Intracellular free calcium concentration in the blowfly retina studied by Fura-2.

The retinal tissue of blowflies was loaded with the fluorescent Ca2+ indicator Fura-2 by incubating cut heads in saline solutions which contained the membrane permeable acetoxymethylester of Fura-2 (Fura-2/AM). The spectral analysis of the tissue fluorescence showed that Fura-2/AM was intracellularly hydrolysed to Fura-2. In order to monitor the intracellular free Ca2+ concentration ([Ca2+]i) the Fura-2 fluorescence was excited by short light flashes. The fluorescence was calibrated by incubating the tissue in Ca2+ buffers of high buffering capacity and subsequent disruption of the cell membranes by freeze/thawing, which gave a dissociation constant for the Ca(2+)-Fura-2 complex of 100 nM. When the extracellular Ca2+ concentration ([Ca2+]o) was altered [Ca2+]i reversibly changed. The changes were most pronounced when [Ca2+]o was varied in the millimolar range, e.g. [Ca2+]i increased from 0.07 microM at [Ca2+]o = 0.1 mM to 1 microM at [Ca2+]o = 10 mM. When extracellular Na+ was replaced by Li+ or other monovalent ions, [Ca2+]i rapidly increased which supports the view that electrogenic Na+/Ca2+ exchange contributes to the control of [Ca2+]i. However, [Ca2+]i decreased again when the tissue was superfused with Na(+)-free media for longer periods, which points to a Ca(2+)-transporting system different from Na+/Ca2+ exchange. Light adaptation had only a small effect on [Ca2+]i. Even after intense stimulation [Ca2+]i increased by a factor of 1.5 only, which is in line with results obtained in the photoreceptors of Balanus and Apis.     

Trypanosoma cruzi: mechanisms of intracellular calcium homeostasis.

Regulation of intracellular Ca2+ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca2+, [Ca2+]o, from 0 to 2 mM, cytosolic Ca2+, [Ca2+]i, increased only from 85 +/- 9 to 185 +/- 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca2+]i. Exposure to monovalent Na+ (monensin)-, K+ (valinomycin, nigericin)-, and divalent Ca2+ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na+/K(+)-ATPase (ouabain), and Ca(2+)-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca2+]o resulted in perturbations of [Ca2+]i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca2+]o, incubation with orthovanadate markedly increased [Ca2+]i, results which are compatible with an active uptake of [Ca2+]i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca2+]o did not influence the relatively smaller increase in [Ca2+]i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca2+]o, exposure to monensin or ouabain, conditions known to decrease the [Na+]o/[Na+]i gradient, upon which the Na+/Ca2+ exchange pathways are dependent, markedly increased [Ca2+]i. In a complementary manner, decreasing the extracellular Na+ gradient with Li+ increased [Ca2+]i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca2+ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca2+]i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [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.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Presence of Na+/Ca2+ exchange activity and its role in regulation of intracellular calcium concentration in bovine adrenal chromaffin cells.

The presence of a Na+/Ca2+ exchanger in bovine adrenal chromaffin cells was demonstrated by measuring the efflux of 45Ca2+ which had been preloaded into cells by a brief depolarization. The efflux of 45Ca2+ was dependent on extracellular Na+ (Na+o); 45Ca2+ efflux was significantly decreased by replacing Na+o with N-methylglucamine (NMG), or Li+. Replacement of Na+o by NMG increased the resting intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated chromaffin cells. This could be reversed by adding Na+, suggesting that Na+/Ca2+ exchanger activity was involved in maintaining [Ca2+]i at its resting level. The initial rate of Na(+)-dependent [Ca2+]i recovery after Ca2+ loading by depolarization was dependent on the level of [Ca2+]i. There was an apparent linear relationship between the activity of the Na+/Ca2+ exchanger and [Ca2+]i both in the presence and absence of Na+o. When cells were treated with other stimuli, including 10 microM DMPP or 40 mM caffeine, the ability of the stimulated cells to decrease [Ca2+]i was significantly reduced upon replacing Na+o with NMG. Our data show that the Na+/Ca2+ exchanger is one of the major pathways for regulating [Ca2+]i in chromaffin cells in both resting and stimulated states.     

Intracellular calcium 'signatures' evoked by different agonists in isolated bovine aortic endothelial cells.

Agonist induced increases in intracellular free calcium, [Ca2+]i, were measured in single Fura-2 loaded bovine aortic endothelial (BAE) cells by dual wavelength microspectrofluorimetry. Low doses of ATP (less than 10 microM) induced complex changes in [Ca2+]i. These changes usually consisted of a large initial transient peak with subsequent fluctuations superimposed upon a maintained rise in [Ca2+]i. Higher doses of ATP (greater than 50 microM) produced much simpler biphasic increases in [Ca2+]i in individual cells. Acetylcholine and bradykinin also elicited increases in [Ca2+]i in single cells in confluent monolayers of endothelial cells. However, only acetylcholine produced complex fluctuations. High doses of acetylcholine evoked simple rises in [Ca2+]i similar to those seen with high doses of ATP. In contrast, bradykinin evoked relatively simple rises in [Ca2+]i at all doses used. These results indicate that the mechanisms responsible for generating agonist induced increases in [Ca2+]i in BAE cells are not homogeneous. ATP and acetylcholine produced more complex Ca2+ changes or 'signatures' in single confluent bovine aortic endothelial cells than bradykinin. All three agonists appeared to release Ca2+ from intracellular stores as well as stimulating Ca2+ influx. The possible mechanisms underlying these phenomena are discussed.     

Effects of antisense oligonucleotides to the cardiac Na+/Ca2+ exchanger on calcium dynamics in cultured cardiac myocytes.

The present study was designed to explore the role of the Na+/Ca2+ exchanger on spontaneous beating of cultured cardiac myocytes. Antisense oligonucleotides (AS) based on the sequence of the cardiac Na+/Ca2+ exchanger were used to decrease expression of this Ca2+ transporting protein in cardiac myocytes. An application of AS (10 microM) caused an increase in beating rate of myocytes within 6-24 h. After 24 h of exposure, AS increased the beating rate from an average rate of 77 beats/min in control and sense-treated myocytes to 103 beats/min. Moreover, myocytes treated for 24 h with 10 microM AS exhibited an increase in diastolic [Ca2+]i levels. The antisense treatment also led to a approximately 20% decrease in expression of Na+/Ca2+ exchanger proteins within 6-24 h. Changes in mRNA levels following AS treatment could not be detected within 3- to 24-h periods. The results of these studies suggest that the Na+/Ca2+ exchanger plays a potentiating role in spontaneous the beating process by regulating [Ca2+]i dynamics and that even a small reduction in the levels of the exchanger protein has marked effects on the handling of [Ca2+]i during the cardiac cycle.     

Mechanisms of lysophosphatidic acid-induced increase in intracellular calcium in vascular smooth muscle cells

Although lysophosphatidic acid (LPA) is known to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMCs), the mechanisms of [Ca2+]i mobilization by LPA are not fully understood. In the present study, the effect of LPA on [Ca2+]i mobilization in cultured A10 VSMCs was examined by Fura-2 fluorescence technique. The expression of LPA receptors was studied by immunostaining. LPA was observed to increase [Ca2+]i in a concentration-dependent manner; this increase was dependent on the concentration of extracellular Ca2+. Both sarcolemmal (SL) Na(+)-Ca2+ exchange inhibitors (amiloride, Ni2+ and KB-R7943) and Na(+)-H+ exchange inhibitor (MIA) as well as SL store-operated Ca2+ channel (SOC) antagonists (SK&F 96365, tyrphostin A9 and gadolinium), unlike SL Ca2+ channel antagonists (verapamil and diltiazem), inhibited the LPA-induced increase in [Ca2+]i. In addition, sarcoplasmic reticulum (SR) Ca2+ channel blocker (ryanodine), SR Ca2+ channel opener (caffeine), SR Ca2+ pump ATPase inhibitor (thapsigargin) and inositol 1,4,5-trisphosphate (InsP3) receptor antagonists (xestospongin and 2-aminoethoxydiphenyl borate) were found to inhibit the LPA-induced Ca2+ mobilization. Furthermore, phospholipase C (PLC) inhibitor (U 73122) and protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) attenuated the LPA-induced increase in [Ca2+]i. These results indicate that Ca2+ mobilization by LPA involves extracellular Ca2+ entry through SL Na(+)-Ca2+ exchanger, Na(+)-H+ exchanger and SL SOCs. In addition, ryanodine-sensitive and InsP(3)-sensitive intracellular Ca2+ pools may be associated with the LPA-induced increase in [Ca2+]i. Furthermore, the LPA-induced [Ca2+]i mobilization in VSMCs seems to be due to the activation of both PLC and PKC.     

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Priming effect of calcium ionophores on phorbol ester-induced respiratory burst in mouse peritoneal neutrophils.

The abilities of three calcium ionophores (A23187, 4-bromo-A23187, and ionomycin) to modulate the respiratory burst of neutrophils induced by phorbol ester and to increase the concentration of free intracellular Ca2+ ([Ca2+]i) were compared. The production of reactive oxygen species (ROS) was determined by luminol-dependent chemiluminescence and [Ca2+]i was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 microM) and ionomycin (0.001-0.5 microM) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burst induced by phorbol ester. The integral response (total production of ROS over 6 min) had a bell-shaped dependence on the concentration of ionomycin and A23187 with increase and decrease at low and high concentrations of the ionophores, respectively. The maximal effect was found at 0.5 microM ionomycin and 2 microM A23187, these concentrations resulting in transient increases in [Ca2+]i to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophores had no effect in calcium-free media, though they increased [Ca2+]i to approximately 400 nM through the mobilization of intracellular Ca2+. In cells with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with phorbol ester amplified twofold the production of ROS. The inhibition of phospholipase A2 with 4-bromophenacyl bromide significantly decreased the production of ROS. Thus, the entrance of Ca2+ and generation of arachidonic acid under the influence of phospholipase A2 are necessary for the ionophore-induced priming of production of ROS during cell activation with phorbol esters.     

Endotoxicosis modulates cytosolic free calcium and basal and ACTH-stimulated lipolysis in rat adipocytes

Lipolytic rates and intracellular Ca2+ concentration ([Ca2+]i) were determined under basal conditions and upon stimulation with adrenocorticotropic hormone (ACTH), norepinephrine (NE) and insulin (I), in adipocytes isolated from control and acutely endotoxin (ET)-treated rats (1 mg/100 g body weight, LD50 at 6 h). [Ca2+]i measurements were done using the fluorescent Ca2(+)-indicator Fura-2. NE and ACTH, but not I, produced a marked increase of [Ca2+]i in cells of both control and ET-treated rats. ET treatment elicited a significant increase in [Ca2+]i of resting cells, and enhanced the ACTH effect on this parameter. The changes in lipolytic activity correlated well with changes of [Ca2+]i induced by ACTH. The results indicate that ET-induced alterations in intracellular calcium homeostasis of adipocytes may contribute to the mediation of effects on fat mobilization during endotoxemia.     

Alpha 1-adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx

The regulation of [Ca2+]i in rat pinealocytes was studied using the fluorescent indicator quin2. Pinealocyte resting [Ca2+]i was approximately 100 nM; this rapidly decreased in low Ca2+ medium (approximately 10 microM), indicating there was a high turnover of [Ca2+]i in these cells. Norepinephrine (NE, 10(-6) M) increased [Ca2+]i to approximately 350 nM within 1 min; [Ca2+]i then remained elevated for 30 min. The relative potency of adrenergic agonists was NE greater than phenylephrine much greater than isoproterenol. Phentolamine (10(-6) M) and prazosin (10(-8) M) blocked the effects of adrenergic agonists; in contrast, propranolol (10(-6) M) or yohimbine (10(-6) M) had little or no effect. These observations indicate NE acts via alpha 1-adrenoceptors to elevate [Ca2+]i. The [Ca2+]i response to NE did not occur when [Ca2+]e was reduced to approximately 10 microM by adding EGTA 5s before NE, indicating an increase in net Ca2+ influx is involved rather than mobilization of Ca2+ from intracellular stores. The effect of NE was not blocked by nifedipine (10(-6) M), which did block a K+-induced increase in [Ca2+]i, presumably involving voltage-sensitive channels. Ouabain (10(-5) M) caused a gradual increase in [Ca2+]i; this increase was not blocked by nifedipine. Together these data indicate that pinealocyte [Ca2+]i may be influenced by mechanisms regulated by alpha 1-adrenoceptors, voltage-dependent Ca2+ channels, and perhaps a Na+/Ca2+ exchange mechanism stimulated by ouabain. These studies indicate that the pinealocyte is an interesting model to use to study the adrenergic regulation of [Ca2+]i because of the rapid and prolonged changes in [Ca2+]i produced by alpha 1-adrenoceptor activation.     

High external Ca2+ levels trigger membrane potential oscillations in mouse pancreatic beta-cells during blockade of K(ATP) channels.

Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.     

Calcium-activated potassium channels in chondrocytes.

The presence of calcium-activated potassium channels in chondrocytes of growing cartilage was tested. Results obtained with fura-2 on cultured resting chondrocytes indicate that the cells respond to an elevation of extracellular calcium concentration ([Ca2+]o) from 0.1 to 2 mM increasing the intracellular concentration of the ion ([Ca2+]i) from 117 to 187 nM. This increment may be blocked by 3 microM La3+. Patch clamp experiments in cell-attached configuration showed that, when [Ca2+]i rises, the open probability (Po) of the K+ channels increases. Increments in both Po and unitary currents of the K+ channels can be obtained after applying 2.5 microM A23187 with 2 mM [Ca2+]o. Hence, the results demonstrate that, in chondrocytes, a class of Ca(2+)-activated K+ channels is present and their activity is related to an increase of [Ca2+]i.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Intracellular calcium stores are involved in growth hormone-releasing hormone signal transduction in rat somatotrophs.

In rat pituitary somatotrophs, the stimulation of growth hormone secretion by growth hormone-releasing hormone (GHRH) is a Ca(2+)-dependent event involving Ca2+ influx. The presence of calcium-induced calcium release (CICR) Ca2+ stores has been suggested in these cells. The aim of our study was to demonstrate the presence of CICR stores in rat somatotrophs and to determine their function in GHRH Ca2+ signalling. To this end we measured cytosolic free Ca2+ concentration ([Ca2+]i), using indo-1 in purified rat somatotrophs in primary culture, while altering intracellular Ca2+ stores. Ionomycin (10 ttM) or 4-bromo-A23187 (10 ItM), used to mobilise organelle-bound Ca2+, raised [Ca2+]i in the absence of extracellular Ca2+. Caffeine (5 to 50 mM), used to mobilise Ca2+ from CICR stores, transiently raised [Ca2+]i in 65% of cells tested. The response to 40 mM caffeine was abolished when Ca2+ stores were depleted, with 1 microM thapsigargin or with 10 microM ryanodine. All cells that responded to 40 mM caffeine responded to 10 nM GHRH. The [Ca2+]i response to 10 nM GHRH was reversible and repeatable. However, the second response was 38% smaller than the first. Ryanodine treatment abolished the reduction in the second [Ca2+]i response, while thapsigargin increased the reduction by 67%. We conclude that rat somatotrophs possess CICR Ca2+ stores and that they account for 30-35% of the GHRH-induced increase in [Ca2+]i, and that their partial depletion is involved in somatotroph desensitization.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

Calcium homeostasis in bovine somatotrophs: calcium oscillations and calcium regulation by growth hormone-releasing hormone and somatostatin.

The free intracellular calcium ion concentration ([Ca2+]i) was measured in single cells of a population containing 65-80% somatotrophs, using the fluorescent Ca(2+)-indicator Fura-2 and digital imaging microscopy. Spontaneous oscillations in [Ca2+]i ranging in frequency up to 1.5 oscillations per minute were observed in 30% of somatotrophs. These Ca2+ oscillations were blocked by the Ca2+ channel blocker CoCl2 and were thus proposed to be the result of influx of Ca2+ into the cell, possibly as the result of spontaneous electrical activity. GHRH (10-100 nM) increased [Ca2+]i in 61% of the cells studied, although the amplitude and dynamics of the response varied from cell to cell. Typically [Ca2+]i rose from 170 +/- 26 nM to 321 +/- 44 nM (n = 13) in response to a challenge with 66 nM GHRH. GHRH also increased the frequency of Ca2+ oscillations in a number of cells, and some previously quiescent cells showed Ca2+ oscillations following addition of GHRH. Forskolin, which raises cAMP levels in bovine anterior pituitary cells, also stimulated a sustained rise in [Ca2+]i in 10 out of 14 cells tested. Somatostatin (SS) (10-80 nM) rapidly reduced basal [Ca2+]i, blocked Ca2+ oscillations, and blocked the [Ca2+]i response to GHRH. The Ca2+ channel blocker CoCl2 (4 mM) had similar actions on [Ca2+]i to those of SS. These results suggest that GHRH and SS may regulate GH release by modulating Ca2+ entry into the cell through the cell membrane. The [Ca2+]i oscillations seen in a proportion of the somatotrophs were modulated in frequency by GHRH and SS, and are probably generated by influx of Ca2+ through channels in the cell membrane. Thus GH secretion may be regulated by changes in the mean level of [Ca2+]i, which in turn, may be influenced by the frequency of [Ca2+]i oscillations in bovine somatotrophs.