Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.     

Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts.

A comparison of the effect of platelet-derived growth factor (PDGF) and bombesin on intracellular Ca2+ stores was carried out in Swiss 3T3 cells loaded with Fura-2. It was found that the tumor promoter thapsigargin (Tg) almost completely inhibited both the PDGF- and the bombesin-induced intracellular Ca2+ concentration ([Ca2+]i) rise, indicating that the two mitogens mobilize Ca2+ from intracellular pool(s) sensitive to the tumor promoter. It was also found that pre-treatment with PDGF almost totally and persistently (up to at least 30 min) inhibited the bombesin-, Tg- and ionomycin-induced rise in [Ca2+]i, whereas pre-treatment with bombesin had only a partial inhibitory effect on the PDGF, Tg and ionomycin [Ca2+]i response, both in the absence and in the presence of external Ca2+. On the other hand, vasopressin and bradykinin, which also stimulate hydrolysis of phosphoinositides in these cells, did not affect the [Ca2+]i response induced by the same agents. These results indicate that, despite the poor production of inositol 1,4,5-trisphosphate (InsP3), PDGF was capable of totally discharging and maintaining discharged the InsP3-sensitive stores of intracellular Ca2+, regardless of whether extracellular Ca2+ was present in the medium. Bombesin only partially caused this effect. On the contrary, bradykinin and vasopressin, after releasing intracellular Ca2+ allowed an almost total refilling of the pools. It is interesting to note that, at variance with PDGF and bombesin, neither bradykinin nor vasopressin are able to induce a mitogenic response in Swiss 3T3 cells.     

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.     

Effect of p-chloroamphetamine on calcium movement and viability in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of p-chloroamphetamine, a neurotoxin that depletes intracellular serotonin, on intracellular Ca2+ concentration ([Ca2+]i) and viability was measured by using the Ca2+-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium. p-Chloroamphetamine (> or = 10 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. p-Chloroamphetamine-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. p-Chloroamphetamine-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which p-chloroamphetamine failed to increase [Ca2+]i; also, pretreatment with p-chloroamphetamine reduced 50% of thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not p-chloroamphetamine)-induced [Ca2+]i rise. Overnight incubation with 1-500 microM p-chloroamphetamine decreased cell viability. These findings suggest that p-chloroamphetamine evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic.     

Mechanisms involved in the increase in intracellular calcium following hypotonic shock in bovine articular chondrocytes

The extracellular osmotic environment of chondrocytes fluctuates during joint loading as fluid is expressed from and reimbibed by the extracellular matrix. Matrix synthesis by chondrocytes is modulated by joint loading, possibly mediated by variations in intracellular composition. The present study has employed the Ca2+-sensitive fluoroprobe Fura-2 to determine the effects of hypotonic shock (HTS) on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a 50% dilution, [Ca2+]i rapidly increased by approximately 250%, a sustained plateau being achieved within 300 s. The effect was inhibited by thapsigargin or by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i reflects both influx from the extracellular medium and release from intracellular stores. Inhibition of the response by neomycin implicates activation of PLC and IP3 synthesis in the mobilisation of Ca2+ from intracellular stores. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels (LVACC) or reverse mode Na+/Ca2+ exchange (NCE) but could be significantly attenuated by ruthenium red, an inhibitor of transient receptor potential vanilloid (TRPV) channels and by Gd3+, a blocker of stretch-activated cation (SAC) channels. The HTS-induced rise in [Ca2+]i was almost completely absent in cells treated with Ni2+, a non-specific inhibitor of Ca2+ entry pathways. We conclude that in response to HTS the opening of SACC and a member of TRPV channel family leads to Ca2+ influx, simultaneously with the release from intracellular stores.     

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.     

Changes in intracellular calcium concentration in response to hypertonicity in bovine articular chondrocytes

Intracellular calcium concentration ([Ca2+]i) in articular chondrocytes changes during mechanical challenges associated with joint movements, because of the fluctuation of the extracellular osmotic environment during joint loading. Matrix synthesis by chondrocytes is modulated by loading patterns, possibly mediated by variations in intracellular composition, including [Ca2+]i. The present study has employed the Ca(2+)-sensitive fluoroprobe Fura-2 to determine the effects of hypertonic shock on intracellular Ca2+ concentration ([Ca2+]i) and to characterise the mechanisms involved in the response for isolated bovine articular chondrocytes. In cells subjected to a hypertonic shock, [Ca2+]i rapidly increased by approximately 300%, reaching a maximal value within 50 s following the hypertonic shock with a recovery of more than 90% towards the initial [Ca2+]i within 5 min. The effect was inhibited by removal of extracellular Ca2+ ions, but not by thapsigargin, indicating that the rise in [Ca2+]i is only a result of influx from the extracellular medium. The rise was insensitive to inhibitors of L-type voltage-activated Ca2+ channels, TRPV channels or stretch-activated cation channels. Non-specific inhibitors of Ca2+ channels like CdCl2, NiCl2, LaCl3 and ZnCl2 significantly attenuated the response, although the extent in which CdCl2 and NiCl2 (both of them inhibitors of annexin-mediated Ca2+ fluxes) inhibited the response was significantly greater. The rise was also sensitive to KBR7943, inhibitor of NCE reverse mode and trifluoperazine, inhibitor of the activity of annexins. Hypertonic shock also produced also hyperpolarisation of chondrocytes (Em measured by means of Di-BA-C4(3), a membrane potential sensitive dye), which was inhibited by TEA-Cl and BaCl, but was not affected by changing the extracellular solution to Ca(2+)-free HBS. Inhibition of hyperpolarisation completely abolished the [Ca2+]i rise following hypertonic shock. Treatment with retinoic acid, which can increase the activity of annexins as Ca2+ transport pathways caused a significant increase in [Ca2+]i. The recovery of [Ca2+] was inhibited by benzamil and was dependent on extracellular Na+, but was unaffected by Na-orthovanadate, an inhibitor of plasma Ca(2+)-ATPase. We conclude that in response to hypertonic shock, NCE reverse mode and annexins are the pathways responsible for the [Ca2+]i increase, while forward mode operation of NCE is responsible for the subsequent extrusion of Ca2+ and recovery of [Ca2+]i towards initial values.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.     

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.     

Spatial and temporal distribution of agonist-evoked cytoplasmic Ca2+ signals in exocrine acinar cells analysed by digital image microscopy.

High resolution digital video imaging has been employed to monitor the spatial and temporal development of agonist-induced cytosolic Ca2+ signals in fura 2-loaded exocrine acinar cells. Enzymatically isolated mouse pancreatic and lacrimal acinar cells or small acinar cell clusters were used. These retain their morphological polarity so that the secretory granules in individual cells are located at one pole, the secretory pole. In acinar cell clusters the granules are located centrally, oriented to surround what would be in situ referred to as the lumen. In pancreatic and lacrimal acinar cells inositol-1,4,5-triphosphate-generating agonists [acetylcholine (ACh) and cholecystokinin octapeptide (CCK) for the pancreas and ACh in the lacrimal gland] give rise to a rapidly spreading Ca2+ signal that is initiated at the secretory pole of the cells. The initial increase in [Ca2+]i in the luminal pole is independent of extracellular Ca2+ indicating that the earliest detectable intracellular Ca2+ release is specifically located at the secretory pole. In lacrimal acinar cells ATP acts as an extracellular agonist, independent of phosphoinositide metabolism to activate a receptor-operated calcium influx pathway which, as for ACh, gives rise firstly to an increase in intracellular Ca2+ concentration in the secretory pole. We propose that this polar rise in intracellular Ca2+ concentration is due to Ca(2+)-induced Ca2+ release. By contrast, when Ca2+ release and Ca2+ influx are induced in the absence of receptor activation by thapsigargin and ionomycin, the Ca2+ signal develops diffusely and slowly with no localization to the secretory pole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine increases intracellular Ca2+ in the rat pituitary folliculostellate cells in primary culture

Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca2+ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 microM) increased intracellular calcium concentration ([Ca2+](i)) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca2+ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca2+](i). These results suggest that ACh increases [Ca2+](i) in FSCs by activating phospholipase C, presumably through activation of M(1) receptors. The rise in [Ca2+](i) could trigger a variety of Ca2+-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.     

Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation.

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.     

A role for Ca2+ in mediating hormone-induced biphasic pepsinogen secretion from the chief cell determined by luminescent and fluorescent probes and X-ray microprobe

In isolated chief cells from the guinea pig, cholecystokinin (10 nM) and a high concentration of ionomycin each caused a biphasic pattern of pepsinogen secretion. The initial fast response to cholecystokinin was not dependent on medium Ca2+ ans was mimicked by low concentration of ionomycin (100 nM). Inositol 1,4,5-trisphosphate caused a similar fast release from permeabilized cells. The slow component of release was dependent on medium Ca2+, however, and was mimicked by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (100 nM) or the diacylglycerol analogue 1-oleoyl-2-acetylglycerol (OAG) (100 microM). Ionomycin (100 nM) and TPA (and/or OAG), when applied together, reproduced the biphasic pattern of pepsinogen secretion, suggesting that the signalling pathways utilized by both types of agonist contribute to the response evoked by cholecystokinin-hormone stimulation. Both fura-2 and aequorin were used to monitor changes of intracellular Ca2+. Three pathways were found to contribute to the Ca2+ transient. A rapid release of Ca2+ from intracellular store(s), a rapid Ca2+ entry from the extracellular space, and a more sustained Ca2+ entry from the extracellular space. Cholecystokinin induced a rapid increase in cytoplasmic Ca2+ ([Ca2+]i) as estimated with fura-2 and aequorin. This rise was reduced but not abolished upon removal of extracellular Ca2+, suggesting that both Ca2+ entry from the extracellular space and Ca2+ mobilization from the intracellular store(s) contribute to the initial, fast component of the Ca2+ transient. A second, more sustained component of the Ca2+ transient induced by cholecystokinin was abolished by lanthanum. TPA and OAG induced a biphasic Ca2+ transient that could be detected only with aequorin. The late, sustained component of this response was again abolished by lanthanum as well as by removal of extracellular Ca2+. It appears that the late component of the Ca2+ transient is dependent on Ca2+ influx from the extracellular space and is too localized to be detected by fura-2. Prestimulation of cells with TPA or OAG prevented the aequorin transient caused by cholecystokinin and vice versa, suggesting that TPA, OAG and cholecystokinin activate the same pathways of Ca2+ entry into the cytosol from the intracellular store(s) or the extracellular space. The stimulation-sensitive Ca2+ pool was examined with electron probe X-ray microanalysis. It appears to be restricted to an area enriched in secretory granules or peripheral endoplasmic reticulum just beneath the apical plasma membrane and in close association with the microtubular-microfilamentous system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells

We studied the effect of thapsigargin on intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca2+]i dose dependently with an EC50 of approximately 0.15 microM. The Ca2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca2+ store with thapsigargin for 7 min abolished the [Ca2+]i increases evoked by bradykinin. Removal of extracellular Ca2+ reduced the thapsigargin response by approximately 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store followed by capacitative Ca2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La3 and Gd3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca2+ store for 30 min with another inhibitor of the internal Ca2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca2+]i, thus suggesting that the thapsigargin-evoked Ca2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La3+ (or Gd3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na+ had no effect. Collectively, our results imply that thapsigargin increased [Ca2+]i in MDCK cells by depleting the internal Ca2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca2+ pump.     

Dynamics of intracellular oxygen in PC12 Cells upon stimulation of neurotransmission

Neurotransmission, synaptic plasticity, and maintenance of membrane excitability require high mitochondrial activity in neurosecretory cells. Using a fluorescence-based intracellular O2 sensing technique, we investigated the respiration of differentiated PC12 cells upon depolarization with 100 mm K+. Single cell confocal analysis identified a significant depolarization of the plasma membrane potential and a relatively minor depolarization of the mitochondrial membrane potential following K+ exposure. We observed a two-phase respiratory response: a first intense spike lasting approximately 10 min, during which average intracellular O2 was reduced from 85-90% of air saturation to 55-65%, followed by a second wave of smaller amplitude and longer duration. The fast rise in O2 consumption coincided with a transient increase in cellular ATP by approximately 60%, which was provided largely by oxidative phosphorylation and by glycolysis. The increase of respiration was orchestrated mainly by Ca2+ release from the endoplasmic reticulum, whereas the influx of extracellular Ca2+ contributed approximately 20%. Depletion of Ca2+ stores by ryanodine, thapsigargin, and 4-chloro-m-cresol reduced the amplitude of respiratory spike by 45, 63, and 71%, respectively, whereas chelation of intracellular Ca2+ abolished the response. Uncoupling of the mitochondria with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone amplified the responses to K+; elevated respiration induced a profound deoxygenation without increasing the cellular ATP levels reduced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Cleavage of synaptobrevin 2 by tetanus toxin, known to reduce neurotransmission, did not affect the respiratory response to K+, whereas the general excitability of d PC12 cells increased.     

Transmembrane calcium influx induced by ac electric fields.

Exogenous electric fields induce cellular responses including redistribution of integral membrane proteins, reorganization of microfilament structures, and changes in intracellular calcium ion concentration ([Ca2+]i). Although increases in [Ca2+]i caused by application of direct current electric fields have been documented, quantitative measurements of the effects of alternating current (ac) electric fields on [Ca2+]i are lacking and the Ca2+ pathways that mediate such effects remain to be identified. Using epifluorescence microscopy, we have examined in a model cell type the [Ca2+]i response to ac electric fields. Application of a 1 or 10 Hz electric field to human hepatoma (Hep3B) cells induces a fourfold increase in [Ca2+]i (from 50 nM to 200 nM) within 30 min of continuous field exposure. Depletion of Ca2+ in the extracellular medium prevents the electric field-induced increase in [Ca2+]i, suggesting that Ca2+ influx across the plasma membrane is responsible for the [Ca2+]i increase. Incubation of cells with the phospholipase C inhibitor U73122 does not inhibit ac electric field-induced increases in [Ca2+]i, suggesting that receptor-regulated release of intracellular Ca2+ is not important for this effect. Treatment of cells with either the stretch-activated cation channel inhibitor GdCl3 or the nonspecific calcium channel blocker CoCl2 partially inhibits the [Ca2+]i increase induced by ac electric fields, and concomitant treatment with both GdCl3 and CoCl2 completely inhibits the field-induced [Ca2+]i increase. Since neither Gd3+ nor Co2+ is efficiently transported across the plasma membrane, these data suggest that the increase in [Ca2+]i induced by ac electric fields depends entirely on Ca2+ influx from the extracellular medium.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.     

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.