Biomedical subjects

O Kifor

Publications and source records attributed to O Kifor.

At least 37 records · Page 2Linked to original sources

PubMed1998

Extracellular calcium (Ca2+(o))-sensing receptor in a murine bone marrow-derived stromal cell line (ST2): potential mediator of the actions of Ca2+(o) on the function of ST2 cells.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+(o)) homeostasis by mediating the actions of Ca2+(o) on parathyroid gland and kidney. Bone marrow stromal cells support the formation of osteoclasts from their progenitors as well as the growth of hematopoietic stem cells by secreting humoral factors and through cell to cell contact. Stromal cells also have the capacity to differentiate into bone-forming osteoblasts. Bone resorption by osteoclasts probably produces substantial local increases in Ca2+(o) that could provide a signal for stromal cells in the immediate vicinity, leading us to determine whether such stromal cells express the CaR. In this study, we used the murine bone marrow-derived, stromal cell line, ST2. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in ST2 cells. We also identified CaR transcripts in ST2 cells by Northern analysis using a CaR-specific probe and by RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of ST2 cells to high Ca2+(o) (4.8 mM) or to the polycationic CaR agonists, neomycin (300 microM) or gadolinium (100 microM), stimulated both chemotaxis and DNA synthesis in ST2 cells. Therefore, taken together, our data strongly suggest that the bone marrow-derived stromal cell line, ST2, possesses both CaR protein and messenger RNA that are very similar if not identical to those in parathyroid and kidney. Furthermore, as ST2 cells have the potential to differentiate into osteoblasts, the CaR in stromal cells could participate in bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local, osteoclast-mediated release of Ca2+(o) and, thereafter, initiating bone formation after their differentiation into osteoblasts.

Amino Acid Sequence↗

PubMed1998

Identification and localization of the extracellular calcium-sensing receptor in human breast.

The extracellular calcium (Ca2+o)-sensing receptor (CaR) plays a critical role in maintaining Ca2+o homeostasis in mammals by virtue of its presence in parathyroid gland and kidney. The breast is well recognized as a Ca(2+)-handling organ, and the effects of altering Ca2+o on the proliferation of breast epithelial cells are well documented. To date there are no data regarding the expression and localization of CaR in breast tissue. In the present study, we assessed the distribution of CaR messenger ribonucleic acid (mRNA) and protein in normal and fibrocystic human breast tissue as well as in ductal carcinoma of the breast using RT-PCR, Northern analysis, and immunohistochemistry with CaR-specific antisera. In all tissues, RT-PCR performed using sense and antisense primers based on the sequence of the human parathyroid CaR complementary DNA amplified a product of the size expected (425 bp) for genuine CaR transcripts. Nucleotide sequencing of RT-PCR products confirmed more than 99% homology with human parathyroid CaR complementary DNA. Although insufficient quantities of mRNA were isolated from normal and fibrocystic tissue for Northern analysis, a single 5.2-kb CaR transcript was expressed in malignant breast tissue similar to the major CaR transcript in human parathyroid. Localization of CaR protein by immunohistochemistry showed specific CaR staining of the ductal epithelial cells of the breast in all three tissue types. These findings indicate the presence of CaR mRNA and protein in the breast, providing indirect evidence that the CaR may have some role(s) in the control of Ca2+ transport, epithelial cell proliferation, and/or other processes in normal and abnormal breast tissue.

Base Sequence↗

PubMed1998

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Animals↗

PubMed1998

Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.

Amino Acid Sequence↗

PubMed1998

Characterization of the phosphatidylinositol-specific phospholipase C isozymes present in the bovine parathyroid and in human kidney HEK293 cells stably transfected with the human parathyroid Ca2+-sensing receptor.

The regulation of parathyroid hormone secretion by the chief cells of the parathyroid is mediated by a 7-transmembrane (7-TM) Ca2+-sensing receptor (CaR), which signals via activation of pertussis toxin-insensitive G proteins, causing stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have identified the PI-PLC isoforms expressed in two model systems utilized for studying CaR signal transduction, i.e. dispersed bovine parathyroid cells and a human embryonic kidney cell line (HEK 293) stably transfected with the human parathyroid CaR-cDNA. All of the eight PI-PLC isozymes examined in this study were found to be expressed to varying extents in the bovine parathyroid gland and in the CaR-transfected HEK cells as assessed by immunoblotting. We localized the expression of the more abundant isozymes (beta1, beta2, beta3, gamma1, gamma2, delta2) to the chief cells of the bovine parathyroid by immunocytochemistry, while the two less abundant isozymes (delta1, beta4) were not detectable in parathyroid sections. G proteins activated by 7-TM receptors are known to activate mainly PI-PLC of the beta class. Therefore, beta1, beta2, beta3 and beta4, all expressed in the bovine parathyroid, are candidate isozymes for coupling to the CaR. A comparison of the levels of expression of PI-PLC isozymes between CaR-transfected HEK cells and non-transfected HEK cells suggested that the expression of the CaR in this human cell line does not cause a significant up-regulation of any of the PLCbeta and PLCgamma isozymes. PLCdelta2, showing predominantly nuclear localization in the parathyroid, was the sole PI-PLC isozyme with higher levels of expression in CaR-transfected HEK cells.

Animals↗

PubMed1998

Extracellular calcium-sensing receptor in rat oligodendrocytes: expression and potential role in regulation of cellular proliferation and an outward K+ channel.

A G protein-coupled, extracellular calcium (Ca(0)2+)-sensing receptor (CaR) cloned from parathyroid, kidney, and brain plays crucial roles in systemic calcium metabolism. In brain, the CaR is located in nerve terminals as well as in fiber tracts, where it may be expressed in glia. Moreover, there is Ca2+- and K+-dependent communication between axons and oligodendroglia. To investigate further the potential role of the CaR in oligodendroglia, we studied expression of CaR mRNA and protein as well as the effects of CaR agonists on cellular proliferation and Ca2+-activated K+ channel activity in immature rat oligodendrocytes in primary culture. Reverse transcriptase polymerase chain reaction and sequencing of CaR transcripts from oligodendrocytes revealed >99% sequence identity with the rat kidney CaR. Northern analysis demonstrated 7.5 and 4.1 kb transcripts in oligodendrocytes, similar to those in rat parathyroid and kidney, while Western analysis and immunocytochemistry with CaR-specific antisera showed the presence of CaR protein. Immunocytochemically, the CaR was colocalized with galactocerebroside in the cultured oligodendrocytes. Raising Ca(0)2+ from 1.8 to 4.8 mM or addition of the polycationic CaR agonist neomycin (300 microM) modestly but significantly increased [3H]-thymidine incorporation into oligodendrocytes. Elevating Ca(0)2+ from 0.75 to 3.0 mM or addition of 100 microM neomycin also produced 2-2.5-fold increases in the open state probability (Po) of an outward K+ channel with a unitary conductance of 88+/-5 pS. Taken together, our data show that the CaR is expressed in immature oligodendrocytes and may be functionally linked to cellular proliferation and an outward K+ channel potentially contributing to local ionic homeostasis in the vicinity of oligodendroglia.

Animals↗

PubMed1997

Calcium-sensing receptor in the rat hippocampus: a developmental study.

The extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR) plays a key role in maintaining near constancy of Ca2+(o) in mammals through its presence in parathyroid gland and kidney. The CaR is also present in brain, and although its role(s) in the brain is not known, it is possible that small changes in Ca2+(o) modify essential physiological and pathological processes, since calcium is crucial for numerous neuronal functions. Northern analysis has revealed that the CaR mRNA is present in hippocampus and several other regions of the brain. The hippocampus is an important site for learning and memory, but the relevance of the CaR to these processes is unknown. Long-term potentiation (LTP), a putative in vitro analog of memory, can only be induced after 7-10 days postnatally in rat hippocampus. Therefore, in the present study we determined the time course for the developmental expression of the CaR in rat hippocampus to assess its relationship to the development of other important hippocampal functions, such as the capacity for induction of LTP. Northern and Western analyses showed that CaR mRNA and protein were expressed at low levels at 5 days postnatally but then increased markedly at 10 days. A high level of receptor expression, due primarily to an increase in a 7.5 kb transcript, persisted until 30 days, when it gradually decreased by 3-fold to reach the adult level of expression. In situ hybridization histochemistry and immunohistochemistry revealed CaR mRNA and protein in pyramidal cells of all the layers of hippocampus and in granule cells of the dentate gyrus. The results show that CaR expression rises at a time when LTP can first be induced in hippocampus and persists at high levels during the time when brain development is proceeding most rapidly. Further studies are needed to determine the role of the CaR in the development of important aspects of the function of hippocampus and other regions of brain, including LTP.

Aging↗

PubMed1997

Expression of extracellular calcium-sensing receptor by human lens epithelial cells.

The extracellular calcium-sensing receptor (CaR) confers the capacity to sense small changes in the extracellular Ca2+ concentration (Ca2+o) not only upon cells involved in maintaining systemic Ca2+ homeostasis but also upon those not directly involved in this process. Since high Ca2+o is known to affect various physiological processes in lens epithelium both in health and in disease states (e.g., the formation of cataracts in hypocalcemic states), we investigated the expression and function of the CaR in these cells. By RT-PCR and immunocytochemistry the CaR is expressed in human lens epithelial cells in culture. In addition, the open state probability of a Ca(2+)-activated potassium (K+) channel with a conductance of 82 +/- 3 pS is significantly increased by elevating Ca2+o to 3.0 mM or by application of 100 microM neomycin, both effective CaR agonists. Therefore, our data suggest that human lens-epithelial cells express the CaR, which may be functionally linked to Ca(2+)-activated K+ channels and, perhaps, to other ion channels involved in ionic homeostasis in the lens.

Base Sequence↗

PubMed1997

Calcium-dependent c-myc proto-oncogene expression and proliferation of Caco-2 cells: a role for a luminal extracellular calcium-sensing receptor.

The human colonic cell line Caco-2 responds to a reduction of ambient [Ca++]o to levels at and below 0.25 mM by a twofold increase in [3H]thymidine labelling of their DNA. [Ca++]o signals sensed preferentially across the luminal aspect of Caco-2 cells, are rapidly (4 h) transduced via PKC activation into up to sixfold increases in c-myc expression. This suggests the presence of a [Ca++]o-sensing membrane receptor (CaR) similar to that described by Brown et al. (1) in parathyroid and kidney cells. By RT-PCR we were able to amplify a 426 bp fragment from Caco-2 mRNA with 98% nucleotide identity to a part of the coding region for the extracellular domain of the parathyroid CaR. Immunohistochemical staining with a monoclonal antiparathyroid CaR antibody demonstrates CaR protein at the plasma membrane of confluent Caco-2 cells. Our results imply that the intestinal CaR is a potential mediator for the transduction of low luminal [Ca++]o into tumor promoting signals in human colonocytes.

Caco-2 Cells↗

PubMed1997

In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia.

We characterized the in vivo, cellular and molecular pathophysiology of a case of neonatal hyperparathyroidism (NHPT) resulting from a de novo, heterozygous missense mutation in the gene for the extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR). The female neonate presented with moderately severe hypercalcemia, markedly undermineralized bones, and multiple metaphyseal fractures. Subtotal parathyroidectomy was performed at 6 wk; hypercalcemia recurred rapidly but the bone disease improved gradually with reversion to an asymptomatic state resembling familial benign hypocalciuric hypercalcemia (FBHH). Dispersed parathyroid cells from the resected tissue showed a set-point (the level of Ca2+(o) half maximally inhibiting PTH secretion) substantially higher than for normal human parathyroid cells (approximately 1.8 vs. approximately 1.0 mM, respectively); a similar increase in set-point was observed in vivo. The proband's CaR gene showed a missense mutation (R185Q) at codon 185, while her normocalcemic parents were homozygous for wild type (WT) CaR sequence. Transient expression of the mutant R185Q CaR in human embryonic kidney (HEK293) cells revealed a substantially attenuated Ca2+(o)-evoked accumulation of total inositol phosphates (IP), while cotransfection of normal and mutant receptors showed an EC50 (the level of Ca2+(o) eliciting a half-maximal increase in IPs) 37% higher than for WT CaR alone (6.3+/-0.4 vs. 4.6+/-0.3 mM Ca2+(o), respectively). Thus this de novo, heterozygous CaR mutation may exert a dominant negative action on the normal CaR, producing NHPT and more severe hypercalcemia than typically seen with FBHH. Moreover, normal maternal calcium homeostasis promoted additional secondary hyperparathyroidism in the fetus, contributing to the severity of the NHPT in this case with FBHH.

Calcium↗

PubMed1997

The Ca2+-sensing receptor: a target for polyamines.

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+ (Ca(o)) but is expressed in a number of tissues that do not have well-established roles in the control of Ca(o), including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+ (Ca(i)), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Ca(i) transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of approximately 500 microM in the presence of 0.5 mM Ca2+, whereas sustained increases in Ca(i) had an EC50 of approximately 200 microM. The order of potency was spermine > spermidine >> putrescine. Elevation of Ca(o) shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 microM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Ca(o). Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 microM spermine, a response similar to that elicited by 2.0 mM Ca(o). These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.

Animals↗

PubMed1997

Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa.

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca(2+)-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Amiloride↗

PubMed1997

Cloning, expression, and tissue localization of the calcium-sensing receptor in chicken (Gallus domesticus).

In previous studies, we characterized an extracellular Ca2+ (Cao(2+))-sensing receptor (CaR) that plays a central role in regulating parathyroid hormone secretion in mammals by sensing Cao2+. In the present study, we have cloned and characterized the chicken (Gallus domesticus) homolog of the CaR. The chicken parathyroid CaR shares a high degree of homology (84% amino acid identity) with the human CaR and displays a similar topology. Moreover, amino acid residues where mutations cause disorders of Cao(2+)-sensing in the human CaR share the wild-type human sequence in the chicken CaR. However, a single region in the extracellular domain of the chicken CaR differs substantially from its mammalian homologs. Xenopus laevis oocytes injected with chicken CaR cRNA respond to elevated ambient levels of Cao2+, extracellular Mg2+, or extracellular Gd3+ with the characteristic activation of inositol trisphosphate-dependent, intracellular Ca(2+)-induced Cl- currents elicited by mammalian CaRs as well as by G protein-linked receptors coupled to activation of phospholipase C. By in situ hybridization, clusters of cells in chicken parathyroid glands were shown to express CaR messenger RNA. Northern analysis and immunohistochemistry demonstrated expression of receptor transcripts and/or protein in kidney tubules and intestine as well as in brain. The close conservation of the amino acid sequence of the chicken CaR with its mammalian homologs as well as its similar tissue distribution suggest that the receptor may also play an important role in avian calcium homeostasis.

Amino Acid Sequence↗

PubMed1997

Expression of an extracellular calcium-sensing receptor in human and mouse bone marrow cells.

The cloning of a G protein-coupled, extracellular calcium (Ca2+e)-sensing receptor (CaR) from bovine parathyroid provided direct evidence that Ca2+e-sensing can occur through receptor-mediated activation of G proteins and their associated downstream regulators of cellular function. CaR transcripts and protein are present in various tissues of humans and other mammals that are involved in Ca2+e homeostasis, including parathyroid, kidney, and thyroidal C-cells. The present study was performed to determine whether bone marrow cells express the CaR, since cells within the marrow space could be exposed to substantial changes in Ca2+e related to bone turnover. Using DNA and RNA probes from the human parathyroid CaR cDNA, we identified CaR transcripts of 5.2 and approximately 4.0 kilobases by Northern analysis of poly(A+) RNA from low-density mononuclear cells isolated from whole human bone marrow that are putatively enriched in marrow progenitor cells, including bone cell precursors. In situ hybridization also identified CaR transcripts in the same cell preparations. Reverse transcription-polymerase chain reaction demonstrated > 99% nucleotide identity between transcripts from human bone marrow cells and the corresponding regions of the human CaR cDNA. Antisera specific for several different regions within the extracellular domain of the CaR were reactive with low-density human marrow cells that were either adherent or nonadherent to plastic. About one-third of the adherent, CaR-immunoreactive cells were also positive for alkaline phosphatase, a nonspecific marker of preosteoblasts, osteoblasts, and assorted cells of the colony-forming unit-fibroblast lineage. In addition, a substantial fraction (approximately 60%) of low density murine marrow cells cultured for 1 week at 4.8 mM Ca2+e expressed both CaR immunoreactivity and nonspecific esterase, an enzyme expressed by monocyte/macrophages and fibroblasts. Finally, erythroid precursors and megakaryocytes from murine marrow as well as blood platelets expressed abundant CaR immunoreactivity, while peripheral blood erythrocytes and most polymorphonuclear leukocytes did not. These studies indicate that the CaR is present in low-density mononuclear bone marrow cells as well as in cells of several hematopoietic lineages and could potentially play a role in controlling the function of various cell types within the marrow space.

Animals↗

PubMed1997

Cloning and characterization of a calcium-sensing receptor from the hypercalcemic New Zealand white rabbit reveals unaltered responsiveness to extracellular calcium.

The extracellular Ca2+ (Ca(0)2+)-sensing receptor (CaR) recently cloned from mammalian parathyroid, kidney, brain, and thyroid plays a central role in maintaining near constancy of Ca(0)2+. We previously showed that the hypercalcemia normally present in New Zealand white rabbits is associated with an elevated set point for Ca(02+)-regulated PTH release (the level of Ca(0)2+ half-maximally inhibiting hormonal secretion). This observation suggested an alteration in the Ca(02+)-sensing mechanism in the rabbit parathyroid, a possibility we have now pursued by isolating and characterizing the rabbit homolog of the CaR. The cloned rabbit kidney CaR (RabCaR) shares a high degree of overall homology (> 90% amino acid identity) with the bovine, human, and rat CaRs, although it differs slightly in several regions of the extracellular domain potentially involved in binding ligands. By Northern analysis and/or immunohistochemistry, a similar or identical receptor is also expressed in parathyroid, thyroid C cells, small and large intestine, and in the thick ascending limb and collecting ducts of the kidney. When expressed transiently in HEK293 cells and assayed functionally through CaR agonist-evoked increases in Ca(i)2+, the rabbit CaR shows apparent affinities for Ca(0)2+, Mg(0)2+, and Gd(0)3+ that are indistinguishable from those observed in studies carried out concomitantly using the human CaR. Therefore, at least as assessed by its ability to increase Ca(i)2+ when expressed in HEK293 cells, the intrinsic functional properties of the rabbit CaR cannot explain the hypercalcemia observed in vivo in the New Zealand white rabbit.

Amino Acid Sequence↗

PubMed1997

The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells.

The extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR) is a G protein-coupled receptor that activates phospholipase C (PLC). In the present studies, we assessed Ca2+(o)-dependent changes in the generation of inositol phosphates (IP), free arachidonic acid (AA), and phosphatidylbutanol (PtdBtOH) by PLC, phospholipase A2 (PLA2), and phospholipase D (PLD), respectively, in bovine parathyroid cells as well as in wild-type or CaR-transfected human embryonic kidney (HEK293) cells (HEK-WT and HEK-CaR, respectively). Elevated Ca2+(o) increased the formation of IPs in parathyroid cells as well in HEK-CaR but not in HEK-WT cells. High Ca2+(o) also elicited time- and dose-dependent increases in PtdBtOH in parathyroid cells and HEK-CaR but not in HEK-WT cells. Brief treatment of parathyroid and HEK-CaR cells with an activator of protein kinase C (PKC), phorbol 12-myristate,13-acetate (PMA), stimulated PLD activity at both low and high Ca2+(o). Moreover, high Ca2+(o)-stimulated PLD activity was abolished following down-regulation of PKC by overnight phorbol myristate acetate (PMA) pretreatment, suggesting that CaR-mediated activation of PLD depends largely upon stimulation of PKC. High Ca2+(o) likewise increased the release of free AA in parathyroid and HEK-CaR but not in HEK-WT cells. Mepacrine, a general PLA2 inhibitor, and AACOCF3, an inhibitor of cytosolic PLA2, reduced AA release in parathyroid cells at high Ca2+(o), suggesting a major role for PLA2 in high Ca2+(o)-elicited AA release. Pretreatment of parathyroid cells with PMA stimulated release of AA at low and high Ca2+(o), while a PKC inhibitor, chelerythrine, reduced AA release at high Ca2+(o) to the level observed with low Ca2+(o) alone. Thus, PKC contributes importantly to the high Ca2+(o)-evoked, CaR-mediated activation of not only PLD but also PLA2. Finally, high Ca2+(o)-stimulated production of IP, PtdBtOH, and AA all decreased substantially in parathyroid cells cultured for 4 days, in which expression of the CaR decreases by 80% or more, consistent with mediation of these effects by the receptor. Thus, the CaR activates, directly or indirectly, at least three phospholipases in bovine parathyroid and CaR-transfected HEK293 cells, providing for coordinate, receptor-mediated regulation of multiple signal transduction pathways in parathyroid and presumably other CaR-expressing cells.

Animals↗

PubMed1997

Ca2+ receptor mRNA and protein increase in the rat parathyroid gland with advancing age.

Parathyroid hormone (PTH) release is regulated by extracellular calcium through a Ca2+ receptor (CaR) located on the surface of the parathyroid cell. With advancing age, the serum concentration of PTH increases, and evidence suggests that the calcium set-point for PTH release may also increase. To determine whether these changes are linked to a change in CaR expression, we quantitated mRNA and protein for the receptor in parathyroid glands of 6-week-, 6-month- and 24-month-old rats. Thyroid and kidney tissue were also studied. Between 6 weeks and 24 months of age, CaR mRNA in the parathyroid gland increased 11.4- and 3.3-fold as measured by competitive reverse transcription PCR and solution hybridization assays respectively. Message levels for the receptor also increased in the thyroid but not in the kidney. Coincident with the increase in message levels, receptor protein concentration in the parathyroid increased 7-fold between 6 weeks and 24 months of age. These results suggest that the altered relationship between extracellular calcium and PTH release observed in aging is associated with dramatic changes in CaR metabolism. That PTH secretion is increased despite increased receptor concentration suggests that aging may impair calcium binding or coupling between the CaR and down-stream effector elements in the pathway regulating PTH release.

Aging↗

PubMed1996

Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (FBH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcaemia (ADH), and studied their function by characterizing intracellular calcium [Ca2+]i transients in response to varying concentrations of extracellular calcium [Ca2+]o or gadolinium [Gd3+]o. The wild type receptor had an EC50 for calcium (EC50[Ca2+]o) (the value of [Ca2+]o producing half of the maximal increase in [Ca2+]i) of 4.0 mM (+/- 0.1 SEM). However, five missense mutations associated with FBH or NSHPT, (P55L, N178D, P221S, R227L, and V817I) had significantly higher EC50[Ca2+]os of between 5.5 and 9.3 mM (all P < 0.01). Another FBH mutation, Y218S, had an EC50[Ca2+]o of > 50 mM but had only a mildly attenuated response to gadolinium, while the FBH mutations, R680C and P747fs, were unresponsive to either calcium or gadolinium. In contrast, three mutations associated with ADH, (F128L, T151M, and E191K), showed significantly reduced EC50[Ca2+]os of between 2.2 and 2.8 mM (all P < 0.01). These findings provide insights into the functional domains of the CaR and demonstrate that mutations which enhance or reduce the responsiveness of the CaR to [Ca2+]o cause the disorders ADH, FBH, and NSHPT, respectively.

Calcium↗