Increase In Cytosolic Calcium Concentration Research Articles

Elevated intracellular Ca2+ functions downstream of mitodysfunction to induce Wallerian-like degeneration and necroptosis in organophosphorus-induced delayed neuropathy

Neurotoxic organophosphorus compounds can induce a type of delayed neuropathy in humans and sensitive animals, known as organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by axonal degeneration akin to Wallerian-like degeneration, which is thought to be caused by increased intra-axonal Ca2+ concentrations. This study was designed to investigate that deregulated cytosolic Ca2+ may function downstream of mitodysfunction in activating Wallerian-like degeneration and necroptosis in OPIDN. Adult hens were administrated a single dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), and then sacrificed at 1 day, 5 day, 10 day and 21 day post-exposure, respectively. Sciatic nerves and spinal cords were examined for pathological changes and proteins expression related to Wallerian-like degeneration and necroptosis. In vitro experiments using differentiated neuro-2a (N2a) cells were conducted to investigate the relationship among mitochondrial dysfunction, Ca2+ influx, axonal degeneration, and necroptosis. The cells were co-administered with the Ca2+-chelator BAPTA-AM, the TRPA1 channel inhibitor HC030031, the RIPK1 inhibitor Necrostatin-1, and the mitochondrial-targeted antioxidant MitoQ along with TOCP. Results demonstrated an increase in cytosolic calcium concentration and key proteins associated with Wallerian degeneration and necroptosis in both in vivo and in vitro models after TOCP exposure. Moreover, co-administration with BATPA-AM or HC030031 significantly attenuated the loss of NMNAT2 and STMN2 in N2a cells, as well as the upregulation of SARM1, RIPK1 and p-MLKL. In contrast, Necrostatin-1 treatment only inhibited the TOCP-induced elevation of p-MLKL. Notably, pharmacological protection of mitochondrial function with MitoQ effectively alleviated the increase in intracellular Ca2+ following TOCP and mitigated axonal degeneration and necroptosis in N2a cells, supporting mitochondrial dysfunction as an upstream event of the intracellular Ca2+ imbalance and neuronal damage in OPIDN. These findings suggest that mitochondrial dysfunction post-TOCP intoxication leads to an elevated intracellular Ca2+ concentration, which plays a pivotal role in the initiation and development of OPIDN through inducing SARM1-mediated axonal degeneration and activating the necroptotic signaling pathway.

The WRKY46–MYC2 module plays a critical role in E-2-hexenal-induced anti-herbivore responses by promoting flavonoid accumulation

Volatile organic compounds (VOCs) play key roles in plant–plant communication, especially in response to pest attack. E-2-hexenal is an important component of VOCs, but it is unclear whether it can induce endogenous plant resistance to insects. Here, we show that E-2-hexenal activates early signaling events in Arabidopsis (Arabidopsis thaliana) mesophyll cells, including an H2O2 burst at the plasma membrane, the directed flow of calcium ions, and an increase in cytosolic calcium concentration. Treatment of wild-type Arabidopsis plants with E-2-hexenal increases their resistance when challenged with the diamondback moth Plutella xylostella L., and this phenomenon is largely lost in the wrky46 mutant. Mechanistically, E-2-hexenal induces the expression of WRKY46 and MYC2, and the physical interaction of their encoded proteins was verified by yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays. The WRKY46–MYC2 complex directly binds to the promoter of RBOHD to promote its expression, as demonstrated by luciferase reporter, yeast one-hybrid, chromatin immunoprecipitation, and electrophoretic mobility shift assays. This module also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes (TT4 and CHIL) and the accumulation of total flavonoids, thereby modulating plant tolerance to insects. Together, our results highlight an important role for the WRKY46–MYC2 module in the E-2-hexenal-induced defense response of Arabidopsis, providing new insights into the mechanisms by which VOCs trigger plant defense responses.

Calcineurin-Dependent Homeostatic Response of C. elegans Muscle Cells upon Prolonged Activation of Acetylcholine Receptors.

Pharmacological adaptation is a common phenomenon observed during prolonged drug exposure and often leads to drug resistance. Understanding the cellular events involved in adaptation could provide new strategies to circumvent this resistance issue. We used the nematode Caenorhabditis elegans to analyze the adaptation to levamisole, an ionotropic acetylcholine receptor agonist, used for decades to treat nematode parasitic infections. Genetic screens in C. elegans identified "adapting mutants" that initially paralyze upon exposure to levamisole as the wild type (WT), but recover locomotion after a few hours whereas WT remain paralyzed. Here, we show that levamisole induces a sustained increase in cytosolic calcium concentration in the muscle cells of adapting mutants, lasting several hours and preceding a decrease in levamisole-sensitive acetylcholine receptors (L-AChR) at the muscle plasma membrane. This decrease correlated with a drop in calcium concentration, a relaxation of the animal's body and a resumption of locomotion. The decrease in calcium and L-AChR content depends on calcineurin activation in muscle cells. We also showed that levamisole adaptation triggers homeostatic mechanisms in muscle cells including mitochondria remodeling, lysosomal tubulation and an increase in autophagic activity. Levamisole adaptation thus provides a new experimental paradigm for studying how cells cope with calcium stress.

Cytosolic Sodium Influx in Mesophyll Protoplasts of Arabidopsis thaliana, wt, sos1:1 and nhx1 Differs and Induces Different Calcium Changes.

The sodium influx into the cytosol of mesophyll protoplasts from Arabidopsis thaliana cv. Columbia, wild type, was compared with the influx into sos1-1 and nhx1 genotypes, which lack the Na+/H+ antiporter in the plasma membrane and tonoplast, respectively. Changes in cytosolic sodium and calcium concentrations upon a 100 mM NaCl addition were detected by use of epifluorescence microscopy and the sodium-specific fluorescent dye SBFI, AM, and calcium sensitive Fura 2, AM, respectively. There was a smaller and mainly transient influx of Na+ in the cytosol of the wild type compared with the sos1-1 and nhx1 genotypes, in which the influx lasted for a longer time. Sodium chloride addition to the protoplasts' medium induced a significant increase in cytosolic calcium concentration in the wild type at 1.0 mM external calcium, and to a lesser extent in nhx1, however, it was negligible in the sos1-1 genotype. LiCl inhibited the cytosolic calcium elevation in the wild type. The results suggest that the salt-induced calcium elevation in the cytosol of mesophyll cells depends on an influx from both internal and external stores and occurs in the presence of an intact Na+/H+ antiporter at the plasma membrane. The Arabidopsis SOS1 more effectively regulates sodium homeostasis than NHX1.

The Mechanism of Action between Pulsed Radiofrequency and Orthobiologics: Is There a Synergistic Effect?

Radiofrequency energy is a common treatment modality for chronic pain. While there are different forms of radiofrequency-based therapeutics, the common concept is the generation of an electromagnetic field in the applied area, that can result in neuromodulation (pulsed radiofrequency—PRF) or ablation. Our specific focus relates to PRF due to the possibility of modulation that is in accordance with the mechanisms of action of orthobiologics. The proposed mechanism of action of PRF pertaining to pain relief relies on a decrease in pro-inflammatory cytokines, an increase in cytosolic calcium concentration, a general effect on the immune system, and a reduction in the formation of free radical molecules. The primary known properties of orthobiologics constitute the release of growth factors, a stimulus for endogenous repair, analgesia, and improvement of the function of the injured area. In this review, we described the mechanism of action of both treatments and pertinent scientific references to the use of the combination of PRF and orthobiologics. Our hypothesis is a synergic effect with the combination of both techniques which could benefit patients and improve the life quality.

Intracellular calcium and inflammatory markers, mediated by purinergic stimulation, are differentially regulated in monocytes of patients with major depressive disorder

The P2X7 receptor (P2X7R) is a ligand-gated ion channel that is being recognized as a major player in neuropsychiatric disorders such as Major Depressive Disorder (MDD). P2X7R activation is triggered by high extracellular ATP concentrations, leading to channel opening and inducing an increase in cytosolic calcium concentration ([Ca2+]c), that activates the inflammatory pathway. Those receptors are expressed not only in CNS cells but also in peripheral blood cells, where they are activated in response to inflammatory molecules such as bacterial lipopolysaccharide (LPS). LPS induced-tissue damage promotes an elevation of extracellular ATP, triggering the NRLP3-inflammasome assembly and activation that, sequentially, induces caspase-1 cleavage and IL-1β processing and secretion. In this context, we attempt to understand the role of P2X7R in [Ca2+]c homeostasis regulation, inflammasome expression and its pharmacological modulation in MDD. For this purpose, monocytes were isolated from peripheral blood of MDD patients and [Ca2+]c was monitored with the intracellular probe Fura-2. Our results point out to P2X7R as the responsible of the Ca2+ imbalance, as well as TNF-α-dependent activation of caspase-1 in MDD patients. In addition, P2X7R blockade with its specific antagonist, JNJ-47965567, reduces the Ca2+ entry upon Bz-ATP exposure. Altogether, our results point that MDD patients have both, Ca2+ homeostasis alteration and an inflammatory status, which promote an independent-inflammasome activation of caspase-1. Therefore, we propose the pharmacological modulation of P2X7R as a therapeutic approach against MDD symptoms.

Exposure to (Z)-11-hexadecenal [(Z)-11-16:Ald] increases Brassica nigra susceptibility to subsequent herbivory

It is well established that plants emit, detect and respond to volatile organic compounds; however, knowledge on the ability of plants to detect and respond to volatiles emitted by non-plant organisms is limited. Recent studies indicated that plants detect insect-emitted volatiles that induce defence responses; however, the mechanisms underlying this detection and defence priming is unknown. Therefore, we explored if exposure to a main component of Plutella xylostella female sex pheromone namely (Z)-11-hexadecenal [(Z)-11-16:Ald] induced detectable early and late stage defence-related plant responses in Brassica nigra. Exposure to biologically relevant levels of vapourised (Z)-11-16:Ald released from a loaded septum induced a change in volatile emissions of receiver plants after herbivore attack and increased the leaf area consumed by P. xylostella larvae. Further experiments examining the effects of the (Z)-11-16:Ald on several stages of plant defence-related responses showed that exposure to 100 ppm of (Z)-11-16:Ald in liquid state induced depolarisation of the transmembrane potential (Vm), an increase in cytosolic calcium concentration [Ca2+]cyt, production of H2O2 and an increase in expression of reactive oxygen species (ROS)-mediated genes and ROS-scavenging enzyme activity. The results suggest that exposure to volatile (Z)-11-16:Ald increases the susceptibility of B. nigra to subsequent herbivory. This unexpected finding, suggest alternative ecological effects of detecting insect pheromone to those reported earlier. Experiments conducted in vitro showed that high doses of (Z)-11-16:Ald induced defence-related responses, but further experiments should assess how specific the response is to this particular aldehyde.

STIM1 Mediates Calcium-Dependent Epigenetic Reprogramming in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) displays a dismal prognosis due to late diagnosis and high chemoresistance incidence. For advanced disease stages or patients with comorbidities, treatment options are limited to gemcitabine alone or in combination with other drugs. While gemcitabine resistance has been widely attributed to the levels of one of its targets, RRM1, the molecular consequences of gemcitabine resistance in PDAC remain largely elusive. Here we sought to identify genomic, epigenomic, and transcriptomic events associated with gemcitabine resistance in PDAC and their potential clinical relevance. We found that gemcitabine-resistant cells displayed a coamplification of the adjacent RRM1 and STIM1 genes. Interestingly, RRM1, but not STIM1, was required for gemcitabine resistance, while high STIM1 levels caused an increase in cytosolic calcium concentration. Higher STIM1-dependent calcium influx led to an impaired endoplasmic reticulum stress response and a heightened nuclear factor of activated T-cell activity. Importantly, these findings were confirmed in patient and patient-derived xenograft samples. Taken together, our study uncovers previously unknown biologically relevant molecular properties of gemcitabine-resistant tumors, revealing an undescribed function of STIM1 as a rheostat directing the effects of calcium signaling and controlling epigenetic cell fate determination. It further reveals the potential benefit of targeting STIM1-controlled calcium signaling and its downstream effectors in PDAC. SIGNIFICANCE: Gemcitabine-resistant and some naïve tumors coamplify RRM1 and STIM1, which elicit gemcitabine resistance and induce a calcium signaling shift, promoting ER stress resistance and activation of NFAT signaling.

Extracellular Ca2+ induces desensitized cytosolic Ca2+ rise sensitive to phospholipase C inhibitor which suppresses root growth with Ca2+ dependence

Calcium (Ca) is an essential element for all organisms. In animal cells, the plasma membrane-localized Ca receptor CaSR coupled to a phospholipase C (PLC)-dependent signaling cascade monitors extracellular Ca2+ concentrations ([Ca2+]ext) and responds with increases in cytosolic calcium concentrations ([Ca2+]cyt). Plant roots encounter variable soil conditions, but how they sense changes in [Ca2+]ext is largely unknown. In this study, we demonstrate that increasing [Ca2+]ext evokes a transient increase in [Ca2+] in the cytosol, mitochondria, and nuclei of Arabidopsis thaliana root cells. These increases were strongly desensitized to repeat applications of [Ca2+]ext, a typical feature of receptor-mediated cellular signaling in animal and plant cells. Treatment with gadolinium (Gd3+), a CaSR activator in animal cells, induced concentration-dependent increases in [Ca2+]cyt in roots, which showed self-desensitization and cross-desensitization to [Ca2+]ext-induced increases in [Ca2+]cyt (EICC). EICC was sensitive to extracellular H+, K+, Na+, and Mg2+ levels. Treatment with the PLC inhibitor neomycin suppressed EICC and Ca accumulation in roots. The inhibitory effect of neomycin on root elongation was fully rescued by increasing [Ca2+]ext but not [Mg2+] or [K+] in the growth medium. These results suggest that [Ca2+]ext and the movement of Ca2+ into the cytosol of plant roots are regulated by a receptor-mediated signaling pathway involving PLC.

Phytic acid attenuates upregulation of GSK-3β and disturbance of synaptic vesicle recycling in MPTP-induced Parkinson's disease models

Heightened activity of glycogen synthase kinase-3β (GSK-3β) is linked to the degeneration of dopaminergic neurons in Parkinson's disease (PD). Phytic acid (PA), a naturally occurring compound with potent antioxidant property, has been shown to confer neuroprotection on dopaminergic neurons in PD. However, the underlying mechanism remains unclear. In the present study, MPTP and MPP+ treatments were used to model PD in mice and SH-SY5Y cells, respectively. We observed reduced tissue dopamine, disrupted synaptic vesicle recycling, and defective neurotransmitter exocytosis. Furthermore, expression of GSK-3β was upregulated while that of β-catenin was downregulated, concentration of cytosolic calcium was increased, and expressions of two dopamine carriers, dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) were decreased. PA treatment attenuated the MPTP-induced upregulation of GSK-3β, increase in cytosolic calcium concentration, decreases in the levels of DAT, VMAT2, tissue dopamine, and synaptic vesicle recycling. Importantly, disturbances in synaptic vesicle recycling are thought to be early events in PD pathology. These findings suggest that PA is a promising therapeutic agent to treat early events in PD.

Boron Deficiency Increases Cytosolic Ca2+ Levels Mainly via Ca2+ Influx from the Apoplast in Arabidopsis thaliana Roots.

Boron (B) is a micronutrient for plant development, and its deficiency alters many physiological processes. However, the current knowledge on how plants are able to sense the B-starvation signal is still very limited. Recently, it has been reported that B deprivation induces an increase in cytosolic calcium concentration ([Ca2+]cyt) in Arabidopsis thaliana roots. The aim of this work was to research in Arabidopsis whether [Ca2+]cyt is restored to initial levels when B is resupplied and elucidate whether apoplastic Ca2+ is the major source for B-deficiency-induced rise in [Ca2+]cyt. The use of chemical compounds affecting Ca2+ homeostasis showed that the rise in root [Ca2+]cyt induced by B deficiency was predominantly owed to Ca2+ influx from the apoplast through plasma membrane Ca2+ channels in an IP3-independent manner. Furthermore, B resupply restored the root [Ca2+]cyt. Interestingly, expression levels of genes encoding Ca2+ transporters (ACA10, plasma membrane PIIB-type Ca2+-ATPase; and CAX3, vacuolar cation/proton exchanger) were upregulated by ethylene glycol tetraacetic acid (EGTA) and abscisic acid (ABA). The results pointed out that ACA10, and especially CAX3, would play a major role in the restoration of Ca2+ homeostasis after 24 h of B deficiency.

Research Progress on the Relationship Between Acute Pancreatitis and Calcium Overload in Acinar Cells.

Acute pancreatitis is a human disease with multiple causes that leads to autodigestion of the pancreas. There is sufficient evidence to support the key role of sustained increase in cytosolic calcium concentrations in the early pathogenesis of the disease. To clarify the mechanism of maintaining calcium homeostasis in the cell and pathological processes caused by calcium overload would help to research directly targeted therapeutic agents. We will specifically review the following: intracellular calcium homeostasis and regulation, the occurrence of calcium overload in acinar cells, the role of calcium overload in the pathogenesis of AP, the treatment strategy proposed for calcium overload.

Mechanisms of BK Channel Activation by Docosahexaenoic Acid in Rat Coronary Arterial Smooth Muscle Cells.

Aim: Docosahexaenoic acid (DHA) is known to activate the vascular large-conductance calcium-activated potassium (BK) channels and has protective effects on the cardiovascular system. However, the underlying mechanisms through which DHA activates BK channels remain unclear. In this study, we determined such mechanisms by examining the effects of different concentrations of DHA on BK channels in freshly isolated rat coronary arterial smooth muscle cells (CASMCs) using patch clamp techniques.Methods and Results: We found that BK channels are the major potassium currents activated by DHA in rat CASMCs and the effects of DHA on BK channels are concentration dependent with a bimodal distribution. At concentrations of <1 μM, DHA activated whole-cell BK currents with an EC50 of 0.24 ± 0.05 μM and the activation effects were abolished by pre-incubation with SKF525A (10 μM), a cytochrome P450 (CYP) epoxygenase inhibitor, suggesting the role of DHA-epoxide. High concentrations of DHA (1–10 μM) activated whole-cell BK currents with an EC50 of 2.38 ± 0.22 μM and the activation effects were unaltered by pre-incubation with SKF525A. Single channel studies showed that the open probabilities of BK channels were unchanged in the presence of low concentrations of DHA, while significantly increased with high concentrations of DHA. In addition, DHA induced a dose-dependent increase in cytosolic calcium concentrations with an EC50 of 0.037 ± 0.01 μM via phospholipase C (PLC)–inositol triphosphate (IP3)–Ca2+ signal pathway, and inhibition of this pathway reduced DHA-induced BK activation.Conclusion: These results suggest that DHA can activate BK channels by multiple mechanisms. Low concentration DHA-induced BK channel activation is mediated through CYP epoxygenase metabolites, while high concentration DHA can directly activate BK channels. In addition, DHA at low and high concentrations can both activate BK channels by elevated cytosolic calcium through the PLC–IP3–Ca2+ signal pathway.

Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

Protective effect of berberine on acute cardiomyopathy associated with doxorubicin treatment.

Doxorubicin (DOX) is a potent and broad-spectrum anthracycline chemotherapeutic agent, but dose-dependent cardiotoxic side effects limit its clinical application. This toxicity is closely associated with the generation of reactive oxygen species (ROS) radical during DOX metabolism. The present study investigated the effects of Berberine (Ber) on DOX-induced acute cardiac injury in a rat model and analysed its mechanism in cardiomyocytes in vitro. Serum creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and malondialdehyde (MDA) levels were significantly increased in the DOX group compared with the control group. This increase was accompanied by cardiac histopathological injury and a decrease in cardiomyocyte superoxide dismutase (SOD) and catalase (CAT). CK, CK-MB and MDA levels decreased and SOD and CAT levels increased in the Ber-treated group compared to the DOX group. Ber ameliorated the DOX-induced increase in cytosolic calcium concentration ([Ca2+]i), attenuated mitochondrial Ca2+ overload and restored the DOX-induced loss of mitochondrial membrane potential in vitro. These results demonstrated that Ber exhibited protective effects against DOX-induced heart tissue free radical injury, potentially via the inhibition of intracellular Ca2+ elevation and attenuation of mitochondrial dysfunction.

Supra-pharmacological concentration of capsaicin stimulates brown adipogenesis through induction of endoplasmic reticulum stress

We previously showed that brown (pre)adipocytes express Trpv1, a capsaicin receptor, and that capsaicin stimulates differentiation of brown preadipocytes in the late stages of brown adipogenesis. The present study revealed that treatment with 100 μM capsaicin stimulates brown adipogenesis by inducing endoplasmic reticulum (ER) stress. Treatment with capsaicin (100 μM) during brown adipogenesis enhanced lipid accumulation and the expression of Ucp1, a gene selectively expressed in brown adipocytes. Capsaicin treatment also caused an increase in the cytosolic calcium concentration even when extracellular calcium was removed. I-RTX, a Trpv1 inhibitor, did not modulate the increase in cytosolic calcium concentration, lipid accumulation or Ucp1 expression. Previous studies revealed that the release of calcium from the ER induces ER stress, leading to the conversion of X-box binding protein 1 (Xbp1) pre-mRNA to spliced Xbp1 (sXbp1) as well as the up-regulation of Chop expression. Capsaicin treatment increased the expression of sXbp1 and Chop in brown preadipocytes and did not enhance lipid accumulation or Ucp1 expression in Xbp1 knockdown cells. The present results describe a novel mechanism of brown adipogenesis regulation via ER stress that is induced by a supra-pharmacological concentration of capsaicin.

Spermine Regulates Pollen Tube Growth by Modulating Ca2+-Dependent Actin Organization and Cell Wall Structure.

Proper growth of the pollen tube depends on an elaborate mechanism that integrates several molecular and cytological sub-processes and ensures a cell shape adapted to the transport of gametes. This growth mechanism is controlled by several molecules among which cytoplasmic and apoplastic polyamines. Spermine (Spm) has been correlated with various physiological processes in pollen, including structuring of the cell wall and modulation of protein (mainly cytoskeletal) assembly. In this work, the effects of Spm on the growth of pear pollen tubes were analyzed. When exogenous Spm (100 μM) was supplied to germinating pollen, it temporarily blocked tube growth, followed by the induction of apical swelling. This reshaping of the pollen tube was maintained also after growth recovery, leading to a 30–40% increase of tube diameter. Apical swelling was also accompanied by a transient increase in cytosolic calcium concentration and alteration of pH values, which were the likely cause for major reorganization of actin filaments and cytoplasmic organelle movement. Morphological alterations of the apical and subapical region also involved changes in the deposition of pectin, cellulose, and callose in the cell wall. Thus, results point to the involvement of Spm in cell wall construction as well as cytoskeleton organization during pear pollen tube growth.

Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca2+]c) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects were almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca2+ channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca2+ entry through activation of TRPV4 channel in synoviocytes.

TRPV4 participates in pressure-induced inhibition of renin secretion by juxtaglomerular cells.

Increase in blood pressure in the renal afferent arteriole is known to induce an increase in cytosolic calcium concentration ([Ca2+ ]i ) of juxtaglomerular (JG) cells and to result in a decreased secretion of renin. Mechanical stimulation of As4.1 JG cells induces an increase in [Ca2+ ]i that is inhibited by HC067047 and RN1734, two inhibitors of TRPV4, or by siRNA-mediated repression of TRPV4. Inhibition of TRPV4 impairs pressure-induced decrease in renin secretion. Compared to wild-type mice, Trpv4-/- mice present increased resting plasma levels of renin and aldosterone and present a significantly altered pressure-renin relationship. We suggest that TRPV4 channel participates in mechanosensation at the juxtaglomerular apparatus. The renin-angiotensin system is a crucial blood pressure regulation system. It consists of a hormonal cascade where the rate-limiting enzyme is renin, which is secreted into the blood flow by renal juxtaglomerular (JG) cells in response to low pressure in the renal afferent arteriole. In contrast, an increase in blood pressure results in a decreased renin secretion. This is accompanied by a transitory increase in [Ca2+ ]i of JG cells. The inverse relationship between [Ca2+ ]i and renin secretion has been called the 'calcium paradox' of renin release. How increased pressure induces a [Ca2+ ]i transient in JG cells, is however, unknown. We observed that [Ca2+ ]i transients induced by mechanical stimuli in JG As4.1 cells were completely abolished by HC067047 and RN1734, two inhibitors of TRPV4. They were also reduced by half by siRNA-mediated repression of TRPV4 but not after repression or inhibition of TRPV2 or Piezo1 ion channels. Interestingly, the stimulation of renin secretion by the adenylate cyclase activator forskolin was totally inhibited by cyclic stretching of the cells. This effect was mimicked by stimulation with GSK1016790A and 4αPDD, two activators of TRPV4 and inhibited in the presence of HC067047. Moreover, in isolated perfused kidneys from Trpv4-/- mice, the pressure-renin relationship was significantly altered. In vivo, Trpv4-/- mice presented increased plasma levels of renin and aldosterone compared to wild-type mice. Altogether, our results suggest that TRPV4 is involved in the pressure-induced entry of Ca2+ in JG cells, which inhibits renin release and allows the negative feedback regulation on blood pressure.

Elevated K+ channel activity opposes vasoconstrictor response to serotonin in cerebral arteries of the Fawn Hooded Hypertensive rat.

Previous studies suggest that middle cerebral arteries (MCAs) of Fawn Hooded Hypertensive (FHH) rats exhibit impaired myogenic response and introgression of a small region of Brown Norway chromosome 1 containing 15 genes restored the response in FHH.1BN congenic rat. The impaired myogenic response in FHH rats is associated with an increase in the activity of the large conductance potassium (BK) channel in vascular smooth muscle cells (VSMCs). The present study examined whether the increased BK channel function in FHH rat alters vasoconstrictor response to serotonin (5-HT). Basal myogenic tone and spontaneous myogenic response of the MCA was attenuated by about twofold and about fivefold, respectively in FHH compared with FHH.1BN rats. 5-HT (0.1 μM)-mediated vasoconstriction was about twofold lower, and inhibition of the BK channel increased the vasoconstrictor response by about threefold in FHH compared with FHH.1BN rats. 5-HT (3 μM) decreased BK channel and spontaneous transient outward currents in VSMCs isolated from FHH.1BN but had no effect in FHH rats. 5-HT significantly depolarized the membrane potential in MCAs of FHH.1BN than FHH rats. Blockade of the BK channel normalized 5-HT-induced depolarization in MCAs of FHH rats. The 5-HT-mediated increase in cytosolic calcium concentration was significantly reduced in plateau phase in the VSMCs of FHH relative to FHH.1BN rats. These findings suggest that sequence variants in the genes located in the small region of FHH rat chromosome 1 impairs 5-HT-mediated vasoconstriction by decreasing its ability to inhibit BK channel activity, depolarize the membrane and blunt the rise in cytosolic calcium concentration.