OBJECTIVE: To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT). METHODS: The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting. RESULTS: Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells (P < 0.001). Despite a significant downregulation of CaSR mRNA (P=0.017) and protein (P=0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations. CONCLUSION Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo.
Humans ; Hyperparathyroidism, Secondary/metabolism* ; Parathyroid Glands/metabolism* ; Parathyroid Hormone ; RNA, Messenger/metabolism* ; Receptors, Calcium-Sensing/metabolism*

Animals ; Apoptosis ; Endotoxins/toxicity* ; Heart/drug effects* ; MAP Kinase Signaling System ; Myocardium/pathology* ; Rats ; Rats, Wistar ; Receptors, Calcium-Sensing/metabolism*

OBJECTIVE: To investigate the change of calcium sensing receptor (CaSR) expression at different time in rat tissue with acute myocardial infarction (AMI) and its effect on cardiomyocyte apoptosis. METHODS: The healthy Wistar rats were randomly divided into Sham and AMI groups, the rat myocardial infarction model was established by ligating left anterior descending coronary artery. The changes of cardiac morphology and hemodynamics were detected at 1, 2 and 4 weeks,respectively. The expressions of CaSR mRNA and protein in myocardial tissue were detected by RT-PCR and Western blot, respectively. The expressions of Bax, Bcl-2, caspase-3 and caspase-9 proteins were detected by Western blot. The serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) activity and cardiac troponin (cTnT) were determined. The apoptosis of cardiomyocytes were tested by TUNEL staining. RESULTS: Compared with the sham group, the expressions of CaSR mRNA and protein, the apoptosis index were increased significantly with the development of AMI (P＜0.05). The ultrastructural damage of cardiomyocytes was serious; the levels of LVSP, +dp/dt and -dp/dt were decreased,while the levels of LVEDP was increased (P＜0.05); In AMI group, the cTnT level, CK and LDH activities were all increased (P＜0.05). With the development of myocardial infarction, the cTnT level and CK activity were gradually decreased, while the activity of LDH was not significantly changed. The expressions of promote apoptosis-related Bax, caspase-3 and caspase-9 were significantly increased, and the expression of inhibited apoptosis-related protein(factor)Bcl-2 was significantly decreased (P＜0.05). CONCLUSION With the development of myocardial infarction,the expressions of CaSR mRNA and protein,the apoptosis index in rat myocardial tissue were increased with time prolongation after AMI. The increased expression of CaSR is involved in rat myocardial infarction, which is related with apoptosis.
Animals ; Apoptosis ; Myocardial Infarction ; metabolism ; Myocardium ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, Calcium-Sensing ; metabolism

Calcium Channels, L-Type ; genetics ; metabolism ; Esophageal Achalasia ; genetics ; metabolism ; Esophagus ; metabolism ; Humans ; Nitric Oxide Synthase ; metabolism ; Nitric Oxide Synthase Type I ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Calcium-Sensing ; genetics ; metabolism

OBJECTIVETo observe the dynamic expression of calcium-sensing receptor(CaSR) in myocardium of diabetic rats.

METHODSThirty male Wistar rats were randomly divided into 3 groups including control, diabetic-4 week and diabetic-8 week groups(n = 10). The type 2 diabetes mellitus models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The cardiac morphology was observed by electron microscope. Western blot analyzed the expression of CaSR, phospholamban (PLN), a calcium handling regulator, and Ca+-ATPase(SERCA) in cardiac tissues.

RESULTSCompared with control group, the expressions of CaSR and SERCA were decreased, while the expression of PLN was significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure.

CONCLUSIONThese results indicate that the CaSR expression of myocardium is reduced in the progression of DCM, and its potential mechanism may be related to the imnaired intracellular calcium homeostasis.

Animals ; Calcium-Binding Proteins ; metabolism ; Diabetes Mellitus, Experimental ; complications ; Diabetes Mellitus, Type 2 ; Diabetic Cardiomyopathies ; metabolism ; physiopathology ; Disease Progression ; Heart ; physiopathology ; Male ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Wistar ; Receptors, Calcium-Sensing ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Streptozocin

OBJECTIVE: To explore the expression levels of three T-type calcium channel receptors (α1G; α1H; α1I) in the cochlea and spiral ganglion neurons of C57BL/6J mice with different ages. METHOD: Thirty cases of C57BL/6J mice were divided into three groups (6-8 W, 24-26 W, 42-44 W) according to the age. The expressions of three T-type calcium channel receptors were quantified by RT-PCR after hearing thresholds measured by ABR. RESULT: Three receptors were detected in the cochlea and spiral ganglion neurons of 6-8 W C57BL/6J mice. The quantitative results showed that the expression levels of α1H and α1I were highest among three receptors in spiral ganglion neurons and in the cochlea respectively. The expression levels of three receptors significantly decreased with age,especially at the age of 4244 W. CONCLUSION The expression of T-type calcium channel receptors reduced with age in the inner ear of C57BL/6J mice.
Animals ; Calcium Channels ; Calcium Channels, T-Type ; biosynthesis ; Cochlea ; metabolism ; Ear, Inner ; Mice ; Mice, Inbred C57BL ; Neurons ; Receptors, Calcium-Sensing ; Spiral Ganglion ; metabolism

This paper aims to investigate the effect of store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC) on Ca(2+)-sensing receptor (CaR)-induced extracellular Ca(2+) influx and nitric oxide (NO) generation in human umbilical vein endothelial cells (HUVEC). SOC blocker, non-selective cation channel blocker, ROC agonist and ROC blocker were used separately and combined. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by Fura-2/AM loading. The activity of endothelial nitric oxide synthase (eNOS) and the production of NO were determined by the DAF-FM diacetate (DAF-FM DA). The results showed that increases of [Ca(2+)]i, eNOS activity and NO generation induced by CaR agonist Spermine were all reduced after single blocking the SOC or ROC, respectively (P < 0.05). ROC agonist can partially abolish the ROC blocker's effect (P < 0.05). The above mentioned effects evoked by CaR agonist Spermine were further reduced when blocking both SOC and ROC than single blocking SOC or ROC in HUVEC (P < 0.05). In conclusion, these results suggest that the SOC and ROC participate in the processes of CaR-evoked extracellular Ca(2+) influx and NO generation by a synergistic manner in HUVEC.
Calcium ; physiology ; Calcium Channel Blockers ; pharmacology ; Calcium Channels ; physiology ; Calcium Signaling ; Fluoresceins ; pharmacology ; Human Umbilical Vein Endothelial Cells ; physiology ; Humans ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type III ; metabolism ; Receptors, Calcium-Sensing ; physiology

Clinical studies reported hypomagnesaemia in long-term omeprazole usage that was probably due to intestinal Mg2+ wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg2+ transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg2+ absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg2+, but not Ca2+, transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg2+ transport-Mg2+ concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg2+ affinity of the paracellular channel. By continually monitoring the apical pH, we showed that omeprazole suppressed apical acid accumulation. Neomycin and spermine had no effect on passive Mg2+ transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg2+ transport in a calcium sensing receptor (CaSR)-independent manner. The results of western blot analysis showed that omeprazole significantly suppressed claudin (Cldn)-7 and -12, but not Cldn-2, expression in Caco-2 cells. By using apical solution of pH 5.5, 6.0, 6.5, and 7.0, we found that apical acidity markedly increased passive Mg2+ transport, Mg2+ affinity of the paracellular channel, and Cldn-7 and -12 expression in Caco-2 monolayers. Apical acidity abolished the inhibitory effect of omeprazole on passive Mg2+ transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg2+ absorption by luminal acidity-induced increase in Cldn-7 and -12 expression.
Absorption/drug effects ; Caco-2 Cells ; Calcium/metabolism ; Claudins/genetics/*metabolism ; Dose-Response Relationship, Drug ; Gene Expression/drug effects ; Humans ; Hydrogen-Ion Concentration ; Magnesium/*metabolism ; Omeprazole/*pharmacology ; Proton Pump Inhibitors/*pharmacology ; Receptors, Calcium-Sensing/metabolism

OBJECTIVETo examine the association of activation of calcium-sensing receptors (CaSR) with apoptosis in cardiomyocytes under simulated ischemia/reperfusion.

METHODSVentricular cardiomyocytes of neonatal rats were incubated in ischemia-mimetic solution for 2 h, then re-incubated in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL assay). The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of Caspase -3 and Bcl-2 was detected by Western blotting.

RESULTThe simulated I/R enhanced the expression of CaSR and cardiomyocyte apoptosis. GdCl(3), a specific activator of CaSR, further increased the expression of CaSR and cardiomyocyte apoptosis, along with upregulation of Caspase-3 and downregulation of Bcl-2.

CONCLUSIONCaSR is associated with I/R injury and apoptosis in neonatal rat ventricular cardiomyocytes via suppressing Bcl-2 and promoting Caspase -3 expression.

Animals ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Cells, Cultured ; Myocardial Reperfusion Injury ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Receptors, Calcium-Sensing ; metabolism ; Signal Transduction

Although the function of extracellular Ca(2+)-sensing receptor (CaR) is known, the regulatory mechanism of the CaR function remains to be clarified. The purpose of the present study was to investigate the effect of caveolin-1 (Cav-1) on CaR-induced extracellular Ca(2+) influx by using acute caveolae disruption with Filipin or siRNA targeted to the Cav-1 in human umbilical vein endothelial cells (HUVECs). Intracellular Ca(2+) concentration ([Ca(2+)](i)) was detected by Fura-2/AM loading. The results showed that different concentrations of extracellular Ca(2+) failed to increase [Ca(2+)](i), while the CaR agonist Spermine (2 mmol/L) resulted in an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) (P<0.05). No matter in buffer with or without 2 mmol/L Ca(2+), the [Ca(2+)](i) increase induced by Spermine in HUVECs was abolished after inhibition of CaR by a negative allosteric modulator Calhex231 (1 μmol/L) (P<0.05), conversely, the effect of Spermine on the increase in [Ca(2+)](i) in HUVECs was further augmented after acute caveolae disruption with Filipin (1.5 μg/mL) or transfection with siRNA targeted to the Cav-1 (P<0.05). This indicated that Cav-1 produced an inhibition of CaR-induced extracellular Ca(2+) influx. As to the biological mechanism of Cav-1-induced inhibition, immunofluorescence technique showed that both CaR and Cav-1 were present in HUVECs, and confocal microscopy supported the co-localization of CaR and Cav-1 on the plasma membrane. Functionally, the Cav-1 protein expression was decreased in HUVECs transfected with siRNA targeted to the Cav-1 (P<0.05); simultaneously, the CaR membrane protein expression was decreased (P<0.05), whereas CaR total protein level was unaffected (P>0.05). In conclusion, the present study suggests that CaR and Cav-1 co-localize on the plasma membrane in HUVECs and CaR-induced Ca(2+) influx is down-regulated by binding with Cav-1, and the mechanism involves the effect of Cav-1 on CaR localization on the plasma membrane and attenuating the CaR response to the agonist.
Calcium ; metabolism ; Calcium Channels ; metabolism ; Caveolin 1 ; agonists ; physiology ; Cells, Cultured ; Down-Regulation ; Filipin ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Receptors, Calcium-Sensing ; physiology ; Spermine ; pharmacology