Effects of estrogen on STIM1/Orai1 in the sublingual gland of ovariectomized rats.

BACKGROUND: Studies have shown that estrogen can protect the function of the sublingual gland, but the specific mechanism is still unclear. Besides, the STIM1/Orai1 pathway is important to secretion in the salivary gland. Here, we explore the possible effects of estrogen on sublingual gland function by observing changes of STIM1 and Orai1 levels in the sublingual glands of ovariectomized rats. METHODS: 42 adult female Sprague-Dawley rats were randomly divided into three groups: SHAM, OVX, and OVX+E (n = 14 per group). Two weeks after ovariectomy, rats were treated with estrogen (ß-estradiol). The expression of STIM1 and Orai1 in the sublingual gland were observed by double label-immunohistochemistry and immunofluorescence. Calcium imaging was conducted to observe changes in cellular Ca²âº levels. RESULTS: IHC and IF showed that the levels of both STIM1 and Orai1 decreased following ovariectomy, but increased to SHAM levels after estrogen treatment. By IF, STIM1 and Orai1 exhibited perfect co-localization. Calcium imaging results showed that the Ca²âº in the cells decreased after ovariectomy. Estrogen intervention returned levels of these proteins and Ca²âº to the same as those in the control group. CONCLUSION: This study demonstrates that low estrogen status significantly reduced the expression of STIM1 and Orai1 in the sublingual gland of rats, along with cellular Ca²âº levels. These data provide insight into the likely mechanisms underlying sublingual gland secretion dysfunction during menopause.

Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex.

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.

Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes In adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.

IL-4 and serum amyloid P inversely regulate fibrocyte differentiation by targeting store-operated Ca2+ channels.

BACKGROUND: Circulating fibrocytes (CFs) have been shown to participate in subepithelial fibrosis of asthma with chronic airflow limitation by acting as an important source of fibroblasts deposited beneath airway epithelia. Serum amyloid P (SAP) is an innate inhibitor of fibrocytes differentiation. Store-operated Ca2+ entry (SOCE) is the major Ca2+ influx of non-excitable cells. In this study, the role of SOCE in the regulation of fibrocytes differentiation and the effects of Th2 cytokine IL-4 and SAP on SOCE of fibrocytes were investigated. METHODS: Peripheral blood mononuclear cells or monocytes were cultured in serum-free medium for 7days to differentiate into fibrocytes; the expression of SOC channels was determined with PCR, SOCE was measured with Ca2+ fluorescence imaging. RESULTS: IL-4 significantly promoted monocyte derived fibrocytes differentiation in vitro. It also increased both SOCE which was induced by thapsigargin or UTP and molecules STIM1 and Orai1 which were related to expression of SOC channels in fibrocytes. Fibrocytes differentiation induced by IL-4 and SOC channels activity could be inhibited by SOC channel blocker SKF-96365. As expected, SAP significantly inhibited IL-4-induced differentiation of fibrocytes, the activity of SOCE and the expression of STIM1 and Orai1 in IL-4-treated fibrocytes. CONCLUSION: IL-4 and SAP reversely regulates cultured fibrocytes differentiation in vitro by respectively promoting or inhibiting the expression and activity of SOC channels in fibrocytes.

High-Salt Intake Augments the Activity of the RhoA/ROCK Pathway and Reduces Intracellular Calcium in Arteries From Rats.

BACKGROUND: We investigated the influence of salt overconsumption on the functionality of the RhoA/Rho-associated kinase (ROCK) pathway and calcium regulation in arteries. METHODS: The aorta and small mesenteric arteries from rats fed a chow containing 2%, 4%, or 8% NaCl were evaluated in organ baths for the activity of the RhoA/ROCK pathway and intracellular calcium mobilization. Components of these pathways and intracellular calcium levels were also assessed in samples from 4% NaCl group. RESULTS: In arteries from animals fed regular chow, the ROCK inhibitor Y-27632 reduced the responses to phenylephrine, even when the smallest concentrations (1 and 3 µM) were tested. However, only higher concentrations of Y-27632 (10 and 50 µM) reduced phenylephrine-induced contraction in vessels from high-salt groups. Immunoblotting revealed augmented phosphorylation of the myosin phosphatase targeting subunit 1 and increased amounts of RhoA in the membrane fraction of aorta homogenates from the 4% NaCl group. Under calcium-free solution, vessels from NaCl groups presented reduced contractile responses to phenylephrine and caffeine, compared with the regular chow group. Moreover, decreased intracellular calcium at rest and after stimulation with ATP were found in aortic smooth muscle cells from 4% NaCl-fed rats, which also showed diminished levels of SERCA2 and SERCA3, but not of IP3 and ryanodine receptors, or STIM1 and Orai1 proteins. CONCLUSIONS: Arteries from rats subjected to high-salt intake are unable to properly regulate intracellular calcium levels and present augmented activity of the calcium sensitization pathway RhoA/ROCK. These changes may precede the development of vascular diseases induced by high-salt intake.

Inhibition of SOCs Attenuates Acute Lung Injury Induced by Severe Acute Pancreatitis in Rats and PMVECs Injury Induced by Lipopolysaccharide.

Acute lung injury (ALI) is a critical complication of the severe acute pancreatitis (SAP), characterized by increased pulmonary permeability with high mortality. Pulmonary microvascular endothelial cells (PMVECs) injury and apoptosis play a key role in ALI. Previous studies indicated that store-operated calcium entry (SOCE) could regulate a variety of cellular processes. The present study was to investigate the effects of SOCE inhibition on ALI induced by SAP in Sprague-Dawley rats, and PMVECs injury induced by lipopolysaccharide (LPS). Rat model of SAP-associated ALI were established by the retrograde infusion of sodium deoxycholate. Serum levels of amylase, TNF-α, and IL-6, histological changes, water content of the lung, oxygenation index, and ultrastructural changes of PMVECs were examined in ALI rats with or without store-operated Ca(2+) channels (SOCs) pharmacological inhibitor (2-aminoethoxydiphenyl borate, 2-APB) pretreatment. For in vitro studies, PMVECs were transiently transfected with or without small interfering RNA (siRNA) against calcium release-activated calcium channel protein1 (Orai1) and stromal interaction molecule1 (STIM1), the two main molecular constituents of SOCs, then exposed to LPS. The viability of PMVECs was determined. The expression of STIM1, Orai1, Bax, and caspase3, both in lung tissue and in PMVECs, were assessed by quantitative real-time PCR and western blot. Administration of sodium deoxycholate upregulated the expression of SOCs proteins in lung tissue. Similarly, the SOCs proteins were increased in PMVECs induced by LPS. 2-APB reduced the serum levels of amylase, TNF-α, and IL-6, and attenuated lung water content and histological findings. In addition, the decreased oxygenation index and ultrastructural damage in PMVECs associated with SAP were ameliorated after administration of 2-APB. Knockdown of STIM1 and Orai1 inhibited LPS-induced PMVECs death. Furthermore, blockade of SOCE significantly suppressed Orai1, STIM1, Bax, and caspase3 expression both in vivo and in vitro. These results suggest that SOCE may play a critical role in SAP-associated ALI and the protective effects of inhibition of SOCs could be mediated, at least partially, by restraining mitochondrial associated apoptosis of PMVECs.