Influence Of Extracellular Calcium Research Articles

Synaptic plasticity rules with physiological calcium levels

Spike-timing-dependent plasticity (STDP) is considered as a primary mechanism underlying formation of new memories during learning. Despite the growing interest in activity-dependent plasticity, it is still unclear whether synaptic plasticity rules inferred from in vitro experiments are correct in physiological conditions. The abnormally high calcium concentration used in in vitro studies of STDP suggests that in vivo plasticity rules may differ significantly from in vitro experiments, especially since STDP depends strongly on calcium for induction. We therefore studied here the influence of extracellular calcium on synaptic plasticity. Using a combination of experimental (patch-clamp recording and Ca2+ imaging at CA3-CA1 synapses) and theoretical approaches, we show here that the classic STDP rule in which pairs of single pre- and postsynaptic action potentials induce synaptic modifications is not valid in the physiological Ca2+ range. Rather, we found that these pairs of single stimuli are unable to induce any synaptic modification in 1.3 and 1.5 mM calcium and lead to depression in 1.8 mM. Plasticity can only be recovered when bursts of postsynaptic spikes are used, or when neurons fire at sufficiently high frequency. In conclusion, the STDP rule is profoundly altered in physiological Ca2+, but specific activity regimes restore a classical STDP profile.

Influence of extracellular magnesium on phagocytosis and free cytosolic Mg levels in differentiated U937 and MH-S cells

The influence of extracellular calcium on phagocytosis is limited, and the involvement of extracellular magnesium is unclear. The role of extracellular calcium and magnesium on phagocytosis efficiency was investigated. Extracellular calcium had no influence on the internalization of 1 μm polystyrene particles by primary monocytes as has been shown before for the human lymphoma-derived, differentiated cell line U937 and murine alveolar macrophage-derived MH-S cells. In contrast, the phagocytosis by differentiated U937 cells was positively affected by the presence of extracellular magnesium whereas that of MH-S cells was not. An extracellular increase in the magnesium level did not cause an increase in the free cytosolic magnesium concentration in either cell line. In contrast to magnesium, extracellular calcium caused an increase in intracellular divalent cation levels in differentiated U937 cells. A phagocytosis-enhancing effect in the extracellular space was observed in relation to extracellular magnesium rather than with an intracellular increase in magnesium levels, indicating that murine and human immune cells might be regulated differently.

Abstract 4003: Influence of extracellular calcium on MAPK signalling cascade in colon cancer cells.

Abstract Background: The calcium sensing receptor (CaSR) is a G-protein coupled receptor, which is capable of sensing even minute changes in extracellular calcium levels. The main role of the CaSR is regulation of calcium homeostasis. Besides the organs responsible for calcium homeostasis the receptor is expressed in a number of tissues including colonic epithelium and can be activated by several ligands, stimulating various downstream signalling pathways. Thus the CaSR is involved in numerous biological processes such as cell proliferation, differentiation and lipid membrane metabolism. Previous studies demonstrated that calcium regulates cell proliferation in colonocytes but the pathways or factors involved in this process still need to be elucidated. Aim: The aim of this study is to investigate the impact of CaSR activation on the Mitogen-Activated Protein Kinase (MAPK) signalling cascade in colon cancer cell lines. We hypothesize that in colonocytes, activated CaSR down regulates proliferative pathways. Methods: We studied the effect of CaSR ligands on downstream pathways through gene expression assays and protein analysis. Results and Conclusion: We could show that the differentiation status of the cells (TC-7 a colon cancer cell line) determines whether a pathway is activated or rather inhibited by calcium treatment. Gene expression studies performed by luciferase assay and protein analysis by western blots reflect that extracellular calcium activated MAPK cascade in subconfluent TC-7 cells. On the other hand, short time (10 to 30 minutes) treatment of confluent/ differentiated cells with extracellular calcium showed no activation of ERK. Treatment for longer time (e.g 8h) inhibited ERK phosphorylation. The treatment of the cells with 100nM R-568, a calcimimetic that act only through the CaSR slightly inhibited ERK phosphorylation compared with the vehicle control, suggesting an anti-proliferative role of the CaSR. Citation Format: Samawansha Tennakoon, Johannes Schmid, Brigitte Marian, Enikö Kallay. Influence of extracellular calcium on MAPK signalling cascade in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4003. doi:10.1158/1538-7445.AM2013-4003

Chondrocyte death in mechanically injured articular cartilage—the influence of extracellular calcium

Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium-free media (approximately 0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium-rich media (2-20 mM, ANOVA at 2.5 h, p = 0.002). In calcium-rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium-free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium-free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium-free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

ICP/OES Application for Assessing Cadmium Uptake (or Toxicity) in Glomerular Cells: Influence of Extracellular Calcium

The risks of metals for health are highlighted by their chemical stability and their persistence in the environment. Chronic exposure to low cadmium (Cd) concentrations results in renal dysfunction mainly. Cd has been regarded primarily as a renal tubular toxicant, but glomerular structures may also be affected. Since the cellular environment may influence metal toxicity, differences concerning Cd uptake and toxicity were evaluated according to calcium (Ca) medium concentrations. An optimized inductively coupled plasma emission spectrometry method (ICP/OES) was developed under defined conditions, as a selective analytical tool to determine cadmium uptake in glomerular mesangial cells. The performance characteristics of the analytical system were evaluated for both Cd and Ca by calibration (50 to 250 μg/L and 1 to 5 mg/L), linearity (r 2 .9968 and .9943), limits of detection (1 μg/L and 0.1 mg/L) and quantitation (3 μg/L and 0.3 mg/L), accuracy with spiking, and repeatability (1.2 and 2.9%) with matrix matched standards. Total intracellular Cd content was significantly threefold lower in 0.175 mM Ca medium (Ca-free Eagle's minumum essential medium [EMEM] medium with 5% fetal bovine serum [FBS]) than in EMEM medium (1.8 mM Ca) with respectively 0.16 and 0.37 μg/mg proteins after 24 h of Cd (1 μM) exposure. Similar differences were obtained in cytotoxicity studies with a fourfold reduction in the mortality index (IC50). Complementary assays using Ca-spiked medium reinforced that Cd cytotoxicity and uptake were significantly dependent on the concentration of extracellular Ca. These findings suggest direct link between Cd uptake and toxicity, underlining the relevance of the analytical method.

Aging-Induced Decrease of Cholinergic Response and Calcium Sensitivity on Rat Jejunum Contractions

The role of aging on contraction or relaxation through muscarinic or alpha-adrenergic receptors, respectively, was studied in isolated rat jejunum. Furthermore, the influence of extracellular calcium was analyzed, through functional and radioligand binding assays. The rank order of potency for selective muscarinic antagonists for M(1), M(2), and M(3) receptor subtypes, measured from affinity (pA(2)) values, was p-fluorohexahydrosiladifenidol (pFHHSiD) (M(3)) > pirenzepine (M(1)) > methoctramine (M(2)), indicating a predominance of M(3) subtype. This order was unchanged with age. Contractions by muscarinic agonist methacholine (MCh) were diminished in aged rats, resulting in lower apparent affinity (pD(2)) values, compared with adult controls. A larger decrease of MCh contractions occurred in aged rats after Ca(2+) withdrawal or after the calcium channel blocker isradipine. Changes were not detected for relaxation by adrenergic agonists. In conclusion, aging caused a decrease of MCh potency, which is probably related to the reduction of calcium sensitivity in jejunum.

Influence of extracellular calcium on flavonoid accumulation by cell cultures of Hypericum androsaemum

Hypericum androsaemum L., commonly known in Portugal as tutsan, is a plant species used in folk medicine for its diuretic and hepatoprotective properties [1]. The diverse flavonoids and phenolic acids present in the plant are supposed to be responsible for such biological activities.

Otolith calcification in Atlantic salmon parr, Salmo salar L. and its relation to photoperiod and calcium metabolism

Otolith calcification in Atlantic salmon parr, Salmo salar, was investigated using a radioisotope of calcium, 45Ca. Otolith calcification was found to be entrained to light‐dark cycles in salmon parr, calcium accumulation on to otoliths declining at night and resuming at dawn. The decline in Otolith calcification at night coincided with a diel decline in plasma calcium concentration. The influence of extracellular calcium on otolith increment formation was considered by inducing hypocalcemia. Induced hypocalcemia resulted in a short‐term net loss of calcium from the otolith. The results are discussed in relation to previous studies of the role of extracellular calcium in otolith formation.

Antiproliferative effect of 1,25-dihydroxyvitamin D 3 and its analogs on human colon adenocarcinoma cells (CaCo-2): Influence of extracellular calcium

Depending on culture in either “low Ca ++” (0.25 mM) or “normal Ca ++” (1.8 mM) medium, human colon adenocarcinoma-derived CaCo-2 cells exhibit differential sensitivity to the antiproliferative action of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and of two side-chain modified analogs, 1,25S,26-trihydroxy-Δ22-vitamin D 3 (Ro 23-4319) and 1,25-dihydroxy-Δ16-23yne-vitamin D 3 (Ro 3-7553). CaCo-2 cells cultured under low Ca ++ conditions exhibit a high proliferative potential, and in these cells, all vitamin D compounds under investigation significantly inhibit [ 3H]thymidine incorporation into cellular DNA at ≥10 −10 M. The rank order of biopotency is: Ro 23-7553≥Ro 23-4319 >1,25(OH) 2D 3. At 1.8 mM Ca ++, only Ro 23-7553 is able to inhibit proliferation of CaCo-2 cells. Parallel to their antiproliferative action, all three vitamin D compounds stimulate akaline phosphatase activity in CaCo-2 cells, indicating their ability to induce differentiated functions at the same time as they reduce neoplastic cell growth.

The mechanism of action of endothelin in human lung

1 The peptides endothelin-1 (ET-1) and endothelin-2 (ET-2) elicited potent and sustained contractions of human isolated bronchus and pulmonary artery. 2 ET-1 is one of the most potent contractile agonists investigated in these tissues with an EC50 value of 18.3 nM (95% confidence interval: 12.9, 25.9 nM: n = 26) in bronchus and 3.2 nM (95% confidence interval: 0.4, 23.9 nM; n = 5) in the arterial preparation. 3 ET-1 is 2.5 times more potent than ET-2 in both the airway and vascular tissues, and both forms of the peptide have geometric mean EC50 values 5 times greater than in the isolated bronchial tissue than in the pulmonary artery. 4 Neither pretreatment with the voltage-dependent calcium (VDC) channel antagonist verapamil (10 microM) nor with indomethacin (25 microM) significantly altered the response curve to ET-1 in human isolated bronchus. Removal of calcium from the Krebs-Henseleit solution did not affect ET-1-induced responses. 5 Specific binding on the smooth muscle of human airway and pulmonary arterial tissue to both ET-1 and ET-2 was detected in autoradiographic studies. There appeared to be no difference between the peptides in the location nor the density of binding sites. 6 We conclude that contraction of human bronchial tissue by ET-1 is not dependent upon influence of extracellular calcium nor release of prostaglandins or thromboxane A2. It is likely that the action of ET-1 in this tissue is due to binding of this peptide to specific receptors located on the smooth muscle.

Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.

Influence of extracellular calcium in the effects of dibekacin and diltiazem on indirectly elicited tetanic responses in the rat phrenic-hemidiaphragm preparation

1. Dibekacin (70 μM-3 mM) produced a decrease of peak tetanic tension in a concentration-dependent manner and this effect was dependent on extracellular calcium (0.3–2.5 mM Ca 2+). Only minimal fade was observed and it was not related with extracellular calcium concentrations. 2. Diltiazem (30–300 μM) decreased peak tetanic tension and produced tetanic fade. Both effects were independent of extracellular calcium, although a significant potentiation was observed at 0.3 mM calcium. 3. It is concluded that tetanic parameters are related differently to extracellular calcium.

Requirement of extracellular calcium in fish pituitary gonadotropin release by gonadotropin hormone-releasing hormone.

Influence of extracellular calcium on gonadotropin hormone-releasing hormone (GnRH)-stimulated gonadotropin hormone (GtH) release from a teleostean fish (Channa punctatus) pituitary was examined in vitro by preparing enzymatically dispersed pituitary cell incubation. Effect of Ca2+ on GnRH-augmented GtH release was evaluated with partially purified C. punctatus GnRH (cGnRH) and synthetic mammalian GnRH (mGnRH). Cells were dispersed by 0.3% collagenase plus 0.05% trypsin in culture medium and a high yield of viable cells were obtained. Addition of cGnRH (10 micrograms/ml) to pituitary cells in Ca2+-free medium resulted in a significant increase in GtH release, but the addition of Ca2+ (2 mM) enhanced it to about four- and threefold over cGnRH and mGnRH, respectively. Increasing concentrations of Ca2+ (0.1-2.0 mM/well) with fixed concentrations of GnRH (10 micrograms/ml) or increasing doses of GnRH (2.5 to 20 micrograms/ml) with fixed amount of Ca2+ (2 mM/well) resulted in a dose dependent increase in GtH release. EDTA or EGTA (2 mM/well) completely suppressed the Ca2+-augmenting effect of GnRH-stimulated GtH release. Addition of lanthanum (La3+, 4 mM/well), a competitive inhibitor of Ca2+, reduced 60% of the Ca2+ (2 mM/well) stimulation. Verapamil, a specific Ca2+ channel blocker, when added in increasing concentrations (1-100 microM/well) to pituitary cell incubations containing GnRH-stimulated GtH release in Ca2+-free medium could be waived by EGTA (2 mM/well), indicating availability of extracellular calcium from tissue sources. The uptake of radioactive Ca2+ by pituitary cells was greatly enhanced by GnRH while the addition of verapamil (10 microM/well) not only inhibited the GnRH-stimulated uptake, but also reduced it below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of extracellular calcium on microvascular tone in the rat cremaster muscle.

In vivo responses of arterioles and venules to changes in bath calcium concentrations were observed in the cremaster muscle of male Sprague-Dawley rats. Small arterioles (2A, 3A) initially exposed to a solution containing calcium (2.55 mM) significantly dilated in response to a 0-calcium bath. Reexposure to calcium (greater than 0.65 mM) caused 2A and 3A arterioles to constrict to diameters similar to the initial control values. In contrast, large arterioles (1A) and all venules (1V, 2V, 3V) were unresponsive to exposure to a 0-calcium solution or to reexposure to calcium (0.65-5.10 mM). Treatment with mefenamic acid (10 micrograms/ml), a prostaglandin synthesis inhibitor, produced marked constriction of arterioles but not of venules, suggesting the involvement of endogenous vasodilator prostaglandins in the regulation of resting diameters of arterioles. In the presence of mefenamic acid, 1A arterioles dilated when exposed to a 0-calcium solution and constricted back to control diameters following reintroduction of calcium into the bath. These data demonstrate heterogeneity in the responsiveness of cremasteric microvessels to changes in extracellular calcium. The small arterioles were most responsive to calcium. The lack of response by the largest arterioles appears to be due to the dilator influences of endogenous prostaglandins.

Divalent cations modulate 5-HT 3 receptor-induced currents in N1E-115 neuroblastoma cells

The influence of extracellular calcium and magnesium ion concentrations upon 5-HT 3 receptor-gated membrane currents in murine N1E-115 neuroblastoma cells has been studied under voltage-clamp conditions. A decrease in the concentration of either Ca 2+ or Mg 2+ from their standard values of 1.0 and 2.0 mM respectively augmented both the amplitude and duration of the 5-HT-induced current, whereas elevating the concentration of either divalent cation produced the opposite effect. Such modulation did not involve a change in the reversal potential of the response.

Protein degradation in bupivacaine-treated muscles: The role of extracellular calcium

The time course of protein degradation and the influence of extracellular calcium and the calcium-channel blocker verapamil were investigated by measuring tyrosine release from the isolated rat soleus muscle exposed to the local anaesthetic agent bupivacaine. Degradation rates were reduced during the first 60–90 min and subsequently increased in muscles treated with bupivacaine concentrations of 1.5 mM or higher. When calcium was omitted from the incubation medium the initial reduction in degradation was greater and the subsequent increase was reduced or prevented. Overall, verapamil (10 −5M or 10 −6M) did not significantly alter the degree or time-course of protein degradation.

Influence of extracellular calcium and nifedipine on alpha 1- and alpha 2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries.

The influence of extracellular Ca2+ and nifedipine on contractile responses to 10 microM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly alpha 2-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly alpha 1-adrenoceptors) and CMA (sensitive to both alpha 1- and alpha 2-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca2+-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K+ was more sensitive to Ca2+ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca2+, can also release Ca2+ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, alpha 1-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca2+ influx and intracellular Ca2+ release, whereas alpha 2-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca2+ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca2+-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca2+, whereas in the other types of artery, Ca2+ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca2+ influx and intracellular Ca2+ release to the contractile activation, but also in the nifedipine sensitivity of the Ca2+ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of alpha-adrenoceptor stimulated by NA and to the type of vessel studied.

The influence of extracellular calcium on the distribution of protein kinase C in non-neoplastic and neoplastic rat liver cells.

Non-neoplastic T51B rat liver epithelial cells cannot proliferate in Ca2+-deficient medium. This proliferative inhibition in Ca2+-deficient medium is accompanied by a large reduction in the amount of cellular EDTA-extractable Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity. By contrast, tumorigenic epithelial cells from several Morris hepatomas proliferate in Ca2+-deficient medium and either maintain or greatly increase their content of EDTA-extractable protein kinase C.

The influence of extracellular calcium on the response of fly photoreceptors

This paper presents a systematic investigation of the influence of the extracellular concentration of calcium ([Ca2+]0) on the electrophysiological response of the fly's photoreceptors (R1–R6) to light. The hemisected heads of flies were perfused with a standard medium containing 10−4 mol/1 CaCl2 and in this medium the intracellularly recorded response of the cell was virtually identical to the normal response obtained in vivo. All the effects of changing the [Ca2+]0 could be reversed within 5 min by perfusing the eye with the standard medium.

The Influence of Extracellular Calcium and Hydrogen Ion Concentration on the Response of Rat Tracheal Smooth Muscle to Verapamil

The Influence of Extracellular Calcium and Hydrogen Ion Concentration on the Response of Rat Tracheal Smooth Muscle to Verapamil