Increase In Extracellular Calcium Concentration Research Articles

Extracellular calcium induces neurite outgrowth through calcium-sensing receptors in PC12 HS cells

PC12 cells have been used extensively to study neuronal differentiation and neurite outgrowth. However, the effects of extracellular Ca2+ on PC12 cells have not yet been sufficiently evaluated. In this study, we investigated the effect of extracellular calcium using PC12 HS cells, which have high sensitivity to nerve growth factor (NGF). The addition of Ca2+ (1 mM) to the medium induced neurite outgrowth and increased the intracellular calcium concentration. In contrast, the calcium-sensing receptor antagonist NPS2143 suppressed neurite outgrowth and increased the intracellular calcium concentration, suggesting that extracellular calcium promotes neurite outgrowth through calcium-sensing receptors. An increase in extracellular calcium concentration increased phosphorylation of Akt and cyclic AMP response element-binding protein (CREB). Semi-quantitative RT-PCR showed that elevated extracellular calcium increased the mRNA expression of brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor (PDGF), which are regulated by CREB activation. Our results suggest that elevated extracellular calcium can promote the phosphorylation of Akt and CREB through calcium-sensing receptors, resulting in the promotion of neurites. PC12 HS cells are a useful model to investigate the regulation of extracellular calcium via calcium-sensing receptors in neuronal cells.

Length-Dependent Prolongation of Force Relaxation Is Unaltered by Delay of Intracellular Calcium Decline in Early-Stage Rabbit Right Ventricular Hypertrophy.

Chronic pressure overload can result in ventricular hypertrophy and eventually diastolic dysfunction. In normal myocardium, the time from peak tension to 50% relaxation of isolated cardiac myocardium is not directly determined by the time for calcium decline. This study aims to determine whether the time for calcium decline is altered with a change in preload in early-stage hypertrophied myocardium, and whether this change in time for calcium decline alters the rate of relaxation of the myocardium. Young New Zealand white rabbits underwent a pulmonary artery banding procedure and were euthanized 10 weeks later. Twitch contractions and calibrated bis-fura-2 calcium transients were measured in isolated thin right ventricular trabeculae at optimal length and with the muscle taut. Systolic calcium, calcium transient amplitude, and time from peak tension to 50% relaxation all increased with an increase in preload for both hypertrophied and sham groups. Time for intracellular calcium decline increased both with an increase in preload and an increase in extracellular calcium concentration in hypertrophied myocardium but not in sham, while time from peak tension to 50% relaxation did not significantly change between groups under either condition. Also, time for intracellular calcium decline generally decreased with an increase in extracellular calcium for both hypertrophied and sham groups, while time from peak tension to 50% relaxation generally did not significantly change in either group. Combined, these results indicate that the mild hypertrophy significantly changes calcium handling, but does not impact on the rate of force relaxation. This implies that the rate-limiting step in force relaxation is not directly related to calcium transient decline.

Effects of extracellular calcium concentration on neurite outgrowth in PC12 cells by staurosporine

Staurosporine as an inhibitor of protein kinases can induce neuronal differentiation in PC12 cells. We investigated the role of extracellular Ca2+ on staurosporine inducing neurite outgrowth in PC12 cells. The cells were cultured during cell differentiation in the presence of 214nM staurosporine with 0.0–0.7 Ca2+mM as treatment media. We obtained the fraction of neurite-bearing cells, total neurite length and the percentage of cytotoxiciy. The results showed that decrease or increase of extracellular calcium ions resulted in correspondingly significant decrease or increase in total neurite length and cell differentiation in treated cells. With an increase of extracellular calcium concentration from 0.0 to 0.7mM, the percentage of cytotoxicity of the PC12 cells decreased (p<0.05). Our data suggest that staurosporine uses the extracellular calcium ions to affect on neurite outgrowth.

Effect of extracellular calcium on the additive effect of theophylline on the cardiac response to catecholamine

At diff erent extracellular calcium concentrations, the positive inotropic eff ect of isoproterenol and isoproterenol in combination with theophylline, a phosphodiesterase inhibitor have been evaluated in the isolated frog heart and the isolated guinea pig left atria to investigate whether extracellular calcium produces any eff ect on the additive eff ect of the theophylline on the cardiac response to catecholamine. Cumulative dose response study of isoproterenol and isoproterenol in presence of theophylline at diff erent extracellular calcium concentration was performed. Th e study revealed an increase in additive eff ect of theophylline on the cardiac response to catecholamine with increase in extracellular calcium concentration, but increase in extracellular calcium concentration decreased the myocardial responsiveness to additive eff ect of theophylline and isoproterenol combination. Th e mechanism of positive inotropic eff ect of isoproterenol and in combination with theophylline involves increase in intracellular CAMP by diff erent pathways and extracellular calcium produces positive inotropic eff ect by initiating the interaction between the contractile proteins actin and myosin.The study revealed that an increase in the concentration of extracellular calcium increased the additive eff ect of theophylline and isoproterenol combination, but a decrease in the myocardial responsiveness was observed. Key words: Additive eff ect, cAMP, isoproterenol, positive inotropic eff ect, theophylline and calcium

Sustained increase of extracellular calcium concentration causes arterial vasoconstriction in humans

Vascular tone is affected by extracellular electrolytes. To evaluate whether a sustained increase in the extracellular calcium concentration may produce vasoconstriction in humans. Cross-sectional data of a cohort study of 65 patients with end-stage renal failure. Arterial tone was obtained from radial artery waveforms. Intracellular stored calcium and sarcoendoplasmic reticulum Ca(2+)-ATPase activity were measured in mononuclear leukocytes using fluorescent dye techniques and oxalate-supported calcium uptake. During the haemodialysis sessions the extracellular calcium concentration increased significantly from 2.28 +/- 0.03 to 2.63 +/- 0.03 mmol/l (n = 65; mean +/- SEM; P < 0.001) and arterial tone increased from 31 +/- 2 to 44 +/- 3 mmHg/ml (n = 65; P < 0.001). Multivariate analysis showed that pre and postdialysis extracellular calcium and pre and postdialysis body weight were the only independent predictors of arterial tone during haemodialysis. Intracellular stored calcium in mononuclear leukocytes significantly declined from 5.1 +/- 1.2 arbitrary units at the start to 2.3 +/- 0.6 arbitrary units at the end of haemodialysis (n = 10; P = 0.01). The activity of the sarcoendoplasmic reticulum Ca(2+)-ATPase significantly decreased from 13.6 +/- 2.6 nmol calcium/mg protein per 5 min at the start to 9.2 +/- 1.6 nmol calcium/mg protein per 5 min at the end of haemodialysis (n = 28; P = 0.01). On the other hand, when a low dialysate calcium concentration was used, the increase in artery tone and reduction in sarcoendoplasmic reticulum Ca(2+)-ATPase activity were reversed. A sustained increase in the extracellular calcium concentration causes arterial vasoconstriction in humans. In addition, a reduction of sarcoendoplasmic reticulum Ca(2+)-ATPase activity and intracellular stored calcium in mononuclear leukocytes was observed.

Extracellular calcium modulates the effects of protamine on rat myocardium.

We studied the effects of protamine (10-300 microg. mL(-1)) as well as its interaction with heparin in rat left ventricular papillary muscles in vitro at calcium concentrations of 0.5 and 1 mM under low (isotony) and high (isometry) loads. Protamine induced a negative inotropic effect that was less pronounced at calcium 0.5 mM (active force at protamine 300 microg/mL, 84 +/- 20 vs 57 +/- 15% of baseline, P: < 0.05); whereas at calcium 1 mM there was a marked contracture of the muscle. For the smallest concentrations of protamine and at calcium 0.5 mM, we observed a moderate positive inotropic effect that was suppressed by nifedipine. Protamine induced a negative lusitropic effect under low load and decreased postrest potentiation, suggesting an impairment in the functions of the sarcoplasmic reticulum. Heparin was able to inhibit and reverse the negative inotropic effect of protamine. The negative inotropic effect of protamine is enhanced by an increase in extracellular calcium concentration. This negative inotropic effect is probably related to calcium overload and impairment in sarcoplasmic reticulum functions, and heparin can block these effects. The negative inotropic effect of protamine is enhanced by an increase in extracellular calcium concentration. This negative inotropic effect is probably related to calcium overload and impairment in sarcoplasmic reticulum functions, and heparin can block these effects.

Ca2+-Induced Increases in Steady-State Concentrations of Intracellular Calcium Are Not Required for Inhibition of Parathyroid Hormone Secretion

It has been well established that increases in extracellular calcium concentration ([Ca2+]) inhibit parathyroid hormone (PTH) secretion. The effects of [Ca2+] are mediated through a G-protein-coupled receptor that has been cloned and characterized. Additionally, it has been demonstrated in parathyroid cells that an increase in [Ca2+] results in an increase in steady-state levels of intracellular calcium ([Ca2+]i). At present, it has not been fully resolved whether changes in [Ca2+]i are related to changes in PTH secretion. In the current study, the effect of increased [Ca2+] on PTH secretion and the connection regarding changes in concentrations of intracellular calcium [Ca2+]i have been examined in primary cultures of bovine parathyroid cells. PTH secretion was measured by radioimmunoassay and intracellular calcium was determined by single cell calcium imaging. Bovine parathyroid cells pre-incubated with either 0.5 or 1 mM calcium responded to rapid increases in [Ca2+] (≥0.5 mM) with an immediate and sustained increase in steady-state levels of [Ca2+]i that persisted for time intervals greater than 15 minutes. Although the magnitude of the sustained increase in [Ca2+]i varied among individual cells (∼40% to >300%), the overall pattern and course of time were similar in all cells examined (n = 142). In all trials, [Ca2+]i immediately returned to baseline levels following the addition of the calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Additional control studies, however, suggest that sustained increases in [Ca2+]i do not correlate with regulation of parathyroid hormone secretion. Sustained elevations of [Ca2+]i were not observed when [Ca2+] was gradually increased by the addition of 0.1 mM increments at 1 minute intervals. Furthermore, the effect on inhibition of PTH secretion was the same regardless of whether [Ca2+] was increased by gradual or rapid addition.

The trabecula culture system: a novel technique to study contractile parameters over a multiday time period.

In the intact heart, various triggers induce alterations in gene expression that impact on contractile function. Because changes in gene expression reflect altered protein expression patterns after 12-48 h, we developed a system in which intact twitching cardiac trabeculae can be studied for multiday periods. Right ventricular trabeculae from pentobarbital sodium anesthetized rabbits were mounted in a sterile, closed muscle chamber. Over the first 48 h, developed force (Fdev) did not significantly change: 102.3 and 98.9% of the initial Fdev was observed after 24 and 48 h, respectively (n = 8). Also, neither diastolic force, time from peak to 50% relaxation (RT50), nor protein synthesis measured by a [3H]leucine incorporation assay changed significantly over time. Contractile response after > 48 h to an increase in extracellular calcium concentration (1.8 to 2.5 mM; Fdev increased 43.5%, n = 2) or to 1 microM isoproterenol (Fdev increased 138.6% and RT50 decreased 34.9%, n = 2) was similar to those observed in freshly dissected preparations. In conclusion, this system can investigate contractile function of multicellular preparations under well-defined physiological conditions after events that alter gene and consequent protein expression.

Effects of dihydropyridines on aldosterone secretion in adrenal capsule preparations from pregnant rats.

The primary aim of this study was to determine when sensitivity in the aldosterone response to extracellular potassium (K+) decreases during pregnancy. Second, it tested the hypothesis that calcium channel alterations occur in the adrenal cortex during pregnancy. The decreased sensitivity to K+, observed at 22 days of gestation, was not evident at 15 days and between 18 and 36 h postpartum. Increases in extracellular calcium concentration heightened sensitivity to K+ in adrenal capsule preparations derived from nonpregnant rats but had no effect in pregnant animals. The influence of nifedipine and BAY K 8644 (blocker and activator, respectively, of voltage-operated calcium channels) on the aldosterone response to K+ and to adrenocorticotropic hormone (ACTH) was studied. Sensitivity to K+ in nonpregnant rats decreased in the presence of nifedipine and became similar to that in pregnant rats. Responses to ACTH were not affected by nifedipine. BAY K 8644 produced a larger increase in sensitivity in adrenal capsule preparations from pregnant than from nonpregnant rats, leading to superposition of the two dose-response curves to K+. These results indicate that voltage-operated calcium channels involved in aldosterone secretion are functionally impaired during pregnancy.

New inotropic concepts: rationale for and differences between calcium sensitizers and phosphodiesterase inhibitors.

Positive inotropic compounds may be harmful in the long-term treatment of chronic congestive heart failure because they may induce a calcium overload, unwanted changes in cross-bridge kinetics and an acceleration in heart rate. As a result of all three alterations, energy consumption would be increased. Different pharmacological modes of action may have different effects on the molecular mechanisms underlying the positive inotropic effect, and hence on myocardial energy consumption. Therefore, we studied the effects of a variety of cardiotonic agents on the heat released from small guinea pig papillary muscles contracting isometrically at an experimental temperature of 21 degrees C and a stimulation frequency of 12 per minute using rapid antimony-bismuth thermopiles. We were able to define the economy of muscle contraction, which was lowest with phosphodiesterase inhibitors and highest with calcium sensitizers. Compared with an increase in extracellular calcium concentration, beta 1-adrenoceptor stimulators and phosphodiesterase inhibitors profoundly decrease the economy of myocardial contraction, and calcium-sensitizers (pimobendan and EMD-53998) slightly increase myocardial economy, whereas ouabain and the calcium channel agonist BAY K 8644 have no effect on this parameter. In addition, we provide evidence that acceleration of heart rate may be harmful not only from an energetic point of view: an increase in heart rate may also decrease the contractility of the failing human myocardium (inverse force-frequency relationship). Taking these observations into consideration, an "optimal' positive inotropic compound should have no, or even negative, chronotropic effects, should not be mediated by increases in calcium transients, and should decelerate, rather than accelerate, cross-bridge kinetics.

Induction of a calcium-dependent long-term enhancement of excitability in the rate olfactory bulb.

We have investigated whether a transient increase in extracellular calcium concentration is able to induce long-term modification of neuronal excitability in the olfactory bulb. High-calcium artificial cerebrospinal fluid containing picrotoxin (Ca-PTX solution) was applied locally near the mitral cell layer through a push-pull device for 10 min in anaesthetized rats. Changes in the neuronal excitability were monitored through electrically-evoked field potentials. Application of the Ca-PTX solution induced a rapid increase in the granule cell response amplitude, whereas mitral/tufted cells response amplitude increased more progressively and reached its maximum within a few hours. The increase in mitral/tufted cells response and granule cells response reached between 30 and 100% in experiments which lasted for 4-8 h. Pre-application of amino-phosphonovalerate (NMDA receptor blocker) potently reduced both short- and long-term enhancement produced by the Ca-PTX solution. Neither application of the high calcium solution alone nor the picrotoxin solution alone induced long-term changes. The results point out the possible importance of Ca2+ and NMDA receptors in persistent forms of olfactory bulb plasticity. Relevance of this phenomenon in normal olfactory bulb physiology remains to be examined.

Influence of formamide on the electrical and mechanical properties of the guinea pig ventricle

1. 1. Guinea pig papillary muscle in vitro upon exposure of Formamide (FMD) exerts a dose dependent positive inotropic effect. 2. 2. The increase in tension developed by papillary muscle is associated with a marked decrease in the duration of the action potential. 3. 3. The increase in extracellular calcium concentration does not modify the positive inotropic effect, but increases the duration of the action potential. 4. 4. FMD markedly potentiate the increase in tension induced by caffeine 3 M suggesting a possible synergetic effect upon the release of Ca 2+ ions from the S.R. 5. 5. These experiments suggest that FMD increases the intracellular calcium concentration by exerting a direct action on intracellular calcium stores.

Defibrotide inhibits Ca2+ dependent neutrophil activation: implications for its pharmacological activity in vascular disorders.

Defibrotide (DEF) is a polydeoxyribonucleotide agent provided with profibrinolytic and antithrombotic properties. Moreover, an antiischemic, cardioprotective effect of the drug has recently been demonstrated in experimental animals. Increasing evidence exists of the important role played by neutrophils in the development of tissue damage during chronic and acute ischemia and in the early phases of reperfusion. In order to evaluate whether the overall cytoprotective effect of DEF could be based, at least in part, on a neutrophil-involving mechanism, the authors studied the in vitro effects of the drug on human neutrophil activation triggered by several specific stimuli. The drug dose-dependently (10-100 microM) inhibited enzyme release, superoxide anion generation, and chemiluminescence induced in neutrophils by the chemoattractant oligopeptide fMLP and by the divalent cation ionophores A23187 and ionomycin. The increase of extracellular calcium concentration from 0.5 to 2.0 mM antagonized the inhibitory effect of DEF. The use of the fluorescent probe Fura 2/AM led them to show that DEF is able to reduce the cytosolic free calcium increase following specific stimulation by affecting extracellular calcium entrance. Such a behavior resembles that of calcium-antagonistic drugs, thus suggesting that DEF works, at least in part, similarly to calcium entry blockers. Such an activity on cell calcium translocation could represent the underlying molecular mechanism of cytoprotection. Finally, the inhibitory action on neutrophil functions may play a role in tissue protection during ischemic injury.

Effects of extracellular sodium on cytosolic calcium, PGE2 and cAMP in papillary collecting tubule cells.

An increase in cytosolic calcium (Cai2+), induced by an increase in extracellular calcium concentration or addition of the calcium ionophore A23187, has been shown to suppress basal and AVP responsive cAMP and inhibit water flow in the collecting tubule. In the present study, the relationships between extracellular Na+ concentration, Cai2+, PGE2 and AVP-responsive cAMP production were examined in cultured rat papillary collecting tubule (RPCT) cells. Reducing extracellular Na+ concentration from 144 to 24 mM increased Cai2+ and PGE2 production approximately below 144 mM on Cai2+ or PGE2 was not due to a change in media osmolality or chloride concentration, since these parameters were maintained at constant levels by addition of tetramethylammonium chloride (TMA) or choline chloride. Exposure of RPCT cells to media containing 24 mM Na+ significantly suppressed basal and AVP responsive cAMP compared to that observed at 144 mM Na+. This suppression was mimicked by the calcium ionophore A23187 which also increased Cai2+ and PGE2. The increase in Cai2+ and PGE2 and the suppression of basal and AVP responsive cAMP, which were observed at 24 versus 144 mM Na+, were abolished in calcium free media and were likely due to influx of extracellular calcium. Indomethacin did not prevent the suppressive effects of reducing extracellular Na+ concentration below 144 mM on basal or AVP responsive cAMP, suggesting that the enhanced production of PGE2 did not mediate the reduction in AVP responsiveness. In contrast to cAMP, reductions in extracellular Na+ concentration from 144 to 24 mM did not influence basal cGMP or the cGMP responses to atrial natriuretic peptide or nitroprusside.(ABSTRACT TRUNCATED AT 250 WORDS)

Putative Mechanism of Blood Pressure Reduction Induced by Increases in Dietary Calcium Intake

An increase in dietary calcium intake lowers blood pressure in spontaneously hypertensive rats and in some patients with arterial hypertension. The mechanisms by which this decrease come about are not clear. A membrane-stabilizing effect wrought by an increase in extracellular calcium would appear unlikely, since the increases in extracellular calcium concentration with increased dietary intake are minimal. Calcium regulatory hormones may be the mediators, and a cybernetic framework has been suggested. Striking defects have been reported in the calcium handling and hormonal household of the spontaneously hypertensive rat. However, a clear cut relationship in terms of a hormonal "template" has not yet been identified in prospective experiments. Data have been presented to show that increased calcium intake has a direct effect on regulatory areas in the brain. However, the mechanisms by which such a response would be mediated are entirely unknown. Increased calcium intake may induce natriuresis. It has been suggested that increased calcium intake helps the "salt sensitive"; however, prospective studies to this effect have not been presented. Increased calcium intake may induce phosphaturia. However, the evidence that blood pressure lowering effects are mediated by phosphate depletion are unconvincing. Some evidence suggests that increased calcium intake may influence local regulatory processes which in turn influences cell integrity and growth. At this point, a unifying hypothesis is not available. However, the clues to various possibilities are intriguing.

High extracellular calcium increases the production of a parathyroid hormone-like activity by cultured Leydig tumor cells associated with humoral hypercalcemia.

Cultured Leydig tumor cells produce a parathyroid hormone (PTH)-like activity, but little is known about the regulation of the release of this factor. In the present work, we investigated the influence of the extracellular calcium concentration on the production of adenylate cyclase-stimulating activity, as evaluated in the osteoblast-like PTH-responsive cell line UMR 106. Medium conditioned in the presence of 0.4 mM or 3 mM Ca elicited a 5.8 +/- 0.4-fold and 10.3 +/- 0.9-fold increase over basal of cAMP production, respectively (p less than 0.001, n = 11 experiments). This effect, which was selective for PTH-like activity, was detectable after 2 h of incubation and maximal at 6-14 h. It was abolished by the protein synthesis inhibitor cycloheximide, but not by actinomycin D or cordycepin, suggesting a post-transcriptional site of action. Thus, the production of a tumoral circulating factor implicated in the pathogenesis of humoral malignant hypercalcemia may be influenced in a positive way by an increase in extracellular calcium concentration.

Cell Membrane in Hypertension

An increase in extracellular calcium concentration over a range of 0 to 5 mM causes membrane "stabilization". This stabilizing action of calcium is less effective in tissues from hypertensive animals, suggesting that less calcium is bound to the membrane of tissues from this source.

Calcium-induced long-term potentiation in the hippocampal slice: Characterization of the time course and conditions

A transient increase in extracellular calcium concentration causes a long-lasting enhancement of radiatum fibers evoked excitatory postsynaptic potential and population spike responses of CA1 pyramidal neurons which resembles long-term potentiation (LTP). The duration of this potentiation is much longer than described previously and is probably limited by the survival of the preparation itself (>8 hr). Therefore, Ca-induced LTP can be used for the investigation of a postulated late phase of LTP. Ca effects were activity-independent, since the subsequently evoked responses were facilitated even when the presynaptic fibers were not concurrently stimulated during or immediately after superfusion with the high Ca medium. In contrast, if too frequent testing of the synaptic input was done during the high Ca pulse, a short lasting depression instead of potentiation was observed. A lower extracellular magnesium concentration in the standard medium (1.3 instead of 2.0 mM MgSO 4) prevents the potentiation of the EPSP at least for the first few hours. Presumably, both tetanus- and Ca-induced LTP share some common mechanisms, since an additional tetanization after Ca induction was not followed by an additional LTP. Compared to the potentiation following tetanization, the Ca-induced LTP was, however, not accompanied by a potentiation of the EPSP/spike ratio within the range of the population spike threshold intensity.

Effects of selective channel blocking agents on contractions and action potentials in K+‐depolarized guinea‐pig atria

Contractions and transmembrane action potentials were induced by 1 microM isoprenaline in K+-depolarized guinea-pig left atria driven at 0.5 Hz. The stability of these responses was significantly increased by doubling the extracellular glucose concentration to 22 mM. Action potential overshoot increased by 28 mV per ten fold increase in extracellular calcium concentration suggesting that the inward current in this preparation is carried by Ca2+. In depolarized driven preparations, nanomolar concentrations of nifedipine and nisoldipine reduced contractility, maximum rate of depolarization (dV/dt max) and action potential height, whereas the fast channel blocking agents tetrodotoxin and mexiletine (in micromolar concentrations) produced little change. Nifedipine also rendered spontaneously beating depolarized right atrial preparations quiescent. In concentrations which reduced dV/dt of normal action potentials, the sodium channel blocking agents quinidine and Org 6001 reduced the amplitude of contractions and reduced the maximum rate of phase 0 depolarization (dV/dt) of action potentials in depolarized tissue. These actions were reversed by Ca2+ and suggest calcium antagonistic activity. However action potential height was not reduced. Like bepridil, both drugs also reduced the frequency of spontaneous contractions in depolarized right atrial preparations. Unlike Org 6001, quinidine failed to produce a shift in calcium log dose-response curves in driven depolarized preparations and induced positive inotropy in the presence of functional sodium channels. Bepridil inhibited contractions in depolarized atria in the absence of a reduction in dV/dt suggesting that any calcium antagonistic action in atrial tissue is mainly located at an intracellular site. In conclusion, action potentials elicited by isoprenaline in potassium-depolarized atria bathed in high glucose appear to be Ca2+ mediated. In concentrations which inhibit the inward Na+ current, both quinidine and Org 6001 exhibit calcium channel blocking properties.

Qualitative characteristics of the inotropic response (IR) in rat papillary muscles to increase of extracellular calcium concentration ([Ca] o)

Qualitative characteristics of the inotropic response (IR) in rat papillary muscles to increase of extracellular calcium concentration ([Ca] o)