Effectors of the frequency of calcium oscillations in HEK-293 cells: wavelet analysis and a computer model

Oscillations of the intracellular concentration of Ca²⁺ in cultured HEK-293 cells, which heterologously expressed the calcium-sensing receptor, were recorded with the fluorophore Fura-2 using fluorescence microscopy. HEK-293 cells are extremely sensitive to small perturbations in extracellular calcium concentrations. Resting cells were attached to cover slips and perifused with saline solution containing physiologically relevant extracellular Ca²⁺ concentrations in the range 0.5–5 mM. Acquired digitized images of the cells showed oscillatory fluctuations in the intracellular Ca²⁺ concentration over the time course, and were processed as a function of the change in Fura-2 excitation ratio and frequency at 12–37°C. Newly developed data processing techniques with wavelet analysis were used to estimate the frequency at which the rectified sinusoidal oscillations occurred; we estimated ~4 min⁻¹ under normal conditions. Temperature variations revealed an Arrhenius relationship in oscillation frequency. A critical Ca²⁺ concentration of ~2 mM was estimated, below which oscillations did not occur. These data were used to develop a kinetic model of the system that was simulated using Mathematica; kinetic parameter values were adjusted to match the experimentally observed oscillations of intracellular Ca²⁺ concentration as a function of extracellular Ca²⁺ concentration, and temperature; and from these, limit cycles were obtained and control coefficients were estimated for all parameters.     

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.

     

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     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

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.

     

The Ca2+-sensing receptor: a target for polyamines

The Ca²⁺-sensing receptor(CaR) is activated at physiological levels of externalCa²⁺(Ca_o) but is expressed in anumber of tissues that do not have well-established roles in thecontrol of Ca_o, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown byour current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic freeCa²⁺(Ca_i), inositol phosphateproduction, and the activity of a nonselective cation channel. Sperminestimulated Ca_i transients inCaR-transfected HEK cells, with a concentration producing ahalf-maximal response (EC₅₀) of ~500µM in the presence of 0.5 mMCa²⁺, whereas sustained increasesin Ca_i had anEC₅₀ of ~200 µM. The order ofpotency was spermine > spermidine >> putrescine. Elevation ofCa_o shifted theEC₅₀ for spermine sharply to theleft, with substantial stimulation below 100 µM. Addition ofsubthreshold concentrations of spermine increased the sensitivity ofCaR-expressing HEK cells to Ca_o.Parathyroid hormone secretion from bovine parathyroid cells wasinhibited by 50% in the presence of 200 µM spermine, a responsesimilar to that elicited by 2.0 mMCa_o. These data suggest thatpolyamines could be effective agonists for the CaR, and severaltissues, including the brain, may use the CaR as a target for theactions of spermine and other endogenous polycationic agonists.

     

Soybean β51–63 peptide stimulates cholecystokinin secretion via a calcium-sensing receptor in enteroendocrine STC-1 cells

We previously demonstrated that intraduodenal administration of an arginine-rich β51–63 peptide in soybean β-conglycinin suppresses food intake via cholecystokinin (CCK) secretion in rats. However, the cellular mechanisms by which the β51–63 peptide induces CCK secretion remain to be clarified. In the present study, we examined whether the extracellular calcium-sensing receptor (CaR) mediates β51–63-induced CCK secretion in murine CCK-producing enteroendocrine cell line STC-1. CCK secretion and changes in intracellular Ca²⁺ concentration in response to β51–63 peptide were measured in STC-1 cells under various extracellular Ca²⁺ concentrations and after treatment with a CaR antagonist. Intracellular Ca²⁺ concentrations in response to β51–63 peptide and extracellular Ca²⁺ were also measured in CaR-expressing human embryonic kidney (HEK-293) cells. The β51-63 peptide induced CCK secretion and intracellular Ca²⁺ mobilization in STC-1 cells under normal (1.2 mM) extracellular Ca²⁺ conditions in a dose-dependent manner. These responses to β51–63 peptide were reduced by the removal of intra- or extracellular Ca²⁺ but enhanced by increasing extracellular Ca²⁺ concentrations. Intracellular Ca²⁺ mobilization induced by extracellular Ca²⁺ was also increased by the pretreatment with β51–63 peptide. Treatment with a specific CaR antagonist (NPS2143) inhibited β51–63-induced CCK secretion and intracellular Ca²⁺ mobilization. In addition, HEK-293 cells transfected with CaR acquired sensitivity to the β51–63 peptide. From these results, we conclude that CaR is the β51–63 peptide sensor responsible for the stimulation of CCK secretion in enteroendocrine STC-1 cells.     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects ofendurance run training onNa⁺-dependentCa²⁺ regulation in rat leftventricular myocytes were examined. Myocytes were isolated fromsedentary and trained rats and loaded with fura 2. Contractile dynamicsand fluorescence ratio transients were recorded during electricalpacing at 0.5 Hz, 2 mM extracellular Ca²⁺ concentration, and 29°C.Resting and peak cytosolic Ca²⁺concentration([Ca²⁺]_c)did not change with exercise training. However, resting and peak[Ca²⁺]_cincreased significantly in both groups during 5 min of continuous pacing, although diastolic[Ca²⁺]_cin the trained group was less susceptible to this elevation ofintracellular Ca²⁺. Run trainingalso significantly reduced the rate of[Ca²⁺]_cdecay during relaxation. Myocytes were then exposed to 10 mM caffeinein the absence of external Na⁺ orCa²⁺ to trigger sarcoplasmicreticular Ca²⁺ release and tosuppress cellular Ca²⁺ efflux.This maneuver elicited an elevated steady-state[Ca²⁺]_c.External Na⁺ was then added, andthe rate of[Ca²⁺]_cclearance was determined. Run training significantly reduced the rateof Na⁺-dependent clearance of[Ca²⁺]_cduring the caffeine-induced contractures. These data demonstrate thatthe removal of cytosolic Ca²⁺ wasdepressed with exercise training under these experimental conditionsand may be specifically reflective of a training-induced decrease inthe rate of cytosolic Ca²⁺ removalviaNa⁺/Ca²⁺exchange and/or in the amount ofCa²⁺ moved across the sarcolemmaduring a contraction.     

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.

     

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids

The Ca²⁺-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca²⁺ or Mg²⁺ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca²⁺ (Ca_i²⁺) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G_q/11 or G_i/o signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca_i²⁺ mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists. cytoskeleton; signaling     

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.

     

Pacing rate, halothane, and BDM affect fura 2reporting of [Ca2+]i in intact rat trabeculae

Experiments weredone on intact trabeculae from rats. Fura 2 in the salt form wasmicroinjected directly into the myoplasm. The experiments wereconducted at 30°C, with 2 mM extracellular Ca²⁺ concentrationand pacing at either 0.5 or 5 Hz. The aims were to establish a newmethod for in vivo calibration of fura 2 and to determine the effect ofautofluorescence changes on intracellular Ca²⁺ concentration([Ca²⁺]_i)reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the maincomponent of autofluorescence in heart. An increase in pacing frequencycaused a decrease in autofluorescence. Both halothane and2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximumfluorescence ratio of fura 2 forCa²⁺ for the in vivo calibrationare 3.4 times larger and 2.6 times smaller, respectively, than thosefound in vitro. Using the parameters obtained in vivo, we found thatthe diastolic and systolic[Ca²⁺]_iof a twitch at 30°C were 0.2 and 2.4 µM, respectively. Proper correction of the autofluorescence change unmasks the[Ca²⁺]_ielevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence needcorrection if fura 2 is used to report[Ca²⁺]_i.

     

Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes

Depletion of Ca²⁺ stores inthe sarcoplasmic reticulum (SR) activates extracellularCa²⁺ influx via capacitativeCa²⁺ entry (CCE). Here, CCE levelsin proliferating and growth-arrested human pulmonary artery smoothmuscle cells (PASMCs) were compared by digital imaging fluorescencemicroscopy. Resting cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)in proliferating PASMCs was twofold higher than that in growth-arrestedcells. Cyclopiazonic acid (CPA; 10 µM), which inhibits SRCa²⁺-ATPase and depletes inositol1,4,5-trisphosphate-sensitiveCa²⁺ stores, transiently increased[Ca²⁺]_cytin the absence of extracellularCa²⁺. The addition of 1.8 mMCa²⁺ to the extracellular solutionin the presence of CPA induced large increases in[Ca²⁺]_cyt,indicative of CCE. The CPA-induced SRCa²⁺ release in proliferatingPASMCs was twofold higher than that in growth-arrested cells, whereasthe transient rise of[Ca²⁺]_cytdue to CCE was fivefold greater in proliferating cells. CCE wasinsensitive to nifedipine but was significantly inhibited by 50 mMK⁺, which reduces the drivingforce for Ca²⁺ influx, and by 0.5 mM Ni²⁺, a putative blocker ofstore-operated Ca²⁺ channels.These data show that augmented CCE is associated with proliferation ofhuman PASMCs and may be involved in stimulating and maintaining cell growth.

     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

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.

     

Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells

Because the activity of thesodium pump (Na-K-ATPase) influences the secretion of aldosterone, wedetermined how extracellular potassium (K_o) and calciumaffect sodium pump activity in rat adrenal glomerulosa cells. Sodiumpump activity was measured as ouabain-sensitive ⁸⁶Rb uptakein freshly dispersed cells containing 20 mM sodium as measured withsodium-binding benzofluran isophthalate. Increasing K_o from4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca_o) stimulated sodium pump activity up to 165% andincreased intracellular free calcium as measured with fura 2. Increasing K_o from 4 to 10 mM in the absence ofCa_o stimulated the sodium pump ~30% and did not increaseintracellular free calcium concentration ([Ca²⁺]_i). In some experiments, addition of1.8 mM Ca_o in the presence of 4 mM K_o increased[Ca²⁺]_i above the levels observed in theabsence of Ca_o and stimulated the sodium pump up to 100%.Ca-dependent stimulation of the sodium pump by K_o andCa_o was inhibited by isradipine (10 µM), a blocker of L-and T-type calcium channels, by compound 48/80 (40 µg/ml) andcalmidizolium (10 µM), which inhibits calmodulin (CaM), and by KN-62(10 µM), which blocks some forms of Ca/CaM kinase II (CaMKII).Staurosporine (1 µM), which effectively blocks most forms of proteinkinase C, had no effect. In the presence of A-23187, a calciumionophore, the addition of 0.1 mM Ca_o increased[Ca²⁺]_i to the level observed in the presenceof 10 mM K_o and 1.8 mM Ca_o and stimulated thesodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mMCa_o was not reduced by isradipine but was blocked by KN-62.Thus, under the conditions that K_o stimulates aldosteronesecretion, it stimulates the sodium pump by two mechanisms: directbinding to the pump and by increasing calcium influx, which isdependent on Ca_o. The resulting increase in[Ca²⁺]_i may stimulate the sodium pump byactivating CaM and/or CaMKII.

     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containingendocrine cells in the gastric mucosa that maintain a negative membranepotential of about 50 mV, largely due to voltage-gated K⁺ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol. 270 (Gastrointest Liver Physiol. 33):G739-G745, 1996]. The current study investigated thepresence of voltage-gated Ca²⁺channels in single ECL cells. ECL cells were isolated from rat fundicmucosa by elutriation, density gradient centrifugation, and primaryculture to a purity >90%. Voltage-gatedCa²⁺ currents were measured insingle ECL cells using the whole cell configuration of the patch-clamptechnique. Depolarization-activated currents were recorded in thepresence of Na⁺ orK⁺ blocking solutions and additionof 20 mM extracellular Ca²⁺. ECLcells showed inward currents in response to voltage steps that wereactivated at a test potential of around 20 mV with maximalinward currents observed at +20 mV and 20 mM extracellular Ca²⁺. The inactivation rate of thecurrent decreased with increasingly negative holding potentials and wastotally abolished at a holding potential of 30 mV. Addition ofextracellular 20 mM Ba²⁺ insteadof 20 mM Ca²⁺ increased thedepolarization-induced current and decreased the inactivation rate. Theinward current was fully inhibited by the specific L-typeCa²⁺ channel inhibitor verapamil(0.2 mM) and was augmented by the L-typeCa²⁺ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activateshigh-voltage-activated Ca²⁺channels in ECL cells. Activation characteristics,Ba²⁺ effects, and pharmacologicalresults imply the presence of L-type Ca²⁺ channels, whereasinactivation kinetics suggest the presence of additional N-typechannels in rat gastric ECL cells.

     

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing thebovine cardiacNa⁺/Ca²⁺exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca²⁺ concentrations([Ca²⁺]_i)that activateNa⁺/Ca²⁺exchange activity. Ba²⁺ influx wasmeasured in fura 2-loaded, ionomycin-treated cells under conditions inwhich the intracellular Na⁺concentration was clamped with gramicidin at ~20 mM.[Ca²⁺]_iwas varied by preincubating ionomycin-treated cells with either theacetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl₂. The rate ofBa²⁺ influx increased in asaturable manner with[Ca²⁺]_i,with the half-maximal activation value of 44 nM and a Hill coefficientof 1.6. When identical experiments were carried out with cellsexpressing a Ca²⁺-insensitivemutant of the exchanger, Ba²⁺influx did not vary with[Ca²⁺]_i.The concentration for activation of exchange activity was similar tothat reported for whole cardiac myocytes but approximately an order ofmagnitude lower than that reported for excised, giant patches. Thereason for the difference in Ca²⁺regulation between whole cells and membrane patches is unknown.

     

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Thenotion that intracellular Ca²⁺ (Ca_i²⁺)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa²⁺ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Ca_i²⁺ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca²⁺ concentration([Ca²⁺]_i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca²⁺. We found thatthreshold [Ca²⁺]_i for high extracellularK⁺ (K_e⁺) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high K_e⁺)-inducedrelease of CA. The same agents produced Ca_i²⁺transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Ca_i²⁺responses elicited by high K_e⁺. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca²⁺]_i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Ca_i²⁺ stores do not playa significant role in the instant-to-instant chemoreception process.