Calcium-sensing Mechanism Research Articles

The Calcium-Sensing Receptor is Upregulated in Post Myocardial Infarct Hearts and Downregulates ANP Expression in Rat Cardiac Myocytes but is Not Expressed in Rat Cardiac Fibroblasts In Vitro

Recently, several reports demonstrated functional expression of calcium-sensing receptor (CaSR) in the heart. Initial reports found the CaSR to be present in the cardiomyocytes, in contrast a recent report have found the CaSR to be present in sheep fibroblasts of the heart. The calcimimetic drug AMG 073 is a pharmacological (allosteric) modulator of CaSR that is in clinical use for the treatment of hyperparathyroidism. Here, we show that CaSR mRNA levels were up- regulated in the hearts from rats having myocardial infarction (MI) compared to sham operated rats. Furthermore, we found that in rat cardiomyocytes AMG 073 in the presence of extracellular Ca 2+ decreased mRNA levels of atrial natriuretic pre-pro peptide (pre-pro-ANP), which is a marker for cardiac hypertrophy. Surprisingly, CaSR mRNA was not detectable in rat neonatal ventricular fibroblasts (RNVF) by reverse transcriptase PCR. Yet, extracellular calcium exerts a biphasic response in DNA synthesis of RNVFs and AMG 073 seems to suppress DNA synthesis in RNVFs. In addition, calcium and calcimimetic activate MEK/ERK signalling in RNVFs that appears to be independent of CaSR activation. From these results it appears that an additional calcium-sensing mechanism may exist in RNVF. Our findings may be of importance in regards to a potential protective role of calcium and perhaps CaSR against cardiac hypertrophy.

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium ( Ca 2 + o ) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca 2 + o stimulates proliferation of the cells. The effects of Ca 2 + o were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca 2 + o -induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca 2 + o -induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

Calcyclin mediates serum response element (SRE) activation by an osteoblastic extracellular cation-sensing mechanism.

The molecular mechanism of sensing extracellular cations in osteoblasts is controversial. Using an expression-cloning strategy, the calcium-binding protein calcyclin was found to mediate the response of MC3T3-E1 osteoblasts to extracellular cations, but not the calcimimetic NPS-568, indicating the presence of another cation-sensing mechanism. Further understanding of calcyclin function in osteoblasts may identify novel targets for regulating bone formation. Extracellular calcium and other cations seem to regulate the function of osteoblasts through a distinct calcium-sensing mechanism that is coupled to activation of c-fos gene transcription. The identity of this calcium-sensing mechanism is unknown. To identify molecules that participate in this extracellular cation-sensing pathway, we developed an expression cloning strategy in COS-7 cells using cation stimulation of a serum response element (SRE) luciferase reporter derived from the c-fos promoter to screen a mouse MC3T3-E1 osteoblast cDNA library. We identified calcyclin (S100A6), a calcium-binding protein of the EF-hand type belonging to the S100 family, as being responsible for transferring a cation-sensing response from osteoblasts to COS-7 cells. Transfection of the calcyclin cDNA into COS-7 and HEK-293 cells confirmed that the overexpression of calcylin caused these cells to gain the ability to sense extracellular cations, including aluminum, gadolinium, calcium, and magnesium. Conversely, we found that an antisense calcyclin construct reduced calcyclin expression and partially inhibited the cation-sensing response in MC3T3-E1 osteoblasts. These results implicate calcyclin in the activation of SRE and establish a role for calcyclin as an accessory protein involved in the cation-sensing pathway in osteoblasts.

Molecular genetics of calcium sensing in bone cells.

The molecular mechanisms regulating bone remodelling are only partially understood. One of the controversial issues discussed during the past few years is the role that calcium signalling plays in this process and, in particular, in the functioning of the osteoclast. Calcium is involved in the recruitment and activation of osteoclasts and their subsequent detachment from bone. Parathyroid hormone and vitamin D are part of a systemic mechanism regulating calcium availability, storage and disposal. But there are conflicting results suggesting the presence of a local calcium-sensing mechanism in osteoclasts, in osteoblasts or in both. If this system could be characterized, it would be of therapeutic relevance for diseases such as postmenopausal osteoporosis and rheumatoid arthritis. Genetic data, animal models and cell-based assays have not yet been used to their full extent in this area. Here we review the available data and outline possible future strategies.

Separate effects of 1,25-dihydroxyvitamin D and calcium on renal calbindin-D28k and intestinal calbindin-D9k.

The effects of normal, low and high plasma concentrations of 1,25-(OH)2vitamin D (1,25-(OH)2D) combined with normal, low and high concentrations of plasma calcium on renal calbindin-D28k and intestinal calbindin-D9k were examined in rats. We found that the expression of renal calbindin-D28k was significantly (P<0.05) increased by high levels of 1,25-(OH)2D, but was not affected by a 50% reduction in 1,25-(OH)2D. In contrast the intestinal calbindin-D9k responded significantly (P<0.005) to both high and low 1,25-(OH)2D levels. The effect of 1,25-(OH)2D on intestinal calbindin-D9k was modulated by plasma calcium concentrations. Increased plasma calcium levels did not affect the renal calbindin-D28k concentrations, but suppressed intestinal calbindin-D9k (P<0.05). The effect of calcium was not mediated by calciotropic hormones. This suggests the existence of a calcium-sensing mechanism in the proximal duodenum. It is concluded that intestinal calbindin-D9k is more sensitive than renal calbindin-D28k to changes in 1,25-(OH)2D and that intestinal calbindin-D9k in contrast to renal calbindin-D28k is sensitive to changes in plasma calcium concentrations.

Alterations in the Sensing and Transport of Phosphate and Calcium by Differentiating Chondrocytes

During endochondral bone formation and fracture healing, cells committed to chondrogenesis undergo a temporally restricted program of differentiation that is characterized by sequential changes in their phenotype and gene expression. This results in the manufacture, remodeling, and mineralization of a cartilage template on which bone is laid down. Articular chondrocytes undergo a similar but restricted differentiation program that does not proceed to mineralization, except in pathologic conditions such as osteoarthritis. The pathogenesis of disorders of cartilage development and metabolism, including osteochondrodysplasia, fracture non-union, and osteoarthritis remain poorly defined. We used the CFK2 model to examine the potential roles of phosphate and calcium ions in the regulatory pathways that mediate chondrogenesis and cartilage maturation. Differentiation was monitored over a 4-week period using a combination of morphological, biochemical, and molecular markers that have been characterized in vivo and in vitro. CFK2 cells expressed the type III sodium-dependent phosphate transporters Glvr-1 and Ram-1, as well as a calcium-sensing mechanism. Regulated expression and activity of Glvr-1 by extracellular phosphate and parathyroid hormone-related protein was restricted to an early stage of CFK2 differentiation, as evidenced by expression of type II collagen, proteoglycan, and Ihh. On the other hand, regulated expression and activity of a calcium-sensing receptor by extracellular calcium was most evident after 2 weeks of differentiation, concomitant with an increase in type X collagen expression, alkaline phosphatase activity and parathyroid hormone/parathyroid hormone-related protein receptor expression. On the basis of these temporally restricted changes in the sensing and transport of phosphate and calcium, we predict that extracellular phosphate plays a role in the commitment of chondrogenic cells to differentiation, whereas extracellular calcium plays a role at a later stage in their differentiation program.

Calcium stimulates parathyroid hormone-related protein production in Leydig tumor cells through a putative cation-sensing mechanism

The production of parathyroid hormone-related protein (PTHrP) is regulated by a variety of hormones and growth factors. Previous research has shown that several PTHrP-producing cells are influenced by extracellular calcium (Ca(2+)(o)) concentration, with elevated levels increasing PTH-like activity released by cultured H500 rat Leydig tumor cells through a post-transcriptional mechanism. We have investigated the hypothesis that calcium stimulates PTHrP production in H500 cells by interacting with a cell membrane-associated cation-sensing receptor. Besides increased Ca(2+)(o) concentration, magnesium and the polycationic antibiotic neomycin also increased PTHrP production in a concentration-dependent manner. In the presence of the calcium ionophore, ionomycin, which markedly elevated cytosolic free calcium, the stimulation by Ca(2+)(o) of PTHrP could still be detected. These results indicate that increasing Ca(2+)(o) stimulates PTHrP production, possibly through a putative cell membrane-associated calcium-sensing mechanism. RT-PCR revealed the presence of a very small amount of calcium-sensing receptor coding mRNA.

The Calcineurin-Dynamin 1 Complex as a Calcium Sensor for Synaptic Vesicle Endocytosis

Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC(50) for calcium in the range of 0.1-0. 4 microM. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calcium-sensing mechanism that facilitates endocytosis.

Characterization of gating and peptide block of mSlo, a cloned calcium-dependent potassium channel.

The 20 amino acid Shaker inactivation peptide blocks mSlo, a cloned calcium-dependent potassium channel. Changing the charge and degree of hydrophobicity of the peptide alters its blocking kinetics. A "triple mutant" mSlo channel was constructed in which three amino acids (T256, S259, and L262), equivalent to those identified as part of the peptide's receptor site in the S4-S5 cytoplasmic loop region of the Shaker channel, were mutated simultaneously to alanines. These mutations produce only limited changes in the channel's susceptibility to block by a series of peptides of varying charge and hydrophobicity but do alter channel gating. The triple mutant channel shows a significant shift in its calcium-activation curve as compared with the wild-type channel. Analysis of the corresponding single amino acid mutations shows that mutation at position L262 causes the most dramatic change in mSlo gating. These results suggest that the three amino acids mutated in the mSlo S4-S5 loop may contribute to, but are not essential for, peptide binding. On the other hand, they do play a critical role in the channel's calcium-sensing mechanism.

Uncoupling of the calcium-sensing mechanism and differentiation in squamous carcinoma cell lines

Increases in the intracellular calcium (Ca i) levels, induced either by extracellular calcium or by calcium ionophores, stimulate the terminal differentiation of normal human keratinocytes in culture (NHK). Despite extensive differences in phenotypic expression, squamous carcinoma cell lines (SCC lines) display only partial terminal differentiation even in the presence of normal extracellular calcium. Therefore, in this study, we evaluated whether the inability of SCC lines to differentiate normally is due to a defect in achieving adequate levels of Ca i. Membrane-bound transglutaminase activity and involucrin levels of the various SCC lines were lower than those of NHK and correlated with their low extent of cornified envelope formation. Ionomycin, a calcium ionophore, acutely increased cornified envelope formation of NHK 60- to 70-fold, but only initiated a 1- to 5-fold increase in SCC lines. Yet resting Ca 1 levels in and the Ca 1 response to various agents of SCC lines were similar or higher than those of NHK. Extracellular calcium evoked a rapid, transient and a slower, sustained increase of Ca i. Extracellular ATP increased Ca i by a rapid release from intracellular sources. Ionomycin, on the other hand, increased Ca i from both intracellular compartments and extracellular sources. Thus, these studies indicate that the abnormalities in differentiation among SCC lines do not appear to involve their calcium-sensing mechanism. An uncoupling of the Ca i changes to the synthesis of the precursor molecules required for differentiation may be responsible for the defect in differentiation displayed by these SCC lines.

Monoclonal antibodies with exclusive reactivity against parathyroid cells and tubule cells of the kidney.

Four monoclonal anti-parathyroid antibodies were generated after immunization of mice with intact cells from human parathyroid tissue. All four monoclonal antibodies reacted in immunohistochemistry with structures present on parathyroid epithelial cells and proximal-tubule cells of the kidney but were unreactive with all other human tissues investigated. Immunofluorescence microscopy on suspended human parathyroid cells showed that the antibodies reacted with structures present on the cell surface. Two of these antibodies efficiently blocked the increase in cytoplasmic calcium concentration of parathyroid cells that is normally associated with increased concentrations of extracellular calcium. The results indicate that these two antibodies interfere with a calcium-sensing mechanism of parathyroid cells--i.e., a potential calcium receptor by which extracellular calcium regulates cytoplasmic calcium and hormone release in these cells.