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.     

Measurement of intracellular Ca2+ in young and old human erythrocytes using 19F-NMR spectroscopy.

Elevated cell calcium has been implicated in functional changes with human erythrocyte aging. However, until recently it has been difficult to measure free ionic intracellular calcium in red cells. We have made use of a fluorinated calcium chelator probe (5,5'-difluoroBAPTA) and fluorine nuclear magnetic resonance (19F-NMR) techniques to measure changes of intracellular Ca2+ concentrations ([Ca2+]i) with cell aging. We have demonstrated in these studies that human erythrocyte [Ca2+]i is significantly elevated as a function of in-vivo aging. Young cells, the least dense fraction of density-separated erythrocytes, contained an average of 62 (+/- 4) nM Ca2+ (+/- S.E.), whereas the oldest, most dense cell fraction contained 221 nM Ca2+ (+/- 25). Mechanisms by which intracellular [Ca2+] increases with in-vivo aging are currently under investigation.     

Calcium affects phosphoinositide turnover in human erythrocytes

Changes in extracellular Ca2+ concentration ([Ca2+]) were observed to affect 32Pi incorporation into polyphosphoinositides (PPI) and phosphatidic acid (PA) of human erythrocytes. A decrease of extracellular [Ca2+] from 1.5 mmol/l to 0.04 mumol/l increased the specific radioactivity (S.A.) of phosphatidylinositol 4,5-bisphosphate to 182% and that of phosphatidylinositol 4-phosphate to 120% of controls. Simultaneously S.A. and concentration of PA decreased. Further decrease of the extracellular [Ca2+] from 0.04 mumol/l to lower values as well as depletion of intracellular Ca2+ using ionophore A 23187 in Ca2(+)-free medium did not accelerate the PPI turnover rates any more. None of the above changes in extracellular [Ca2+] had any effect on the phosphorylation pattern of erythrocyte membrane proteins. Isolated erythrocyte membranes were incubated in the presence of [gamma-32P]ATP in media with various [Ca2+]. The decrease of [Ca2+] from 0.04 mumol/l (physiological concentration inside the cell) to lower values did not influence the turnover of PPI and PA monoester phosphates. Only after [Ca2+] was increased to 1-5 mumol/l an increase of PPI and PA turnover was observed. Our data suggest that the changes in extracellular [Ca2+] affect the metabolism of PPI and PA (despite the intracellular location of the latter) and may thus influence the properties of red cell plasma membrane.     

Extracellular calcium is required for the maintenance of plasma membrane integrity in nucleated cells

In contrast to rat and human erythrocytes, nucleated erythrocytes from two fish species (Cyprinus carpio and Salmo trutta) underwent almost complete haemolysis in 20 min of EDTA addition. Using Ca2+/Mg2+ EGTA-citrate buffer, we observed that half-maximal haemolysis of fish erythrocytes occurs at [Ca2+]o approximately 10 microM independently of extracellular Mg2+ concentration. Attenuation of [Ca2+]o with EGTA also decreased stability of the plasma membrane of vascular smooth muscle cells (VSMC) and HeLa cells, indicated by a three- to five-fold elevation of lactate dehydrogenase release and passive permeability of plasma membrane for Na+. In VSMC, EGTA lowered [Ca2+]i by approximately 20%. This effect was absent in VSMC-loaded with the intracellular Ca2+ chelator BAPTA. In contrast to EGTA, BAPTA did not affect haemoglobin release from fish erythrocytes and passive permeability for Na+ in VSMC. Viewed collectively, our data show that in nucleated cells, extracellular Ca2+ plays a crucial role in the maintenance of plasma membrane integrity.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin

The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.     

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.     

Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae)

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca2+] is maintained around 20 nM and the cells contain membrane-bound Ca2+ pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca2+ ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca2+ both in the presence and in the absence of extracellular Ca2+. In addition to the ER, an acidic compartment appears to be involved in Ca2+ storage, as collapse of intracellular pHgradients by monensin, a Na+ -H+ exchanger, and nigericin, a K+ -H+ exchanger, induce the release of Ca2+ from internal pools. A vacuolar H+ pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca2+ pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c in the cells from both lizard species, mostly by mobilization of the cation from internal stores.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.     

Cytokinin increases intracellular Ca2+ in Funaria: detection with Indo-1.

Changes in intracellular [Ca2+] ([Ca2+]i) after cytokinin-treatment in protonema cells of the moss Funaria hygrometrica have been measured using the pentapotassium salt of Indo-1. The extent of dye loading strongly depended on lowering the pH of the incubation medium to 5.0. Exposing dye-loaded cells briefly with Mn2+ did not quench fluorescence suggesting that the source of fluorescence is from the cytoplasm and not from the cell wall. Indo-1 remains responsive to changes in [Ca2+]i in Funaria cells. The [Ca2+]i in quiescent cells (with and without extracellular Ca2+) is 250 nM, which is within the range of reported [Ca2+]i of other plant cells. Treatment of cells with extracellular cytokinin in 4 mM Ca2+ induced a three-fold increase in [Ca2+]i to 750 nM in target caulonema cells. This increase was not observed in Ca(2+)-free medium. These target cells respond to cytokinin treatment by an asymmetrical division, while non-target chloronema cells do not divide. Cytokinin appears to increase [Ca2+]i by extracellular Ca2+ uptake. However, non-target chloronema cells and tip cells also respond to cytokinin treatment by increasing [Ca2+]i. The differential physiological response of these cell types to hormonal stimulation must lie further down the signal transduction chain.     

Recycling of transferrin receptors and heparan sulfate proteoglycans in a rat parathyroid cell line.

We examined recycling of heparan sulfate (HS) proteoglycans and transferrin receptor (Tf-R) in a rat parathyroid cell line. While extracellular Ca2+ concentration ([Ca2+]e) regulates the recycling of HS proteoglycans in parathyroid cells, such that HS proteoglycans only recycle when [Ca2+]e is lowered below physiological levels, recycling of Tf-R occurs equally well both in 0.05 mM (low) and 2 mM (high) [Ca2+]e. Inhibiting endocytosis chemically with phenylarsine oxide or at low temperature (4 degrees C) did not abolish the effects of changing [Ca2+]e on HS proteoglycans in the recycling compartment even though transport of HS proteoglycans from the Golgi complex to the cell surface was inhibited in low [Ca2+]e. Microtubules are not involved in the recycling of HS proteoglycans or of Tf-R since nocodazole did not affect these processes. Inhibiting the increase of intracellular Ca2+ by an intracellular Ca2+ chelator sustained recycling of HS proteoglycans even in the presence of high [Ca2+]e. These observations show that the exocytosis pathway of HS proteoglycans in the recycling compartment is specifically regulated by [Ca2+]e, whereas that for constitutive secretion is not. Therefore, the recycling of HS proteoglycans may be directly related to some functions of parathyroid cells regulated by [Ca2+]e. Although the mechanism by which [Ca2+]e regulates the exocytosis and recycling of HS proteoglycans is uncertain, it is suggested that an increase of intracellular Ca2+ is necessary, but not necessarily sufficient, for inhibiting their exocytosis.     

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.     

The role of extracellular Ca2+ in the response of the hepatocyte to Ca2+-dependent hormones

The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ [( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in [Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting [Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in [Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase [Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of [Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of [Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular [Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of [Ca2+]i.     

Differences in intracellular calcium mobilization by interferon-beta and interferon-gamma in RPMI-4788 cells

Human interferon (IFN) stimulates a 1.5- to 1.7-fold transient increase in the concentration of cytoplasmic-free calcium ion ([Ca2+]i) within 10-20 s upon exposure of RPMI-4788 cells to IFN. This early event of IFN-induced [Ca2+]i mobilization was measurable by loading the cells with Fura-2AM, a fluorescent Ca2+ indicator. The mobilization induced by IFN-beta or IFN-gamma was dependent on the concentration of each IFN. The increased [Ca2+]i gradually returned to its resting level within 60 s. The addition of EGTA (0.5-10 mM) to medium induced a marked decrease in the amount of [Ca2+]i mobilized by IFN-beta and a partial decrease by IFN-gamma. This finding suggests that the mechanisms of [Ca2+]i mobilization by IFN-beta and IFN-gamma might be different. While IFN-beta-induced mobilization may be mainly from an influx of the extracellular calcium ion ([Ca2+]o), IFN-gamma-induced mobilization may be a summation of an influx of [Ca2+]o and a release from intracellular Ca2+ stores.     

Calcium efflux across the plasma membrane of rat parotid acinar cells is unaffected by receptor activation or by the microsomal calcium ATPase inhibitor, thapsigargin

The rate of Ca2+ extrusion across the plasma membrane of rat parotid acinar cells was determined by measuring the decay of the intracellular calcium concentration, [Ca2+]i, following the addition of EGTA to agonist stimulated cells. In the presence of extracellular Ca2+, the muscarinic cholinergic receptor agonist, methacholine, rapidly increased [Ca2+]i (peaking within 5 s), which then decreased to a higher steady state level. This elevated steady state level was dependent on extracellular Ca2+ concentration. Likewise, thapsigargin, a non-phorbol ester tumor promoter that does not increase inositol phosphates, gradually increased [Ca2+]i, peaking within 1 min and then declining to a new elevated plateau level which was also dependent on extracellular Ca2+. [Ca2+]i, elevated by methacholine or thapsigargin, was rapidly decreased by the addition of EGTA by a process the kinetics of which depended on the value of [Ca2+]i before the addition of EGTA. That is, [Ca2+]i increased as a function of the extracellular Ca2+ concentration and also the apparent half-time for Ca2+ extrusion following the addition of EGTA to cells was increased as the [Ca2+]i increased. This presumably reflects the saturable nature of the Ca2+ extrusion mechanism. The steady state [Ca2+]i in cells stimulated with methacholine or thapsigargin in nominally Ca2+ free medium was similar to the steady state [Ca2+]i in unstimulated cells in normal, Ca2(+)-containing medium. Under these similar [Ca2+]i conditions, stimulated and unstimulated cells showed a similar time course of decay upon addition of EGTA. In addition, neither methacholine nor phorbol myristate acetate decreased the sustained elevation of [Ca2+]i induced by ionomycin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose-dependency in spatial dynamics of [Ca2+]c in adrenal chromaffin cells

The spatial dynamics of cytosolic Ca2+ concentration, [Ca2+]c, in guinea pig adrenal chromaffin cells was monitored by a digital image analysing technique using Fura-2. When a freshly isolated cluster of cells was stimulated with lower concentrations of carbachol (CCh; 0.3-1 microM), the [Ca2+]c began to increase in the region beneath the plasma membrane facing the extracellular environment. The [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCh at a higher concentration (100 microM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. These results are compatible with the view that the receptor activation with a physiological concentration of secretagogue accelerates Ca2+ entry, and that stimulation with a higher concentration of the secretagogue induces small transient Ca2+ release from intracellular stores and predominant continuous Ca2+ entry.     

The effects of Ca2+ and Sr2+ on Ca2+-sensitive biochemical changes in human erythrocytes and their membranes.

1. The Ca2+-dependency of K+ efflux, microvesiculation and breakdown of polyphosphoinositides and of ankyrin have been measured in intact human erythrocytes exposed to ionophore A23187 and HEDTA [N'-(2-hydroxyethyl)ethylenediamine NNN'-triacetate]-Ca2+ buffers. Half-maximal responses were observed at pCa values of 6.4, 4.1, 5.0 and 4.8 respectively. 2. The Ca2+ dependencies of K+ efflux and breakdown of polyphosphoinositides and ankyrin measured in erythrocyte ghosts without addition of ionophore showed almost identical values with those seen in whole cells treated with ionophore. 3. We conclude that ionophore A23187 is able to cause rapid equilibration of extracellular and intracellular [Ca2+] in intact cells and that in the presence of a suitable Ca2+ buffer, ionophore A23187 can be used to precisely fix the intracellular concentration of Ca2+ in erythrocytes. 4. The relatively high concentration of Ca2+ required to produce microvesiculation in intact cells may indicate that microvesiculation could be at least partly dependent on a direct interaction of Ca2+ with phospholipid. 5. Results obtained with Sr2+ paralleled those with Ca2+, although higher Sr2+ concentrations were required to achieve the same effects as Ca2+. Mg2+ produced none of the changes seen with Ca2+ or Sr2+.     

Hg2+ signaling in trout hepatoma (RTH-149) cells: involvement of Ca2+-induced Ca2+ release

Mercury is a non-essential heavy metal affecting intracellular Ca2+ dynamics. We studied the effects of Hg2+ on [Ca2+]i in trout hepatoma cells (RTH-149). Confocal imaging of fluo-3-loaded cells showed that Hg2+ induced dose-dependent, sustained [Ca2+]i transient, triggered intracellular Ca2+ waves, stimulated Ca2+-ATPase activity, and promoted InsP3 production. The effect of Hg2+ was reduced by the Ca2+ channel blocker verapamil and totally abolished by extracellular GSH, but was almost unaffected by cell loading with the heavy metal chelator TPEN or esterified GSH. In a Ca2+-free medium, Hg2+ induced a smaller [Ca2+]i transient, that was unaffected by TPEN, but was abolished by U73122, a PLC inhibitor, and by cell loading with GDP-betaS, a G protein inhibitor, or heparin, a blocker of intracellular Ca2+ release. Data indicate that Hg2+ induces Ca2+ entry through verapamil-sensitive channels, and intracellular Ca2+ release via a G protein-PLC-InsP3 mechanism. However, in cells loaded with heparin and exposed to Hg2+ in the presence of external Ca2+, the [Ca2+]i rise was maximally reduced, indicating that the global effect of Hg2+ is not a mere sum of Ca2+ entry plus Ca2+ release, but involves an amplification of Ca2+ release operated by Ca2+ entry through a CICR mechanism.     

NMR studies of intracellular sodium ions in mammalian cardiac myocytes

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.     

Caffeine-Sensitive Calcium Stores in Bovine Adrenal Chromaffin Cells

Caffeine was used to study the intracellular Ca2+ pools of bovine chromaffin cells. Its effects on cytosolic Ca2+ concentration ([Ca2+]i) were examined using fura-2. Caffeine caused a transient increase in [Ca2+]i in the presence or absence of extracellular Ca2+. In the former case, the caffeine-induced [Ca2+]i increase was higher and stayed above the basal value for several minutes. In the latter case, the [Ca2+]i rise was lower and fell to the basal level within 1 min. These results suggest that caffeine increases [Ca2+]i by causing both Ca2+ influx and Ca2+ release from intracellular pools. In the absence of extracellular Ca2+, ionomycin but not caffeine caused a further increase in [Ca2+]i in cells that had been treated with caffeine. Apparently there are at least two intracellular Ca2+ pools, only one of which is sensitive to caffeine. The caffeine-induced [Ca2+]i rise became smaller when the cells were pretreated with the inositol trisphosphate-generating agonists, methacholine and bradykinin. In addition, methacholine was unable to initiate a [Ca2+]i transient after the cells had been treated with caffeine. The results indicate that the caffeine-sensitive Ca2+ pools overlap with the inositol trisphosphate-sensitive pool and that the size of the latter pool is smaller than that of the former. The caffeine-sensitive Ca2+ pools were refilled after high K+ treatment, which suggests that the caffeine-sensitive Ca2+ pools may be important in buffering the cytosolic Ca2+. The effect of caffeine on [Ca2+]i is not due to inhibition of phosphodiesterase. Our results support a Ca2+ entry model in which depletion of intracellular Ca2+ pools controls the rate of Ca2+ entry across the plasma membrane.