Channel Protein Expression Research Articles

ClC-3 chloride channels are involved in estradiol regulation of bone formation by MC3T3-E1 osteoblasts.

Evidence has been reported by us and others supporting the important roles of chloride channels in a number of osteoblast cell functions. The ClC-3 chloride channel is activated by estradiol binding to estrogen receptor alpha on the cell membranes of osteoblasts. However, the functions of these chloride channels in estrogen regulation of osteoblast metabolism remain unclear. In the present study, the roles of chloride channels in estrogen regulation of osteoblasts were investigated in the osteoblastic cell line MC3T3-E1. Estrogen 17β-estradiol enhanced collagen I protein expression, alkaline phosphatase activity, and mineralization were inhibited, by chloride channel blockers. Estradiol promoted ClC-3 chloride channel protein expression. Silencing of ClC-3 chloride channel expression prevented the elevation of osteodifferentiation in osteoblasts, which were regulated by estrogen. These data suggest that estrogen can regulate bone formation by activating ClC-3 chloride channels and the activation of ClC-3 chloride channels can enhance the osteodifferentiation in osteoblasts.

Atrial fibrillation and electrophysiology in transgenic mice with cardiac-restricted overexpression of FKBP12.

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.

Gene and Protein Expression Profile of Selected Molecular Targets Mediating Electrophysiological Function in Pgc-1α Deficient Murine Atria.

Increases in the prevalence of obesity, insulin resistance, and metabolic syndrome has led to the increase of atrial fibrillation (AF) cases in the developed world. These AF risk factors are associated with mitochondrial dysfunction, previously modelled using peroxisome proliferator activated receptor-γ (PPARγ) coactivator-1 (Pgc-1)-deficient murine cardiac models. We explored gene and protein expression profiles of selected molecular targets related to electrophysiological function in murine Pgc-1α−/− atria. qPCR analysis surveyed genes related to Na+-K+-ATPase, K+ conductance, hyperpolarisation-activated cyclic nucleotide-gated (Hcn), Na+ channels, Ca2+ channels, and indicators for adrenergic and cholinergic receptor modulation. Western blot analysis for molecular targets specific to conduction velocity (Nav1.5 channel and gap junctions) was performed. Transcription profiles revealed downregulation of molecules related to Na+-K+-ATPase transport, Hcn-dependent pacemaker function, Na+ channel-dependent action potential activation and propagation, Ca2+ current generation, calsequestrin-2 dependent Ca2+ homeostasis, and adrenergic α1D dependent protection from hypertrophic change. Nav1.5 channel protein expression but not gap junction expression was reduced in Pgc-1α−/− atria compared to WT. Nav1.5 reduction reflects corresponding reduction in its gene expression profile. These changes, as well as the underlying Pgc-1α−/− alteration, suggest potential pharmacological targets directed towards either upstream PGC-1 signalling mechanisms or downstream ion channel changes.

Retraction Note: Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation

This Article has been retracted.

PO-156 Effects of acute hypoxic exercise on TRPV4 channels in prefrontal cortex of rats

Objective At the early stage of high altitude training, the decrease of motor ability caused by hypoxia makes it impossible for the training to be carried out normally. Since 90s, researchers have focused more on the decrease of the cardiopulmonary function and muscle oxygen utilization capacity, but there are few studies on cerebral hemodynamic factors, and no related mechanism studies. This study will take this as a starting point, to study the acute hypoxic exercise and its biological mechanism for brain hemodynamic effects of prefrontal cortex, providing a new target in order to improve the athletes at the beginning of the plateau duration and decreased exercise capacity problems.
 Methods In order to explore the changes of the TRPV4 channels protein in rat prefrontal cortex induced by exercise under hypoxic exposure, this study used HE staining and immunohistochemical method to analyze the pathological changes and TRPV4 channel protein expression of prefrontal cortex in rats during incremental exercise in different oxygen concentration environment. 
 Results The results showed that cortex lesions characterized by venous congestion and neuronophagia, when exercise to level 5 under the condition of the simulated 4500 meters altitude, which was similar to normoxia exercise to level 8. With the increasing of exercise load, the expressions of TRPV4 in rat prefrontal cortex were significantly increased. And the TRPV4 expressions of every groups in the simulated 4500 meters altitude were significantly higher than those in normoxia groups. 
 Conclusions The results indicate that increased load exercise in acute hypoxia can lead to pathological changes of brain tissue much earlier that in normoxia condition, and increase the expression of TRPV4 channel protein in brain tissue. This study will provide valuable data for the study of the role of Ca2+ channels in the membrane of central nervous system in the phenomenon of the centers fatigue appearing earlier in hypoxic exercise.

Ischemic postconditioning confers cerebroprotection by stabilizing VDACs after brain ischemia

Ischemic postconditioning provides robust neuroprotection, therefore, determining the molecular events may provide promising targets for stroke treatment. Here, we showed that the expression of functional mitochondrial voltage-dependent anion channel proteins (VDAC1, VDAC2, and VDAC3) reduced in rat vulnerable hippocampal CA1 subfield after global ischemia. Ischemic postconditioning restored VDACs to physiological levels. Stabilized VDACs contributed to the benefits of postconditioning. VDAC1 was required for maintaining neuronal Ca2+ buffering capacity. We found that microRNA-7 (miR-7) was responsible for postischemic decline of VDAC1 and VDAC3. Notably, miR-7 was more highly expressed in the peripheral blood of patients with acute ischemic stroke compared to healthy controls. Inhibition of miR-7 attenuated neuronal loss and ATP decline after global ischemia, but also diminished the infarct volume with improved neurological functions after focal ischemia. Thus, ischemic postconditioning protects against mitochondrial damage by stabilizing VDACs. MiR-7 may be a potential therapeutic target for ischemic stroke.

Chronic stimulation of the sigma-1 receptor ameliorates autonomic nerve dysfunction and atrial fibrillation susceptibility in a rat model of depression.

The present study aimed to assess the effect of sigma-1 receptor (S1R) stimulation on autonomic nerve dysfunction and susceptibility to atrial fibrillation (AF) in a rat depression model. Male rats were randomly divided into one of the following four treatment groups: saline [control (CTL)]; saline + intragastric administration of SA4503, an agonist of S1R (CTS); chronic unpredictable mild stress (CUMS) to produce depression (MDD); and CUMS + intragastric administration of SA4503 (MDS). Depression-like behaviors, such as reduced sucrose preference, decreased body weight gain, and increased immobility time during forced swimming, improved in the MDS group after 4 wk of SA4503 treatment. Compared with rats in the CTL group, rats in the MDD group showed significantly augmented sympathetic activity, reduced parasympathetic activity, decreased heart rate variability, and lowered S1R expression in the atrium and hippocampus (all P < 0.01). However, rats in the MDS group showed mitigated aforementioned alterations and improved electrical remodeling compared with rats in the MDD group (all P < 0.01). Furthermore, rats in the MDS group showed shortened activation latencies, increased effective refractory periods, and lowered frequency of AF incidence duration and fibrosis compared with rats in the MDD group (all P < 0.01). The results indicate that S1R stimulation reduces sympathetic activity and susceptibility to AF by improving depressive behaviors, modulating cardiac autonomic nerve balance, lightening nerve remodeling, and upregulating S1R and ion channel protein expression. NEW & NOTEWORTHY Chronic stimulation of the sigma-1 receptor (S1R) ameliorates depression-induced autonomic nerve dysfunction by modulating the imbalance between overactivated sympathetic activity and decreased vagal activity. Chronic S1R stimulation alleviates atrial electrical remodeling, fibrosis, and susceptibility to atrial fibrillation (AF). The S1R agonist may target the underlying mechanisms related to AF occurrence. The results indicate that the S1R could be a potential clinical target for atrial arrhythmia, especially when it is combined with major depressive disorders.

Opening of the CLC-3 chloride channel induced by dihydroartemisinin contributed to early apoptotic events in human poorly differentiated nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a specific type of head and neck cancer that is prevalent in Southeast Asia. Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has specific anticancer activity. Here, we aimed to investigate the role of the CLC-3 chloride channel in the anticancer effect of DHA in poorly differentiated NPC CNE-2Z cells. First, we observed that DHA could specifically inhibit the proliferation, induce apoptosis, and increase cleaved caspase-3 expression in the CNE-2Z cells. Then, we found that DHA could activate chloride channels, which led to Cl- efflux and apoptotic volume decrease (AVD) in the early stage in the CNE-2Z cells. DHA also specifically increased CLC-3 chloride channel protein expression in the CNE-2Z cells. Silencing of the CLC-3 protein expression depleted the Cl- currents, and decreased the AVD capacity and cell apoptosis induced by DHA. Finally, we revealed that the [Ca2+ ]i increased after around 6 hours of treatment with DHA, which was also inhibited by silencing of the CLC-3 protein expression. Our data demonstrated that the selective antitumor activities of DHA in NPC may occur through the specific activation of the CLC-3 Cl- channel, leading to Cl- efflux, and induced AVD, then led to [Ca2+ ]i accumulation and caspase-3 activation, and finally induced apoptosis. The activation of the CLC-3 chloride channel played an essential and proximal upstream role in the antitumor activities of DHA.

An efficient nonlinear hybridization chain reaction-based sensitive fluorescent assay for in situ estimation of calcium channel protein expression on bone marrow cells

A sensitive and highly efficient approach to monitor the expression of proteins on live cells was urgently needed to demonstrate its factor and mechanism and most important for clinical diagnostics and molecular biology. Herein, we developed a simple and highly efficient strategy, nonlinear hybridization chain reaction (nonlinear HCR), for the sensitive determination of proteins on live cells with transient receptor potential vanilloid 4 (TRPV4) and RAW264.7 cells as a model. Unlike the normal hybridization chain reaction (HCR) with multiplicative amplification, an exponential amplified fluorescent response could be obtained in theory based on the proposed nonlinear HCR. As a result, the nonlinear HCR generated a significant enhancement about 3 times compared with the normal HCR and 10 times compared with the directly immunofluorescence assay. Based on the proposed nonlinear HCR, the fluorescent signals increased with the concentration of TRPV4 in the range from 10 pg/mL to 100 ng/mL with a detection limit of 2.8 pg/mL, which would be useful for the sensitive detection of proteins in cell lysis or on cell surface. At the same time, the significant improvements via nonlinear HCR were achieved in the fluorescent imaging system compared with traditional immunofluorescence staining and normal HCR, proving the significant value of nonlinear HCR-based amplification strategy. Success in the establishment of the highly efficient nonlinear HCR strategy offered a simple and sensitive approach to demonstrate the concentration of special proteins on cell and other proteins and nucleotide potentially, revealing a simple and efficient technology for research fields of clinical diagnostics and molecular biology.

Chronic exercise impairs nitric oxide pathway in rabbit carotid and femoral arteries.

Some of the beneficial effects of exercise in preventing vascular related diseases are mediated by the enhancement of endothelial function where the role of nitric oxide (NO) is well documented, although the relevance of calcium activated potassium channels is not fully understood. The impact of oxidative stress induced by training on endothelial function remains to be clarified. By evaluating different endothelial vasodilator pathways on two vascular beds in a rabbit model of chronic exercise, we found a decreased NO bioavailability and endothelial nitric oxide synthase expression in both carotid and femoral arteries. Physical training induced carotid endothelial dysfunction as a result of an increase in oxidative stress and a reduction in superoxide dismutase expression. In the femoral artery, the lower production of NO was counteracted by an increased participation of large conductance calcium activated potassium channels, preventing endothelial dysfunction. The present study aimed to evaluate the effects of chronic exercise on vasodilator response in two different arteries. Rings of carotid and femoral arteries from control and trained rabbits were suspended in organ baths for isometric recording of tension. Endothelial nitric oxide synthase (eNOS), Cu/Zn and Mn-superoxide dismutase (SOD), and large conductance calcium activated potassium (BKCa) channel protein expression were measured by western blotting. In the carotid artery, training reduced the relaxation to ACh (10-9 to 3×10-6 m) that was reversed by N-acetylcysteine (10-3 m). l-NAME (10-4 m) reduced the relaxation to ACh in both groups, although the effect was lower in the trained group (in mean ± SEM, 39±2% vs. 28±3%). Physical training did not modify the relaxation to ACh in femoral arteries, although the response to l-NAME was lower in the trained group (in mean ± SEM, 41±5% vs. 17±2%). Charybdotoxin (10-7 m) plus apamin (10-6 m) further reduced the maximal relaxation to ACh only in the trained group. The remaining relaxation in both carotid and femoral arteries was abolished by KCl (2×10-2 m) and BaCl2 (3×10-6 m) plus ouabain (10-4 m) in both groups. Physical training decreased eNOS expression in both carotid and femoral arteries and Cu/Zn and Mn-SOD expression only in the carotid artery. BKCa channels were overexpressed in the trained group in the femoral artery. In conclusion, chronic exercise induces endothelial dysfunction in the carotid artery as a result of oxidative stress. In the femoral artery, it modifies the vasodilator pathways, enhancing the participation of BKCa channels, thus compensating for the impairment of NO-mediated vasodilatation.

Neuropilin 1 ameliorates electrical remodeling at infarct border zones in rats after myocardial infarction.

Electrical remodeling at infarct border zone (IBZ) has been shown to contribute to the occurrence of ventricular arrhythmias after myocardial infarction (MI). Sema3A has been demonstrated to reduce the inducibility of ventricular arrhythmias. Neuropilin 1 (NRP1) is the receptor of Sema3A. In the present study, we investigated whether treatment with NRP1 can ameliorate electrical remodeling at IBZ after MI. Wistar rats underwent sham operation (n = 20), the ligation of left coronary artery (MI group, n = 30), MI with control adenovirus (Ad group, n = 30), and MI with NRP1 adenovirus (NRP1 group, n = 30). Eight weeks after treatment, electrophysiological properties including heart rate variability (HRV), monophasic action potential duration (MAPD), effective refractory period (ERP) and the inducibility of ventricular arrhythmias and the expression of arrhythmia-related ion channel proteins including Kv4.2, Kv4.3, KChIP2 and Kir2.1 at the IBZ of the left ventricle were examined. Compared with the MI or Ad group, NRP1 significantly increased HRV and shortened MAPD and ERP (all P < 0.05). Inducibility of VT by electrophysiological study was significantly lower in the NRP1 group than in the MI or Ad group (P < 0.05). The expression levels of Kv4.2, Kv4.3, KChIP2 and Kir2.1 proteins were significantly decreased in MI group and Ad group. In contrast, the expression levels of these proteins were restored in NRP1 group, which may represent the molecular basis of the NRP1-mediated inhibition of electrical remodeling. NRP1 can ameliorate electrical remodeling at IBZ after MI.

Downregulation of BK channel function and protein expression in coronary arteriolar smooth muscle cells of type 2 diabetic patients.

Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) channels, composed of four pore-forming α subunits (BK-α), and four regulatory β1 subunits (BK-β1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK channel function and protein expression, and BK channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Atrial tissues were obtained from 16 patients with T2D and 25 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the channel open probability. Functional studies showed that BK channel activation by dehydrosoyasaponin-1 was diminished and that BK channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-α and BK-β1 subunits were significantly downregulated, but the ratio of BK-α/BK-β1 was unchanged in the coronary arterioles of T2D patients. Our results demonstrated for the first time that BK channel function and BK channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK channels.

G protein pathway suppressor 2 enhanced the renal large-conductance Ca2+-activated potassium channel expression via inhibiting ERK1/2 signaling pathway.

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+-activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity ( NPo), increasing BK open probability ( Po), and channel number ( N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

Long non-coding RNA CACNA1G-AS1 promotes calcium channel protein expression and positively affects human keloid fibroblast migration.

Keloids are a type of benign hyperplasia that cause dermatologic dysfunction and esthetic deformity by invading adjacent normal tissues. Little is known about their etiology, therefore, they are a challenge to treat using plastic surgery. In a previous study, it was demonstrated that the expression of the long non-coding RNA CACNA1G-AS1 (CAS1) is high in keloid tissue, suggesting that CAS1 is involved in keloid formation. In the present study, the aim was to identify potential keloid target proteins by exploring CAS1 biological function during cell proliferation and migration, cytokine secretion, collagen secretion and the control of calcium channel protein expression in human keloid fibroblasts. Three biopsy samples were collected from each patient with keloids at The Peking Union Medical College Hospital, which were then used to investigate the role of CAS1 in cell proliferation and migration. CAS1 silencing was also carried out using small interfering RNA; cell factors, collagen and calcium channel protein levels were compared with control cells. The interference of CAS1 expression reached 50% compared with the control group. CACNA1G and type I collagen expression was significantly downregulated by CAS1 knockdown, while the expression of transforming growth factor-β and type III collagen was not affected. Wound healing time was longer in the CAS1-knockdown group, but there was no visible change in cell proliferation. In conclusion, CAS1 appeared to promote calcium channel protein and type I collagen expression, and to have a positive effect on cell migration in human keloid fibroblasts. Therefore it has potential as a novel therapeutic target for keloids.

S-adenosyl methionine regulates calcium channels and inhibits uterine smooth muscle contraction in rats with infectious premature delivery through the transient receptor protein 3/protein kinase Cβ/C-kinase-activated protein phosphatase-1 inhibitor of 17 kDa signaling pathway.

The aim of the present study was to investigate the effects of S-adenosyl methionine (SAMe) on infectious premature inflammatory factors and uterine contraction, and to further explore its mechanism of action via the transient receptor protein 3 (TRPC3)/protein kinase Cβ (PKCβ)/C-kinase-activated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) signaling pathway, following intervention by a TRPC3 inhibitor. A rat model of premature delivery induced by lipopolysaccharide (LPS) was established. Following treatment with SAMe and inhibiting TRPC3 expression, rat serum and uterus were isolated. Hematoxylin and eosin staining was used to observe the histopathological changes in the uterus. Uterine muscle strips in vitro were selected to measure the changes in muscle tension. ELISA was utilized to measure the changes in serum inflammatory factor and oxidative stress indexes. Immunohistochemistry, western blot assay and reverse transcription-quantitative polymerase chain reaction were applied to detect calcium channel protein expression in the uterus. Western blot analysis was employed to measure the expression of TRPC3/PKCβ/CPI-17 signaling pathway-related proteins. TRPC3 was highly expressed in the uterus of rat models of premature delivery induced by LPS. Following treatment with SAMe, inflammatory cell infiltration markedly reduced in the uterus and the tension of in vitro uterine muscle strips significantly decreased. SAMe treatment suppressed inflammatory reaction and oxidative stress, and diminished L-type and T-type calcium channel protein expression. TRPC3/PKCβ/CPI-17 signaling pathway-related protein expression was also reduced. When TRPC3 expression was suppressed, the effects of SAMe against inflammation and oxidative stress were diminished. TRPC3/PKCβ/CPI-17 signaling pathway-related protein expression significantly increased. SAMe was able to reduce inflammatory reaction and oxidative stress in the uterus of rat model of infectious premature delivery induced by LPS, prolong delivery time, reduce the mortality rate of offspring rats, and serve a therapeutic role. This effect is likely achieved via the regulation of uterine contractions and childbirth through the TRPC3/PKCβ/CPI-17 signaling pathway.

Pharmacological inhibition of TRPV4 channels protects against early renal hypoperfusion induced by ischemia‐reperfusion in neonatal pigs

Renal ischemia‐reperfusion (IR) remains a significant cause of acute kidney injury (AKI) in neonates. Early renal vasoconstriction in ischemic AKI induces hypoperfusion and a rapid decline in kidney function. Mechanisms that underlie post‐ischemic renal hypoperfusion are poorly understood, but possibilities include alterations in ion channel‐mediated renal vasoregulation. Here, we show that pharmacological activation of TRPV4 channels promotes renal vasoconstriction and hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short‐term reperfusion increased TRPV4 channel protein expression and activity in preglomerular microvessels and vascular smooth muscle cells (SMCs), respectively. Renal IR caused oliguria and elevated serum or urine levels of AKI biomarkers. Renal IR also increased renal vascular resistance (RVR) and attenuated renal blood flow and glomerular filtration rate in the pigs. However, TRPV4 channel blockers HC 067047 and RN 1734 mitigated renal IR‐induced oliguria, hypoperfusion, and alterations in AKI biomarkers. Furthermore, renal IR increased serum angiotensin II (ANG II) concentration, but not renal vascular expression of type 1 ANG II receptors in the pigs. TRPV4 channel blockade reduced ANG II‐induced renal vasoconstriction, RVR increase, and renal hypoperfusion in the pigs. Together, our data suggest that increased renal vascular SMC TRPV4 channel expression, ANG II‐induced TRPV4 activation, and subsequent increase in RVR contribute to hypoperfusion and kidney insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels regulates neonatal renal hemodynamics in health and disease.Support or Funding InformationR01DK101668This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPC3 Overexpression Promotes the Progression of Inflammation-Induced Preterm Labor and Inhibits T Cell Activation

Background/Aims: To detect the expression of the TRPC3 channel protein in the tissues of women experiencing preterm labor and investigate its interaction with T lymphocytes, providing a theoretical basis for the clinical prevention of threatened preterm labor and the development of drug-targeted therapy. Methods: Forty-seven women experiencing preterm labor and 47 women experiencing normal full-term labor were included in this study. All included women underwent delivery via cesarean section; uterine samples were obtained at delivery. The expression of TRPC3 in uterine tissue was detected by immunohistochemistry, real-time quantitative reverse transcription-PCR, and western blot assay. Activation of T lymphocytes in peripheral blood and uterine tissue were detected by flow cytometry. A TRPC3^-/- mouse model of inflammation-induced preterm labor was established; expression of TRPC3, Cav3.1, and Cav3.2 were analyzed in mouse uterine tissue. Activation of T lymphocytes in female mouse and human peripheral blood samples was determined using flow cytometry. Results: In women experiencing preterm labor, expression of TRPC3 and the Cav3.1 and Cav3.2 proteins was significantly increased; in addition, the percentage of CD3⁺, CD4⁺, and CD8⁺ T cells in peripheral blood was significantly decreased. TRPC3 knockout significantly delayed the occurrence of preterm labor in mice. The muscle tension of ex vivo uterine strips was lower, Cav3.1 and Cav3.2 protein expression was lower, and the percentage of CD8⁺ T lymphocytes was significantly increased in wild-type mice subjected to an inflammation-induced preterm labor than in wild-type mice experiencing normal full-term labor. Conclusion: TRPC3 is closely related to the initiation of labor. TRPC3 relies on Cav3.1 and Cav3.2 proteins to inhibit inflammation-induced preterm labor by inhibiting the activation of T cells, in particular CD8⁺ T lymphocytes.

Magnesium sulfate attenuates brain edema by lowering AQP4 expression and inhibits glia-mediated neuroinflammation in a rodent model of eclampsia

Eclampsia is characterized by high morbidity and mortality wordwide. Magnesium sulfate (MgSO4) is used frequently as a prophylaxis for eclamptic seizure in clinical settings. However, the underlying mechanism is less studied, we have previously demonstrated that MgSO4 pretreatment decreases eclampsia-like seizure threshold. Here, we further evaluated the hypothesis that MgSO4 exert neuroprotective actions in eclampsia-like rats model by ameliorating neuroinflammation and brain edema. In this study, the eclampsia-like model was established by administering lipopolysaccharide plus pentylenetetrazol in pregnant Sprague-Dawley rats. Rats were given MgSO4 from gestation day14–19. Then, Iba-1 (a marker for microglia) and S100-B (a marker for astrocytes) expression levels in the hippocampus CA3 region were detected by Enzyme-linked immunosorbent assay. Cerebrospinal fluid (CSF) levels of inflammatory cytokines were measured by Luminex assays. Aquaporin-4 (a transmembrane water channel protein) expression levels in cortex were analyzed using immunohistochemistry. Astrocyte and microglia expressions were detected by immunofluorescence, neuronal damage were evaluated by Nissl staining, and changes in neuronal number in the hippocampal CA3 region (CA3) among different groups were detected by neuronal nuclei staining. Our results demonstrated that MgSO4 effectively attenuated astrocyte and microglia activation and promoted the neuronal survival in the CA3. Additionally, MgSO4 significantly reduced inflammatory cytokines response in the CSF, and decreased the expression of AQP-4 protein in the cortex. Collectively, the findings of this study indicated that MgSO4 has a neuroprotective role in eclampsia-like seizure rats through its anti-neuroninflammatory and brain edema-attenuating properties.

LEFTY2 inhibits endometrial receptivity by downregulating Orai1 expression and store-operated Ca2+ entry

Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-β family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca2+ entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca2+ ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca2+-sensitive receptivity genes.Key messages•Endometrial receptivity is negatively regulated by LEFTY2.•LEFTY2 inhibits the expression of key murine receptivity genes, including Cox2, Bmp2and Wnt4, and blocks embryo implantation.•LEFTY2 downregulates the expression of Orai1 and inhibits SOCE.•LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, and YM-58483 inhibit COX2, BMP2, and WNT4 expression in endometrial cells.•Targeting LEFTY2 and Orai1 may represent a novel approach for treating unexplained infertility.

Increased myoendothelial feedback is associated with increased connexin37 and IK1 channel expression in mesenteric arteries of diet-induced hyperhomocysteinemic mice.

Previously, we found that diet-induced HHcy in mice caused decreased eNOS expression and signaling in mesenteric arteries, but greatly enhanced non-NOS, non-prostacyclin-dependent vasodilation, which involves MEJ communication. To further assess whether HHcy enhances MEJ communication, this study examined endothelium-dependent attenuation of phenylephrine-induced vasoconstriction (myoendothelial feedback) and key molecules involved. Myoendothelial feedback was examined in isolated mouse mesenteric arteries, after 6-weeks diet-induced HHcy, using pressure myography. Gap junction (Cx37, Cx40, Cx43), NOS (eNOS, nNOS, iNOS), and potassium channel (IK1) protein expression were measured with immunoblots, and connexin mRNAs with real-time PCR. Contribution of nNOS+iNOS to vasomotor responses was assessed using the drug TRIM. Myoendothelial feedback was significantly (P<.05) enhanced in HHcy arteries compared to control, coincident with significantly greater Cx37 and IK1 protein and Cx37 mRNA. Cx43 protein, but not mRNA, was significantly less in HHcy, and Cx40 was not different. eNOS protein was significantly less in HHcy. nNOS and iNOS were not different. TRIM had little effect on vasomotor function. Diet-induced HHcy enhanced myoendothelial feedback, and increased Cx37 and IK1 expression may contribute. nNOS or iNOS did not upregulate to compensate for decreased eNOS, and they had little involvement in vasomotor function.