Ca2+ uptake and release properties of a thapsigargin-insensitive nonmitochondrial Ca2+ store in A7r5 and 16HBE14o- cells

In a previous study we overexpressed the thapsigargin (tg)-insensitive Pmr1 Ca(2+) pump of the Golgi apparatus of Caenorhabditis elegans in COS-1 cells and studied the properties of the Ca(2+) store into which it was integrated. Here we assessed the properties of an endogenous tg-insensitive nonmitochondrial Ca(2+) store in A7r5 and 16HBE14o- cells, which express a mammalian homologue of Pmr1. The tg-insensitive Ca(2+) store was considerably less leaky for Ca(2+) than the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA)-containing Ca(2+) store. Moreover like for the worm Pmr1 Ca(2+) pump expressed in COS-1 cells, Ca(2+) accumulation into the endogenous tg-insensitive store showed a 2 orders of magnitude lower sensitivity to cyclopiazonic acid than the SERCA-mediated transport. 2,5-Di-(tert-butyl)-1,4-benzohydroquinone was only a very weak inhibitor of the tg-insensitive Ca(2+) uptake in A7r5 and 16HBE14o- cells and in COS-1 cells overexpressing the worm Pmr1. Inositol 1,4,5-trisphosphate released 11% of the Ca(2+) accumulated in permeabilized A7r5 cells pretreated with tg with an EC(50) that was 5 times higher than for the SERCA-containing Ca(2+) store but failed to release Ca(2+) in 16HBE14o- cells. In the presence of tg, 15% of intact A7r5 cells responded to 10 microm arginine-vasopressin with a small rise in cytosolic Ca(2+) concentration after a long latency. In conclusion, A7r5 and 16HBE14o- cells express a Pmr1-containing Ca(2+) store with properties that differ substantially from the SERCA-containing Ca(2+) store.     

Ca(2+) signals in Pmr1-GFP-expressing COS-1 cells with functional endoplasmic reticulum

We studied the role of the Pmr1-containing Ca(2+) store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100 microM ATP with baseline Ca(2+) spiking, while non-transfected cells produced an initial Ca(2+) peak followed by a long-lasting plateau. The Ca(2+) signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca(2+) with a regenerative Ca(2+) signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca(2+) concentration. These results demonstrate the importance of the Pmr1-containing Ca(2+) store in generating or modifying cellular Ca(2+) signals.     

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)

Intracellular pH (pHi) and Ca(2+) regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+). The sources of the Ca(2+) increase are from the endoplasmic reticulum (ER) Ca(2+) pools as well as from Ca(2+) influx. The store-mobilization component of the Ca(2+) increase induced by the pHi rise was not sensitive to antagonists for either IP(3)-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to depletion of the ER Ca(2+) store. We further showed that the physiological consequence of depletion of the ER Ca(2+) store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca(2+) influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+) leak from ER pools followed by Ca(2+) influx via SOCs.     

Utilization of purified myometrium cell plasma membrane Ca2+, Mg2+-ATPase for comparative estimation of the efficiency of energy-dependent Ca2+-transport inhibitors

With the aim of comparative estimation of efficacy of well-known inhibitors of energy-dependent Ca(2+)-transporting systems their effects were investigated on the activity of purified Ca2+, Mg(2+)-ATPase of the myometrium cell plasma membranes. From the approved inhibitors (eosin Y, o-vanadate, thapsigargin, cyclopiazonic acid, ruthenium red, sodium azide) only eosin Y and o-vanadate are potent inhibitors of myometrium sarcolemma Ca(2+)-pump: the values of Ki equal 0.8 and 4.7 microM, respectively. Thapsigargin and cyclopiazonic acid as well as ruthenium red in concentrations inhibiting, respectively, endo(sarco)plasmic reticulum Ca(2+)-pump and energy-dependent Ca(2+)-transport in mitochondria had no effect on the Ca2+, Mg(2+)-ATPase of the uterus smooth muscle cell plasma membrane. Sodium azide (10 mM) blocking completely Ca(2+)-transport in mitochondria inhibited activity of the plasma membrane Ca(2+)-transporting ATPase by 14%.     

Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.     

Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca(2+). The VR1 agonist capsaicin induced Ca(2+) mobilization from intracellular stores in the absence of extracellular Ca(2+), and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca(2+) mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca(2+) release receptor. The main part of the capsaicin-releasable Ca(2+) store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca(2+) store. In addition, VR1 was observed to behave as a store-operated Ca(2+) influx channel. In DRG neurons, capsazepine attenuated Ca(2+) influx following thapsigargin-induced Ca(2+) store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca(2+) influx and inward currents following Ca(2+) store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca(2+) with the plasma membrane-associated form behaving as a store-operated Ca(2+) influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca(2+) release receptor in sensory neurons.     

Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.     

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

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.     

NAADP mobilizes calcium from the endoplasmic reticular Ca(2+) store in T-lymphocytes

The target calcium store of nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent endogenous calcium-mobilizing compound known to date, has been proposed to reside in the lysosomal compartment or in the endo/sarcoplasmic reticulum. This study was performed to test the hypothesis of a lysosomal versus an endoplasmic reticular calcium store sensitive to NAADP in T-lymphocytes. Pretreatment of intact Jurkat T cells with glycyl-phenylalanine 2-naphthylamide largely reduced staining of lysosomes by LysoTracker Red and abolished NAADP-induced Ca(2+) signaling. However, the inhibitory effect was not specific since Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate and cyclic ADP-ribose was abolished, too. Bafilomycin A1, an inhibitor of the lysosomal H(+)-ATPase, did not block or reduce NAADP-induced Ca(2+) signaling, although it effectively prevented labeling of lysosomes by LysoTracker Red. Further, previous T cell receptor/CD3 stimulation in the presence of bafilomycin A1, assumed to block refilling of lysosomal Ca(2+) stores, did not antagonize subsequent NAADP-induced Ca(2+) signaling. In contrast to bafilomycin A1, emptying of the endoplasmic reticulum by thapsigargin almost completely prevented Ca(2+) signaling induced by NAADP. In conclusion, in T-lymphocytes, no evidence for involvement of lysosomes in NAADP-mediated Ca(2+) signaling was obtained. The sensitivity of NAADP-induced Ca(2+) signaling toward thapsigargin, combined with our recent results identifying ryanodine receptors as the target calcium channel of NAADP (Dammermann, W., and Guse, A. H. (2005) J. Biol. Chem. 280, 21394-21399), rather suggest that the target calcium store of NAADP in T cells is the endoplasmic reticulum.     

Role of endothelial Ca2+ stores in the regulation of hydraulic conductivity of Rana microvessels in vivo

Vascular permeability is regulated by endothelial cytosolic Ca(2+) concentration ([Ca(2+)](i)). To determine whether vascular permeability is dependent on extracellular Ca(2+) influx or release of Ca(2+) from stores, hydraulic conductivity (L(p)) was measured in single perfused frog mesenteric microvessels in the presence and absence of Ca(2+) influx and store depletion. Prevention of Ca(2+) reuptake into stores by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibition increased L(p) in the absence of extracellular Ca(2+) influx. L(p) was further increased when Ca(2+) influx was restored. Depletion of the Ca(2+) stores with ionomycin and SERCA inhibition increased L(p) in the presence and the absence of extracellular Ca(2+) influx. However, store depletion in itself did not significantly increase L(p) in the absence of active Ca(2+) release from stores into the cytoplasm. There was a significant positive correlation between baseline permeability and the magnitude of the responses to both Ca(2+) store release and Ca(2+) influx, indicating that the Ca(2+) regulating properties of the endothelial cells may regulate the baseline L(p). To investigate the role of Ca(2+) stores in regulation of L(p), the relationship between SERCA inhibition and store release was studied. The magnitude of the L(p) increase during SERCA inhibition significantly and inversely correlated with that during store release by Ca(2+) ionophore, implying that the degree of store depletion regulates the size of the increase on L(p). These data show that microvascular permeability in vivo can be increased by agents that release Ca(2+) from stores in the absence of Ca(2+) influx. They also show that capacitative Ca(2+) entry results in increased L(p) and that the size of the permeability increase can be regulated by the degree of Ca(2+) release.     

Microtubules regulate local Ca2+ spiking in secretory epithelial cells

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.     

Differential involvement of vacuolar H(+)-ATPase in the refilling of thapsigargin- and agonist-mobilized Ca(2+) stores

Our objective was to evaluate the role of vacuolar H(+)-ATPase and proton gradients in the refilling of Ca(2+) stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca(2+) stores were replenished with a Ca(2+)-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca(2+) pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H(+)-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca(2+)](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca(2+) stores which are differently operated by vacuolar proton ATPase.     

The secretory pathway Ca2+/Mn2+-ATPase 2 is a Golgi-localized pump with high affinity for Ca2+ ions

Accumulation of Ca(2+) into the Golgi apparatus is mediated by sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) and by secretory pathway Ca(2+)-ATPases (SPCAs). Mammals and birds express in addition to the housekeeping SPCA1 (human gene name ATP2C1, cytogenetic position 3q22.1) a homologous SPCA2 isoform (human gene name ATP2C2, cytogenetic position 16q24.1). We show here that both genes present an identical exon/intron layout. We confirmed that hSPCA2 has the ability to transport Ca(2+), demonstrated its Mn(2+)-transporting activity, showed its Ca(2+)- and Mn(2+)-dependent phosphoprotein intermediate formation, and documented the insensitivity of these functional activities to thapsigargin inhibition. The mRNA encoding hSPCA2 showed a limited tissue expression pattern mainly confined to the gastrointestinal and respiratory tract, prostate, thyroid, salivary, and mammary glands. Immunocytochemical localization in human colon sections presented a typical apical juxtanuclear Golgi-like staining. The expression in COS-1 cells allowed the direct demonstration of (45)Ca(2+) (K(0.5) = 0.27 microm) or (54)Mn(2+) transport into an A23187-releasable compartment.     

Identification of a Ca2+-ATPase in brown adipose tissue mitochondria: regulation of thermogenesis by ATP and Ca2+

In brown adipose tissue (BAT) adrenaline promotes a rise of the cytosolic Ca(2+) concentration from 0.05 up to 0.70 mum. It is not known how the rise of Ca(2+) concentration activates BAT thermogenesis. In this report we compared the effects of Ca(2+) in BAT and liver mitochondria. Using electron microscopy and immunolabeling we identified a sarco/endoplasmic reticulum (ER) Ca(2+)-ATPase bound to the inner membrane of BAT mitochondria. A Ca(2+)-dependent ATPase activity was detected in BAT mitochondria when the respiratory substrates malate and pyruvate were included in the medium. ATP and Ca(2+) enhanced the amount of heat produced by BAT mitochondria during respiration. The Ca(2+) concentration needed for half-maximal activation of the ATPase activity and rate of heat production were the same and varied between 0.1 and 0.2 mum. Heat production was partially inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and abolished by thapsigargin, a specific ER Ca(2+)-ATPase inhibitor, and by both rotenone and KCN, two substances that inhibit the electron transfer trough the mitochondrial cytochrome chain. In liver mitochondria Ca(2+) did not stimulate the ATPase activity nor increase the rate of heat production. Thapsigargin had no effect on liver mitochondria. In conclusion, this is the first report of a Ca(2+)-ATPase in mitochondria that is BAT-specific and can generate heat in the presence of Ca(2+) concentrations similar to those noted in the cell during adrenergic stimulation.     

Ca2+-transport in sea urchin unfertilized eggs: regulation by endogenous sulfated polysaccharides and K+

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca(2+) by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K(+) leading to an enhancement of the Ca(2+) transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca(2+) at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K(+) but not by Li(+) and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K(+). These data suggest that the regulation of the SERCA pump by K(+) and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.