Effect Of Extracellular Calcium Research Articles

Osteoclasts from medullary bone of egg‐laying Japanese quail do not express the putative calcium ‘receptor’

The present study reports the contrasting effects of extracellular calcium ([Ca2+]e) elevation on cytosolic free calcium levels ([Ca2+]i) of osteoclasts, freshly isolated either from medullary bone of the egg-laying Japanese quail or from rat cortical bone. [Ca2+]i was measured in single osteoclasts using the Ca(2+)-sensitive fluorochrome, Indo-1. We found that elevation of [Ca2+]e failed to induce a rise of [Ca2+]i in quail osteoclasts, whilst causing an elevation of [Ca2+]i in rat osteoclasts. The calcium ionophore, ionomycin, led to a sustained elevation of [Ca2+]i in both cell types. These findings suggest that osteoclasts isolated from egg-laying quail do not possess the calcium sensor or 'receptor' that appears to be vital for the survival and function of rat osteoclasts.

Endothelin-induced changes in intracellular calcium concentration in rat vascular smooth muscle cells: effects of extracellular calcium and atrial natriuretic factor.

The purpose of this study was to examine whether different mechanisms might underlie the changes in intracellular calcium concentration ([Ca2+]i) stimulated by high and low concentrations of endothelin, and whether atrial natriuretic factor (ANF) has an inhibitory effect on endothelin-induced [Ca2+]i changes in cultured rat vascular smooth muscle cells (VSMCs). In calcium-replete buffer, cultured monolayers of rat VSMCs superfused with endothelin at a high concentration (10 nM) exhibited a marked transient rise in [Ca2+]i, followed by a sustained elevation, whereas a low concentration of endothelin (0.1 nM) induced a sustained monophasic elevation. When calcium-free buffer was used, 10 nM endothelin induced a transient rise in [Ca2+]i of lesser amplitude, whereas 0.1 nM endothelin did not produce a significant rise. Pretreatment of VSMCs with ANF and cosuperfusion with endothelin failed to inhibit either transient or sustained endothelin-induced changes in [Ca2+]i in calcium-replete buffer.

Indirect inhibitory effect of succinylcholine on acetylcholine-activated channel activities and its modulation by external Ca2+ in mouse skeletal muscles.

1. The effect of extracellular calcium on single acetylcholine (ACh)-activated channel activities when desensitizing concentrations of succinylcholine (SuCh) were applied to the surrounding endplate membrane was investigated by the cell-attached patch-clamp technique at endplates of single skeletal muscle (flexor digitorum brevis) fibres of adult mice. 2. Bath-applied SuCh (0.1-3 microM, in 2.5 mM Ca2+) increased in a concentration-dependent manner the mean open time of ACh-activated channel currents recorded at membrane potentials which cancelled the SuCh-induced depolarizations. 3. In the presence of 0.5 and 2.5 mM external Ca2+, SuCh (3 microM) applied outside the patch pipette prolonged the mean open time of ACh-activated channel currents in a time-dependent manner (by 45% and 52%, respectively), and simultaneously significantly decreased the single channel conductance (by 14% and 10%, respectively). These SuCh-induced effects did not occur in a nominally Ca(2+)-free extracellular medium. 4. Under the same conditions, SuCh (3 microM) augmented the time-dependent decline in the opening frequency of ACh-activated channel currents obtained in nominally Ca(2+)-free medium. 5. These results suggest that external calcium ions act to modulate nicotinic ACh receptor channel activity, and accelerate desensitization of the receptor.

Regulation of secretion of the teleost fish hormone stanniocalcin: Effects of extracellular calcium

The release in vivo and in vitro of stanniocalcin (STC) from the corpuscles of Stannius (CS) of the rainbow trout and the European eel was studied. Intraperitoneal injection of CaCl 2 (2.45 mmol · kg −1 fish) leads to an elevation of both ionic and total calcium in the plasma and results in the release of STC from the CS into the blood. Release of STC in vitro is not affected at “physiological” (1.0–1.5 m M) or lower Ca 2+ levels in the incubation medium. High levels of Ca 2+ (2.5 m M and higher), however, stimulate the release of STC, in particular that of stored STC. We hypothesize that variations in extracellular Ca 2+ in the normocalcaemic range do not directly regulate STC release.

Extracellular calcium and luteinizing hormone effects on 22-hydroxycholesterol used for testosterone production in mouse Leydig cells.

The effect of extracellular calcium on testosterone synthesis in response to luteinizing hormone (LH) or 22-hydroxycholesterol (22-OH-C) by isolated adult mouse Leydig cells was studied. Leydig cells were isolated by linear density gradient centrifugation. The cells were incubated in minimum essential medium with or without calcium (1.36 mmol/L) in an atmosphere of 95% air and 5% carbon dioxide at 37 degrees C for 3 hours with or without LH (10 ng/sample), or with or without 22-OH-C (10 mumol/L). Testosterone production in response to LH was significantly lower (P less than 0.02) in the absence of extracellular calcium and in the presence of verapamil (10 mumol/L), a calcium channel blocking agent. Extracellular calcium did not significantly (P greater than 0.05) affect testosterone production in cells incubated with 22-OH-C in either the presence or absence of LH. The results suggest that steps in steroidogenesis from 22-OH-C to testosterone are unaffected by extracellular calcium content and that extracellular calcium affects the use of intracellular cholesterol by the cholesterol side-chain cleavage enzyme.

Developmental Aspects of Bladder Contractile Function: Sensitivity to Extracellular Calcium

The urinary bladders of 1-day and 1-week-old rabbits generate higher intravesical pressures in response to bethanechol and field stimulation than bladders isolated from mature 8-week-old rabbits. Yet the density of cholinergic receptors in the rabbit bladder does not change with maturation (1 day to 8 weeks). In an effort to better understand the molecular mechanisms by which newborn rabbit bladders generate greater pressures than the bladders of adult rabbits, we studied the effect of maturation on the relationship between extracellular calcium and contraction. Our results showed quite clearly that (1) at physiologic concentrations of calcium (2.5 mumol/l), isolated bladder strips of 1-day- and 1-week-old rabbits contracted in response to bethanechol to 98% of their maximal tension as opposed to 68% for their 8-week-old counterparts, (2) the ED50 (for calcium) for the 1-day and 1-week bladders was 0.4 mmol/l whereas the ED50 for the adult bladder strips was 2.2 mmol/l, and (3) the neonatal bladders demonstrated a much greater sensitivity to diltiazem than the adult bladders. The contractile response to calcium of the neonatal bladders was significantly inhibited by 1 mumol/l diltiazem whereas the 8-week-old bladders showed no inhibition at this concentration. In a second series of experiments, the effect of extracellular calcium on concentration was correlated with the intracellular concentration of free calcium using the calcium fluoride FURA-2 and surface spectrofluorometry. These studies confirmed that the increased contractile response of the neonatal bladder strips to calcium or cholinergic agonists was associated with an increase in the maximal intracellular free calcium concentration.

The effect of extracellular calcium on the contractile action of endothelin

The tension developed by rat aortic strips in response to endothelin-1 is determined by three types of mechanisms: a [Ca 2+]o independent mechanism, L-type Ca 2+ channels and a [Ca 2+]o dependent, verapamil insensitive, mechanism. Their relative contributions to the tension recorded 30 minutes after the addition of 50 nM endothelin-1 were 43%, 34% and 23%. Upon longer exposures to endothelin-1, the whole tension could be abolished by reducing [Ca 2+]o to 20 nM. Endothelin-1 induced contractions were highly sensitive to changes in free [Ca 2+]o. The EC50 value for the [Ca 2+]o dependence of endothelin-1 induced contractions was 600 nM, a value 400 times lower than the corresponding value found for KCl induced contractions (250 μM). These results suggest that extracellular Ca 2+ is necessary for full tension development in response to endothelin-1 but that a major action of endothelin-1 is to increase the sensitivity of pharmacomechanical coupling mechanisms to Ca 2+.

Stimulatory effects of extracellular calcium on chorionic gonadotrophin and placental lactogen release by human placental explants

The effects of sudden modifications of extracellular calcium ion concentration on human chorionic gonadotrophin (hCG) and human placental lactogen (hPL) release were investigated using term placental explants incubated in Krebs Ringer solution. The hCG and hPL releases were both stimulated when the extracellular Ca2+ concentration was increased. The hCG and hPL secretions elicited by the addition of extracellular Ca2+ were larger when placental explants were preincubated in alpha-calcium (no added calcium + EGTA) than in normo-calcium (1.5 mM). Removal of extracellular Ca2+ from the medium also elicited an increase in hCG and hPL release. However, this stimulatory effect of Ca2+ omission was partly suppressed by washing the explants prior to incubation in the alpha-calcium medium, and was completely abolished when alpha-calcium medium was supplemented with 1 mM cobalt. Our results indicate that changes in Ca2+ modify hCG and hPL release from term placental explants in a manner concordant with the 'stimulus-secretion coupling' concept.

Augmentation of forskolin-induced cAMP production by ouabain in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca 2+) on the cellular action of forskolin was studied using a Na +, K +-ATPase inhibitor ouabain in rat renal papillary collecting tubule cells in culture. Forskolin-induced cAMP production was enhanced by the pretreatment of cells with ouabain, providing that a dose-dependent curve with forskolin shifted to the left. The enhancement by ouabain of cellular cAMP production in response to forskolin was totally blunted by cotreatment with cobalt, verapamil, or Ca 2+-free medium containing 1 mM EGTA. In addition, two dissimilar antagonists of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W - 7), attenuated the ouabain's effect on cAMP production in response to forskolin. These results therefore indicate that ouabain enhances the activation of adenylate cyclase by forskolin, mediated through cellular free Ca 2+, in renal papillary collecting tubule cells, and that extracellular Ca 2+ is an important source for cellular Ca 2+ mobilization by ouabain.

Extracellular calcium alleviates cell toxicity due to hepatotoxins that induce lipid peroxidation, but has no effect on toxins that do not cause lipid peroxidation: A study in isolated rat hepatocytes

The effect of extracellular calcium on cell death, induced by hepatotoxins that induce lipid peroxidation [diethyl maleate (DEM), allyl alcohol (AA) and bromoisovalerylurea (BIU)] and hepatotoxins that do not induce lipid peroxidation [disulfiram (DSF), N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF) and tetrahydroaminoacridine (THA)] was studied in freshly isolated rat hepatocytes. Extracellular calcium strongly delayed the onset of toxicity of DEM, AA and BIU as detected by lipid peroxidation, depletion of free protein thiol groups and cell death. This protective effect of calcium was decreased at higher concentrations of the toxic compounds. In contrast, no effect of calcium was observed on toxicity induced in the absence of lipid peroxidation by DSF, N-OH-AAF and THA. Addition of calcium was also without effect on the protein thiol depletion. These results indicate that calcium only alleviates cytotoxicity which is induced by thiol depletion resulting from lipid peroxidation. Cytotoxicity as a result of protein thiol depletion through disulfide formation is not affected by extracellular calcium.

Effect of extracellular calcium and potassium depolarization on the chorionic gonadotropin and placental lactogen releases by human placental explants

Effect of extracellular calcium and potassium depolarization on the chorionic gonadotropin and placental lactogen releases by human placental explants

Effect of ouabain on cellular free calcium and cellular cyclic AMP production in response to arginine vasopressin in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using an Na+, K+-ATPase inhibitor in rat renal papillary collecting tubule cells in culture. The pretreatment of cells with ouabain enhanced basal and AVP-induced cAMP production in a dose-dependent manner. The augmentation by ouabain of cellular cAMP production in response to AVP was totally abolished by co-treatment with cobalt, lanthanum, verapamil or Ca2+-free medium containing 1 mmol EGTA/l, each blocking cellular Ca2+ uptake by different mechanisms. Two other findings indicated that ouabain directly stimulated cellular Ca2+ mobilization; namely, that ouabain significantly increased 45Ca2+ influx and cellular free Ca2+ concentration [( Ca2+]i) determined by Fura-2 fluorescence. The ouabain-induced increase in [Ca2+]i was completely blocked by either cobalt or Ca2+-free medium containing 1 mmol EGTA/l. AVP at 0.1 mumol/l increased [Ca2+]i to 177.1 +/- 26.2 nmol/l from 92.2 +/- 8.0 nmol/l (P less than 0.01) in renal papillary collecting tubule cells, and ouabain significantly enhanced the AVP-induced increase in [Ca2+]i. The increase of cellular free Ca2+ induced by ouabain probably binds to calmodulin to form an active complex of Ca2+-calmodulin in the cell, since two chemically dissimilar antagonists of calmodulin attenuated the enhancement by ouabain of cAMP production in response to AVP. These results therefore indicate that ouabain increases cellular Ca2+ uptake and enhances AVP-induced cellular free Ca2+ mobilization and its own second messenger cAMP production in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for ouabain-mobilized cellular Ca2+.

Effect of extracellular calcium and calcium channel antagonists on ATP and field stimulation induced contractions of the mouse urinary bladder

1. The dependence on extracellular calcium of the responses to the electrical stimulation, ATP and high K induced contractions has been studied in the mouse urinary bladder. 2. The responses to ATP, field stimulation and K induced depolarization were eliminated in calcium free EGTA medium. However, a small remanent of these responses was observed in the absence of calcium in the superfusing medium. 3. The calcium antagonists verapamil, nifedipine and diltiazem decreased in a dose dependent manner the contractions induced by ATP and electrical stimulation of the mouse urinary bladder. 4. The responses of the mouse urinary bladder to high K concentration were antagonized by verapamil and diltiazem, but nifedipine was less effective in decreasing the tonic component of the contraction induced by K in the muscle. 5. The responses of the mouse urinary bladder to electrical stimulation, ATP and high concentration of K are mainly dependent on the extracellular calcium.

Effects of extracellular calcium and sodium on cadmium uptake in guinea-pig and rabbit aorta

1. 1. Effects of Na + and Ca 2+ on Cd 2+ uptake in the guinea-pig and rabbit aorta were investigated. 2. 2. The Cd 2+ fraction can be divided with EDTA and Zn 2+. The EDTA fraction may be intracellular fraction and the Zn 2+ fraction may be loosely bound to surface membrane. 3. 3. In Na + deficient solution the Zn 2+ fraction was markedly increased and the EDTA fraction also increased while the external Ca 2+ concentrations scarcely affect the Cd 2+ contents in both fractions. 4. 4. Cd 2+ did not affect the cellular Na + and K + contents. 5. 5. These results suggest that Na + strongly relates with these fractions but Na + pump does not contribute to these phenomena.

Enhancement by Veratridine and 60 mM KC1 of Vasopressin-Induced 3′,5′-Cyclic Adenosine Monophosphate Production and Cellular Free Calcium Concentration in Rat Renal Papillary Collecting Tubule Cells in Culture*

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using depolarizing agents in rat renal papillary collecting tubule cells in culture. One-hour exposure of cells to veratridine enhanced AVP-induced cAMP production in a dose-dependent manner. This enhancement by veratridine of cellular cAMP production in response to AVP was totally blunted by cotreatment with 5 X 10(-4) M verapamil, 3 X 10(-3) M cobalt, or Ca2+-free medium containing 1 X 10(-3) M EGTA. These agents block cellular Ca2+ uptake by different mechanisms. Similarly, 60 mM KCl enhanced AVP-induced cAMP production, and this effect was blocked by pretreatment with verapamil, cobalt or Ca2+-free medium containing 1 X 10(-3) M EGTA. When cellular free Ca2+ concentrations [Ca2+]i were measured by the fluorescence dye fura-2, both 1 X 10(-4) M veratridine and 60 mM KCl significantly increased [Ca2+]i from 67.6 to 141.8 nM and from 74.6 to 166.2 nM, respectively. Such rises in [Ca2+]i depended on extracellular Ca2+ since the increase in [Ca2+]i was completely blocked in Ca2+-free medium or in the presence of 3 X 10(-3) M cobalt. In addition, veratridine and 60 mM KCl significantly augmented the AVP-induced increase in [Ca2+]i. The possible mechanisms by which depolarizing agents induce cellular Ca2+ mobilization include the opening of voltage-sensitive Ca2+ channels in the plasma membrane. The present results indicate that veratridine and 60 mM KCl enhance AVP-induced cAMP production and cellular free Ca2+ concentration through cellular Ca2+ uptake in renal papillary collecting tubule.

Release of calcitonin gene-related peptide from rat enteric nerves is Ca 2+-dependent but is not induced by K+ depolarization

The effect of extracellular calcium on the release of calcitonin gene-related peptide (CGRP) induced by electrical field stimulation from enteric nerves of isolated rat ileum was studied; the effect of high potassium, veratridine and caffeine was also examined. Release of endogenous substance P from enteric nerves was also measured for comparison. Electrical field stimulation (10 Hz, 0.3 ms for 2 min) of the ileum preparation caused a significant ( P < 0.0001) increase in the release of CGRP and substance P from enteric nerves. The evoked, but not the basal, release of both CGRP and substance P was inhibited in the presence of tetrodotoxin (TTX). The release of CGRP and substance P induced by electrical stimulation was abolished in Ca 2+-free medium containing CDTA and also in normal medium containing the calcium channel blocker cadmium chloride (CdCl 2), with no change in the level of the basal release of both peptides. However, potassium depolarization (76 and 110 mM) failed to evoke an increase in the release of endogenous CGRP, although it did cause an increase in the release of substance P. Caffeine (10 mM) induced the release of CGRP, suggesting that CGRP release can be induced by mobilization of calcium from intracellular Ca 2+ stores. Veratridine, on the other hand, did not cause an increase in CGRP release, although substance P and VIP release was induced by veratridine from the same preparations. The results of the present study have demonstrated that CGRP release from enteric nerves requires the presence of extracellular calcium but, unlike substance P and most other transmitters reported to show calcium-dependent release, potassium depolarization does not induce CGRP release from enteric nerves of rat ileum. The lack of release of CGRP by veratridine suggests that it is unlikely that activation of Na + channels is directly responsible for the release. These findings will be discussed in relation to different possible mechanisms of release of gut peptides; it is suggested that the neural release of CGRP is likely to involve receptor-operated rather than voltage-gated calcium channels.

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

Calcium efflux was measured in desheathed rabbit vagus nerves loaded with 45Ca2+. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of 45Ca2+ efflux profiles. The 45Ca2+ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min-1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min-1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm-2 sec-1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.

Effects of extracellular calcium on calcium movements of excitation‐contraction coupling in frog skeletal muscle fibres.

1. The effect of low extracellular free calcium ion concentration ([Ca2+]o) on the transient changes in cytoplasmic [Ca2+] associated with membrane depolarization (Ca2+ transients) was studied on single cut skeletal muscle fibres of the frog, voltage clamped in a double-Vaseline-gap chamber. The Ca2+ transients were monitored with the dye Antipyrylazo III diffused intracellularly. 2. The Ca2+ transients were substantially reduced in external salines with low [Ca2+] (10(-5) M or less and Mg2+ substituted for Ca2+). This decrease was more noticeable at late times during 100 ms or longer depolarizing pulses. 3. The rates of the processes that remove Ca2+ from the myoplasmic solution were not altered by the low [Ca2+]o. This implies that the input flux of Ca2+ into the myoplasm was reduced. 4. The Ca2+ input flux, equal to release flux from the sarcoplasmic reticulum (SR) plus Ca2+ influx via the T-tubule membrane Ca2+ channel, was derived from the Ca2+ transient. In low [Ca2+]o the peak input flux was reduced by 45% (n = 16 fibres) and decayed more rapidly during a depolarizing pulse. 5. The reduction in Ca2+ influx via the T-tubule membrane Ca2+ channel due to the reduced [Ca2+]o could not account for more than 5% of the reduction in Ca2- input flux, which was thus interpreted as an actual reduction of release from the SR. 6. The inward (T-tubular) Ca2+ current was not associated with this effect of extracellular Ca2+ as the effect was voltage independent at high intracellular voltages at which the Ca2+ inward current was strongly voltage dependent. 7. Low [Ca2+]o made Ca2+ release more readily inactivatable; the effect of low [Ca2-]o is best described as a left shift by 29 mV of the 'inactivation curve' of Ca2+ release, relating peak release flux to membrane holding potential. 8. The reduction of Ca2+ release by low [Ca2+]o was not accompanied by changes in the voltage dependence of Ca2+ release or in the threshold voltage for just-detectable release. 9. The results are consistent with a primary effect of Ca2+ on the T-tubular-membrane voltage sensor of excitation-contraction coupling.

Effects of extracellular calcium and other divalent cations on mechanical response of frog skeletal muscle.

In order to investigate the mechanism by which elevated extracellular Ca ions decrease twitch and tetanus tension in frog skeletal muscle we made mechanical and electrophysiological measurements on single fibers or small bundles from twitch muscles. High concentration of Ca caused a hyperpolarization and an increase in the duration of action potential. The mechanical threshold, estimated by using the strength-duration curve, was shifted upward by adding Ca ions. These effects were fully reversible. Steady state twitch tension was slightly increased by replacing Mg and Ni with Ca and decreased by elevating their concentrations, although Ba resulted in a marked twitch augmentation and a positive correlation with the ion concentration. By contrast, the strength-duration curve was shifted upward by Ni while Mg and Ba showed no shift. These evidences point to a failure of the early step of excitation-contraction coupling, including the T-membrane depolarization, as the primary mechanism of action of high concentration of Ca, Mg, and Ni ions, whereas Ba ion has an additional intracellular potentiating effect.

Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation.

Optimal proliferation of cultured hepatocytes from normal rats occurs in response to epidermal growth factor (EGF) at an extracellular calcium concentration of 0.4 mM, whereas physiological concentrations of calcium have been shown to decrease hepatocyte proliferation. Exposure of hepatocytes in vivo to phenobarbital (PB, 0.1% in the drinking water) reduced significantly the ability of physiological levels of calcium to suppress hepatocyte proliferation. An increased relative ability of hepatocytes to proliferate at physiological calcium concentrations versus that at 0.4 mM calcium was first seen after 3 days of in vivo PB treatment and this effect was maintained during 2 months of exposure. Hepatocytes from short-term PB-exposed animals (i.e. 3-28 days) proliferated at physiological Ca2+ concentrations 2-3 times better than those from control animals. However, after 2 months of continuous PB exposure, cell growth was reduced significantly at all extracellular Ca2+ concentrations investigated. EGF binding studies demonstrated that the biphasic kinetic effect of PB on hepatocyte proliferation was not correlated directly with the steady decline in EGF receptor number or the lack of significant change in receptor binding affinity with duration of PB exposure. These results imply that the effectiveness of PB in tumour promotion may result from its ability to reduce the absolute magnitude of normal hepatocyte proliferation and to alter the growth regulatory effect of extracellular calcium. Further, the results argue that PB effects on hepatocyte proliferation are not mediated simply through regulation of EGF surface receptor number or binding affinity. Additional events of the EGF-induced cascade necessary for hepatocyte proliferation appear to be modified by PB.