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1.
Drug Dev Res ; 82(6): 784-788, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33687087

RESUMO

Over the recent decades, a number of new pathogens have emerged within specific and diverse populations across the globe, namely, the Nipah virus, the Ebola virus, the Zika virus, and coronaviruses (CoVs) to name a few. Recently, a new form of coronavirus was identified in the city of Wuhan, China. Interestingly, the genomic architecture of the virus did not match with any of the existing genomic sequencing data of previously sequenced CoVs. This had led scientists to confirm the emergence of a new CoV strain. Originally, named as 2019-nCoV, the strain is now called as SARS-CoV-2. High serum levels of proinflammatory mediators, namely, interleukin-12 (IL-12), IL-1ß, IL-6, interferon-gamma (IFNγ), chemoattractant protein-1, and IFN-inducible protein, have been repeatedly observed in subjects who were infected with this virus. In addition, the virus demonstrated strong coagulation activation properties, leading to further the understanding on the SARS-CoV2. To our understanding, these findings are unique to the published literature. Numerous studies have reported anomalies, namely, decline in the number of lymphocytes, platelets and albumins; and a rise in neutrophil count, aspartate transaminase, alanine aminotransaminase, lactate dehydrogenase, troponins, creatinine, complete bilirubin, D-dimers, and procalcitonin. Supplementation of calcium during the SARS CoV-2 associated hyperactive stage of calcium-sensing receptors (CaSR) may be harmful to the cardio-renal system. Thus, pharmacological inhibition of CaSR may prevent the increase in the levels of intracellular calcium, oxidative, inflammatory stress, and cardio-renal cellular apoptosis induced by high cytokines level in COVID-19 infection.


Assuntos
COVID-19/metabolismo , Proteínas do Envelope de Coronavírus/metabolismo , Receptores de Detecção de Cálcio/metabolismo , SARS-CoV-2/metabolismo , Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , COVID-19/virologia , Cálcio/metabolismo , Humanos , Terapia de Alvo Molecular , Receptores de Detecção de Cálcio/efeitos dos fármacos , Tratamento Farmacológico da COVID-19
2.
Am J Physiol Lung Cell Mol Physiol ; 319(6): L1010-L1020, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32964725

RESUMO

Phenylalanine levels are associated with pulmonary hypertension in metabolic profiling clinical studies. However, the pathophysiological role of phenylalanine on pulmonary circulation is still unclear. We experimentally addressed the direct impact of phenylalanine on pulmonary circulation in rats and explored the underlying molecular pathway. Phenylalanine was injected intraperitoneally into Sprague-Dawley rats (400 mg/100 g body wt) as a single dose or daily in a chronic manner for 2, 3, and 4 wk. Chronic injection of phenylalanine induced pulmonary hypertension with time-dependent severity, evidenced by elevated pulmonary artery pressure and pulmonary vascular resistance as well as pulmonary artery and right ventricular hypertrophy. Using tandem mass spectrometry analysis, we found a quick twofold increase in blood level of phenylalanine 2 h following injection. This increase led to a significant accumulation of phenylalanine in lung after 4 h, which remained sustained at up to a threefold increase after 4 wk. In addition, a cellular thermal shift assay with lung tissues from phenylalanine-injected rats revealed the binding of phenylalanine to the calcium-sensing receptor (CaSR). In vitro experiments with cultured pulmonary arterial smooth muscle cells showed that phenylalanine activated CaSR, as indicated by an increase in intracellular calcium content, which was attenuated or diminished by the inhibition or knockdown of CaSR. Finally, the global knockout or lung-specific knockdown of CaSR significantly attenuated phenylalanine-induced pulmonary hypertension. Chronic phenylalanine injection induces pulmonary hypertension through binding to CaSR and its subsequent activation. Here, we demonstrate a pathophysiological role of phenylalanine in pulmonary hypertension through the CaSR. This study provides a novel animal model for pulmonary hypertension and reveals a potentially clinically significant role for this metabolite in human pulmonary hypertension as a marker, a mediator of disease, and a possible therapeutic target.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Hipertensão Pulmonar/metabolismo , Fenilalanina/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Animais , Sinalização do Cálcio/fisiologia , Hipertensão Pulmonar/induzido quimicamente , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/metabolismo
3.
Expert Rev Endocrinol Metab ; 15(5): 299-310, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32552012

RESUMO

INTRODUCTION: The calcium-sensing receptor is an important treatment target for secondary hyperparathyroidism (SHPT) in patients undergoing dialysis. In addition to vitamin D receptor activator, cinacalcet has recently been widely used for SHPT management, and the significant suppression of parathyroid hormone (PTH) with better control of serum calcium and phosphorus has been reported. However, low adherence and insufficient dose escalation mainly due to frequent gastrointestinal adverse events, still remain as major issues. To overcome these unmet needs, we have developed a new oral calcimimetic agent evocalcet, which has recently been approved by the Pharmaceutical Affairs Act in Japan. AREAS COVERED: PubMed was searched from inception until April 2020 with the word evocalcet to summarize the development of this new calcimimetic agent, its pharmacokinetics, and the results of clinical trials, along with an overview of the differences among calcimimetic agents. This review also includes the management of SHPT with a focus on calcimimetics. EXPERT OPINION: Evocalcet evoked fewer gastrointestinal-related adverse events while suppressing PTH at a lower dose than cinacalcet. These data suggest evocalcet may contribute to better adherence and sufficient dose escalation in patients with SHPT. Whether or not evocalcet improves clinical outcomes remains to be elucidated.


Assuntos
Calcimiméticos/uso terapêutico , Cinacalcete/uso terapêutico , Hiperparatireoidismo Secundário/tratamento farmacológico , Naftalenos/uso terapêutico , Pirrolidinas/uso terapêutico , Calcimiméticos/efeitos adversos , Cálcio/sangue , Cinacalcete/efeitos adversos , Gastroenteropatias/induzido quimicamente , Humanos , Hiperparatireoidismo Secundário/sangue , Japão , Naftalenos/efeitos adversos , Fósforo/sangue , Pirrolidinas/efeitos adversos , Receptores de Detecção de Cálcio/efeitos dos fármacos , Diálise Renal , Insuficiência Renal Crônica/tratamento farmacológico
4.
Am J Physiol Cell Physiol ; 318(5): C954-C968, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32186932

RESUMO

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.


Assuntos
Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.5/genética , Hipertensão Arterial Pulmonar/genética , Receptores de Detecção de Cálcio/genética , Molécula 2 de Interação Estromal/genética , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estrenos/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Proteína Jagged-1/genética , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Pirrolidinonas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores Notch/genética , Análise de Célula Única
5.
Biomed Pharmacother ; 125: 109931, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32066040

RESUMO

κ-opioid receptor (κ-OR) plays a key role in preventing hypoxic pulmonary hypertension (HPH) development after activated by exogenous agonist U50,488H. Calcium sensing receptor (CaSR) activation induces HPH by promoting vasoconstriction and vascular remodeling. The activated κ-OR is reported to inhibit the expression of CaSR in pulmonary artery smooth muscle cells (PASMCs). Thus, in this study, we aimed to explore the effect of activated κ-OR on the role of CaSR in preventing HPH development. An HPH rat model was constructed using Sprague-Dawley rats. Changes in mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) mediated by κ-OR agonist U50,488H and CaSR inhibitor NPS2143 were observed. The effects of CaSR agonist spermine and inhibitor NPS2143 on pulmonary artery tension were tested. The expression and localization of κ-OR and CaSR were measured in isolated PASMCs. A cell-counting kit-8 assay was performed to evaluate the effect of spermine in PASMC proliferation. Expression of proliferating cell nuclear antigen (PCNA), Erk, and p-Erk was evaluated by western blot analysis. Results showed that κ-OR and CaSR were co-expressed and colocalized in PASMCs under normoxic and hypoxic conditions. Interactions between κ-OR and CaSR were also observed. Spermine improved vasoconstriction in the pulmonary artery in HPH rats, which was abolished by U50,488H. RVP and mPAP were significantly increased in HPH rats under CaSR stimulation, but were significantly reduced when the rats were pretreated with U50,488H and NPS2143 (P < 0.01). Spermine treatment significantly promoted PASMC proliferation, which was significantly inhibited by U50,488H, p38 inhibitor SB203580, JNK inhibitor SP600125, Erk inhibitor SCH772984, and MEK inhibitor U0126, especially Erk inhibitor (P < 0.01). Spermine significantly increased PCNA and P-Erk expression in hypoxic conditions, which was inhibited by U50,488H and NPS2143. κ-OR stimulation prevented HPH development via the CaSR/MAPK signaling pathway.


Assuntos
(trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Hipertensão Pulmonar/prevenção & controle , Naftalenos/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Receptores Opioides kappa/agonistas , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Hipóxia/complicações , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Espermina/farmacologia , Remodelação Vascular/efeitos dos fármacos
7.
Nat Rev Endocrinol ; 15(1): 33-51, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30443043

RESUMO

The Ca2+-sensing receptor (CaSR) is a dimeric family C G protein-coupled receptor that is expressed in calcitropic tissues such as the parathyroid glands and the kidneys and signals via G proteins and ß-arrestin. The CaSR has a pivotal role in bone and mineral metabolism, as it regulates parathyroid hormone secretion, urinary Ca2+ excretion, skeletal development and lactation. The importance of the CaSR for these calcitropic processes is highlighted by loss-of-function and gain-of-function CaSR mutations that cause familial hypocalciuric hypercalcaemia and autosomal dominant hypocalcaemia, respectively, and also by the fact that alterations in parathyroid CaSR expression contribute to the pathogenesis of primary and secondary hyperparathyroidism. Moreover, the CaSR is an established therapeutic target for hyperparathyroid disorders. The CaSR is also expressed in organs not involved in Ca2+ homeostasis: it has noncalcitropic roles in lung and neuronal development, vascular tone, gastrointestinal nutrient sensing, wound healing and secretion of insulin and enteroendocrine hormones. Furthermore, the abnormal expression or function of the CaSR is implicated in cardiovascular and neurological diseases, as well as in asthma, and the CaSR is reported to protect against colorectal cancer and neuroblastoma but increase the malignant potential of prostate and breast cancers.


Assuntos
Calcimiméticos/uso terapêutico , Hipercalcemia/congênito , Hipercalciúria/genética , Hipocalcemia/genética , Hipoparatireoidismo/congênito , Nefrolitíase/genética , Receptores de Detecção de Cálcio/genética , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença/epidemiologia , Humanos , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Hipercalcemia/fisiopatologia , Hipercalciúria/tratamento farmacológico , Hipercalciúria/fisiopatologia , Hipocalcemia/tratamento farmacológico , Hipocalcemia/fisiopatologia , Hipoparatireoidismo/tratamento farmacológico , Hipoparatireoidismo/genética , Hipoparatireoidismo/fisiopatologia , Incidência , Masculino , Mutação/genética , Nefrolitíase/tratamento farmacológico , Nefrolitíase/fisiopatologia , Prognóstico , Receptores de Detecção de Cálcio/efeitos dos fármacos , Medição de Risco , Resultado do Tratamento
8.
Clin Respir J ; 12(4): 1518-1525, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28862394

RESUMO

BACKGROUND: Despite using vasoactive and pulmonary hypertension (PH) specific therapies, the in-hospital mortality of severe PH with right heart failure (RHF) is high. We conducted a prospective analysis evaluating the efficacy and safety of levosimendan in PH patients with severe acute RHF. METHODS: Forty-five PH patients hospitalized between January 2016 and November 2016 were recruited into a single arm, prospective, open-label study. Levosimendan was administered at the rate of .05-0.1 µg/kg/min, up to a total dose of 12.5 mg. The primary endpoints were changes of World Health Organization Function Class (WHO-FC) and Borg dyspnoea scores. Secondary endpoints included changes in 6-min walk distance (6-MWD), biochemical markers and right heart structure and function together with adverse events on day 7 and incidence of major cardiovascular events (death or readmission due to RHF) on day 30. RESULTS: Forty-five PH patients were enrolled. On the 7th day after levosimendan infusion, seven out of 13 PH patients with WHO-FC IV improved by one class (P = .008). Borg dyspnoea scores, 6-MWD and NT-proBNP improved significantly (P < .001). Compared with baseline, the right atrial transverse dimension, end-systolic eccentricity index and tricuspid annular plane systolic excursion improved significantly (58.8 ± 13.1 mm vs 53.7 ± 12.4 mm; 1.50 ± 0.27 vs 1.38 ± 0.23; 15.0 (13.0, 16.0) mm vs 15.8 (14.0, 17.4) mm, P < .005, respectively). One patient occurred sudden death after discharge during follow-up. CONCLUSIONS: Intravenous levosimendan can effectively improve severe RHF of PH patients in hospital and well tolerated.


Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Hidrazonas/administração & dosagem , Hipertensão Pulmonar/tratamento farmacológico , Piridazinas/administração & dosagem , Receptores de Detecção de Cálcio/efeitos dos fármacos , Função Ventricular Direita , Doença Aguda , Adolescente , Adulto , Idoso , Cardiotônicos/administração & dosagem , China/epidemiologia , Relação Dose-Resposta a Droga , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/mortalidade , Humanos , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/fisiopatologia , Injeções Intravenosas , Pessoa de Meia-Idade , Estudos Prospectivos , Pressão Propulsora Pulmonar/fisiologia , Simendana , Taxa de Sobrevida/tendências , Resultado do Tratamento , Adulto Jovem
9.
Reprod Fertil Dev ; 30(2): 391-398, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28712411

RESUMO

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.


Assuntos
Blastocisto/metabolismo , Sinalização do Cálcio , Fase de Clivagem do Zigoto/metabolismo , Fertilização in vitro , Oócitos/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Espermatozoides/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Calcimiméticos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Fase de Clivagem do Zigoto/efeitos dos fármacos , Técnicas de Cultura Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Oócitos/efeitos dos fármacos , Fenetilaminas/farmacologia , Propilaminas/farmacologia , Quinoxalinas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores de Detecção de Cálcio/genética , Espermatozoides/efeitos dos fármacos , Sus scrofa
10.
Vascul Pharmacol ; 96-98: 53-62, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28867591

RESUMO

Stimulation of calcium-sensing receptors (CaSR) by increasing the external calcium concentration (Ca2+]o) induces endothelium-dependent vasorelaxation through nitric oxide (NO) production and activation of intermediate Ca2+-activated K+ currents (IKCa) channels in rabbit mesenteric arteries. The present study investigates the potential role of heteromeric TRPV4-TRPC1 channels in mediating these CaSR-induced vascular responses. Immunocytochemical and proximity ligation assays showed that TRPV4 and TRPC1 proteins were expressed and co-localised at the plasma membrane of freshly isolated endothelial cells (ECs). In wire myography studies, increasing [Ca2+]o between 1 and 6mM induced concentration-dependent relaxations of methoxamine (MO)-induced pre-contracted tone, which were inhibited by the TRPV4 antagonists RN1734 and HC067047, and the externally-acting TRPC1 blocking antibody T1E3. In addition, CaSR-evoked NO production in ECs measured using the fluorescent NO indicator DAF-FM was reduced by RN1734 and T1E3. In contrast, [Ca2+]o-evoked perforated-patch IKCa currents in ECs were unaffected by RN1734 and T1E3. The TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent relaxation of MO-evoked pre-contracted tone and increased NO production, which were inhibited by the NO synthase inhibitor L-NAME, RN1734 and T1E3. GSK activated 6pS cation channel activity in cell-attached patches from ECs which was blocked by RN1734 and T1E3. These findings indicate that heteromeric TRPV4-TRPC1 channels mediate CaSR-induced vasorelaxation through NO production but not IKCa channel activation in rabbit mesenteric arteries. This further implicates CaSR-induced pathways and heteromeric TRPV4-TRPC1 channels in regulating vascular tone.


Assuntos
Células Endoteliais/metabolismo , Artéria Mesentérica Superior/metabolismo , Óxido Nítrico/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPV/metabolismo , Vasodilatação , Animais , Sinalização do Cálcio , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Técnicas In Vitro , Masculino , Potenciais da Membrana , Artéria Mesentérica Superior/efeitos dos fármacos , Coelhos , Receptores de Detecção de Cálcio/efeitos dos fármacos , Canais de Cátion TRPC/efeitos dos fármacos , Canais de Cátion TRPV/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-28431775

RESUMO

Cinacalcet hydrochloride is a calcimimetic agent that increases the sensitivity to the extracellular calcium of the calcium-sensing receptors of the parathyroid gland which regulates parathyroid hormone secretion. This comprehensive profile on cinacalcet hydrochloride starts with a description: nomenclature, formulae, chemical structure, elemental composition, and appearance. The uses and applications of the drug are included. The methods of preparation of cinacalcet hydrochloride are described and their respective schemes are outlined. The physical characterization of the drug is: ionization constant, solubility, X-ray powder diffraction (XRPD) pattern, crystal polymorphs, melting point, and differential scanning calorimetry. The spectral characteristics of the drug include: ultraviolet spectrum, vibrational spectrum, 1H and 13C nuclear magnetic resonance spectra, and the mass spectrum. The methods of analysis of the drug include: spectrophotometry, electrophoresis, fluorimetry, and high-performance liquid chromatography alone or with mass spectrometry. The stability of the drug in various media and storage conditions are reported. Biological studies on the drug include: the metabolism pharmacokinetics and pharmacodynamics. More than 100 references are listed at the end of the chapter.


Assuntos
Calcimiméticos , Cinacalcete , Calcimiméticos/química , Calcimiméticos/farmacologia , Calcimiméticos/uso terapêutico , Cinacalcete/química , Cinacalcete/farmacologia , Cinacalcete/uso terapêutico , Humanos , Hipercalcemia/tratamento farmacológico , Hiperparatireoidismo Secundário/tratamento farmacológico , Estrutura Molecular , Receptores de Detecção de Cálcio/efeitos dos fármacos
12.
J Cell Physiol ; 232(9): 2508-2518, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27662515

RESUMO

Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 (ET-1) system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here, we hypothesized that ET-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1 , ETB2 , and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of ET-1 on cardiomyocytes. Indeed, ET-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions, cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Inhibition of ETA and ETB1 receptors led to ET-1-dependent reduction in cell shortening and attenuated up-regulation of CaR. Down-regulation of RAMP-1 reduced CaR responsiveness. In conclusion, ET-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA and/or ETB1 receptors. J. Cell. Physiol. 232: 2508-2518, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Cardiomegalia/metabolismo , Endotelina-1/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Receptores de Detecção de Cálcio/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Adaptação Fisiológica , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Preparação de Coração Isolado , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Comunicação Parácrina , Interferência de RNA , Ratos Wistar , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Receptor de Endotelina A/agonistas , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/agonistas , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Regulação para Cima
13.
Oxid Med Cell Longev ; 2016: 5203801, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27965733

RESUMO

Objective. To investigate whether calcium-sensing receptor (CaSR) plays a role in calcium-oxalate-induced renal injury. Materials and Methods. HK-2 cells and rats were treated with calcium oxalate (CaOx) crystals with or without pretreatment with the CaSR-specific agonist gadolinium chloride (GdCl3) or the CaSR-specific antagonist NPS2390. Changes in oxidative stress (OS) in HK-2 cells and rat kidneys were assessed. In addition, CaSR, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 expression was determined. Further, crystal adhesion assay was performed in vitro, and the serum urea and creatinine levels and crystal deposition in the kidneys were also examined. Results. CaOx increased CaSR, ERK, JNK, and p38 protein expression and OS in vitro and in vivo. These deleterious changes were further enhanced upon pretreatment with the CaSR agonist GdCl3 but were attenuated by the specific CaSR inhibitor NPS2390 compared with CaOx treatment alone. Pretreatment with GdCl3 further increased in vitro and in vivo crystal adhesion and renal hypofunction. In contrast, pretreatment with NPS2390 decreased in vitro and in vivo crystal adhesion and renal hypofunction. Conclusions. CaOx-induced renal injury is related to CaSR-mediated OS and increased mitogen-activated protein kinase (MAPK) signaling, which subsequently leads to CaOx crystal adhesion.


Assuntos
Oxalato de Cálcio/toxicidade , Células Epiteliais/efeitos dos fármacos , Rim/efeitos dos fármacos , Nefrolitíase/metabolismo , Receptores de Detecção de Cálcio/efeitos dos fármacos , Receptores de Detecção de Cálcio/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Linhagem Celular , Cristalização , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Etilenoglicol , Gadolínio/farmacologia , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nefrolitíase/induzido quimicamente , Nefrolitíase/patologia , Nefrolitíase/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Quinoxalinas/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
14.
Oncotarget ; 7(13): 16112-29, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26893368

RESUMO

The calcium-sensing receptor is a G protein-coupled receptor that exerts cell-type specific functions in numerous tissues and some cancers. We have previously reported that this receptor exhibits tumor suppressor properties in neuroblastoma. We have now assessed cinacalcet, an allosteric activator of the CaSR approved for clinical use, as targeted therapy for this developmental tumor using neuroblastoma cell lines and patient-derived xenografts (PDX) with different MYCN and TP53 status. In vitro, acute exposure to cinacalcet induced endoplasmic reticulum stress coupled to apoptosis via ATF4-CHOP-TRB3 in CaSR-positive, MYCN-amplified cells. Both phenotypes were partially abrogated by phospholipase C inhibitor U73122. Prolonged in vitro treatment also promoted dose- and time-dependent apoptosis in CaSR-positive, MYCN-amplified cells and, irrespective of MYCN status, differentiation in surviving cells. Cinacalcet significantly inhibited tumor growth in MYCN-amplified xenografts and reduced that of MYCN-non amplified PDX. Morphology assessment showed fibrosis in MYCN-amplified xenografts exposed to the drug. Microarrays analyses revealed up-regulation of cancer-testis antigens (CTAs) in cinacalcet-treated MYCN-amplified tumors. These were predominantly CTAs encoded by genes mapping on chromosome X, which are the most immunogenic. Other modulated genes upon prolonged exposure to cinacalcet were involved in differentiation, cell cycle exit, microenvironment remodeling and calcium signaling pathways. CTAs were up-regulated in PDX and in vitro models as well. Moreover, progressive increase of CaSR expression upon cinacalcet treatment was seen both in vitro and in vivo. In summary, cinacalcet reduces neuroblastoma tumor growth and up-regulates CTAs. This effect represents a therapeutic opportunity and provides surrogate circulating markers of neuroblastoma response to this treatment.


Assuntos
Antígenos de Neoplasias/biossíntese , Antineoplásicos/farmacologia , Cinacalcete/farmacologia , Neuroblastoma/patologia , Animais , Antígenos de Neoplasias/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
16.
PLoS One ; 9(12): e115178, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25506941

RESUMO

INTRODUCTION: Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants. METHODS: All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914. RESULTS: All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants. CONCLUSION: The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.


Assuntos
Síndrome de Bartter/genética , Sinalização do Cálcio/efeitos dos fármacos , Hipercalciúria/genética , Hipocalcemia/genética , Hipoparatireoidismo/congênito , Naftalenos/farmacologia , Quinazolinonas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio/genética , Células HEK293 , Humanos , Hipoparatireoidismo/genética , Mutação , Receptores de Detecção de Cálcio/genética
17.
Cardiovasc Res ; 101(2): 256-65, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24217682

RESUMO

AIMS: Vascular calcification (VC) contributes to morbidity and mortality in patients with chronic kidney disease (CKD). Allosteric modulators of the calcium (Ca)-sensing receptor (CaSR) may slow the progression of VC in CKD patients either by reducing serum parathyroid hormone (PTH), Ca, and phosphate levels or by a direct effect on the vessel wall. The aim of this study was to examine the effects of calcimimetics on CaSR expression, cell phenotype, and mineral deposition in human vascular smooth muscle cells (h-VSMCs). METHODS AND RESULTS: Primary h-VSMCs were exposed for 14 days to increasing concentrations of Ca(2+) (from 1.8 to 5 mmol/L) in the presence or absence of calcimimetics R-568 or AMG 641 (0.1 µmol/L). Mineralization was detected by Alizarin red staining, and the cell phenotype was assessed using immunocytochemistry and qRT-PCR. CaSR expression was evaluated using flow cytometry. Short- and long-term exposure (1 day to 14 days) of h-VSMCs to calcimimetics promoted CaSR protein transport from the endoplasmic reticulum to the plasma membrane with enhanced CaSR expression on the cell surface, together with an increase in total cell CaSR expression due to enhanced biosynthesis. In pro-mineralizing conditions, exposure to calcimimetics counteracted the Ca(2+)-dependent reduction of CaSR expression, decreased matrix collagen secretion, and mineral deposition by ~90%. These effects involved CaSR activation since it could be inhibited by CaSR siRNA, but not scrambled siRNA. CONCLUSIONS: The calcimimetic-dependent increase in biosynthesis and activation of the CaSR in h-VSMCs probably play a key role in the protection against calcium-induced VC.


Assuntos
Compostos de Anilina/farmacologia , Compostos de Bifenilo/farmacologia , Calcimiméticos/farmacologia , Cálcio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fenetilaminas/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Calcificação Vascular/prevenção & controle , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Propilaminas , Transporte Proteico , Interferência de RNA , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima , Calcificação Vascular/genética , Calcificação Vascular/metabolismo
18.
Circ Res ; 112(4): 640-50, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23300272

RESUMO

RATIONALE: An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH). OBJECTIVE: Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC. METHODS AND RESULTS: The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt). CONCLUSIONS: The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.


Assuntos
Bloqueadores dos Canais de Cálcio/efeitos adversos , Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Hipertensão Pulmonar/induzido quimicamente , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/efeitos adversos , Artéria Pulmonar/citologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/ultraestrutura , Citosol/metabolismo , Progressão da Doença , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Fosfatos de Inositol/fisiologia , Masculino , Monocrotalina/toxicidade , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Naftalenos/farmacologia , Naftalenos/uso terapêutico , Nifedipino/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Regulação para Cima/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos
19.
Reprod Sci ; 20(8): 882-90, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23287098

RESUMO

Ligands for extracellular calcium-sensing (CaS) receptors inhibit oxytocin-induced contractions of the rat's uterus. In this study, we investigated whether the CaS receptor ligands calindol, cinacalcet, and calhex 231 have similar effects on pregnant human myometrium. We compared their effects to those of the calcium-channel blocker nifedipine. In conventional concentration-effect experiments, both the mean contractile force (MCF) and the maximum amplitude of contractions induced by 1 nmol/L oxytocin were inhibited by nifedipine. Calindol and cinacalcet were ineffective as inhibitors, while calhex-231 produced partial inhibition. When single 10 µmol/L doses were applied calhex-231 produced a slowly developing inhibition, reducing the MCF to 38%, and amplitude to 34%, of vehicle controls after 1 hour. In similar experiments, calindol was ineffective while cinacalcet weakly inhibited only the amplitude. Immunohistochemistry revealed sparse expression of CaS receptors in pregnant human myometrium.


Assuntos
Benzamidas/farmacologia , Cicloexilaminas/farmacologia , Indóis/farmacologia , Miométrio/efeitos dos fármacos , Naftalenos/farmacologia , Receptores de Detecção de Cálcio/efeitos dos fármacos , Contração Uterina/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Cinacalcete , Relação Dose-Resposta a Droga , Feminino , Humanos , Técnicas In Vitro , Ligantes , Miométrio/metabolismo , Nifedipino/farmacologia , Ocitócicos/farmacologia , Ocitocina/farmacologia , Gravidez , Receptores de Detecção de Cálcio/metabolismo , Fatores de Tempo
20.
Am J Physiol Renal Physiol ; 304(3): F248-56, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23220722

RESUMO

The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.


Assuntos
Inositol 1,4,5-Trifosfato/metabolismo , Sistema Justaglomerular/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Renina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo , Animais , Calcimiméticos/farmacologia , Cálcio/metabolismo , Células Cultivadas , Cinacalcete , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Sistema Justaglomerular/citologia , Sistema Justaglomerular/efeitos dos fármacos , Camundongos , Modelos Animais , Naftalenos/farmacologia , Toxina Pertussis/farmacologia , Pirrolidinonas/farmacologia , Receptores de Detecção de Cálcio/agonistas , Receptores de Detecção de Cálcio/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores
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