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.

     

Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle

The role of Na⁺/Ca²⁺ exchange inregulating intracellular Ca²⁺ concentration([Ca²⁺]_i) in isolated smooth muscle cellsfrom the guinea pig urinary bladder was investigated. Incrementalreduction of extracellular Na⁺ concentration resulted in agraded rise of [Ca²⁺]_i; 50-100 µMstrophanthidin also increased [Ca²⁺]_i. Asmall outward current accompanied the rise of[Ca²⁺]_i in low-Na⁺ solutions(17.1 ± 1.8 pA in 29.4 mM Na⁺). The quantity ofCa²⁺ influx through the exchanger was estimated from thecharge carried by the outward current and was ~30 times that which isnecessary to account for the rise of [Ca²⁺]_i,after correction was made for intracellular Ca²⁺ buffering.Ca²⁺ influx through the exchanger was able to loadintracellular Ca²⁺ stores. It is concluded that the levelof resting [Ca²⁺]_i is not determined by theexchanger, and under resting conditions (membrane potential 50 to60 mV), there is little net flux through the exchanger. However, asmall rise of intracellular Na⁺ concentration would besufficient to generate significant net Ca²⁺ influx.

     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.

     

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.

     

Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor

We determined the effect of aromatic aminoacid stimulation of the human extracellular Ca²⁺-sensingreceptor (CaR) on intracellular Ca²⁺ concentration([Ca²⁺]_i) in single HEK-293 cells. Additionof L-phenylalanine or L-tryptophan (at 5 mM)induced [Ca²⁺]_i oscillations from a restingstate that was quiescent at 1.8 mM extracellular Ca²⁺concentration ([Ca²⁺]_e). Each[Ca²⁺]_i peak returned to baseline values, andthe average oscillation frequency was ~1 min¹ at37°C. Oscillations were not induced or sustained if the[Ca²⁺]_e was reduced to 0.5 mM, even in thecontinued presence of amino acid. Average oscillation frequency inresponse to an increase in [Ca²⁺]_e (from 1.8 to 2.5-5 mM) was much higher (~4 min¹) than thatinduced by aromatic amino acids. Oscillations in response to[Ca²⁺]_e were sinusoidal whereas those inducedby amino acids were transient. Thus both amino acids andCa²⁺, acting through the same CaR, produce oscillatoryincreases in [Ca²⁺]_i, but the resultantoscillation pattern and frequency allow the cell to discriminate whichagonist is bound to the receptor.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

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     

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     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

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.

     

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca²⁺]_i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, which was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also upregulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump     

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     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

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     

Changes in intracellular Ca2+ and pH in response to thapsigargin in human glioblastoma cells and normal astrocytes

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca²⁺ ([Ca²⁺]_i) and intracellular pH (pH_i) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) is a major regulator of [Ca²⁺]_i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca²⁺]_i and pH_i in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca²⁺]_i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [Ca²⁺]_i increase than in normal astrocytes. This increase was prevented in nominally Ca²⁺-free buffer and by 2-APB, an inhibitor of store-operated Ca²⁺ channels. In addition, TG-activated Ca²⁺ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pH_i was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na⁺/H⁺ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca²⁺ homeostasis and pH regulation in tumor cells compared with normal human astrocytes. fura-2; BCECF; store-operated calcium channels     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

Activation of Ca2+-activated K+ (maxi-K+) channel by angiotensin II in myocytes of the guinea pig ileum

We investigatedthe regulation of theCa²⁺-activatedK⁺(maxi-K⁺) channel by angiotensinII (ANG II) and its synthetic analog, [Lys²]ANG II, infreshly dispersed intestinal myocytes. We identified amaxi-K⁺ channel population in theinside-out patch configuration on the basis of its conductance (257 ± 4 pS in symmetrical 150 mM KCl solution), voltage andCa²⁺ dependence of channelopening, lowNa⁺-to-K⁺andCl-to-K⁺permeability ratios, and blockade by externalCs⁺ and tetraethylammoniumchloride. ANG II and[Lys²]ANG II caused anindirect, reversible, Ca²⁺- anddose-dependent activation ofmaxi-K⁺ channels in cell-attachedexperiments when cells were bathed inhigh-K⁺ solution. This effect wasreversibly blocked by DUP-753, being that it is mediated by theAT₁ receptor.Evidences that activation of themaxi-K⁺ channel by ANG II requiresa rise in intracellular Ca²⁺concentration([Ca²⁺]_i)as an intermediate step were the shift of the open probability of thechannel-membrane potential relationship to less positive membranepotentials and the sustained increase in[Ca²⁺]_iin fura 2-loaded myocytes. The preservation of the pharmacomechanical coupling of ANG II in these cells provides a good model for the studyof transmembrane signaling responses to ANG II and analogs in thistissue.