Receptor-operated Ca2 Research Articles

TRPC3 promotes tumorigenesis of gastric cancer via the CNB2/GSK3β/NFATc2 signaling pathway.

The transient receptor potential canonical (TRPC) channels have been implicated in various types of malignancies including gastric cancer (GC). However, the detailed mechanisms of TRPC channels underlying cell proliferation and apoptosis of GC cells remain largely unknown. Here, we report that TRPC3 was highly expressed in clinical GC specimens and correlated with GC malignant progression and poor prognosis. Forced expression of TRPC3 in GC cells enhanced both receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) and promoted the nuclear factor of activated T cell 2 (NFATc2) nuclear translocation by AKT/GSK-3β and CNB2 signaling. Pharmacological inhibition of TRPC3 or CRISPR/Cas9-mediated TRPC3 knockout effectively inhibited the growth of GC cells both in vitro and in vivo. These effects were reversible by the rescue of TRPC3 expression. Furthermore, we confirmed the role of TRPC3 and the ROCE-AKT/GSK3β-CNB2/NFATc2 signaling cascade in regulating cell cycle checkpoint, apoptosis cascade, and intracellular ROS production in GC. Overall, our findings suggest an oncogenic role of TRPC3 in GC and may highlight a potential target of TRPC3 for therapeutic intervention of GC and its malignant progression.

Role of transient receptor potential channel 6 in the osteogenesis of periodontal ligament cells

Transient receptor potential channel 6 (TRPC6) is a receptor-operated Ca2+ channel that plays an important role in Ca2+ influx in the majority of non–excitable cells and influences calcium signalling and cellular responses. Therefore, the purpose of the present study was to gain insight into the role of TRPC6 in the osteogenesis of periodontal ligament cells (PDLCs). By western blot and immunohistochemical staining, the protein level of TRPC6 was found to be increased in a time-dependent manner during osteoblastic differentiation of PDLCs. In addition, the TRPC6 inhibitor SKF96365 was used to block the function of TRPC6 and inhibit osteoblastic differentiation of PDLCs. The TRPC6 activator hyperforin dicyclohexylammonium salt (hyperforin DCHA) was used to activate TRPC6 and promote osteoblastic differentiation of PDLCs. In vivo, wild-type mice showed better bone regeneration than TRPC6−/− mice, suggesting that TRPC6 has notable osteogenic induction properties and is important for bone defect repair. In conclusion, the current data demonstrated that TRPC6 plays a significant role in osteoblastic differentiation of PDLCs, suggesting that it may be a promising therapeutic target in osteogenesis.

Halofuginone, a promising drug for treatment of pulmonary hypertension.

Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH). Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments. Halofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 μM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice. Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

Amelioration of circadian disruption and calcium-handling protein defects by choline alleviates cardiac remodeling in abdominal aorta coarctation rats

The key pathophysiological process leading to heart failure is cardiac remodeling, a term referring to cardiac hypertrophy, fibrosis, and apoptosis. We explored circadian rhythm disruption and calcium dyshomeostasis in cardiac remodeling and investigated the cardioprotective effect of choline. The experiments were conducted using a model of cardiac remodeling by abdominal aorta coarctation (AAC) in Sprague–Dawley rats. In vitro cardiomyocyte remodeling was induced by exposing neonatal rat cardiomyocytes to angiotensin II. The circadian rhythms of the transcript levels of the seven major components of the mammalian clock (Bmal1, Clock, Rev-erbα, Per1/2, and Cry1/2) were altered in AAC rat hearts during a normal 24 h light/dark cycle. AAC also upregulated the levels of proteins that mediate store-operated Ca2+ entry/receptor-operated Ca2+ entry (stromal interaction molecule 1 [STIM1], Orai1, and transient receptor potential canonical 6 [TRPC6]) in rat hearts. Moreover, choline ameliorated circadian rhythm disruption, reduced the upregulated protein levels of STIM1, Orai1, and TRPC6, and alleviated cardiac dysfunction and remodeling (evidenced by attenuated cardiac hypertrophy, fibrosis, and apoptosis) in AAC rats. In vitro analyses showed that choline ameliorated calcium overload, downregulated STIM1, Orai1, and TRPC6, and inhibited thapsigargin-induced store-operated Ca2+ entry and 1-oleoyl-2-acetyl-sn-glycerol-induced receptor-operated Ca2+ entry in angiotensin II-treated cardiomyocytes. In conclusion, choline attenuated AAC-induced cardiac remodeling and cardiac dysfunction, which was related to amelioration of circadian rhythm disruption and attenuation of calcium-handling protein defects. Modulation of vagal activity by choline targeting the circadian rhythm and calcium homeostasis may have therapeutic potential for cardiac remodeling and heart failure.Choline attenuates abdominal aortic coarctation-induced cardiac remodeling and cardiac dysfunction, by amelioration of circadian rhythm disruption and attenuation of calcium-handling protein defects. Modulation of vagal activity by choline may have therapeutic potential for cardiac remodeling and heart failure.

Effects Of F-18 On KCL And Phenylephrine - Induced Contractions Of Rat Aorta

The mechanism of action of the alkaloid 1-(2´-bromine-4´,5´-dimethoxyphenyl) - 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (F-18) on the functional activity of smooth muscle cells of the rat aorta was studied. Isometric contraction activity was recorded using a Grass FT – 03 (Grass Instrument, USA) mechanotron. The relaxant effect of the F-18 alkaloid was found to be associated with blockade of Ca2 + (IP3R) channels in the SR, along with voltage- dependent and receptor-operated Ca2+channels in the aorta smooth muscle cell plasmalemma.

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.

Vasorelaxant Effects of Coriandrum SativumL. Extract on Rat Isolated Aorta

Aim: Coriander(Coriandrum sativum L.) is an annual plant belonging to the family Umbelliferae, which distributed in Central and Western Europe, India, Bangladesh, Thailand, China, and other Asian regions. Besides being edible, coriander is an important traditional medicine in India and China, and is used to treat circulatory disorders, such as respiratory, urinary tract, and skin diseases. In this study, we investigated the vasorelaxant effects of extracts from coriander and further studied to clarify their action mechanisms. Method: The aerial part of coriander, which analyzed the rutin content as its quality evaluand using a chromatographic method (HPLC), was cut and extracted with ethyl acetate or hot water. The extracts were concentrated under the reduced pressure. Vasorelaxant effects of these extracts were assessed on rat isolated aorta. The aorta was placed in a well-oxygenated bath of modified Krebs-Henseleit solution and the mechanical tension was measured isometrically. Results: These extracts showed vasorelaxant effects on aorta precontracted with 3×10-7 M norepinephrine (NE). The ethyl acetate extract showed biphasic vasorelaxation (fast and slowly developing relaxations) on isolated rat aortic rings with endothelium. Fast relaxation disappeared in deendothelialized or pre-administration of 10-4 M L-NG-monomethylarginine. Furthermore, the hot water extract showed only slowly developing relaxations independent in endothelium. After treatment with the hot water extract, NE-induced phasic vasoconstriction was not inhibited. While the hot water extract inhibited vasoconstrictions induced by a high concentration (60 mM) of K+ and also showed inhibitory effect on NE-induced vasoconstriction in the presence of nicardipine. Conclusion: These results suggest that the vasorelaxant effect of ethyl acetate extract of coriander on NE-induced vasoconstriction may be attributed to Nitric Oxide (NO) releasing dependent on endothelium. And the hot water extract of coriander showed vasorelaxant activities attributed to blocking of Ca2+ influx via voltage-depended Ca2+ channels (VDCs) and receptor-operated Ca2+ channels (ROCs), but not competing for the adrenergic receptor. Keywords: Coriandrum sativum; quality evaluation; vasorelaxant effects; aorta; endothelium; Ca2+ channel

Revisiting the mechanism of hypoxic pulmonary vasoconstriction using isolated perfused/ventilated mouse lung.

Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O2 saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca2+ signaling and Ca2+ influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca2+ or Ca2+ influx through various Ca2+-permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca2+ abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca2+ channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca2+-sensing receptors (by 30 µM NPS2143, an allosteric Ca2+-sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca2+ influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca2+ entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca2+-mediated activation of Ca2+-sensing receptors and the cell–cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.

Store-operated Ca2+ entry in primary murine lung fibroblasts is independent of classical transient receptor potential (TRPC) channels and contributes to cell migration

Stromal interaction molecules (STIM1, 2) are acting as sensors for Ca2+ in intracellular stores and activate Orai channels at the plasma membrane for store-operated Ca2+ entry (SOCE), while classical transient receptor potential (TRPC) channel mediate receptor-operated Ca2+ entry (ROCE). Several reports, however, indicate a role for TRPC in SOCE in certain cell types. Here, we analyzed Ca2+ influx and cell function in TRPC1/6-deficient (TRPC1/6−/−) and STIM1/2- deficient (STIM1/2ΔpmLF) primary murine lung fibroblasts (pmLF). As expected, SOCE was decreased in STIM1/2- deficient pmLF and ROCE was decreased in TRPC1/6−/− pmLF compared to control cells. By contrast, SOCE was not significantly different in TRPC1/6−/− pmLF and ROCE was similar in STIM1/2-deficient pmLF compared to Wt cells. Most interestingly, cell proliferation, migration and nuclear localization of nuclear factor of activated T-cells (NFATc1 and c3) were decreased after ablation of STIM1/2 proteins in pmLF. In conclusion, TRPC1/6 channels are not involved in SOCE and STIM1/2 deficiency resulted in decreased cell proliferation and migration in pmLF.

Hypoxic Pulmonary Vasoconstriction in Isolated Mouse Lungs

BackgroundHypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism of lung to ensure maximal gas exchange and blood oxygenation. Persistent and global alveolar hypoxia leads to sustained vasoconstriction and vascular remodeling causing pulmonary hypertension. It has been demonstrated that acute HPV is initiated by an increase in cytosolic [Ca2+] ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) and modulated by factors released from endothelium. Acute HPV is well reserved in isolated perfused/ventilated lungs, whereas HPV in isolated pulmonary arterial rings shows different pattern in comparison to HPV in intact animals and humans. In this study, we aimed to use isolated perfused/ventilated mouse lungs to further study potential mechanisms involved in development of HPV.MethodsC57BL/6 mice (male, 8–10 weeks old) were anesthetized by pentobarbital sodium (120 mg/kg). After tracheostomy, isolated lungs were immediately ventilated with normoxic gas (21% O2/5% CO2) using a rodent ventilator (80 breaths/min with a tidal volume of 250 μl). The mice were then placed in an isolated lung system chamber with a heated water jacket at 37°C. After tracheal intubation, the chest was opened and a catheter was inserted into the main PA via the right ventricle to measure PAP. The lung vasculature was consistently superfused with physiological salt solution (PSS) via a pump while lung airway and alveoli were ventilated with normoxic or hypoxic gas. Before experimentation, the isolated lung was first superfused with the 40 mM K+‐containing PSS (40K) to stabilize the basal PAP. The lung was then repetitively challenged by ventilation of hypoxic gas (1% O2 in N2, for 4 min) to induce an increase in PAP (due to HPV). Pharmacological effects of various inhibitors on HPV were examined by superfusion of PSS containing the inhibitors while the lung was ventilated with hypoxic gas.ResultsOur results show that HPV was dependent on Ca2+ influx through a) nifedipine‐sensitive L‐type voltage‐dependent Ca2+ channels; b) SKF‐sensitive non‐selective cation channels; and c) TRP channels like TRPC6, TRPV1 and TRPP. HPV in isolated perfused lung was also significantly inhibited by blocking Ca2+‐sensing receptors (CaSR) and Notch receptors and by extracellular Mg2+ and Mn2+. Interestingly, blockade of Ca2+‐activated Cl− channels and inhibition of muscarinic receptors and endothelin receptors had little effect on HPV. Furthermore, inhibition of nitric oxide synthase (eNOS) potentiated HPV.ConclusionAn increase in [Ca2+]cyt due to Ca2+ influx through various Ca2+‐permeable cation channels in PASMC influx is required for the initiation of acute HPV. Both voltage‐gated Ca2+ channels (e.g., CaV1.2) and receptor‐operated Ca2+ channels (TRPC6/V1/P) are involved in mediating Ca2+ influx to induce HPV. G protein‐coupled receptor/CaSR‐mediated synthesis of intracellular signaling molecules (e.g., diacylglycerol and inositol 1, 4, 5‐trisphosphate) and Notch‐associated cell‐to‐cell interaction also play important roles in HPV. These data indicate that multiple mechanisms are involved in the initiation and regulation of HPV.Support or Funding InformationNIH NIHLBI HL135807

Notch enhances Ca2+ entry by activating calcium-sensing receptors and inhibiting voltage-gated K+ channels.

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.

Moringa oleifera leaf extract induces vasorelaxation via endothelium-dependent hyperpolarization and calcium channel blockade in mesenteric arterial beds isolated from L-NAME hypertensive rats

ABSTRACT Background An aqueous extract of Moringa oleifera leaves (MOE) is known to cause relaxation of mesenteric resistance arteries of rats in which hypertension has been induced by the administration of L-NAME, but the mechanism(s) of action of MOE remains unclear. The purpose of this study was to investigate these mechanisms in mesenteric arterial beds isolated from L-NAME induced hypertensive rats. Methods: An investigation of vascular reactivity was conducted on isolated mesenteric arterial beds by measuring the changes in perfusion pressure using an in vitro system. Results MOE (0.001–3 mg in 0.1 ml injection volume) caused a dose-dependent relaxation in methoxamine (5 µM) pre-contracted arterial beds, which was partially abolished by endothelium removal. The endothelium-dependent component of vasorelaxation was insensitive to both L-NAME (100 µM) and indomethacin (10 µM), while completely inhibited in high KCl (45 mM)-induced contraction. MOE (1 and 3 mg/ml) showed a dose-dependent inhibitory effect on CaCl2-induced contractions of denuded preparations in Ca2+-free medium containing a high KCl (60 mM) or methoxamine (10 µM). In Ca2+-free medium, MOE (3 mg/ml) also inhibited phenylephrine-induced contractions of denuded preparations. Conclusion: These findings suggest that MOE relaxes mesenteric arterial beds of L-NAME hypertensive rats via both endothelium-dependent and endothelium-independent mechanisms. The endothelium-dependent action occurred via endothelium-derived hyperpolarizing factor-mediated hyperpolarization. The endothelium-independent action was related to blocking the entry of extracellular Ca2+ via voltage-operated and receptor-operated Ca2+ channels, and inhibiting mobilization of sarcolemmal Ca2+ via inositol trisphosphate receptor Ca2+ channels. MOE may be potentially useful as a natural vasodilator against hypertension.

Pathophysiological roles of TRPC6 channels in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive and lethal disease of the pulmonary artery. The pathogenesis of PAH is mainly sustained vasoconstriction and vascular remodeling of the pulmonary artery. These pathogeneses cause progressive elevations in pulmonary vascular resistance and pulmonary arterial pressure in PAH patients. Elevated pulmonary arterial pressure leads to right heart failure and finally death. The vascular remodeling is caused by the enhanced proliferation and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Excitable abnormality in the pulmonary artery of PAH patients is mostly mediated by an elevated cytosolic Ca2+ concentration. PASMCs express several Ca2+-permeable channels including voltage-dependent Ca2+ channels, store-operated Ca2+ (SOC) channels, and receptor-operated Ca2+ (ROC) channels. The activation and upregulation of these Ca2+ channels have been reported in PASMCs from PAH patients. Here, we analyzed pathophysiological functions of enhanced Ca2+ signaling mediated by SOC and ROC channels using PASMCs from idiopathic PAH patients and animal PAH models. Notch signal enhanced transient receptor potential canonical 6 (TRPC6) "SOC" channels via direct (non-genomic and stimulatory) and indirect (genomic and upregulating) effects in PAH. On the other hand, the activation of Ca2+-sensing receptors evoked Ca2+ influx through TRPC6 "ROC" channels in PAH. In addition, TRPC6 channel blocker and TRPC6 gene deletion inhibited the development of PAH. Specifically, TRPC6 channels potentially form both ROC and SOC channels in PASMCs, which are involved in the pathophysiological events in PAH. Therefore, targeting TRPC6 channels in PASMCs may help develop novel therapeutic approach for PAH.

L-ornithine activates Ca2+ signaling to exert its protective function on human proximal tubular cells

Oxidative stress and reactive oxygen species (ROS) generation can be influenced by G-protein coupled receptor (GPCR)-mediated regulation of intracellular Ca2+ ([Ca2+]i) signaling. ROS production are much higher in proximal tubular (PT) cells; in addition, the lack of antioxidants enhances the vulnerability to oxidative damage. Despite such predispositions, PT cells show resiliency, and therefore must possess some inherent mechanism to protect from oxidative damage. While the mechanism in unknown, we tested the effect of l-ornithine, since it is abundantly present in PT luminal fluid and can activate Ca2+-sensing receptor (CaSR), a GPCR, expressed in the PT luminal membrane. We used human kidney 2 (HK2) cells, a PT cell line, and performed Ca2+ imaging and electrophysiological experiments to show that l-ornithine has a concentration-dependent effect on CaSR activation. We further demonstrate that the operation of CaSR activated Ca2+ signaling in HK-2 cells mediated by the transient receptor potential canonical (TRPC) dependent receptor-operated Ca2+ entry (ROCE) using pharmacological and siRNA inhibitors. Since PT cells are vulnerable to ROS, we simulated such deleterious effects using genetically encoded peroxide-induced ROS production (HyperRed indicator) to show that the l-ornithine-induced ROCE mediated [Ca2+]i signaling protects from ROS production. Furthermore, we performed cell viability, necrosis and apoptosis assays, and mitochondrial oxidative gene expression to establish that presence of l-ornithine rescued the ROS-induced damage in HK-2 cells. Moreover, l-ornithine-activation of CaSR can reverse ROS production and apoptosis via mitogen-activated protein kinase p38 activation. Such nephroprotective role of l-ornithine can be useful as the translational option for reversing kidney diseases involving PT cell damage due to oxidative stress or crystal nephropathies.

Effects of Prosopis farcta Root Extract in the Vascular Reactivity of Isolated Goat Coronary Artery

Prosopis species is a medicinal plant, well-known for its beneficial effects in treating various smooth muscles disorders, and its phytochemical analysis revealed the presence of different bioactive compounds in different parts of the plant, most of which show a great role in reducing cardiovascular risks. In the present study, the cardiovascular effect of Prosopis farcta Root Extract (PFRE) was investigated in vitro for possible mechanisms of the extract effect in the vascular reactivity of isolated goat coronary artery (CA) using Organ bath and PowerLab Data Acquisition system. The results of the recording and analyzing of the effect of the PFRE in isolated CA, showed the negative inotropic activity of the extract in CA rings with intact-endothelium, while in CA ring with hyperglycemic-induced endothelium dysfunction the extract tended to vasodilate the CA ring non-significantly, and the extract induced dose-dependent vasodilation in CA rings pre-constricted with high concentration of (30 mM)KCl and showed no effects on contractions induced by (1X10-3-1X10-5) Phenylephrine PE, which is an indicator for its blockade activity on L-type voltage-dependent Ca+2 channel and non-interfering of the extract with the receptor-operated Ca+2 channel. The mechanical recording of the CA ring activities, revealed different potassium (K+) channels including selective calcium-activated potassium channel, ATP-sensitive potassium channel, and different endothelium-derived relaxing factors (EDRF) including nitric oxide and Prostacyclin (PGI2 ) seems to have no role in the relaxation effects of the extract, while the endothelium derived-hyperpolarizing factor (EDHF); epoxy eicosatrienoic acid (EET) showed significant participation in the vasodilation effects of the extract. On the other hand, the extract tended to relax the CA rings through its antagonizing of Ca+2, reducing and inhibiting Ca+2 influx and release from internal stores and interfering with the voltage-operated Ca+2 channels through its alkaloid and flavonoid active compounds which suggested to be the most predicted mechanisms for the maintenance of vasodilating tone and coronary circulation of the PFRE in coronary artery.

Abrogation of store-operated Ca2+ entry protects against crystal-induced ER stress in human proximal tubular cells

Calcium crystal internalization into proximal tubular (PT) cells results in acute kidney injury, nephrocalcinosis, chronic kidney disease (CKD), and kidney-stone formation. Ca2+ supersaturation in PT luminal fluid induces calcium crystal formation, leading to aberrant crystal internalization into PT cells. While such crystal internalization produces reactive oxygen species (ROS), cell membrane damage, and apoptosis; the upstream signaling events involving dysregulation of intracellular Ca2+ homeostasis and ER stress, remain largely unknown. We have recently described a transepithelial Ca2+ transport pathway regulated by receptor-operated Ca2+ entry (ROCE) in PT cells. Therefore, we examined the pathophysiological consequence of internalization of stone-forming calcium crystals such as calcium phosphate (CaP), calcium oxalate (CaOx), and CaP + CaOx (mixed) crystals on the regulation of intracellular Ca2+ signaling by measuring dynamic changes in Ca2+ transients in HK2, human PT cells, using pharmacological and siRNA inhibitors. The subsequent effect on ER stress was measured by changes in ER morphology, ER stress-related gene expression, endogenous ROS production, apoptosis, and necrosis. Interestingly, our data show that crystal internalization induced G-protein-coupled receptor-mediated sustained rise in intracellular Ca2+ concentration ([Ca2+]i) via store-operated Ca2+ entry (SOCE); suggesting that the mode of Ca2+ entry switches from ROCE to SOCE following crystal internalization. We found that SOCE components—stromal interacting molecules 1 and 2 (STIM1, STIM2) and ORAI3 (SOCE) channel were upregulated in these crystal-internalized cells, which induced ER stress, ROS production, and cell death. Finally, silencing those SOCE genes protected crystal-internalized cells from prolonged [Ca2+]i rise and ER stress. Our data provide insight into the molecular mechanism of crystal-induced Ca2+ dysregulation, ER stress, and PT cell death and thus could have a translational role in treating crystal nephropathies including kidney stones. Taken together, modulation of Ca2+ signaling can be used as a tool to reverse the pathological consequence of crystal-induced conditions including cardiovascular calcification.

Pharmacological inhibition of TRPV4 channels protects against ischemia-reperfusion-induced renal insufficiency in neonatal pigs.

Renal vasoconstriction, an early manifestation of ischemic acute kidney injury (AKI), results in renal hypoperfusion and a rapid decline in kidney function. The pathophysiological mechanisms that underlie ischemia-reperfusion (IR)-induced renal insufficiency are poorly understood, but possibilities include alterations in ion channel-dependent renal vasoregulation. In the present study, we show that pharmacological activation of TRPV4 channels constricted preglomerular microvessels and elicited renal hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short-term reperfusion increased TRPV4 protein expression in resistance size renal vessels and TRPV4-dependent cation currents in renal vascular smooth muscle cells (SMCs). Selective TRPV4 channel blockers attenuated IR-induced reduction in total renal blood flow (RBF), cortical perfusion, and glomerular filtration rate (GFR). TRPV4 inhibition also diminished renal IR-induced increase in AKI biomarkers. Furthermore, the level of angiotensin II (Ang II) was higher in the urine of IR- compared with sham-operated neonatal pigs. IR did not alter renal vascular expression of Ang II type 1 (AT1) receptors. However, losartan, a selective AT1 receptor antagonist, ameliorated IR-induced renal insufficiency in the pigs. Blockade of TRPV4 channels attenuated Ang II-evoked receptor-operated Ca2+ entry and constriction in preglomerular microvessels. TRPV4 inhibition also blunted Ang II-induced increase in renal vascular resistance (RVR) and hypoperfusion in the pigs. Together, our data suggest that SMC TRPV4-mediated renal vasoconstriction and the ensuing increase in RVR contribute to early hypoperfusion and renal insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels controls neonatal renal microcirculation in health and disease.

Increased caveolin-1 expression enhances the receptor-operated Ca2+ entry in the aorta of two-kidney, one-clip hypertensive rats.

What is the central question of this study? The aim was to examine and compare the contributions of caveolin-1 to the contractile responses mediated by L-type voltage-dependent calcium channels, store-operated Ca2+ channels and receptor-operated Ca2+ channels in two different types of arteries from two-kidney, one-clip hypertensive rats. What is the main finding and its importance? We demonstrated that the density of caveolae and caveolin-1 expression were significantly upregulated in the aorta of two-kidney, one-clip hypertensive rats, but not in the third-order branches of mesenteric arteries. We highlight that caveolin-1 plays an important role in aortic constriction by enhancing receptor-operated Ca2+ entry in the hypertensive rat model. Calcium and its multiple regulatory mechanisms are crucial for the development of hypertension. Among these regulatory mechanisms, store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE) mediate agonist-induced calcium influx, contributing to vascular contraction. The SOCE and ROCE are regulated by a variety of mechanisms involving caveolin-1 (Cav1), which has been found to be strongly associated with hypertension in gene polymorphism. In the present study, we investigated the role of Cav1 during the enhanced activity of calcium channels in hypertensive arteries. We demonstrated that the expression level of Cav1 was significantly increased in the aorta of two-kidney, one-clip (2K1C) hypertensive rats. The disruption of caveolae by methyl-β-cyclodextrin did not cause a marked difference in agonist-induced vasoconstriction in the third-order branches of the mesenteric arteries but strongly suppressed the aortic contractile response to endothelin-1 in the 2K1C group, which was not found in the control group. The increase in Cav1 by introduction of Cav1 scaffolding domain enhancing peptide promoted the 1-oleoyl-2-acetyl-glycerol-induced ROCE in hypertensive aortic smooth muscle cells but did not enhance the cyclopiazonic acid-induced SOCE. In the resistance arteries, similar changes were not observed, and no statistical changes of Cav1 expression were evident in the third-order branches of the mesenteric arteries. Our results indicate that increased Cav1 expression might promote the altered [Ca2+ ]i -induced aortic vasoreactivity by enhancing ROCE and be involved in the pathogenesis of hypertension.

Redox Regulation of Ion Channels and Receptors in Pulmonary Hypertension.

Significance: Pulmonary hypertension (PH) is characterized by elevated vascular resistance due to vasoconstriction and remodeling of the normally low-pressure pulmonary vasculature. Redox stress contributes to the pathophysiology of this disease by altering the regulation and activity of membrane receptors, K+ channels, and intracellular Ca2+ homeostasis. Recent Advances: Antioxidant therapies have had limited success in treating PH, leading to a growing appreciation that reductive stress, in addition to oxidative stress, plays a role in metabolic and cell signaling dysfunction in pulmonary vascular cells. Reactive oxygen species generation from mitochondria and NADPH oxidases has substantial effects on K+ conductance and membrane potential, and both receptor-operated and store-operated Ca2+ entry. Critical Issues: Some specific redox changes resulting from oxidation, S-nitrosylation, and S-glutathionylation are known to modulate membrane receptor and ion channel activity in PH. However, many sites of regulation that have been elucidated in nonpulmonary cell types have not been tested in the pulmonary vasculature, and context-specific molecular mechanisms are lacking. Future Directions: Here, we review what is known about redox regulation of membrane receptors and ion channels in PH. Further investigation of the mechanisms involved is needed to better understand the etiology of PH and develop better targeted treatment strategies.

Pyrazolo[1,5-a]pyrimidine TRPC6 antagonists for the treatment of gastric cancer

Transient receptor potential canonical 6 (TRPC6) proteins form receptor-operated Ca2+-permeable channels, which have been thought to bring benefit to the treatment of diseases, including cancer. However, selective antagonists for TRPC channels are rare and none of them has been tested against gastric cancer. Compound 14a and analogs were synthesized by chemical elaboration of previously reported TRPC3/6/7 agonist 4o. 14a had very weak agonist activity at TRPC6 expressed in HEK293 cells but exhibited strong inhibition on both 4o-mediated and receptor-operated activation of TRPC6 with an IC50 of about 1 μM. When applied to the culture media, 14a suppressed proliferation of AGS and MKN45 cells with IC50 values of 17.1 ± 0.3 and 18.5 ± 1.0 μM, respectively, and inhibited tube formation and migration of cultured human endothelial cells. This anti-tumor effect on gastric cancer was further verified in xenograft models using nude mice. This study has found a new tool compound which shows excellent therapeutic potential against human gastric cancer most likely through targeting TRPC6 channels.