Activation and inactivation of the osteoclast Ca2+ receptor by the trivalent cation, La3+.

We report changes in the cytosolic Ca2+ concentration ([Ca2+]i) of single rat osteoclasts in response to Ca2+ receptor activation by micromolar concentrations of the lanthanide metal cation, La3+. The extracellular application of La3+ induced a concentration-dependent elevation of cytosolic [Ca2+]. Prior conditioning of osteoclasts with La3+ resulted in a concentration-dependent reduction of the response to a subsequent application of a maximally effective concentration of Ni2+, a known agonist of the osteoclast Ca2+ receptor. The results establish that the osteoclast Ca2+ receptor is highly sensitive to activation and inactivation by the trivalent cation, La3+.     

Alpha 1-adrenergic receptor activation mobilizes cellular Ca2+ in a muscle cell line

Regulation of cellular Ca2+ movements by alpha 1-adrenergic receptors has been studied using 45Ca2+ flux techniques in monolayer cultures of intact BC3H-1 cells. Unidirectional 45Ca2+ efflux from BC3H-1 cells reveals multiphasic kinetics, with a major fraction of cellular Ca2+ residing in a slowly exchanging intracellular compartment. Stimulation of alpha 1-adrenergic receptors by the agonist phenylephrine substantially increases 45Ca2+ unidirectional efflux, accompanied by a far smaller increase in 45Ca2+ influx. The selective enhancement of 45Ca2+ unidirectional efflux upon alpha 1-adrenergic receptor activation results in a net 30-40% decline in total cell Ca2+ content, measured either by radioisotopic equilibrium techniques or by atomic absorption spectroscopy. The relatively large pool of Ca2+ responsive to alpha-adrenergic stimulation is not displaced by La3+ but can be depleted with the Ca2+ ionophore A-23187. These results indicate that alpha 1-adrenergic receptor activation predominantly mobilizes Ca2+ from intracellular stores, together with a much smaller increase in transmembrane Ca2+ permeability. This interpretation is supported by comparative 45Ca2+ flux studies using a sister clone of BC3H-1 cells possessing surface nicotinic acetylcholine receptors but no alpha 1-adrenergic receptors. Agonist stimulation of the cholinergic receptor opens a well characterized transmembrane ion permeability gate. Cholinergic receptor activation greatly enhances the observed 45Ca2+ unidirectional influx relative to efflux, leading to net elevation of cellular Ca2+ content as Ca2+ moves down its inwardly directed concentration gradient.     

Stimulation of the T3-T cell receptor complex induces a membrane-potential-sensitive calcium influx

Three monoclonal antibodies selected for their recognition of parts of the T3-T cell receptor complex on human T lymphocytes were found to induce an increase in cytoplasmic free Ca2+ (Ca2+i) in the T cell leukemia line HPB-ALL as measured by Quin2 fluorescence. These reagents are directed against T3 (OKT3), a nonvariable T3-associated structure (WT-31) and the variable region of the T3-associated antigen receptor (T40/25) of this cell line. The rise in Ca2+i was dependent on the presence of extracellular Ca2+, occurred within 30 sec of stimulation, and was sustained for at least 10 min. Fab fragments of OKT3 also caused a rapid increase in Ca2+i, indicating that cross-linking is not necessary to induce a Ca2+ response. Alterations in plasma membrane potential and La3+ blocked the Ca2+ influx induced by OKT3 and T40/25. These data suggest that the T3-T cell receptor complex of human T lymphocytes may be an antigen-regulated Ca2+ channel.     

Requirement for Ca2+ signaling in the mechanism of thrombin-induced increase in endothelial permeability

We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40-50% decrease in transendothelial monolayer electrical resistance and a twofold increase in 125I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular Ca2+ concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. La3+ reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in 125I-albumin permeability in HUVECs. Because the absence of Ca2+ signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular Ca2+ concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased 125I-albumin permeability. These results indicate that Ca2+ influx and activation of Ca2+-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability.     

The role of intracellular Ca2+ in the regulation of the plasma membrane Ca2+ permeability of unstimulated rat lymphocytes

The mechanism responsible for the increase in cytosolic free Ca2+ concentration ([Ca2+]i) during mitogenic stimulation of lymphocytes has been widely investigated. By contrast, little is known about the processes underlying Ca2+i homeostasis in resting (unstimulated) cells. It has been suggested that [Ca2+]i is an important determinant of the rate of Ca2+ influx following mitogenic activation. Using rat thymic lymphocytes, we investigated whether the resting influx pathway is similarly controlled by [Ca2+]i. Otherwise untreated cells were Ca(2+)-depleted by loading with Ca2+ chelators while suspended in Ca(2+)-free solution. Ca2+ depletion induced an 8-fold increase in the rate of unidirectional Ca2+ uptake. The depletion-activated flux was voltage-sensitive and was blocked by La3+ and by compound SK&F 96365, a receptor-operated Ca2+ channel blocker. Upon reintroduction to Ca(2+)-containing solution, the increased influx brought about a rapid recovery of [Ca2+]i. Detailed analysis of the magnitude of the 45Ca2+ flux during this recovery indicated that [Ca2+]i is not the primary determinant of the plasmalemmal Ca2+ permeability. Instead, depletion of an internal thapsigargin-sensitive store correlates with and appears to be responsible for the increased permeability of the plasma membrane. Accordingly, the Ca2+ fluxes induced by intracellular Ca2+ depletion and by thapsigargin were pharmacologically indistinguishable. Mitogenic lectins also released Ca2+ from a thapsigargin-sensitive store and activated a plasmalemmal Ca2+ permeability displaying identical pharmacology. The data support the existence of a coupling process whereby the degree of filling of an internal Ca2+ store dictates the Ca2+ permeability of the plasma membrane. This coupling mechanism is important not only in mediating the effects of mitogens and other agonists, as suggested before, but seemingly also in the control of resting Ca2+i homeostasis in unstimulated cells.     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

Characterization of the calcium signaling system in the submandibular cell line SMG-C6

Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, alpha1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Consistently, 1, 4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.     

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

Dynamic changes of [Ca2+]i in cerebellar granule cells exposed to pulsed electric fields

Intracellular free Ca2+ concentration ([Ca2+]i) in embryonic chick cerebellar granule cells loaded with fluo-3/AM and exposed to a single pulsed electric field was investigated using a confocal laser scanning microscope and fluorescent microscope equipped with CCD video imaging system.The results showed that [Ca2+]i increased immediately and rose to the peak rapidly as the cells exposed to a single pulsed electric field.The amplitude and rate of the increases of [Ca2+]i depend on the intensity of external electric field.In the presence of Ca2+ chelant EGTA or Ca2+ channels blocker La3+ in the pulsation solutions,the increase of [Ca2+]i was still observable.It was also observed that [Ca2+]i of different intracellular areas in the cell elevated simultaneously while the peak of the increase of [Ca2+]i in the poles of the cell preceded to the peak in its somata and recovered to a plateau within a short time.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Study on biological effect of La3+ on Escherichia coli by atomic force microscopy

The biological effects of rare-earth metal ions on the organism have been studied using La3+ as a probe ion and Escherichia coli cell as a target organism. Atomic force microscopy (AFM) studies reveal that La3+ substantially changes the structure of the outer cell membrane responsible for the cell permeability. Significant damages of the outer cell membrane are observed using scanning electron microscopy (SEM) after the introduction of La3+. In result, the cell becomes easily attacked by lysozyme. Moreover, inductively coupled plasma-mass spectrometry (ICP-MS) measurements show considerable amount of Ca2+ and Mg2+ in the supernatant from the La3+ exposed cells. It is proposed that La3+ can replace Ca2+ from the binding sites because of their close ionic radii and similar ligand specificities. Lipopolysaccharide (LPS), which forms the outer membrane of Gram-negative bacteria, could not serve as the cellular envelope steadily after Ca2+ and Mg2+ released from their binding sites on the LPS patches.     

Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics

Measurement of fura-2 fluorescence and 45Ca2+ uptake was used to evaluate Ca2+ influx in cultured bovine aortic endothelial cells (BAECs) stimulated by bradykinin (BK). The BK-stimulated influx pathway was characterized with respect to its 1) sensitivity to extracellular Ca2+, 2) inhibition by membrane depolarization, and 3) permeability to Ba2+ and Sr2+. The results indicate that the activity of the influx pathway is a saturable function of extracellular Ca2+ and that membrane depolarization inhibits Ca2+ influx by changing the apparent affinity and maximal capacity of the pathway for Ca2+. Fura-2 fluorescence was used to compare the profiles for BK-stimulated changes in cytosolic Ca2+, Sr2+, and Ba2+ (Ca2+i, Ba2+i, and Sr2+i). Addition of Ca2+ and Sr2+ to Ca2+-depleted cells in the presence of BK produced a transient increase in Ca2+i and Sr2+i. Following the peak of the response, Ca2+i and Sr2+i declined within 2 min to a steady elevated level. Blockade of influx by the addition of La3+ at the peak of the response to Ca2+ and Sr2+ immediately reduced Ca2+i and Sr2+i to basal levels. Addition of Ba2+ to Ca2+-depleted cells in the presence of BK produced an increase in Ba2+i which continued to rise with time to a steady level. Addition of La3+ after Ba2+, however, did not reduce Ba2+i. These results suggest that 1) Ca2+ and Sr2+ (but not Ba2+) are sequestered by intracellular mechanisms and that the declining phase of the Ca2+ and Sr2+ response reflects a time and divalent cation-dependent inactivation of the influx pathway. The inactivation of the influx pathway was further demonstrated by measuring the kinetics of BK-stimulated 45Ca2+ uptake into BAECs. The results of these experiments demonstrate that BK stimulates a 100- to 150-fold increase in Ca2+ permeability of the BAEC but that the influx pathway turns off or inactivates within 2 min. The magnitude of the flux, the voltage sensitivity, and the ability to conduct Ca2+, Sr2+, and Ba2+ are suggestive of a channel mechanism.     

Effect of olvanil (N-vanillyl-cis-9-octadecenoamide) on cytosolic Ca2+ increase in renal tubular cells

In canine renal tubular cells, effect of olvanil, a presumed cannabinoid and vanilloid receptor modulator, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Olvanil (5-100 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. Olvanil-induced [Ca2+]i rise was prevented by 70 and 90% by removal of extracellular Ca2+ and La3+, respectively, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of olvanil on [Ca2+]i was abolished; also, pretreatment with olvanil partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phoispholipase C, abrogated ATP-, but partly inhibited olvanil-, induced [Ca2+]i rise. Two cannabinoid receptor antagonists (AM251 and AM281; 5 microM) and a vanilloid receptor antagonist (capsazepine; 100 microM) did not alter olvanil (50 microM)-induced [Ca2+]i rise. These results suggest that olvanil rapidly increases [Ca2+]i in renal tubular cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via mechanism(s) independent of stimulation of cannabinoid and vanilloid receptors.     

Lanthanum reduces the excitation efficiency in fly photoreceptors

Lanthanum (La3+), a known inhibitor of Ca2+ binding proteins, was applied to the extracellular space of fly retina. Shot noise analysis indicated that a combination of intense light and La3+ caused a large (down to zero) reduction in the rate of occurrence of the quantal responses to single photons (quantum bumps) which sum to produce the photoreceptor potential. Light in the presence of La3+ also increased the effective bump duration. These effects are very similar to the effects of the mutations trp of Drosophila and nss of Lucilia flies on the quantum bump rate and duration. La3+ applied to the nss mutant caused only a small reduction in the bump rate, suggesting that La3+ may affect the nss gene product which is deficient in the mutant. The close similarity in the properties of the receptor potential of the La(3+)-treated photoreceptor of the wild type and of the nss mutant together with existing evidence for the highly reduced intracellular Ca2+ ([Ca2+]i) level in nss photoreceptors suggest that both La3+ and the mutation cause a severe reduction in [Ca2+]i. This effect may arise from an inhibition of a Ca2+ transporter protein located in the surface membrane that normally replenishes Ca2+ pools in the photoreceptors, a process essential for light excitation.     

Calcium influx in a rat mast cell (RBL-2H3) line. Use of multivalent metal ions to define its characteristics and role in exocytosis

An increase in concentration of cytosolic Ca2+ ([Ca2+]i) is associated with an accelerated influx of 45Ca2+ when cultured RBL-2H3 cells are stimulated with either antigen or analogs of adenosine although these agents act via different receptors and coupling proteins (Ali, H., Cunha-Melo, J.R., Saul, W.F., and Beaven, M.A. (1990) J. Biol. Chem. 265, 745-753). The same mechanism probably operates for basal Ca2+ influx in unstimulated cells and for the accelerated influx in stimulated cells. This influx had the following characteristics. 1) It was decreased when cells were depolarized with high external K+; 2) it was blocked by other cations (La3+ greater than Zn2+ greater than Cd2+ greater than Mn2 = Co2+ greater than Ba2+ greater than Ni2+ greater than Sr2+) either by competing with Ca2+ at external sites (e.g. La3+ or Zn2+) or by co-passage into the cell (e.g. Mn2+ or Sr2+); and 3) the inhibition of influx by K+ and the metal ions had exactly the same characteristics whether cells were stimulated or unstimulated even though influx rates were different. The dependence of various cellular responses on influx of Ca2+ was demonstrated as follows. The stimulated influx of Ca2+, rise in [Ca2+]i, and secretion, could be blocked in a concentration-dependent manner by increasing the concentration of La3+, but concentrations of La3+ (greater than 20 microM) that suppressed influx to below basal rates of influx markedly suppressed the hydrolysis of inositol phospholipids (levels of inositol 1,4,5-trisphosphate were unaffected). Some metal ions, e.g. Mn2+ and Sr2+, however, supported the stimulated hydrolysis of inositol phospholipid and some secretion in the absence of Ca2+. Thus a basal rate of influx of Ca2+ was required for the full activation of inositol phospholipid hydrolysis, but in addition an accelerated influx was necessary for exocytosis.     

Inhibition of glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical neurones

Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca2+]i followed by a delayed, irreversible [Ca2+]i rise governed by rapid kinetics, with Ca2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca2+ rise is unknown. Here, we report that the delayed Ca2+ entry in cortical neurones is diminished by 2-aminoethoxydiphenyl borate (2-APB: IC50 = 62 +/- 9 microm) and La3+ (IC50 = 7.2 +/- 3 microm), both known to inhibit transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca2+]i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate-induced delayed Ca2+ deregulation. In basal Ca2+ entry experiments, La3+ and 2-APB modulated the rapid rise in [Ca2+]i caused by exposure of neurones to Ca2+ after pre-incubating in a calcium-free medium. This basal Ca2+ influx was mitigated by extracellular Mg2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2-APB and La3+ influence non-store-operated Ca2+ influx in cortical neurones and that this route of Ca2+ entry is involved in glutamate-induced delayed Ca2+ deregulation.     

Characterization of volume-sensitive, calcium-permeating pathways in the osteosarcoma cell line UMR-106-01

Measurements of cell volume changes, free cytosolic Ca2+ concentration [( Ca2+]i) with Fura 2 and cell membrane potential with 3,3'-dipropylthiodicarbocyanine iodide were used to study the effect of cell volume change on Ca2+ influx and the membrane potential of the osteoblastic osteosarcoma cell line, UMR-106-01. Swelling the cells by hypo-osmotic stress was followed by reduction in cell volume which was markedly impaired by removal of medium Ca2+. Accordingly, cell swelling resulted in [Ca2+]i increase only in the presence of medium Ca2+. The cell swelling-activated Ca2+ entry pathway was active at resting membrane potentials, and Ca2+ influx through this pathway markedly increased upon cell hyperpolarization. A linear relationship between Ca2+ entry and the potential across the plasma membrane was observed. Thus, the volume-activated Ca2+ permeating pathway in UMR-106-01 cells has conductive properties. These pathways do not spontaneously inactivate with time when the cells are not allowed to volume regulate. The pathway can be blocked by micromolar concentrations of nicardipine and La3+ but display very low sensitivity to diltiazem and verapamil. Activation of the volume-sensitive, Ca2+ permeating pathway was not dependent on an increase in [Ca2+]i. Likewise, activation of the pathway was independent of a change in membrane potential between -85 and -3 mV. The increase in [Ca2+]i resulted in hyperpolarization of the cells, probably due to activation of Ca2+-activated K+ channels. The volume-sensitive pathways were partially active under isotonic conditions. Their activity was inhibited by cell shrinkage and increased by cell swelling. The pathways were sensitive to small changes in cell volume, particularly around a medium osmolarity of 310 mosM.     

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.