A Case of Neonatal Severe Hyperparathyroidism: Challenges in Management

Neonatal severe hyperparathyroidism is a rare disorder arising from inherited defects in the calcium sensing receptor (CaSR) that presents early in life with severe hypercalcemia, failure to thrive, and developmental retardation. The authors describe an infant with neonatal severe hyperparathyroidism due to homozygous CaSR gene mutation presenting with recurrent episodes of severe hypercalcemia, growth retardation, and developmental delay. Medical management served as an efective bridge therapy to surgery. Total parathyroidectomy with right hemithyroidectomy was performed at 7 mo of age and resulted in successful cure and normalization of growth and developmental milestones. Timely medical and surgical management can help prevent mortality and morbidity in the form of neurodevelopmental sequelae. Life-long monitoring and treatment is mandatory for the resultant hypoparathyroidism.

Cultivation and characterization of primary human parathyroid cells from patients with severe secondary hyperparathyroidism / 南方医科大学学报

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.

Clinical characteristics and genetic analysis of familial hypocalciuric hypercalcemia type 1 / 中华内分泌代谢杂志

A case of familial hypocalciuric hypercalcemia type 1 (FHH1) was reported detailing the course of diagnosis and treatment. The main clinical manifestations of the patient were recurrent pancreatitis with moderate hypercalcemia and low urinary calcium. The C→T heterozygous missense mutation at nucleotide 2 393 with conversion of codon Pro798 to Leu (p.P155L) in CaSR gene was identified. Serum calcium and parathyroid hormone levels of the patient were decreased significantly after treatment with cinacalcet.

Role of calcium sensing receptors in proliferation and migration of renal artery smooth muscle cells under insulin resistance / 中国药理学通报

To investigate the role and mechanism of calcium-sensing receptor (CaSR) in the proliferation and migration of renal artery smooth muscle cells (RASMCs) under insulin resistance. Methods RAMSCs in the logarithmic growth stage were randomly divided into control, pure model, model + GdCl

Role of Gq-PLC-IP3 signal transduction pathway in calcium-sensing receptor-mediated proliferation of A549 cells / 解剖学报

Objective: To explore the signal transduction pathway of calcium-sensing receptor (CaSR) mediating hypoxia-induced proliferation of A549 cells of human non-small cell lung cancer. Methods: The A549 cells were randomly divided into several groups which conclude control group, hypoxia group (H), hypoxia + CaSR agonist group (H + Gd), hypoxia + CaSR inhibitor group (H + NPS) and phospholipase C(PLC) pathway inhibitor group (H + Gd +U73122). Expression of CaSR and proliferating cell nuclear antigen (PCNA) in A549 cells under different treatments was analyzed by Western blotting. The changes of intracellular calcium ion concentration were detected by confocal laser scanning microscope. The effects of cell proliferation cycle and proliferation index were gauged by flow cytometry under different drugs. HE staining was used to observe the changes of cell number with different drugs. Results: The expression levels of CaSR and PCNA in A549 cells increased by hypoxia. Meanwhile, cell proliferation index and cell number were also upregulated. GdCl3(CaSR agonist) could amplify the effect of hypoxia, and NPS2390 (CaSR inhibitor) could reduce the effect of hypoxia. All effects mentioned above could be inhibited by U73122 (PLC pathway inhibitor). Conclusion: Hypoxia-induced CaSR can mediate the proliferation of A549 cells through Gq-PLC-IP, signal transduction pathway.

Role of PI3K/Akt pathway in hypoxic-activated calcium sensing receptor mediated A549 cell metastasis / 中国药理学通报

Aim: To explore the role of PI3K/Akt signaling pathway in A549 and A549/DDP cells metastasis mediated by hypoxic-activated calcium sensing receptor (CaSR). Methods: The A549 and A549/DDP cells in the logarithmic growth stage were randomly divided into control, hypoxia, hypoxia + GdCl3 (CaSR agonist), hypoxia + NPS2143 (CaSR inhibitor) and hypoxia + LY294002 (PI3K inhibitor) + GdCl3 group. The protein levels of CaSR, MMP-2 and p-Akt were analyzed by Western blot in A549 and A549/DDP cells under different treatment conditions. The effects of different treatment factors on the ability of cell migration and invasion were measured by wound scratch assay and transwell migration assay. The effects of different treatment factors on the secretion of matrix metalloproteinase-2 (MMP-2) protein by A549 and A549/DDP cells was analyzed by ELISA. Results: Compared with control group, hypoxia increased the protein expression of CaSR, enhanced cell migration ability, increased MMP-2 protein expression in cells and culture medium, and promoted Akt protein phosphorylation in A549 and A549/DDP cells. NPS2143 reduced the effect of hypoxia, GdCl3 amplified the effect of hypoxia, and LY294002 inhibited effect of hypoxia and GdCl3. Conclusions: Hypoxic-activated CaSR promotes A549 cell metastasis, and its mechanism may involve PI3K/Akt pathway.

Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma

Background: As histopathological findings of parathyroid carcinoma are not certain, the diagnosis of tumors with degenerative changes may be difficult. In these cases, immunohistochemical markers are beneficial. We aimed to research the acceptability of calcium-sensing receptor (CaSR), Galactin-3, Cyclin D1, and Ki-67 as helpful markers in parathyroid tumors in cases which are difficult to diagnose. Materials and Methods: Those cases who had been diagnosed with atypical parathyroid adenoma and parathyroid carcinoma between 2010 and 2015 were reevaluated. ?mmunohistochemical markers were applied to this cases. Results: About 21 cases were parathyroid adenoma, 14 were atypical adenoma, and 10 cases were parathyroid carcinoma. According to the immunohistochemical results, global loss of CaSR staining was seen in 50% (5/10) of the patients with carcinoma while there was no loss of staining in those with parathyroid adenoma (P = 0,001). Global loss of CaSR staining was found in only one out of 14 cases with atypical adenoma. The expression of Galactin-3 was found to be positive in 40% (4/10) of carcinoma cases, 71.4% (10/14) of those with atypical adenoma, and 14.3% (3/21) of those with adenoma (P = 0,002). Cyclin D1 expression was determined to be positive in 70% (7/10) of patients with carcinoma, 71.4% (10/14) of atypical adenoma cases, and 23.8% (5/21) of those with adenoma. The Ki-67 proliferation index was seen to be above 5% in 50% (5/10) of carcinoma cases and 35,7% (5/14) of those with atypical adenoma. Conclusion: In these studies, it has been emphasized that the global loss of CaSR staining was used as a negative marker in the diagnosis of carcinoma. In this study, we have also confirmed that the global loss of CaSR staining is a useful marker to determine potential increased malignancy.

Expression of CaSR in neurons and cardiomyocytes incubated with magnesium-free extracellular fluid and its relationship with the MAPK pathway / 医学研究生学报

Objective At present, studies on the calcium sensing receptor (CaSR) in the pathogenesis of epilepsy are carried out in animal models in vivo and in single cells cultured in vitro. This study was to investigate the expression of CaSR and its relationship with the MAPK pathway in the rat model of epilepsy.Methods The neurons and cardiomyocytes of 3-day-old Wistar rats were cultured for 10 days and randomly divided into groups A (control), B (magnesium-free), C (magnesium free+spermine), D (magnesium free+calhex231), and E (magnesium free+spermine+calhex231). The model of epilepsy was made by abnormal discharge of the neurons induced by coculturing magnesium-free extracellular fluid with cardiomyocytes. The morphological changes of the cells were observed by HE staining and transmission electron microscopy, their survival rate detected by MTT, and the expressions of the CaSR, Bcl-2, P-ERK, P-JNK and P-P38 proteins in the cocultured cells determined by Western blot.Results Compared with the cells in group B, those in group C were swollen and broken with nuclear fragmentation, those in group D showed a relative integrity, and those in group E were also swollen and broken but improved in comparison with those in group C. The survival rates of the cells were (61.08±15.44)%, (82.80±14.37)% and (82.04±17.37)% in groups C, D and E, respectively, all significantly lower than in A (\[100.00±0.00\]%, P<0.01) and B (\[88.88±9.85\]%, P<0.01). The expression of CaSR was markedly higher in group B than in A (\[0.73±0.19\] vs \[0.45±0.12\], P<0.01) but lower than in C (1.32±0.15) and E (1.19±0.12) (P<0.01). The expression levels of Bcl-2 and P-ERK were remarkably lower in group B than in A but higher than in C (P<0.01), and those of P-JNK and P-P38 significantly higher in group B than in A and lower than in C and E (P<0.05).Conclusion Magnesium-free extracellular fluid can damage neurons and cardiomyocytes, increase the expression of CaSR, participate in the MAPK signaling pathway, and mediate the apoptosis of neurons and cardiomyocytes, while CaSR inhibitors can relieve the CaSR agonist-induced damage to the cells.

One pedigree analysis of a familial hypocalciuric hypercalcemia caused by a new heterozygous mutation in calcium-sensing receptor gene / 中华内分泌代谢杂志

Medical history and physical examinations were performed to assess the clinical manifestations and growth of one patient with familial hypocalciuric hypercalcemia(FHH). Clinical data, including histories of his parents and 3 maternal relatives were collected. Serum parathyroid hormone(PTH), calcium, phosphorus, 24-hour urinary calcium, and 24-hour urinary calcium to creatinine ratio(UCCR)were measured or calculated. Meanwhile, after peripheral blood samples were collected and genomic DNA was extracted, the whole exome sequencing to detect gene mutations of the proband was performed. Further family screenings were also performed by Sanger sequencing to assess the relationship between genotype and phenotype. The results showed that the proband with motor developmental delays had severe hypercalcemia(4.20 mmol/L), while his mother without clinical symptoms had a higher blood calcium within the normal range(2.57 mmol/L). However, their urinary calcium levels were both low(UCCR< 0.01). The C→T heterozygous missense mutation was found by exome sequencing at nucleotide 1243 within exon 4 of calcium sensing receptor(CaSR)gene in the proband, which caused a substitution of Arginine to Tryptophan(R415W). Sanger sequencing confirmed the same mutation in his mother. There was no mutation in other family members. (Chin J Endocrinol Metab, 2018, 34: 583-586)

Calcium-sensing receptor-mediated L-tryptophan-induced secretion of cholecystokinin and glucose-dependent insulinotropic peptide in swine duodenum

This study aimed to elucidate the effect of tryptophan (Trp) on gut hormone secretion as well as the roles of the calcium-sensing receptor (CaSR) and its downstream signaling pathway in gut hormone secretion by assessing swine duodenal perfusion in vitro. Swine duodenum was perfused with Krebs-Henseleit buffer as a basal solution. Various concentrations (0, 10, and 20 mM) of Trp were applied to investigate its effect on gut hormone secretion. A CaSR antagonist was used to detect the involvement of CaSR and its signal molecules. The 20 mM Trp concentration promoted the secretion of cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP), elevated the mRNA level of CaSR, and upregulated the protein levels of CaSR, protein kinase C (PKC), and inositol trisphosphate receptor (IP3R). However, NPS 2143, an inhibitor of CaSR, attenuated the CCK and GIP release, reduced the mRNA level of CaSR, and decreased the protein levels of CaSR, PKC, and IP3R with 20 mM Trp perfusion. The results indicate that CCK and GIP secretion can be induced by Trp in swine duodenum in vitro, and the effect is mediated by CaSR and its downstream signal molecules PKC and IP3R.

Hipercalcemia hipocalciurica familiar en una paciente con mutación del receptor de calcio: forma atípica de presentación y tratamiento con cinacalcet / Familial hypocalciuric hypercalcemia in a patient with calcium-sensing receptor mutation: atypical clinical presentation and treatment with cinacalcet

El hiperparatiroidismo familiar y la hipercalcemia hipocalciúrica familiar (HHF) constituyen un subgrupo heterogéneo de trastornos con herencia mendeliana, que representan en conjunto el 5% de las causas de hipercalcemia PTH dependiente. La HHF se asocia con mutaciones del gen del receptor sensor de calcio (CaSR). Esta entidad se manifiesta, en la mayoría de los casos, con la presentación asintomática y familiar de hipercalcemia e hipocalciuria y valores elevados o normales de hormona paratiroidea (PTH). Los avances en la biología molecular han contribuido al diagnóstico, evaluación del fenotipo de cada entidad y elección del tratamiento. Se describe el caso de una paciente con hipercalcemia estudiada a partir de una tumoración de cuello asociada con una glándula paratiroides quística. Luego de un exhaustivo proceso diagnóstico se halló en el estudio genético una mutación inactivante en el gen CaSR. Teniendo en cuenta la presencia de la relación clearance calcio/clearance creatinina <0,01 y la falta de respuesta al tratamiento quirúrgico, se consideró la entidad de HHF con forma de presentación atípica. La paciente, sin tratamiento, presentaba un progresivo incremento de la calcemia luego de la cirugía de las glándulas paratiroides, que no se controló con el uso de bifosfonatos y evolucionó con episodios de mareos y desmayos frecuentes sin causa neurológica o cardiovascular detectada. Por lo tanto, se inició el tratamiento con cinacalcet, con el cual se obtuvo una buena respuesta terapéutica: descenso de la calcemia y mejoría de la sintomatología luego de un año de su comienzo. El cinacalcet es una herramienta terapéutica de importancia en estos raros casos de HHF. (AU)

Familial hyperparathyroidism including familial hypocalciuric hypercalcemia (FHH) is an heterogeneous subgroup of disorders with Mendelian inheritance, that account for 5% of PTH dependent hypercalcemia. FHH is associated with mutations of the calcium receptor (CaSR) gene. This entity is manifested by hypercalcemia with hypocalciuria and high or normal levels of parathyroid hormone (PTH) generally asymptomatic and with familial presentation. Advances in molecular biology have contributed to the diagnosis, evaluation of the phenotype of each entity and the choice of treatment. We describe a patient with hypercalcemia diagnosed following the finding of a neck tumor associated with cystic parathyroids. After an exhaustive diagnostic process, an inactivating mutation in the CaSR gene was found. Considering the presence of a ratio clearance calcium / clearance creatinine <0.01 and the lack of response to surgical treatment, HHF entity with atypical presentation was considered. The patient exhibited progressive increase in serum calcium following parathyroid surgery, which was not controlled with the use of bisphosphonates and evolved into episodes of frequent dizziness and fainting, without neurological or cardiovascular causes. Treatment with cinacalcet was initiated, with a good therapeutic response. The use of cinacalcet is a useful therapeutic tool in these rare cases of FHH. (AU)

Calcemic response to burns differs between adults and children: A review of the literature

OBJECTIVES: The calcemic and parathyroid hormone (PTH) responses to severe burn injury appear to differ between children and adults. In our limited studies children exhibited hypocalcemic hypoparathyroidism consistent with up-regulation of the parathyroid calcium-sensing receptor (CaSR) while adults did not, suggesting a developmental cutoff in cytokine-mediated up-regulation of the CaSR. This difference may be clinically important as published studies indicate that extracellular calcium (Ca) may stimulate the inflammatory response. The aim of this study was to examine the existing literature on burns to see if the differences between pediatric and adult calcemic and PTH responses to burn supported our findings providing stronger evidence to support this developmental difference. METHODS: We reviewed the National Library of Medicine database using the terms burns, PTH and ionized calcium and found 9 articles from 8 different medical centers; one was eliminated due to mixing of adults and children. RESULTS: There were 245 burn patients reported from the literature, 178 pediatric and 67 adults. The data are mostly consistent with our reported findings. Of the 10 pediatric patients with severe burns that we studied, mean ionized Ca concentration was below the lower limit of normal of 1.10 mM. The 67 adult burn patients reported in the literature had a mean blood ionized Ca concentration that was within the adult normal range or was lower than normal but with secondary hyperparathyroidism. Moreover, serum PTH concentrations were uniformly low in the 178 children in the burn literature but normal or mildly elevated in the 67 adults. CONCLUSIONS: These results support the hypothesis that the difference between pediatric and adult victims is consistent with an age-related CaSR response to cytokine stimulation and may be consistent with a lower level of inflammation in children. Ionized Ca and PTH might serve as possible therapeutic targets to lower the inflammatory response in burn victims.

Functions and characteristics of calcium-sensing receptorsand effects of CaSR on characteristics of adult stem cells / 中国药理学通报

Calcium-sensing receptor(CaSR) is a widely distributed G-protein coupled receptor.The activated CaSR plays an important role in many kinds of signaling pathway regulation, such as Ca2+ signaling pathway.It not only maintains the body calcium balance, but also is involved in the regulation of a variety of cell secretion, proliferation, apoptosis, and ion channel opening processes.CaSR expression is involved in stem cell migration, adhesion and homing in hematopoietic stem cells and mesenchymal stem cells.The activated CaSR also regulated the function of itself and characteristics in stem cells through a variety of cell signaling pathways.We introduce the functions and characteristics of CaSR, the relationship between CaSR and disease, and review the effects of the biological characteristics on hematopoietic stem cells and mesenchymal stem cells.

Triangle relationship of PTH, intracellular calcium and CaSR in myocardial damage / 中国病理生理杂志

Parathyroid hormone ( PTH) is an important hormone secreted by parathyroid cells , and regulates the metabolism of calcium and phosphorus in the body .In recent years , the toxic effect of PTH on myocardium has been re-ported.Calcium-sensing receptor (CaSR), a member of G protein-coupled receptor family, can feel the subtle change of extracellular calcium concentration and regulate intracellular calcium concentration through multifarious ways in order to control the secretion of PTH .The expression of CaSR is observed in parathyroid cells , renal tubular epithelial cells , myo-cardial cells, etc.Intracellular calcium, as a second messenger, participates in various cell functions , such as excitation-contraction coupling , fertilization and so on .The injury of myocardial cells is intimately linked with high concentrations of PTH and intracellular calcium , and high expression of CaSR .

Calcium-sensing receptor diseases and their molecular pathogenesis / 中华内分泌代谢杂志

[Summary] Extracellular calcium is essential for the regulation of a variety of biological processes. Calcium-sensing receptor (CaSR) plays a central role in maintaining Ca2+ homeostasis, while G-protein α-11 (Gα11 ) subunit and adaptor-related protein complex 2 sigma (AP2σ) are also involved in CaSR signaling transduction. Loss- or gain-of-function mutations of these encoding genes cause different types of familial hypocalciuric hypercalcaemia (FHH) and autosomal dominant hypocalcaemia (ADH). Calcimimetic and calcilytic drugs are useful in treating these FHH and ADH disorders. The current paper is a Chinese translation of a review entitled as “Disorder of the calcium-sensing receptor and partner proteins: insights into the molecular basis of calcium homeostasis” published in 《 Journal of Molecular Endocrinology》(2016,57:R127-R142) with the permission from the author and the journal.

Effects of CaSR on vascular relaxation/constriction in rats / 局解手术学杂志

Objective To investigate the effects of calcium sensing receptor (CaSR)on vasorelaxation/vasoconstriction of superior mes-enteric artery (SMA)in rats and its relationship to endothelium.Methods With endothelium-intact and endothelium-denuded SMA rings of rats,the effects of CaSR-specific allosteric modulator cinacalcet on the SMA rings pre-contracted with norepinephrine (NE),and vascular contractile response /relaxation reactivity were observed.Results Cinacalcet had no effects on resting tension of SMA rings with or without endothelium.Cinacalcet caused a significant relaxation in the endothelium-intact SMA rings pre-contraction with NE in a dose-dependent manner.Endothelium denudation abolished cinacalcet-induced vasorelaxation.Pretreatment with cinacalcet for 30 minutes decreased the con-tractile response of endothelium-intact SMA rings to NE,but had no significant influence on relaxation reactivity.In the endothelium-denuded SMA rings,cinacalcet did not affect both vasoconstriction and vasorelaxation.Conclusion CaSR plays an important role in the regulation of the vascular reactivity,and this effect is endothelium-dependent.

The calcium-sensing receptor participates in testicular damage in streptozotocin-induced diabetic rats / 亚洲男科学杂志(英文版)

Male infertility caused by testicular damage is one of the complications of diabetes mellitus. The calcium-sensing receptor (CaSR) is expressed in testicular tissues and plays a pivotal role in calcium homeostasis by activating cellular signaling pathways, but its role in testicular damage induced by diabetes remains unclear. A diabetic model was established by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg-1 ) in Wistar rats. Animals then received GdCl 3 (an agonist of CaSR, 8.67 mg kg-1 ), NPS-2390 (an antagonist of CaSR, 0.20 g kg-1 ), or a combination of both 2 months after STZ injection. Diabetic rats had significantly lower testes weights and serum levels of testosterone compared to healthy rats, indicating testicular damage and dysfunction in STZ-induced diabetic rats. Compared with healthy controls, the testicular tissues of diabetic rats overexpressed the CaSR protein and had higher levels of malondialdehyde (MDA), lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and higher numbers of apoptotic germ cells. The testicular tissues from diabetic rats also expressed lower levels of Bcl-2 and higher levels of Bax and cleaved caspase-3 in addition to higher phosphorylation rates of c-Jun NH 2 -terminal protein kinase (JNK), p38, and extracellular signaling-regulated kinase (ERK) 1/2. The above parameters could be further increased or aggravated by the administration of GdCl 3 , but could be attenuated by injection of NPS-2390. In conclusion, the present results indicate that CaSR activation participates in diabetes-induced testicular damage, implying CaSR may be a potential target for protective strategies against diabetes-induced testicular damage and could help to prevent infertility in diabetic men.

Effect of calcium-sensing receptor on intracellular calcium, cell proliferation and migration of SGC-7901 cell line / 临床与实验病理学杂志

Purpose To observe the functional expression of calcium sensing receptor ( CaSR) in human gastric cancer SGC-7901 cell line, the effect of CaSR on intracellular calcium, cell proliferation and migration of SCG-7901. Methods The expression and distribu-tion of CaSR were detected by Western blotting and immunofluorescence observation in SGC-7901. The intracellular concentration of free calcium ( [ Ca2+] i ) was determined by confocal laser scanning microscopy. MTT, flow cytometry and scratch test were used to an-alyze the impact of CaSR the proliferation and the migration capabilities of SGC-7901 cell. Results CaSR protein was expressed in SGC-7901. Extracellular calcium or calindol significantly increased the expression of [Ca2+]i, CaSR and E-cadherin;In addition, the migration capabilities were decreased. Conclusion CaSR is expressed in SGC-7901. The activation of CaSR induces the expression of E-cadherin, and decreases migration ability.

Effect of suppressing calcium-sensing receptor on rat myocardial H9c2 cell hypertrophy induced by angiotensin Ⅱ / 中华地方病学杂志

Objective To explore the effects and possible mechanism of calcium-sensing receptor(CaSR) in rat myocardial H9c2 cells hypertrophy model using angiotensin Ⅱ (Ang Ⅱ).Methods Cardiac hypertrophy model was established by treating cultured H9c2 cells with Ang Ⅱ in vitro.Hypertrophic H9c2 cells were treated with gadolinium chloride (GdCl3,a specific agonist of CaSR) and/or with Calhex231 (a specific inhibitor of CaSR) and 3-methyladenine (3-MA,a specific inhibitor of autophagy) to divided into 5 groups (six in each group):control,Ang Ⅱ,GdCl3 + Ang lⅡ,GdCl3 + Calhex231 + Ang Ⅱ,GdCl3 + 3-MA + Ang Ⅱ groups.To evaluate the status of H9c2 cells hypertrophy,protein content was determined through a coomassie brilliant blue protein kit and the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and the phosphorylation form (pCaMK Ⅱ/CaMK Ⅱ) was analyzed by Western blotting.The protein expression of CaSR,autophagy maker [Beclin-1,micmtubule-associated protein 1 light chain 3(LC3)Ⅱ/LC3 Ⅰ,P62] and Ca2+/calmodulin-dependent-protein kinase-kinase-β (CaMKKβ)-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway was analyzed by Western blotting.Results ①GdCl3 further increased H9c2 cells protein content [control group:(2.52 ± 0.84) g/L,Ang Ⅱ group:(8.72 ± 3.60) g/L GdCl3 + Ang Ⅱ group:(14.17 ± 4.49) g/L,all P ＜ 0.05] and the expression of CaSR (control group:0.22 ± 0.04,Ang Ⅱ group:0.43 ± 0.02,GdCl3 + Ang Ⅱ group:0.63 ± 0.08,all P ＜ 0.05) and pCaMK Ⅱ/CaMKⅡ (control group:0.25 ± 0.05,AngⅡ group:0.51 ± 0.03,GdCl3 + AngⅡ group:0.77 ± 0.06,all P＜ 0.05) induced by Ang Ⅱ.Calhex231 suppressed the increasing of hypertrophy indicators induced by GdCl3 [GdCl3 + Calhex231 + AngⅡ group,CaSR:0.41 ± 0.16,protein content:(9.92 ± 2.54) g/L,pCaMK Ⅱ/CaMKⅡ:0.58 ± 0.08,all P ＜ 0.05].②GdCl3 promoted the effect of Ang Ⅱ in regulation of autophagy such as Beclin-1 protein increased (control group:0.31 ± 0.06,AngⅡ group:0.55 ± 0.09,GdCl3 + AngⅡ group:0.74 ± 0.08,all P ＜ 0.05),LC3 Ⅱ/LC3 Ⅰ increased (control group:0.28 ± 0.06,Ang Ⅱ group:0.56 ± 0.10,GdCl3 + Ang Ⅱ group:1.00 ± 0.15,all P ＜ 0.05) and P62 protein decreased (control group:0.54 ± 0.03,AngⅡ group:0.34 ± 0.02,GdCl3 + AngⅡ group:0.15 ± 0.03,all P ＜ 0.05).Moreover,Calhex231 suppressed autophagy induced by GdCl3 (GdCl3 + Calhex231 + Ang Ⅱ group,Beclin-1:0.53 ± 0.14,LC3 Ⅱ/LC3 Ⅰ:0.57 ± 0.12,P62:0.28 ± 0.05,all P ＜ 0.05).③GdCl3 increased pCaMKKβ/CaMKKβ (control group:0.43 ± 0.09,AngⅡ group:0.76 ± 0.12,GdCl3 + AngⅡ group:1.19 ± 0.21,all P ＜ 0.05),pAMPK/AMPK (control group:0.38 ± 0.11,AngⅡ group:0.68 ± 0.08,GdCl3 + AngⅡ group:1.18 ± 0.08,all P ＜ 0.05) and decreased pmTOR/mTOR (control group:0.90 ± 0.10,Ang Ⅱ group:0.54 ± 0.04,GdCl3 + AngⅡ group:0.29 ± 0.09,all P ＜ 0.05).Furthermore,Calhex231 blocked the effect of GdCl3 on the above-mentioned proteins changes (GdCl3 + Calhex231 + Ang Ⅱ group,pCaMKKβ/CaMKKβ:0.75 ± 0.06,pAMPK/AMPK:0.57 ± 0.05,pmTOR/mTOR:0.51 ± 0.08,all P ＜ 0.05).Conclusion Inhibiting calcium-sensing receptor expression has reversed H9c2 cell hypertrophy induced by Ang Ⅱ,which may be related to suppressing autophagy and suppressing CaMKKβ-AMPK-mTOR pathway.

Calcium-sensing receptor modulates pulmonary artery tension through G-protein-PLC-IP3 pathways / 中国病理生理杂志

AIM:To observe the role of calcium-sensing receptor (CaSR) in the regulation of pulmonary artery tension.METHODS:The intracellular calcium concentration ([Ca2+]i) was detected by laser-scanning confocal micros-copy, and the pulmonary artery tension was determined by the pulmonary arterial ring technique .RESULTS: Increased levels of [Ca2+]o or Gd3+(an agonist of CaSR) induced the increase in [Ca2+]i and pulmonary artery constriction in a concentration-dependent manner.Additionally, the effects of Ca2+and Gd3+were inhibited by U73122 and D609 (specific inhibitor of PLC), and 2-APB and heparin (specific antagonist of IP3 receptor).However, U73343 (U73122 inactive ana-logue) did not take effect.CONCLUSION: CaSR may be involved in the regulation of pulmonary artery tension by in-creasing [Ca2+]i through G-protein-PLC-IP3 pathway.