RESUMEN
To investigate the association between store-operated Ca2+ entry (SOCE) and reactive oxygen species (ROS) during hypoxia, this study determined the changes of transient receptor potential canonical 1 (TRPC1) and Orai1, two candidate proteins for store-operated Ca2+ (SOC) channels and their gate regulator, stromal interaction molecule 1 (STIM1), in a hypoxic environment and their relationship with ROS in pulmonary arterial smooth muscle cells (PASMCs). Exposure to hypoxia caused a transient Ca2+ spike and subsequent Ca2+ plateau of SOCE to be intensified in PASMCs when TRPC1, STIM1, and Orai1 were upregulated. SOCE in cells transfected with specific short hairpin RNA (shRNA) constructs was almost completely eliminated by the knockdown of TRPC1, STIM1, or Orai1 alone and was no longer affected by hypoxia exposure. Hypoxia-induced SOCE enhancement was further strengthened by PEG-SOD but was attenuated by PEG-catalase, with correlated changes to intracellular hydrogen peroxide (H2O2) levels and protein levels of TRPC1, STIM1, and Orai1. Exogenous H2O2 could mimic alterations of the interactions of STIM1 with TRPC1 and Orai1 in hypoxic cells. These findings suggest that TRPC1, STIM1, and Orai1 are essential for the initiation of SOCE in PASMCs. Hypoxia-induced ROS promoted the expression and interaction of the SOC channel molecules and their gate regulator via their converted product, H2O2.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Peróxido de Hidrógeno/farmacología , Hipoxia/metabolismo , Miocitos del Músculo Liso/metabolismo , Arteria Pulmonar/patología , Animales , Catalasa/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Hipoxia/genética , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Polietilenglicoles/metabolismo , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Superóxido Dismutasa/metabolismo , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Pepsinogen A (PGA)/pepsin A is often used as a diagnostic marker of extra-gastroesophageal reflux. We aimed to explore whether its positivity in upper aerodigestive tract (UADT) was specific enough to diagnose reflux. METHODS: PGA/pepsin A protein levels were examined in 10 types of tissues and 10 types of body fluid by immunological staining, western blot or Elisa, using three different commercially available brands simultaneously. Liquid chromatography-tandem mass spectrometry parallel reaction monitoring (LC-MS/MS PRM) served as a gold reference for the detection of PGA/pepsin A proteins. PGA gene expression was analyzed by reverse transcriptase sequencing methods for tissue samples. Specifically, 24 hour pH monitoring technique was conducted for patients who donated saliva samples. RESULTS: Eight out of ten types of human tissue samples (stomach, esophagus, lung, kidney, colon, parotid gland, nasal turbinate and nasal polyps) were confirmed positive for PGA/pepsin A gene and protein by genetic and PRM technique, respectively. Two out of ten types of body fluid samples (gastric fluid, urine) were confirmed positive for PGA/pepsin A protein by PRM technique. The consistence rates of PGA/pepsin A positivity among three commercial antibody brands and Elisa kit were poor, and Elisa results of salivary did not match with 24-hour pH monitoring. CONCLUSIONS: Multiple tissues and body fluid could be detected baseline expression levels of PGA/pepsin A gene and protein. However, those commercially available PGA/pepsin A antibodies achieved poor sensitivity and specificity, therefore, relying on the detection of PGA/pepsin A in UADT by single antibodies to diagnose extra-gastroesophageal reflux without a specific positive cut-off value is unreliable.