Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)     

Predicted changes in concentrations of free and bound ATP and ADP during intracellular Ca2+ signaling

High Ca²⁺ concentrationscan develop near Ca²⁺ sourcesduring intracellular signaling and might lead to localized regulationof Ca²⁺-dependent processes. Byshifting the amount of Ca²⁺ andother cations associated with ATP, local highCa²⁺ concentrations might alsoalter the substrate available for membrane-associated and cytoplasmicenzymes. To study this, simultaneous equations were solved over a rangeof Ca²⁺ andMg²⁺ concentrations to determinethe general effects of Ca²⁺ on theconcentrations of free and Ca²⁺-and Mg²⁺-bound forms of ATP. Toobtain a more specific picture of the changes that might occur insmooth muscle cells, mathematical models ofCa²⁺ diffusion and regulation wereused to predict the magnitude and time course of near-membraneCa²⁺ transients and their effectson the free and bound forms of ATP near the membrane. The results ofthis work indicate that changes in freeCa²⁺ concentration over the rangeof 50 nM-100 µM would result in significant changes in free ATPconcentration, MgATP concentration, and the CaATP-to-MgATPconcentration ratio.

     

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]_M) and the rate of cytosolic Ca²⁺ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca²⁺ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca²⁺ concentration ([Ca²⁺]_M) and, consequently, the Ca²⁺-stimulated increase in [ATP]_M, thus allowing studies of how the increase in [ATP]_M and the cytosolic Ca²⁺ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca²⁺]_M and [ATP]_M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca²⁺ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca²⁺ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca²⁺ removal during stimulation with a Ca²⁺-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca²⁺ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca²⁺ pumps. This work reveals the usefulness of rhodamine-based Ca²⁺ indicators in examining the role of intramitochondrial Ca²⁺ in mitochondrial (patho) physiology. human skin fibroblast; OXPHOS disease; calcium ion extrusion; rhod-2; CGP-37157     

Relations Between Indoleacetic Acid, Calcium Ions and Ethylene in the Regulation of Growth and Cell Wall Composition in Picea abies

Effects of indoleacetic acid, calcium ions and ethylene on thegrowth of and deposition of different cell wall fractions inthe hypocotyl of Norway spruce (Picea abies (L.) Karst.) seedlingswere investigated. Indoleacetic acid progressively stimulated cellulose depositionas the amount of added Ca²⁺ increased. In contrast, indoleaceticacid promoted lignification and the deposition of non-cellulosicpolysaccharides only in the absence of added Ca²⁺ . When Ca²⁺was added, the indoleacetic acid effect disappeared. Similarly,indoleacetic acid promoted non-cellulosic polysaccharide depositiononly in the absence of ethylene. At increasing ethylene levelsthe effect of indoleacetic acid on non-cellulosic polysaccharidedeposition disappeared and indoleacetic acid instead promotedcellulose deposition. The response to indoleacetic acid depended on the Ca²⁺ concentrationand on the rate of ethylene production. The relationship betweenindoleacetic acid and Ca²⁺ seemed complex, but clearly indoleaceticacid could partially overcome a Ca²⁺ deficiency. The resultssuggest that ethylene may be a factor of particular importancefor the type of polysaccharide deposition during cell wall formation. Key words: Calcium, cell wall, conifers, ethylene, indoleacetic acid     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function

The role of theNa⁺/Ca²⁺exchanger in intracellular Ca²⁺regulation was investigated in freshly dissociated catfish retinalhorizontal cells (HC).Ca²⁺-permeable glutamate receptorsand L-type Ca²⁺ channels as wellas inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitiveintracellular Ca²⁺ stores regulateintracellular Ca²⁺ in these cells.We used the Ca²⁺-sensitive dyefluo 3 to measure changes in intracellularCa²⁺ concentration([Ca²⁺]_i)under conditions in whichNa⁺/Ca²⁺exchange was altered. In addition, the role of theNa⁺/Ca²⁺exchanger in the refilling of the caffeine-sensitiveCa²⁺ store followingcaffeine-stimulated Ca²⁺ releasewas assessed. Brief applications of caffeine (1-10 s) producedrapid and transient changes in[Ca²⁺]_i.Repeated applications of caffeine produced smallerCa²⁺ transients until no furtherCa²⁺ was released. Store refillingoccurred within 1-2 min and required extracellularCa²⁺. Ouabain-induced increases inintracellular Na⁺ concentration([Na⁺]_i)increased both basal free[Ca²⁺]_iand caffeine-stimulated Ca²⁺release. Reduction of external Na⁺concentration([Na⁺]_o)further and reversibly increased[Ca²⁺]_iin ouabain-treated HC. This effect was not abolished by the Ca²⁺ channel blocker nifedipine,suggesting that increases in[Na⁺]_ipromote net extracellular Ca²⁺influx through aNa⁺/Ca²⁺exchanger. Moreover, when[Na⁺]_owas replaced by Li⁺, caffeine didnot stimulate release of Ca²⁺ fromthe caffeine-sensitive store afterCa²⁺ depletion. TheNa⁺/Ca²⁺exchanger inhibitor 2',4'-dimethylbenzamil significantlyreduced the caffeine-evoked Ca²⁺response 1 and 2 min after store depletion.

     

Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart

The Na⁺/Ca²⁺ exchanger is the major Ca²⁺ extrusion mechanism in cardiac myocytes. The activity of the cardiac Na⁺/Ca²⁺ exchanger is dynamically regulated by intracellular Ca²⁺. Previous studies indicate that Ca²⁺ binding to a high-affinity Ca²⁺-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to yellow and cyan fluorescent protein. Ca²⁺ binding to CBD1 induces conformational changes, as detected by fluorescence resonance energy transfer. With this transgenic fish model, we were able to monitor conformational changes of the Ca²⁺ regulatory domain of Na⁺/Ca²⁺ exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca²⁺ transients and Ca²⁺-induced changes in fluorescence resonance energy transfer. The results indicate that Ca²⁺ regulation of the Na⁺/Ca²⁺ exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na⁺/Ca²⁺ exchanger in vivo. Ca²⁺-binding domain; sodium/calcium exchange; zebrafish; fluorescence resonance energy transfer     

Upregulation of Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca²⁺]_cyt in human PASMC. The resting [Ca²⁺]_cyt and Ca²⁺ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na⁺/Ca²⁺ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na⁺/Ca²⁺ exchanger operates in a forward (Ca²⁺ exit) and reverse (Ca²⁺ entry) mode. By activating the reverse mode of Na⁺/Ca²⁺ exchange, removal of extracellular Na⁺ caused a rapid increase in [Ca²⁺]_cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca²⁺ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through store-operated Ca²⁺ channels but also mediated a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, but did not accelerate Ca²⁺ extrusion via the forward mode of Na⁺/Ca²⁺ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange are an additional mechanism responsible for the elevated [Ca²⁺]_cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation     

Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca²⁺] ([Ca²⁺]_m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca²⁺] ([Ca²⁺]_em) to >0.5 µM resulted in a [Ca²⁺]_em-dependent increase in the rate of mitochondrial Ca²⁺ accumulation ([Ca²⁺]_em resulting in half-maximal rate of Ca²⁺ accumulation = 4.4 µM) via Ca²⁺ uniporter. Ca²⁺ uptake was sensitive to the Ca²⁺ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca²⁺]_m increase and recovery were dependent on the extramitochondrial [Na⁺] ([Na⁺]_em) due to Ca²⁺ extrusion via mitochondrial Na⁺/Ca²⁺ exchanger. The maximal rate of Ca²⁺ extrusion was observed with [Na⁺]_em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of [Ca²⁺]_em between 0 and 100 µM simulated rapid beat-to-beat changes in [Ca²⁺]_i (with [Ca²⁺]_i transient duration of 100–500 ms). No [Ca²⁺]_m oscillations were observed, either under conditions of maximal rate of Ca²⁺ uptake (100 µM [Ca²⁺]_em, 0 [Na⁺]_em) or with maximal rate of Ca²⁺ removal (0 [Ca²⁺]_em, 40 mM [Na⁺]_em). The slow frequency-dependent increase of [Ca²⁺]_m argues against a rapid transmission of Ca²⁺ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca²⁺]_m changes elicited by continuous or pulsatile exposure to elevated [Ca²⁺]_em showed no difference in mitochondrial Ca²⁺ uptake. Thus in cardiac myocytes fast [Ca²⁺]_i transients are integrated by mitochondrial Ca²⁺ transport systems, resulting in a frequency-dependent net mitochondrial Ca²⁺ accumulation. mitochondrial Ca²⁺; excitation-contraction coupling; cardiomyocytes     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry     

Bidirectional Ca2+ coupling of mitochondria with the endoplasmic reticulum and regulation of multimodal Ca2+ entries in rat brown adipocytes

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca²⁺ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca²⁺ or Mg²⁺ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) produced by thapsigargin was abolished by removing extracellular Ca²⁺, depressed by depleting extracellular Na⁺, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca²⁺]_i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca²⁺-free EGTA solution, and an Na⁺-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca²⁺ and fully blocked by oligomycin but not by thapsigargin or an Na⁺-deficient solution, was accompanied by a rise in cytoplasmic Mg²⁺ concentration, and was summated with a high pH-induced rise in [Ca²⁺]_i, whereas the extracellular Ca²⁺-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca²⁺ but not by thapsigargin, depressed by decreasing Na⁺, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca²⁺]_i in a Ca²⁺-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca²⁺ release, activating Ca²⁺ release from the ER and store-operated Ca²⁺ entry, and directly elicits a novel plasmalemmal Ca²⁺ entry, whereas Ca²⁺ release from the ER activates Ca²⁺ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP     

Intracellular Calcium Increase in Gerbil Taste Cell by Amino Acid Sweeteners

Gustatory transduction mechanisms for sucrose and amino acidsweeteners in gerbil taste cells were studied with Ca²⁺ imagingand whole cell recording techniques. A 100 mM sucrose stimuluswith Ca²⁺ increased the intracellular Ca²⁺ concentration ([Ca²⁺]i)in sweet-sensitive taste cells of the taste bud, but the sucrosestimulus without Ca²⁺ did not change the [Ca²⁺]i. A 10 mM D-phenylalaninesweet stimulus with or without Ca²⁺ similarly increased the[Ca²⁺]i in the taste bud. The addition of 5 µM ionomysin,a Ca²⁺-ionophore, without Ca²⁺ greatly increased the [Ca²⁺]iin the taste bud. The application of 10 mM D-phenylalanine stimuluswithout Ca²⁺ enhanced the outward K⁺ current in isolated tastecells. These results suggest that a sugar sweetener such assucrose induces a depolarization in gerbil taste cells whichactivates voltage-dependent Ca²⁺ channels and that a non-sugarsweetener such as D-phenylalanine releases Ca²⁺ from the internalstores without a depolarization. Chem. Senses 22: 83–91,1997.     

Differential regulation of Ca(2+) sparks and Ca(2+) waves by UTP in rat cerebral artery smooth muscle cells

Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activatesphospholipase C, shifted Ca²⁺ signaling from sparks towaves in the smooth muscle cells of rat cerebral arteries. UTPdecreased the frequency of Ca²⁺ sparks and transientCa²⁺-activated K⁺ (K_Ca) currentsand increased the frequency of Ca²⁺ waves. The UTP-inducedreduction in Ca²⁺ spark frequency did not reflect adecrease in global cytoplasmic Ca²⁺, Ca²⁺influx through voltage-dependent Ca²⁺ channels (VDCC), orCa²⁺ load of the sarcoplasmic reticulum (SR), since globalCa²⁺ was elevated, blocking VDCC did not prevent theeffect, and SR Ca²⁺ load did not decrease. However,blocking protein kinase C (PKC) with bisindolylmaleimide I did preventUTP reduction of Ca²⁺ sparks and transient K_Cacurrents. UTP decreased the effectiveness of caffeine, which increasesthe Ca²⁺ sensitivity of ryanodine-sensitiveCa²⁺ release (RyR) channels, to activate transientK_Ca currents. This work supports the concept thatvasoconstrictors shift Ca²⁺ signaling modalities fromCa²⁺ sparks to Ca²⁺ waves through the concertedactions of PKC on the Ca²⁺ sensitivity of RyR channels,which cause Ca²⁺ sparks, and of inositol trisphosphate(IP₃) on IP₃ receptors to generateCa²⁺ waves.

     

Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic

AlF₄^-is known to generate oscillations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF₄^--evoked [Ca²⁺]_i oscillations were reported to be dependent on extracellular Ca²⁺, which contrasts with the [Ca²⁺]_i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF₄^- generates extracellular Ca²⁺-dependent [Ca²⁺]_i oscillations in rat pancreatic acinar cells. AlF₄^--induced [Ca²⁺]_i oscillations were stopped rapidly by the removal of extracellular Ca²⁺ and were abolished on the addition of 20 mM caffeine and 2 µM thapsigargin, indicating that Ca²⁺ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store is also required. The amount of Ca²⁺ in the intracellular Ca²⁺ store was decreased as the AlF₄^--induced [Ca²⁺]_i oscillations continued. Measurement of ⁴⁵Ca²⁺ influx into isolated microsomes revealed that AlF₄^-directly inhibited sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). The activity of plasma membrane Ca²⁺-ATPase during AlF₄^- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺. This study shows that AlF₄^- induces [Ca²⁺]_i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca²⁺ as a result of the partial inhibition of SERCA. cholecystokinin; plasma membrane adenosine 5'-triphosphatase; G proteins; caffeine     

Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca²⁺ (Ca_i²⁺) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Ca_i²⁺ (to micromolar concentrations) observed after the addition of ionophore plus Ca²⁺ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Ca_i²⁺, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Ca_i²⁺ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Ca_i²⁺ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Ca_i²⁺. calcium; connexin43; lens gap junctions     

Time-dependent modulation of capacitative Ca2+ entry signals by plasma membrane Ca2+ pump in endothelium

In vascular endothelial cells, depletion of intracellularCa²⁺ stores elicited capacitativeCa²⁺ entry (CCE) that resulted inbiphasic changes of intracellular Ca²⁺ concentration([Ca²⁺]_i)with a rapid initial peak of[Ca²⁺]_ifollowed by a gradual decrease to a sustained plateau level. Weinvestigated the rates of Ca²⁺entry, removal, and sequestration during activation of CCE and theirrespective contributions to the biphasic changes of[Ca²⁺]_i.Ca²⁺ buffering by mitochondria,removal byNa⁺/Ca²⁺exchange, and a fixed electrical driving force forCa²⁺ (voltage-clamp experiments)had little effect on the CCE signal. The rates of entry ofMn²⁺ andBa²⁺, used as unidirectionalsubstitutes for Ca²⁺ entry throughthe CCE pathway, were constant and did not follow the concomitantchanges of[Ca²⁺]_i.Pharmacological inhibition of the plasma membraneCa²⁺ pump, however, abolished thesecondary decay phase of the CCE transient. The disparity between thebiphasic changes of[Ca²⁺]_iand the constant rate of Ca²⁺entry during CCE was the result of a delayed,Ca²⁺-dependent activation of thepump. These results suggest an important modulatory role of the plasmamembrane Ca²⁺ pump in the netcellular gain of Ca²⁺ during CCE.

     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca²⁺ stores opens store-operated Ca²⁺ channels (SOC) and causes Ca²⁺ entry. Transient receptor potential (TRP) cation channels that are permeable to Na⁺ and Ca²⁺ are believed to form functional SOC. Because sarcolemmal Na⁺/Ca²⁺ exchanger has also been implicated in regulating [Ca²⁺]_cyt, this study was designed to test the hypothesis that the Na⁺/Ca²⁺ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca²⁺]_cyt by contributing to store depletion-mediated Ca²⁺ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na⁺, which switches the Na⁺/Ca²⁺ exchanger from the forward (Ca²⁺ exit) to reverse (Ca²⁺ entry) mode, significantly increased [Ca²⁺]_cyt, whereas inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Store depletion also induced a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Removal of extracellular Na⁺ or inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 attenuated the store depletion-mediated Ca²⁺ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange contributes to store depletion-mediated increase in [Ca²⁺]_cyt, and that blockade of the Na⁺/Ca²⁺ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle     

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger (NCX) play crucial roles in regulating [Ca²⁺]_i and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca²⁺ overload by maintaining Ca²⁺ homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca²⁺ transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca²⁺ transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca²⁺]_i levels and attenuated the depression of the Ca²⁺ transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca²⁺ release channels and/or ryanodine receptors (RyRs) and SR Ca²⁺ pump ATPase (SERCA2) and SR Ca²⁺ release and uptake. In addition, a delayed decay rate time constant of Ca²⁺ transients and cell shortening of Ca²⁺ transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca²⁺ homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R. intracellular Ca²⁺ concentration; Ca²⁺ transients; Ca²⁺ transporters; myofilament Ca²⁺ sensitivity