Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells

The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45-50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic screening would provide a powerful approach for defining the molecular details of oscillatory Ca2+ signaling. However, electrophysiological characterization of the intestinal epithelium has not been possible because of its relative inaccessibility. We developed primary intestinal epithelial cell cultures that circumvent this problem. Intestinal cells express two highly Ca2+-selective, voltage-independent conductances. One conductance, IORCa, is constitutively active, exhibits strong outward rectification, is 60-70-fold more selective for Ca2+ than Na+, is inhibited by intracellular Mg2+ with a K1/2 of 692 microM, and is insensitive to Ca2+ store depletion. Inhibition of IORCa with high intracellular Mg2+ concentrations revealed the presence of a small amplitude conductance that was activated by passive depletion of intracellular Ca2+ stores. Active depletion of Ca2+ stores with IP3 or ionomycin increased the rate of current activation approximately 8- and approximately 22-fold compared with passive store depletion. The store-operated conductance, ISOC, exhibits strong inward rectification, and the channel is highly selective for Ca2+ over monovalent cations with a divalent cation selectivity sequence of Ca2+ > Ba2+ approximately Sr2+. Reversal potentials for ISOC could not be detected accurately between 0 and +80 mV, suggesting that PCa/PNa of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both IORCa and ISOC reversibly. Our studies provide the first detailed electrophysiological characterization of voltage-independent Ca2+ conductances in C. elegans and form the foundation for ongoing genetic and molecular studies aimed at identifying the genes that encode the intestinal cell channels, for defining mechanisms of channel regulation and for defining their roles in oscillatory Ca2+ signaling.     

Removal of the MDCK Cell Primary Cilium Abolishes Flow Sensing

The hypothesis that cell primary cilium is solely responsible for the flow-induced Ca2+ response in MDCK cells was tested by removal of the cilia from mature, responsive cells. Incubation of the cells with 4 mM chloral hydrate for 68 hours resulted in the complete loss of the primary cilia and in disorganization of microtubules, as visualized by immunofluorescence. When intracellular Ca2+ concentration was measured with Fluo-4, the elevation that normally accompanies an increase in fluid flow was abolished after 20 hours exposure to chloral hydrate. At this time, the primary cilia still remained attached to the cells but had become twisted and flexible. Twenty-four hours after return of the deciliated cells to normal medium, intracellular microtubule organization appeared normal, but primary cilia had not yet been expressed. The cells failed to increase intracellular Ca2+ in response to fluid flow until after they had been in normal medium for 120 hours, at which time the primary cilia were 3-4 microm long. Chloral hydrate did not impair the Ca2+ mobilization machinery, as the Ca2+ response to mechanical contact and the spread to neighboring cells was unaffected by the drug. We conclude that the primary cilium is the only sensor for the flow-induced Ca2+ response in MDCK cells and estimate that a single mechanically sensitive channel in the cilium could provide the requisite Ca2+ influx.     

Regulation of Transmembrane Signaling by Ganglioside GM1 :

Interaction of antibodies to ganglioside GM1 with Neuro2a cells was studied to investigate the role of GM1 in cell signaling. Binding of anti-GM1 to Neuro2a cells induced the formation of 3H-inositol phosphates (3H-IPs) and elevated the intracellular Ca2+ concentration [Ca2+]i. The rise in [Ca2+]i was due to the influx of Ca2+ from the extracellular medium and release from intracellular Ca2+ pools. The Ca2+ influx pathway did not allow the permeation of Na+ or K+. The influx was inhibited by amiloride, a specific blocker of T-type Ca2+ channels, whereas nifedipine and diltiazem, blockers of L-type Ca2+ channels, did not have any effect. Thus, anti-GM1 appears to activate a T-type Ca2+ channel in Neuro2a cells. The intracellular Ca2+ release was inhibited by pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis toxin (PTx), which also inhibited the 3H-IP formation in Neuro2a cells. Addition of caffeine neither elevated the [Ca2+]i nor affected the anti-GM1-induced [Ca2+]i rise. The data reveal that the binding of anti-GM1 to Neuro2a cells activates phospholipase C via a PTx-sensitive G protein, which leads to formation of IPs and release of Ca2+ from inositol trisphosphate-sensitive pool of endoplasmic reticulum. Anti-GM1 also arrested the differentiation of Neuro2a cells in culture and significantly stimulated their proliferation. This stimulatory effect of anti-GM1 on cell proliferation was blocked by amiloride but not by PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.     

Ca2+-dependent and Ca2+-independent excretion modes of salicylic acid in tobacco cell suspension culture.

14C-salicylic acid (SA) was used to monitor SA metabolism and its regulation in tobacco cell suspension culture. Two SA concentrations (20 microM and 200 microM) were used for comparison. SA was quickly taken up in both treatments, and the 200 microM-treated cells absorbed approximately 15 times that of 20 microM-treated cells within 5 min. More than 85% and 50% of the absorbed SA were excreted in free form to the culture medium within 5 h from cells treated with 200 microM and 20 microM SA, respectively. SA excretion was significantly inhibited by EGTA and the inhibition could be reversed by the addition of exogenous Ca2+ to the culture medium in the 200 microM SA treatment. However, EGTA had little or no effect on SA excretion in the 20 microM SA treatment. The data suggest that tobacco suspension-cultured cells may contain both Ca2+-dependent and Ca2+-independent pathways for SA excretion. Reduced glutathione (an active oxygen species scavenger), staurosporine (a protein kinase inhibitor), and cycloheximide (an inhibitor of de novo protein synthesis) also blocked intracellular SA excretion to the culture medium in the 200 microM but not in the 20 microM SA treatment. These data support the existence of alternative SA excretion pathways in tobacco suspension-cultured cells. Tobacco cells may use both Ca2+-dependent and Ca2+-independent excretion pathways to cope with different intracellular SA status, and the pathway influenced by EGTA, reduced glutathione, staurosporine, and cycloheximide is activated by SA at 200 microM, but not at 20 microM.     

Induction of calcium influx from extracellular fluid by beauvericin in human leukemia cells

Beauvericin, a cyclic hexadepsipeptide, is a mycotoxin that can induce cell death in human lymphoblastic leukemia CCRF-CEM cells. Our previous data have shown that beauvericin induces cell death in CCRF-CEM cells in a dose- and time-dependent manner, and that this beauvericin-induced cell death can be prevented by administration of intracellular calcium chelator-BAPTA. Therefore, the intracellular Ca2+ concentration ([Ca2+]i) may play an important role in beauvericin-induced cell death in CCRF-CEM cells. In this study, the effect of beauvericin on [Ca2+]i and the possible mechanism responsible for the changes of [Ca2+]i in CCRF-CEM cells were investigated. Beauvericin caused a rapid and sustained [Ca2+]i rise in a dose-dependent manner. Excess extracellular Ca2+ facilitated beauvericin-induced [Ca2+]i rise by adding 1 mM CaCl2 in the bathing medium. On the other hand, beauvericin-induced [Ca2+]i rise was prevented in Ca2+-free Tyrode's solution by 200 microM EGTA. In addition, beauvericin-induced [Ca2+]i rise was also attenuated by intracellular Ca2+ chelator-BAPTA/AM. It is worthy to note that neither the voltage-dependent Ca2+ channel blocker, nimodipine, nor depletion of intracellular Ca2+ with thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, has any effect on beauvericin-induced [Ca2+]i rise. The data from present study indicate that beauvericin acts as a potent Ca2+ mobilizer by stimulating extracellular Ca2+ influx CCRF-CEM cells.     

Effect of methylglyoxal on intracellular calcium levels and viability in renal tubular cells

Methylglyoxal (2-oxopropanal), a physiological glucose metabolite, is a highly reactive dicarbonyl compound that can induce stress in cells and cause apoptotic cell death. This study examines the early signaling effects of methylglyxal on renal cells. It was found that methylglyoxal caused a slow and sustained rise of intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (EC50=1.8 mM). Methylglyoxal also induced a [Ca2+]i rise when extracellular Ca2+ was removed, but the magnitude was reduced by 80%. Depletion of intracellular Ca2+ stores with thapsigargin (TG), an endoplasmic reticulum (ER) Ca2+ pump inhibitor, did not affect methylglyoxal's effect. In Ca2+-free medium, the methylglyoxal-induced [Ca2+]i rise was abolished by depleting stored Ca2+ with carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Methylglyoxal-caused [Ca2+]i rise in the Ca2+-containing medium was not affected by modulation of protein kinase C activity, presence of voltage-gated Ca2+ channel blockers, or preincubation with thiol-containing antioxidants. U73122, an inhibitor of phospholipase C, abolished ATP (but not methylglyoxal)-induced [Ca2+]i rise. Furthermore, the [Ca2+]i-elevating effect of methylglyoxal was cell type-dependent, because methylglyoxal failed to cause [Ca2+]i rises in CHO-K1, neutrophils, or platelets. Pretreatment with methylglyoxal for 0-24 h decreased cell viability in a concentration- and time-dependent manner. Meanwhile, methylglyoxal-induced cell death involved apoptotic and necrotic events, the former being the dominant. These findings suggest that methylglyoxal induced a significant rise in [Ca2+]i in Madin-Darby canine kidney (MDCK) renal tubular cells by stimulating both extracellular Ca2+ influx and CCCP-sensitive intracellular Ca2+ release via as yet unidentified mechanisms. The cell type-specific Ca2+ signaling may play an important role in the early process of cytotoxic action of methylglyoxal.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.     

Cytosolic free magnesium in cardiac myocytes: identification of a Mg2+ influx pathway

Regulation of intracellular Mg2+ activity in the heart is not well characterized. Cardiac myocytes were prepared as primary cultures from 7 day old chick embryo hearts and intracellular Mg2+ concentration [( Mg2+]i) was determined in single ventricular cells with mag-fura-2. Basal [Mg2+]i was 0.48 +/- 0.03 mM in normal culture medium. There was no correlation of basal [Mg2+]i with cellular contraction or intracellular [Ca2+]i (determined with fura-2). Cardiocytes cultured (16 hr) in low Mg (0.16 mM) media contained 0.21 +/- 0.05 mM Mg2+ which returned to normal levels when placed in Mg media with a refill time of 20 min. Basal [Ca2+]i (121 +/- 11 nM) and stimulated [Ca2+]i (231 +/- 41 nM) was similar to control cells. Verapamil, 25 microM, reversibly blocked Mg2+ refill. In conclusion, the basal [Mg2+]i of isolated cardiomyocytes is considerably below the Mg2+ electrochemical equilibrium allowing passive Mg2+ influx. The influx pathway for Mg2+ is inhibited by verapamil and appears to be independent of Ca2+ as assessed by fura-2.     

NaF-induced Ca2+ mobilization is dependent upon the culture density in a parathyroid hormone-responsive osteoblast-like cell line

The effect of NaF on cytosolic free Ca2+ concentrations [( Ca2+]i) was examined in a clonal osteoblast-like cell line (MOB 3-4) loaded with Fura 2. MOB 3-4 cells in a sparse culture, which exhibited neither alkaline phosphatase (ALP) activity nor the response to parathyroid hormone (PTH), responded to NaF (0.1-10 mM) to increase [Ca2+]i transiently. In contrast, the cells in a dense culture, which exhibited both ALP activity and the response to PTH, responded to NaF (above 4 mM) to increase [Ca2+]i slowly. [Ca2+]i in osteoblasts in primary culture slowly increased in response to both NaF (above 4 mM) and PTH (3 U/ml). Thus, the sensitivity and the response of MOB 3-4 cells to NaF and PTH varied with the culture density, and high culture density matured the cells like osteoblasts in primary culture. These NaF-induced Ca2+ mobilizations were not dependent upon external Ca2+ and were enhanced by Al3+ (1 microM), whereas the PTH-induced Ca2+ mobilizations were due to Ca2+ influx. These results suggest that the maturation of MOB 3-4 cells, dependent upon the culture density, modulates intracellular signal transduction pathways and thereby alters the NaF-induced Ca2+ mobilization, and that the culture density must be taken into consideration in studying Ca2+ mobilization in such an osteoblast-like cells line as MOB 3-4 cell line.     

Alteration of brown adipocyte Ca2+ responses in culture by adrenergic activation

Thermogenic capability of brown adipose tissue is controlled by norepinephrine. Interaction of norepinephrine with adipocyte at- and P3-adrenergic receptors results in the increase of Ca2+ and cAMP concentrations. The [Ca2+]i changes initiated by norepinephrine and selective agonists of alpha1- and beta-adrenergic receptors, cirazolin and isoproterenol, were recorded in single cells of primary culture on the 1st, 3rd and 6th days in vitro. On the first day, isoproterenol-induced [Ca2+]i changes as compared to cirazolin-induced ones were characterized by greater amplitude and lesser impulse duration over the entire range of physiological concentrations used. These differences were negligible after 3 days and kinetic differences were practically absent after 6 days of cultivation. The agonist-induced [Ca2+]i changes in proliferating and differentiated cells differed significantly: in the process of cell growth in culture, the amplitude of calcium response increased, the duration of impulse signal decreased and the sensitivity to adrenergic agonists increased. The Ca2+ store in endoplasmic reticulum increased during the cell growth and development in culture, according to thapsigargin-induced Ca2+ response amplitude increase in Ca2+ free medium. The rate of Ca2+ pumping out of cell characterizing PMCA-activity also increased.     

Regulation of Intracellular Calcium in Cerebellar Granule Neurons: Effects of Depolarization and of Glutamatergic and Cholinergic Stimulation

The regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was investigated by microfluorimetry in single cerebellar granule neurons exposed to various treatments (high K+, glutamate, or acetylcholine) and drugs. The responses to the treatments developed asynchronously during cell culture, with high K+ and glutamate reaching their maxima at 6 and 7 days in vitro and acetylcholine at 9 days in vitro. The biphasic [Ca2+]i transients induced by high K+ (an initial peak, followed by a plateau 30-40% of the peak, both sustained by dihydropyridine-sensitive voltage-gated Ca2+ channels) were dissipated by washing with fresh medium or, more rapidly, by addition of excess EGTA (t1/2 = 11 +/- 2 and 3 +/- 0.6 s, respectively). Compared to those induced by high K+, the [Ca2+]i transients induced by glutamate administered in Mg2(+)-free medium were much more variable. An initial peak, sustained by voltage-gated Ca2+ channels, was visible in only approximately 50% of the cells and disappeared when multiple glutamate pulses were administered. In the rest of the population, the transients were monophasic, with persistent plateaus sustained only in part (30-40%) by voltage-gated Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Effect of selenium-supplement on the calcium signaling in human endothelial cells

Intracellular Ca2+ signaling controls many cellular functions. Understanding its regulation by selenoproteins is essential for understanding the role of selenoproteins in regulating cell functions. The activity of thioredoxin reductase (TrxR), thioredoxin (Trx) content, and the activity of glutathione peroxidase (GPx) in the human endothelial cells cultured in selenium-supplemented medium (refer as Se+ cells) was found 70%, 40%, and 20% higher, respectively than those in the cells cultured in normal medium (refer as Se0 cells). The intracellular Ca2+ signaling initiated by inositol 1,4,5-trisphosphate (IP3), histamine, thapsigargin (TG), carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone (FCCP), and cyclosporin A (CsA) was investigated in both Se+ and Se0 cells. It was interestingly found that the higher activity of selenoproteins reduced the sensitivity of IP3 receptor to the IP3-triggered Ca2+ release from intracellular stores, but enhanced activation of the receptor-coupled phospholipase C in histamine-stimulated Se+ cells by showing much more generation of IP3 and higher elevation of cytosolic Ca2+. The higher selenoprotein activity also reduced susceptibility of the uniporter to the mitochondrial uncoupler, susceptibility of the permeability transition pore (PTP) to its inhibitor, and the vulnerability of endoplasmic reticulum (ER) Ca2+-ATPase to its inhibitor in selenium-supplementing cells. The results suggest that cell calcium signaling is subjected to thiol-redox regulation by selenoproteins.     

Serum-induced cytosolic calcium movements and mitogenesis in cultured preosseous chondrocytes

The differentiation of preosseous chondrocytes begins with the proliferation of resting cells and results in the expression of the hypertrophic phenotype. The effect of fetal calf serum on chondrocyte mitogenesis and intracellular Ca2+ concentration was studied in resting and hypertrophic cells in primary culture. Resting chondrocytes respond to the growth stimulus with immediate release of Ca2+ from intracellular stores and with opening of the plasma membrane Ca2+ channels. These events may be related to the elevated [3H]thymidine incorporation observed after serum exposure. In contrast, in hypertrophic chondrocytes the lower rate of DNA synthesis seems to be coupled with a lower activity of the Ca2+ signaling mechanism and, probably, with reduced intracellular calcium stores. It is proposed that expression of the Ca2+ signaling mechanism may be modulated during the differentiation of preosseous chondrocytes.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth.

The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.     

Regulation of nitric oxide-responsive recombinant soluble guanylyl cyclase by calcium.

Calcium (Ca2+) and cyclic GMP (cGMP) subserve antagonistic functions that are reflected in their coordinated reciprocal regulation in physiological systems. However, molecular mechanisms by which Ca2+ regulates cGMP-dependent signaling remain incompletely defined. In this study, the inhibition of recombinant nitric oxide (NO)-stimulated soluble guanylyl cyclase (SGC) by Ca2+ was demonstrated. The alpha- and beta-subunits of recombinant rat SGC were heterologously coexpressed in HEK 293 cells which do not express NO synthase, whose Ca2+-stimulated activity can confound the effects of that cation on SGC. Ca2+ inhibited basal and NO-stimulated SGC in a concentration- and guanine nucleotide-dependent fashion. This cation inhibited SGC in crude cell extracts and immunopurified preparations. Ca2+ lowered both the Vmax and Km of SGC via an uncompetitive mechanism through direct interaction with the enzyme. In intact HEK 293 cells, increases in the intracellular Ca2+ concentration induced by ionomycin, a Ca2+ ionophore, and thapsigargin, which releases intracellular stores of that cation, inhibited NO-stimulated intracellular cGMP accumulation. Similarly, carbachol-induced elevation of the intracellular Ca2+ concentration inhibited NO-stimulated intracellular cGMP accumulation in HEK 293 cells. These data demonstrate that SGC behaves as a sensitive Ca2+ detector that may play a central role in coordinating the reciprocal regulation of Ca2+- and cGMP-dependent signaling mechanisms.     

The Na+/Ca2+ exchanger regulates cytolysin/perforin-induced increases in intracellular Ca2+ and susceptibility to cytolysis.

The Ca2+ dependency of NK cell-mediated and cytolysin-mediated cytolysis may be related to increases in target cell intracellular Ca2+. In a previous study we hypothesized that the Na+/Ca2+ exchanger can act as a counter-lytic mechanism by regulating the damaging increases in intracellular free calcium ([Ca2+]i) produced by cytolysin. We found that conditions said to inhibit Ca2+ extrusion by Na+/Ca2+ exchange, namely low extracellular Na+ or the presence of certain amiloride analogs which block Na+/Ca2+ exchange, enhanced the cytolysin-mediated cytolysis of YAC-1 lymphoma cells. In the present work we have confirmed the above hypothesis by measuring the [Ca2+]i of fura-2- or aequorin-labeled YAC-1 cells treated with cytolysin and low Na+ medium or amiloride analogs. YAC-1 cells appear to have a Na+/Ca2+ exchange system: low Na+ medium caused gradual increases in [Ca2+]i, and this effect was reversed in Na(+)-replete medium. Cytolysin purified from NK cell granules caused rapid dose-dependent increases in [Ca2+]i, and low Na+ medium enhanced these cytolysin-mediated increases. The Na+/Ca2+ exchange system appeared to be more active in cytolysin-challenged cells: amiloride analogs, which inhibit Na+/Ca2+ exchange in other systems, acted synergistically with cytolysin to cause large increases in [Ca2+]i, but had little effect, if any, on their own. 5-(N-4-Chlorobenzyl)-2',4'-dimethylbenzamil, the amiloride analog which has the greatest specificity for the Na+/Ca2+ exchanger and which previously was found to be the most potent enhancer of cytolysin-mediated cytolysis, was the most potent enhancer of cytolysin-mediated increases in [Ca2+]i. The above results suggest that Na+/Ca2+ exchange may be one of the target cell mechanisms of resistance to cytolysin and NK cell-mediated cytolysis.     

Epidermal growth factor-stimulated DNA synthesis requires an influx of extracellular calcium

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)-induced rat liver epithelial (T51B) cell DNA synthesis. Fura-2-loaded T51B cells responded with an increase in [Ca2+]i to EGF (5-50 ng/ml) that was blocked by low (25 microM) extracellular Ca2+ or by pretreatment with 50 microM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1-50 ng/ml) dose-dependently incorporated [3H]-thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF-stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.