.09 mM used in this study) as demonstrated by the ability of Ag+ treated cercariae to mature successfully into adult worms (8.8% maturation compared to 10.2% of untreated controls, n = 5) after subcutaneous injection. When Ag+ was added to cercarial suspensions, penetration into linolenic-impregnated agar was significantly inhibited (80.8%). Washing cercariae free of Ag+ reversed this inhibition. These data, as well as observations that both argentophilic papillae and cercarial response to FAs disappeared within 3 to 4 hr after mechanical conversion to schistosomules, implicate argentophilic papillae on cercariae as chemoreceptors for lipid stimulation.     

Mitochondrial Calcium Transport Systems: Properties, Regulation, and Taxonomic Features

Currently available information on properties and regulation of mitochondrial Ca2+ transporting systems in eukaryotic cells is summarized. We describe in detail kinetic properties and effects of inhibitors and modulators on the energy-dependent Ca2+ uptake through the Ca2+ uniporter, as well as on Na+-dependent and Na+-independent pathways for Ca2+ release in mammalian mitochondria. Special emphasis is placed on Ca2+ transport systems (for ion uptake and release) in mitochondria of higher plants, algae, and yeasts. Potential physiological implications of mitochondrial Ca2+ fluxes (influx and efflux), e.g., regulation of activity of Ca2+-dependent enzymes of the Krebs cycle, maintaining of cellular Ca2+ homeostasis, and engagement in pathophysiological processes, are discussed.     

Synaptosomal calcium influx is activated by sodium fluoride

Neuronal calcium channels can be modulated by changes in membrane potential or by activation of channel-associated receptors. The latter may be modulated by guanine nucleotide binding proteins. NaF, which activates guanine nucleotide binding proteins, caused a large stimulation of 45Ca2+ uptake by synaptosomes prepared from rat brain. Stimulation of 45Ca2+ influx by NaF (i) was apparent in media containing either 5 mM-K+ or 50 mM-K+, (ii) was slower than the fast-phase of voltage-dependent 45Ca2+ influx but continued for a longer period of time than did depolarization-induced 45Ca2+ influx, and (iii) was not mimicked or modified by a number of drugs, including ouabain, dinitrophenol, sodium azide or sodium vanadate. These results are consistent with the hypothesis that NaF activates a guanine nucleotide binding protein associated with receptor-coupled calcium channels, resulting in stimulation of calcium influx.     

Neurotransmitter release from bradykinin-stimulated PC12 cells. Stimulation of cytosolic calcium and neurotransmitter release.

The effect of bradykinin on intracellular free Ca2+ and neurotransmitter secretion was investigated in the rat pheochromocytoma cell line PC12. Bradykinin was shown to induce a rapid, but transient, increase in intracellular free Ca2+ which could be separated into an intracellular Ca2+ release component and an extracellular Ca2+ influx component. The bradykinin-induced stimulation of intracellular free Ca2+ displayed a similar time course, concentration dependencies and extracellular Ca2+ dependence as that found for neurotransmitter release, indicating an association between intracellular free Ca2+ levels and neurotransmitter secretion. The selective BK1-receptor antagonist des-Arg9,[Leu8]BK (where BK is bradykinin) did not significantly affect the stimulation of intracellular free Ca2+ or neurotransmitter release. In contrast, these effects of bradykinin were effectively blocked by the selective BK2-receptor antagonist [Thi5,8,D-Phe7]BK, and mimicked by the BK2 partial agonist [D-Phe7]BK in a concentration-dependent manner. The stimulation of intracellular free Ca2+ and neurotransmitter release induced by bradykinin was shown not to involve voltage-sensitive Ca2+ channels, since calcium antagonists had no effect on either response at concentrations which effectively inhibit depolarization-induced responses. These results indicate that bradykinin, acting through the interaction with the BK2 receptor, stimulates an increase in intracellular free Ca2+ leading to neurotransmitter secretion. Furthermore, bradykinin-induced responses involve the release of intracellular Ca2+ and the influx of extracellular Ca2+ that is not associated with the activation of voltage-sensitive Ca2+ channels.     

Regulation of store-operated calcium channels by the intermediary metabolite pyruvic acid

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses, including neurotransmitter release, muscle contraction, and cell growth and proliferation. A major route for Ca(2+) influx is through store-operated Ca(2+) channels. One important intracellular target for Ca(2+) entry through store-operated channels is the mitochondrion, which increases aerobic metabolism and ATP production after Ca(2+) uptake. Here, we reveal a novel feedback pathway whereby pyruvic acid, a critical rate-limiting substrate for mitochondrial respiration, increases store-operated entry by reducing inactivation of the channels. Importantly, the effects of pyruvic acid are manifest at physiologically relevant concentrations and membrane potentials. The reduction in the inactivation of calcium release-activated calcium (CRAC) channels by pyruvate is highly specific in that it is not mimicked by other intermediary metabolic acids, does not require its metabolism, is independent of its Ca(2+) buffering action, and does not involve mitochondrial Ca(2+) uptake or ATP production. These results reveal a new and direct link between intermediary metabolism and ion-channel gating and identify pyruvate as a potential signaling messenger linking energy demand to calcium-channel function.