K+-depolarization induces a direct release of Ca2+ from intracellular storage sites in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, effects of K+-depolarization on cytosolic free calcium concentrations were recorded microfluorometrically in rat aorta vascular smooth muscle cells in primary culture. When the cells were exposed to high extracellular K+ in Ca+-free media containing 2mM EGTA, there was a transient and dose-dependent elevation of cytosolic Ca2+ concentrations. However, the concentration of the cytosolic Ca2+ was not elevated when the intracellularly stored Ca2+ was depleted by the repetitive treatment with caffeine prior to the application of high K+. Thus depolarization of plasma membrane, per se, directly induces a release of Ca2+ from intracellular storage sites in vascular smooth muscle cells, and the main fraction of this released Ca2+ is derived from the caffeine sensitive storage sites; perhaps from the sarcoplasmic reticulum.     

Carmustine augments the effects of tert-butyl hydroperoxide on calcium signaling in cultured pulmonary artery endothelial cells

The effects of oxidant stress and inhibition of glutathione reductase on the bradykinin-stimulated changes in cytosolic free Ca2+ concentration ([Ca2+]i) of calf pulmonary artery endothelial cells were determined using the intracellular fluorescent probe, fura-2. Changes in [Ca2+]i upon stimulation with bradykinin were measured after incubation of cells with the chemical oxidant tert-butyl hydroperoxide (0.4 mM) for various times. After 60 min, bradykinin-stimulated Ca2+ influx was significantly decreased. With more prolonged incubations with the peroxide, bradykinin had little effect on cytosolic calcium concentration. Preincubation of cells with the glutathione reductase inhibitor, carmustine, led to elevated basal [Ca2+]i, yet the cells remained responsive to bradykinin. However, incubation of carmustine-treated cells with tert-butyl hydroperoxide for 30 min dramatically reduced both bradykinin-stimulated release of Ca2+ from internal stores and influx of Ca2+ from the extracellular space. These results suggest that inhibition of glutathione reductase alters cytosolic Ca2+ homeostasis and enhances the effects of oxidative stress on signal transduction in vascular endothelial cells.     

Histamine activates H1-receptors to induce cytosolic free calcium transients in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, the effects of histamine on cytosolic free calcium concentrations in rat aortic vascular smooth muscle cells in primary culture were recorded, microfluorometrically. When the cells were exposed to histamine, both in the presence and the absence of extracellular Ca2+, there was a rapid, transient and dose-dependent elevation of cytosolic Ca2+ concentrations, with a similar time course. This elevation of cytosolic Ca2+ was dose-dependently inhibited by mepyramine, but not by cimetidine. Thus, histamine activates H1- but not H2- receptors to mediate a release of Ca2+ from the store sites, and there is a rapid and transient elevation of cytosolic Ca2+.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

Abnormalities in the regulation of blood platelet free cytosolic calcium in malignant hyperthermia. I. Human platelets.

The effect of halothane on the regulation of blood platelet free cytosolic calcium was investigated in Quin-2-loaded cells from patients susceptible to Malignant Hyperthermia (MH) and healthy controls, respectively. The resting level of free cytosolic calcium was slightly, but statistically significantly, enhanced in platelets from patients (90 +/- 10 nM vs 110 +/- 35 nM). Halothane induced a dose-dependent, rapid Ca2+ release from intracellular stores both in normal and in MH derived cells, but the resulting increase in cytosolic calcium was significantly higher in the latter (2 mM halothane: [Ca2+]i = 117 +/- 12 nM vs 218 +/- 117 nM; 4 mM halothane: 225 +/- 35 nM vs. 417 +/- 201 nM). Whereas in platelets from healthy donors a complete reversibility of the halothane effect could be observed within 30-45 min, the cytosolic Ca2+ transients in platelets from patients were different from those in normals either in a higher initial peak or in a diminished decline velocity or in both. The basal Ca2+ permeability of the platelet plasma membrane was very low. Generally, halothane caused a dose-dependent increase in Ca2+ permeability. However, the influx of external calcium was significantly higher in platelets from patients than in controls (2 mM halothane: delta [Ca2+]i = 69 +/- 12 nM vs 135 +/- 63 nM; 4 mM halothane: 127 +/- 33 nM vs. 258 +/- 111 nM). Combining the results, the suggestion can be made that susceptibility to MH is characterized by a generalized membrane defect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.     

Effects of low extracellular sodium on cytosolic ionized calcium. Na+-Ca2+ exchange as a major calcium influx pathway in kidney cells

The effects of extracellular Na+ (Na+o) on cytosolic ionized calcium (Ca2+i) and on calcium and sodium fluxes were measured in monkey kidney cells (LLC-MK2). Ca2+i was measured with aequorin and the ion fluxes with 45Ca and 22Na. Na+-free media rapidly increased Ca2+i from 60 to a maximum of about 700 nM in 2-3 min. After the peak, Ca2+i declined and reached a plateau of about twice the resting Ca2+i. The peak Ca2+i was inversely proportional to Na+o and directly proportional to the extracellular calcium concentration (Ca2+o). On the other hand, a pH of 6.8 reduced and Ca2+o substitution with Sr2+ completely blocked the Ca2+i response to low Na+o. A Na+-free medium stimulated calcium efflux from the cells 4-5-fold, a response which was abolished in the absence of extracellular Ca2+. Na+-free media also stimulated calcium influx and sodium efflux. The cell calcium content, however, was not increased. These results indicate that removal of extracellular Na+ increases Ca2+i by stimulating calcium influx and not by inhibiting calcium efflux; the increased calcium influx takes place on the Na+-Ca2+ antiporter operating in the reverse mode in exchange for sodium efflux. The increased calcium efflux occurs as a consequence of the rise in Ca2+i and presumably takes place on the (Ca2+-Mg2+) ATPase-dependent calcium pump.     

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.     

Calcium signalling and cell-fate choice in B cells

Alterations in the cytosolic concentration of calcium ions (Ca2+) transmit information that is crucial for the development and function of B cells. Cytosolic Ca2+ concentration is determined by a balance of active transport and gradient-driven Ca2+ fluxes, both of which are subject to the influence of multiple receptors and environmental sensing pathways. Recent advances in genomics have allowed for the compilation of an increasingly comprehensive list of Ca2+ transporters and channels expressed by B cells. The increasing understanding of the function and regulation of these proteins has begun to shift the frontier of Ca2+ physiology in B cells from molecular analysis to determining how diverse inputs to cytosolic Ca2+ concentration are integrated in specific immunological contexts.     

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+ stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in the Drosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ ionophore (monensin, 10 microM) evoked cytosolic alkalinization followed by Ca2+ release from acidic intracellular Ca2+ stores. Pretreatment of S2 cells with either thapsigargin (1 microM), an inhibitor of endoplasmic reticulum Ca(2+)-ATPases, or with the Ca2+ ionophore ionomycin (10 microM) was without effect on the amplitude of Ca2+ release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 microM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+ store but failed to affect the amplitude of alkalinization-evoked Ca2+ release. Glycyl-L-phenylalanine-beta-naphthylamide (200 microM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+ release from internal stores, while application of brefeldin A (10 microM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+ stores did not elicit depletion-operated Ca2+ entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o = 2 mM), but not by thapsigargin-induced [Ca2+]i elevation in Ca(2+)-free saline.     

Recombinant human tumour necrosis factor increases cytosolic free calcium in murine fibroblasts and stimulates inositol phosphate formation in L-M and arachidonic acid release in 3T3 cells

Stimulation of murine L-M and 3T3 fibroblasts with human recombinant tumour necrosis factor (rTNF) resulted in an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). In 3T3 cells rTNF also induced release and metabolization of arachidonic acid, whereas in L-M cells rTNF provoked rapid increases in the levels of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3). In these cells the Ca2+ response was also observed in Ca2+ free medium, suggesting that rTNF promotes mobilization of Ca2+ from intracellular stores. In 3T3 cells, however, Ca2+ originated from the extracellular space, since the response was abolished in medium containing 1 mM EGTA. Both rTNF-induced calcium responses were inhibited by a specific rabbit IgG antibody to rTNF but not by 1-verapamil, a blocker potential-operated calcium channels. These results suggest that increased formation of inositol phosphates, arachidonic acid release and increased cytosolic free Ca2+ are involved in the biological effects of rTNF. However, rTNF generate these signals by different mechanisms depending upon the target cell.     

Structural Association of Endoplasmic Reticulum with Other Membrane Systems in Populus deltoides Apical Bud Cells and Its Alterations During the Short Day-induced Dormancy

A comparative study was carried out on the EM-cytochemical localization of calcium and Ca2+-ATPase activity in the suspension-cultured cells between the chilling-sensitive maize (Zea mays L. cv. Black Mexican Sweet) and chilling-insensitive Trititrigia (Triticum sect. Trititrigia mackey) at 4 ℃ chilling. When maize and Tyititrigia cells were cultured at 26 ℃, electron microscopic observations revealed that the electron-dense calcium antimonate deposits, an indication of the calcium localization, were localized mainly in the vacuoles, and few was found in the cytosol and nuclei. The electron-dense cerium phosphate deposits, an indication of Ca2+-ATPase activity, were abundantly distributed on the plasma membrane (PM). When the cells from both species were cultured at 4 ℃ for 1 and 3 h, an elevation of Ca2+ level in the cytosol and nuclei was observed, whereas the cerium phosphate deposits on the PM showed no quantitative difference from those of the 26 ℃-cultured cells, indicating that the enzymatic activities were not altered during these chilling periods. However, there was a distinct difference in the dynamics of the Ca2+ distribution and the PM Ca2+-ATPase activity between maize and Trititrigia when chilled at 4 ℃ for 12, 24 and 72 h. In maize cells, a large number of Ca2+ deposits still existed in the cytosol and nuclei, and the PM Ca2+-ATPase became less and less active, and even inactive at all. In Trititrigia cells, the increased cytosolic and nuclear Ca2+ ions decreased after 12 h chilling. By chilling up to 24 and 72 h, the intracellular Ca2+ concentration had been restored to a similar low level as those of the warm temperature-cultured cells, while the activity of the PM Ca2+-ATPase maintained high. The transient cytosolic and nuclear Ca2+ increase and the activities of PM Ca2+-ATPase during chilling are discussed in relation to plant cold hardiness.     

Mitochondrial uncoupling does not influence the stability of the intracellular signal activating plasma membrane calcium channels.

The effects of various concentrations of thapsigargin, a specific inhibitor of Ca2+-ATPase in the endoplasmic reticulum (ER) membrane, on calcium homeostasis in lymphoidal T cells (Jurkat) were investigated. Preincubation of these cells suspended in nominally calcium-free medium with 0.1 microM thapsigargin resulted in a complete release of Ca2+ from intracellular calcium stores. When the medium was supplemented with 3 mM CaCl2 the cells maintained constantly elevated level of cytosolic Ca2+. However, thapsigargin applied at lower concentration produced only a partial depletion of the stores. For example, in the cells pretreated with 1 nM thapsigargin and suspended in calcium-free medium approximately 75% of the calcium content was released from the intracellular stores. The addition of 3 mM CaCl2 to such cell suspension led to a transient increase in cytosolic calcium concentration, followed by a return to a lower steady-state. This phenomenon, related to the refilling of the ER by Ca2+, allowed to estimate the half-time for the process of cell recovery after activation of store-operated calcium channels. By this approach we have found that carbonyl cyanide m-chlorophenylhydrazone, which has been documented to inhibit calcium entry into Jurkat cells, does not influence the stability of the intracellular signal involved in the activation of store-operated calcium channels.     

Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study

To better understand the relation between cell calcium and exocytotic secretion, a quantitative dependence of adrenal catecholamine secretion on cytosolic free calcium has been determined for isolated, intact, bovine chromaffin cells, using the fluorescent probe Quin-2. The cells required a threshold of 250-300 nM cytosolic calcium to be reached before detectable secretion occurred and half-maximal secretion occurred near 2 microM cytosolic calcium. Nicotinic receptors mediated an increase of cytosolic calcium from resting levels near 100 nM to levels in the 1-10 microM range within seconds followed by a decay back to resting levels over several minutes. Muscarinic receptors mediated a smaller rise in cytosolic free calcium from 100 to about 200 nM, within seconds. The nicotinic response required extracellular calcium, while the muscarinic response was largely independent of extracellular calcium, suggesting the latter mobilizes intracellular calcium. The acetylcholine-evoked rise in cytosolic calcium decayed by at least two kinetically distinct processes with half-time constants: t1 = 0.6 min and t2 = 3.2 min. Extracellular Na+ deprivation caused a more prolonged elevation of the acetylcholine-evoked calcium transient, suggesting a possible role of Na+/Ca2+ exchange and/or other Na+ -dependent processes in lowering cytosolic calcium following stimulation. The possible perturbing effects of Quin-2 on resting and stimulated cytosolic calcium levels and on secretion were examined and a novel use of Quin-2 to measure membrane calcium flux was demonstrated.     

Proliferation-associated changes of Ca2+ transport in myeloid leukemic cell lines

Proliferation-associated changes in calcium metabolism were investigated employing the promyelocytic HL-60 and monoblastic U-937 cell lines. The cells were stimulated to proliferate employing mitogenic factors as follows. 1) Transferrin or insulin: HL-60 cells were adjusted for growth in serum-free medium, and 24 h prior to the experiment, the cells were deprived of transferrin or insulin. The re-addition of either one of them stimulated cell proliferation as was evident by increased [3H]-tymidine incorporation activity. Cell proliferation was associated with an enhanced Ca2+ influx rate, measured by 45Ca2+ uptake activity. 2) Granulocyte-monocyte colony-stimulating factor (GM-CSF): addition of GM-CSF to proliferating or quiescent HL-60 cells resulted in increased cell proliferation, which was also accompanied by increased rate of Ca2+ influx. 3) Serum: HL-60 and U-937 were grown for 24 h in serum-depleted medium. Re-addition of serum to the cells was not associated with immediate or delayed change in calcium influx rate but rather with an immediate increase in the cytosolic free calcium concentration, measured employing the fluorescent probe, fura-2AM. This increase was independent of extracellular calcium, unaffected by verapamil, diltiazem, and lanthanum, and associated with enhanced 45Ca2+ efflux. Thus, in all three cases evoked cell proliferation was accompanied by quantitative changes in Ca2+ metabolism. While the transferrin-, insulin-, and GM-CSF-stimulated cell proliferation was accompanied by delayed increases in 45Ca2+ influx, the serum-stimulated cell proliferation was accompanied by an immediate elevation of free cytosolic Ca2+.     

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.     

Calcium sequestration in the liver: Review article

Hepatic parenchymal cells maintain intracellular total and cytosolic free Ca2+ levels by: entry of Ca2+ through channels, extrusion of Ca2+ by an outwardly directed Ca2+ pump, and controlled sequestration into intracellular pools. The mechanism of Ca2+ inflow is poorly characterized. The plasma membrane Ca2+ channels seem to share some of the characteristics of Ca2+ channels in excitable cells, but also differ from them. The outwardly directed plasma membrane Ca2(+)-ATPase is a calmodulin independent, P-type enzyme. Ca2+ uptake into the endoplasmic reticulum is due to the activity of a different Ca2(+)-ATPase, which is similar in molecular weight and shares antigenic determinants with the sarcoplasmic reticulum enzyme. In addition, mitochondria and nuclei also take up calcium. The exact mechanism by which Ca2+ is released from intracellular organelles is not well known. Several mechanisms for Ca2+ release from the endoplasmic reticulum were reported, including IP3 and GTP-induced. The most effective identified way of eliciting Ca2+ release from microsomal fraction is by the oxidation of critical -SH groups. This mechanism is likely to be involved in the rise of cytosolic Ca2+ observed in many situations of hepatocellular injury. In addition to being sequestered into subcellular organelles, some of the intracellular Ca2+ is bound to specific Ca2+ binding proteins. Both calmodulin and members of the annexin family were identified in the liver. Stimulation of the liver with gluconeogenic hormones results in increased Ca2+ entry into the cell, the release of Ca2+ from intracellular pools, and an oscillatory increase in free cytosolic Ca2+ levels. Extensive research is still needed for the elucidation of the exact mechanisms by which these events occur.     

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles.     

Parvalbumin is expressed in normal and pathological human parathyroid glands.

The parathyroid glands are of major importance in calcium homeostasis. Small changes in the plasma calcium (Ca2+) concentration induce rapid changes in parathyroid hormone (PTH) secretion to maintain the extracellular Ca2+ levels within the physiological range. Extracellular Ca2+ concentration is continuously measured by a G-protein-coupled Ca2+-sensing receptor, which influences the expression and secretion of PTH. The mechanism of signal transduction from receptor sensing to PTH secretion is not well understood, but changes in PTH secretion are tightly linked to changes in the cytosolic Ca2+ concentration. Using immunohistochemistry and Western blot analysis, we detected the EF Ca2+ binding protein parvalbumin (PV) in normal and in hyperplastic and adenomatous human parathyroid glands. The strongest PV signal was present in chief cells and water clear cells, whereas in oxyphilic cells only a weak signal was observed. Immunohistochemistry and in situ hybridization of the PTH indicated a co-localization of PV and PTH in the same cell types. Because changes in the cytosolic Ca2+ concentration are believed to influence the process of PTH secretion, a possible role of PV as a modulator of this Ca2+ signaling is envisaged.