Calcium as an extracellular signalling molecule: perspectives on the Calcium Sensing Receptor in the brain

Calcium acts as a universal signal that is responsible for controlling a spectrum of cellular processes ranging from fertilization to apoptosis. For a long time, calcium was regarded solely as an intracellular second messenger. However, the discovery that calcium can also act as an external ligand together with the molecular cloning of its cell surface receptor, the Calcium Sensing Receptor (CaSR), demonstrated that calcium also acts as an important extracellular or first messenger. Here, we give an overview of the main structural, pharmacological and physiological features of the CaSR and provide an assessment of its functions and cellular and molecular mechanisms of action. In addition, we propose possible avenues for future research into the trafficking of CaSR and the role(s) of this receptor in the central nervous system.     

Effect of the calcium sensing receptor on rat bone marrow-derived mesenchymal stem cell proliferation through the ERK1/2 pathway

Migration and proliferation of bone marrowderived mesenchymal stem cells (BMSCs) is critical to treatment of ischemic injury. The calcium sensing receptor (CaSR) has an important role in maintaining systemic calcium homeostasis, which is related to cell proliferation, apoptosis and paracrine signaling. We hypothesize that CaSR may enhance BMSC proliferation. Rat BMSCs were incubated with various calcium concentrations for 48 h in vitro to activate CaSR. To investigate potential mechanisms responsible for growth enhancement by calcium, the rat BMSC cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), and induction of apoptosis confirmed by cytofluorimetric analysis using propidium iodide and Annexin V-FITC double staining. Since the mitogen-activated protein kinase (MAPK) signaling pathway was one of the most significantly affected by CaSR, MAPK activation was measured by western blotting. Calcium exposure significantly enhanced rat BMSCs proliferation, as well as the proportion of the population in S phase, in a dose-dependent manner, effects which were abolished by NPS2390 (a CaSR antagonist) and U0126 (a MEK1/2 inhibitor). These results demonstrate that CaSR is involved in rat BMSC proliferation, as seen by an increased proliferation index, decreased apoptosis, and ERK1/2 activation, and provide important insight into the cellular and molecular mechanisms by which CaSR affects cell proliferation. A CaSR agonist may prove useful to enhance BMSC survival during transplantation.     

Effects of Chan Su,a traditional Chinese medicine,on the calcium transients of isolated cardiomyocytes: cardiotoxicity due to more than Na,K-ATPase blocking

Extracts of Chan Su, a traditional Chinese medication used as a topical anesthetic and cardiac medication, were incubated with cardiomyocytes that had been loaded with a calcium specific fluorescent probe. Calcium transients were measured by real-time fluorescence spectrophotometry following treatment. The transients were rapidly abolished following addition of a moderate concentration of the extract (400 ng/ml), resulting in high levels of intracellular calcium, while the lower amount (40 ng/ml) blocked the sodium-potassium adenosine triphosphatase. Treatments with ouabain and nifedipine were also made, either prior to, or after the addition of the Chan Su, in an attempt to better delineate the site(s) of action. The moderate concentration of Chan Su (400 ng/ml) extract caused the myocytes to cease beating within seconds of addition, even in experiments when saturating concentrations of nifedipine or ouabain had been previously added to the cells. As expected bufalin, the active component of Chan Su has similar effects. Our findings indicate that this compound is extremely cardiotoxic, even in small dose and acts rapidly to alter intracellular calcium stores in cardiomyocytes and possibly acts at sites other than the Na(+)+K(+) ATPase, either directly, or indirectly via changes in calcium concentrations.     

Obesity-associated proinflammatory cytokines increase calcium sensing receptor (CaSR) protein expression in primary human adipocytes and LS14 human adipose cell line

Obesity-associated health complications are thought to be in part due to the low-grade proinflammatory state that characterizes this disease. The calcium sensing receptor (CaSR), which is expressed in human adipose cells, plays an important role in diseases involving inflammation. To assess the relevance of this protein in adipose pathophysiology, we evaluated its expression in adipocytes under obesity-related proinflammatory conditions. As in primary adipose cells, we established that LS14, a recently described human adipose cell line, expresses the CaSR. Differentiated LS14 and primary adipose cells were exposed overnight to cytokines typically involved in obesity-related inflammation (interleukin (IL)1β, IL6 and tumor necrosis factor (TNF)α). The cytokines increased CaSR abundance in differentiated adipocytes. We incubated LS14 cells with medium previously conditioned (CM) by adipose tissue from subjects with a wide range of body mass index (BMI). Cells exposed to CM from subjects of higher BMI underwent a greater increase in CaSR protein, likely resulting from the greater proinflammatory cytokines secreted from obese tissue. Our observations that proinflammatory factors increase CaSR levels in adipocytes, and the reported ability of CaSR to elevate cytokine levels, open new aspects in the study of obesity inflammatory state pathophysiology, providing a potential novel therapeutic prevention and treatment target.     

钙在控制水果采后病害中的应用

钙在果实生长发育中起着非常重要的作用。果实的许多生理失调和病害都与钙在水果组织中的含量有关。钙处理与气调、热处理、生物防治等联用在减少果实采后病害方面发挥了显著的作用。主要论述钙在控制水果采后病害中的作用机理、钙处理的方法、影响果实钙吸收的因素、以及钙与其它防病方法联用的效果。对于钙处理中可能出现的问题 ,提出了一些可行的解决方法     

Plasma levels and urinary excretion of amino acids by subjects with renal calculi

Plasma levels and urinary amino acid excretions were estimated by high-performance liquid chromatography in 15 control subjects and 36 stone formers (SFs) classified according to the stone type: (1) 22 cases with calcium oxalate stones; (2) four cases with pure uric acid stones; (3) 10 cases with magnesium-ammonium phosphate stones, either pure or mixed with apatite. Some types of stones (namely oxalate and uric acid calculi) are mainly formed as a result of a metabolic deficiency that may affect the amino acid metabolism, and thus may be reflected in the urinary amino acid pattern. Data demonstrated clearly that there is a general tendency towards decreased amino acid excretions in all SFs with all types of stones. As a whole, one can observe a higher percentage of patients with calcium oxalate and phosphate calculosis, who have low urine excretions of amino acids; about 50% are the SFs with lower urine excretion of serine, glycine, taurine and i-leucine; the high percentage of patients with CaOX calculi shows lower urine excretions of tyrosine and ornithine.     

钙敏感受体(CaSR)基因986、990多态性与尿石症的相关性研究

目的:探讨钙敏感受体(Ca SR)基因单核昔酸多态性与泌尿系结石的关系。方法:选取90例黑龙江地区的泌尿系结石患者及90例健康对照者外周血标本中的基因组DNA,采用PCR(聚合酶链反应)结合DNA测序,检测并分析Ca SR基因的单核苷酸多态性位点的分布。结果:泌尿系结石组和对照组Ca SR基因第986位、990位频率分布符合Hardy-Weinberg定律,其基因型分布频率在泌尿系结石患者和健康对照者中差异无统计学意义(P0.05),但在泌尿系结石患者组内Ca SR第990位GG纯合子和RG杂合子出现频率明显偏高,差异有统计学意义(P0.05)。结论:Ca SR基因第7外显子第986、990多态性位点与泌尿系结石的形成无直接相关性,但第7外显子第990位A/G单核苷酸多态性可能与泌尿系结石的形成密切相关。     

Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release <Emphasis Type="Italic">in vivo</Emphasis>

Background  

The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium.     

PGE2 and LTB4 tissue levels in benign and cancerous prostates

PGE2 and LTB4 are involved in inflammation and carcinogenesis in several tissues but have not been studied in prostate cancer and hyperplasia until now. We therefore measured PGE2 and LTB4 productions in a total of 206 prostate tissues from 116 patients including benign hyperplastic (90), pericancerous (106) and cancerous samples (10). We also analysed the influence of inflammation levels, prostate volume and glandular to epithelial ratio. PGE2 and LTB4 concentrations were measured using specific enzyme immunoassay kits. There was a correlation between PGE2 level, prostatic volume, inflammation score, and decreased glandular surface. By contrast, there was no correlation between LTB4 levels and inflammation or PGE2 production. Cancerous samples had higher LTB4 levels than pericancerous samples, but there was no difference in PGE2 levels. PGE2 and inflammation may be associated to stromal benign prostatic hyperplasia whereas LTB4 may play a role in prostate carcinogenesis.     

PGE2 and LTB4 tissue levels in benign and cancerous prostates

PGE2 and LTB4 are involved in inflammation and carcinogenesis in several tissues but have not been studied in prostate cancer and hyperplasia until now. We therefore measured PGE2 and LTB4 productions in a total of 206 prostate tissues from 116 patients including benign hyperplastic (90), pericancerous (106) and cancerous samples (10). We also analysed the influence of inflammation levels, prostate volume and glandular to epithelial ratio. PGE2 and LTB4 concentrations were measured using specific enzyme immunoassay kits. There was a correlation between PGE2 level, prostatic volume, inflammation score, and decreased glandular surface. By contrast, there was no correlation between LTB4 levels and inflammation or PGE2 production. Cancerous samples had higher LTB4 levels than pericancerous samples, but there was no difference in PGE2 levels. PGE2 and inflammation may be associated to stromal benign prostatic hyperplasia whereas LTB4 may play a role in prostate carcinogenesis.     

Role of inflammation in benign prostatic hyperplasia

Inflammation of the prostate may represent a mechanism for hyperplastic changes to occur in the prostate. There are a variety of growth factors and cytokines that may lead to a proinflammatory process within the prostate. There are several proposed mechanisms that lead to both the intrinsic and extrinsic basis of inflammation. Prostatic inflammation may represent an important factor in influencing prostatic growth and progression of symptoms. This article reviews the recent literature on inflammation leading to chronic prostatic diseases, such as benign prostatic hyperplasia.     

Kidney Stones

The prevalence of kidney stones has steadily risen during this century; passage of a calculus and a positive family history increase the probability of recurrence. Findings from recent studies on the cause of renal calculi have stressed crystallization and crystal aggregation of stone minerals from supersaturated urine, rather than excessive organic matrix. Absence of normal urine inhibitors of calcium salts is also stressed. Formation of calcium oxalate stones is the major problem. Therapy with decreased calcium and oxalate intake, thiazides, phosphate salts and allopurinol in various combinations has substantially decreased the prevalence of recurrent stones. The rationale for the use of allopurinol is that uric acid salts enhance the tendency for calcium oxalate to crystallize from supersaturated urine. The hypercalciuria seen in 30 percent to 40 percent of patients with oxalate stones is usually caused by intestinal hyperabsorption of calcium. Although patients with uric acid calculi constitute only a small fraction of those in whom stones form, they represent a group in whom good medical therapy, based on sound physiologic principles, has proved extremely successful. Renal tubular syndromes lead to nephrocalcinosis and lithiasis through hypercalciuria, alkaline urine and hypocitraturia, the latter an inhibitor of calcium salt precipitation. Recent advances in surgical techniques are discussed, including the rationale for removing staghorn calculi. The ileal ureter and coagulum pyelolithotomy deserve special emphasis.     

The extracellular calcium-sensing receptor (CaSR) on human esophagus and evidence of expression of the CaSR on the esophageal epithelial cell line (HET-1A)

Gastrointestinal reflux disease and eosinophilic esophagitis are characterized by basal cell hyperplasia. The extracellular calcium-sensing receptor (CaSR), a G protein-coupled receptor, which may be activated by divalent agonists, is expressed throughout the gastrointestinal system. The CaSR may regulate proliferation or differentiation, depending on cell type and tissue. The current experiments demonstrate the expression of the CaSR on a human esophageal epithelial cell line (HET-1A) and the location and expression of the CaSR in the human esophagus. CaSR immunoreactivity was seen in the basal layer of normal human esophagus. CaSR expression was confirmed in HET-1A cells by RT-PCR, immunocytochemistry, and Western blot analysis. CaSR stimulation by extracellular calcium or agonists, such as spermine or Mg(2+), caused ERK1 and 2 activation, intracellular calcium concentration ([Ca(2+)](i)) mobilization (as assessed by microspecfluorometry using Fluo-4), and secretion of the multifunctional cytokine IL-8 (CX-CL8). HET-1A cells transiently transfected with small interfering (si)RNA duplex against the CaSR manifested attenuated responses to Ca(2+) stimulation of phospho- (p)ERK1 and 2, [Ca(2+)](i) mobilization, and IL-8 secretion, whereas responses to acetylcholine (ACh) remained sustained. An inhibitor of phosphatidylinositol-specific phospholipase C (PI-PLC) (U73122) blocked CaSR-stimulated [Ca(2+)](i) release. We conclude that the CaSR is present on basal cells of the human esophagus and is present in a functional manner on the esophageal epithelial cell line, HET-1A.     

Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion

Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.     

The extracellular calcium-sensing receptor is required for cholecystokinin secretion in response to L-phenylalanine in acutely isolated intestinal I cells

The extracellular calcium-sensing receptor (CaSR) has recently been recognized as an L-amino acid sensor and has been implicated in mediating cholecystokinin (CCK) secretion in response to aromatic amino acids. We investigated whether direct detection of L-phenylalanine (L-Phe) by CaSR results in CCK secretion in the native I cell. Fluorescence-activated cell sorting of duodenal I cells from CCK-enhanced green fluorescent protein (eGFP) transgenic mice demonstrated CaSR gene expression. Immunostaining of fixed and fresh duodenal tissue sections confirmed CaSR protein expression. Intracellular calcium fluxes were CaSR dependent, stereoselective for L-Phe over D-Phe, and responsive to type II calcimimetic cinacalcet in CCK-eGFP cells. Additionally, CCK secretion by an isolated I cell population was increased by 30 and 62% in response to L-Phe in the presence of physiological (1.26 mM) and superphysiological (2.5 mM) extracellular calcium concentrations, respectively. While the deletion of CaSR from CCK-eGFP cells did not affect basal CCK secretion, the effect of L-Phe or cinacalcet on intracellular calcium flux was lost. In fact, both secretagogues, as well as superphysiological Ca(2+), evoked an unexpected 20-30% decrease in CCK secretion compared with basal secretion in CaSR(-/-) CCK-eGFP cells. CCK secretion in response to KCl or tryptone was unaffected by the absence of CaSR. The present data suggest that CaSR is required for hormone secretion in the specific response to L-Phe by the native I cell, and that a receptor-mediated mechanism may inhibit hormone secretion in the absence of a fully functional CaSR.     

14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling

Calcium sensing receptors (CaSR) interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.     

Calcium Modulation of Activation and Desensitization of Nicotinic Receptors from Mouse Brain

Abstract: The effects of extracellular calcium on functional properties of nicotinic receptors from mouse thalamus were investigated. Previous studies have reported that calcium modulates the function of several neuronal nicotinic receptors. A ⁸⁶Rb⁺ ion efflux assay was developed to measure nicotinic receptor function from brain tissue, and data indicate that α₄β₂ receptors may mediate this response. Using the ⁸⁶Rb⁺ efflux assay, calcium effects on receptor activation, desensitization induced by high, activating and low, subactivating concentrations of agonist, and recovery from desensitization were examined. Effects of calcium on the kinetics of ligand binding were also investigated. Calcium modulated receptor activation by increasing the maximal response to nicotine in a concentration-dependent manner, without affecting the EC₅₀ of nicotine. Barium, but not magnesium, mimicked the effects of calcium on receptor activation. The increase in receptor activation could not be explained by changes in the ratio of activatable to desensitized receptors as assessed by the kinetics of ligand binding. Desensitization following activation was unaffected by calcium. Calcium, barium, and magnesium, however, increased the potency of nicotine for desensitization induced by exposure to low, subactivating concentrations of nicotine. Recovery from desensitization was not modulated by calcium. These data suggest that calcium modulates various functional aspects of nicotinic receptors from mouse brain and may do so via different mechanisms.     

Activation of Protein Kinase C in Permeabilized Human Neuroblastoma SH-SY5Y Cells

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.