Stretch-activated Cation Channels Research Articles

TRPV4 subserves physiological and pathological elevations in intraocular pressure.

Ocular hypertension (OHT) caused by mechanical stress and chronic glucocorticoid exposure reduces the hydraulic permeability of the conventional outflow pathway. It increases the risk for irreversible vision loss, yet healthy individuals experience nightly intraocular pressure (IOP) elevations without adverse lifetime effects. It is not known which pressure sensors regulate physiological vs. pathological OHT nor how they impact the permeability of the principal drainage pathway through the trabecular meshwork (TM). We report that OHT induced by the circadian rhythm, occlusion of the iridocorneal angle and glucocorticoids requires activation of TRPV4, a stretch-activated cation channel. Wild-type mice responded to nocturnal topical administration of the agonist GSK1016790A with IOP lowering, while intracameral injection of the agonist elevated diurnal IOP. Microinjection of TRPV4 antagonists HC067047 and GSK2193874 lowered IOP during the nocturnal OHT phase and in hypertensive eyes treated with steroids or injection of polystyrene microbeads. Conventional outflow-specific Trpv4 knockdown induced partial IOP lowering in mice with occluded iridocorneal angle and protected retinal neurons from pressure injury. Indicating a central role for TRPV4-dependent mechanosensing in trabecular outflow, HC067047 doubled the outflow facility in TM-populated steroid-treated 3D nanoscaffolds. Tonic TRPV4 signaling thus represents a fundamental property of TM biology as a driver of increased in vitro and in vivo outflow resistance. The TRPV4-dependence of OHT under conditions that mimic primary and secondary glaucomas could be explored as a novel target for glaucoma treatments.

Intermediate conductance Ca2+-activated potassium channels are activated by functional coupling with stretch-activated nonselective cation channels in cricket myocytes.

Cooperative gating of localized ion channels ranges from fine-tuning excitation-contraction coupling in muscle cells to controlling pace-making activity in the heart. Membrane deformation resulting from muscle contraction activates stretch-activated (SA) cation channels. The subsequent Ca2+ influx activates spatially localized Ca2+-sensitive K+ channels to fine-tune spontaneous muscle contraction. To characterize endogenously expressed intermediate conductance Ca2+-activated potassium (IK) channels and assess the functional relevance of the extracellular Ca2+ source leading to IK channel activity, we performed patch-clamp techniques on cricket oviduct myocytes and recorded single-channel data. In this study, we first investigated the identification of IK channels that could be distinguished from endogenously expressed large-conductance Ca2+-activated potassium (BK) channels by adding extracellular Ba2+. The single-channel conductance of the IK channel was 62 pS, and its activity increased with increasing intracellular Ca2+ concentration but was not voltage-dependent. These results indicated that IK channels are endogenously expressed in cricket oviduct myocytes. Second, the Ca2+ influx pathway that activates the IK channel was investigated. The absence of extracellular Ca2+ or the presence of Gd3+ abolished the activity of IK channels. Finally, we investigated the proximity between SA and IK channels. The removal of extracellular Ca2+, administration of Ca2+ to the microscopic region in a pipette, and application of membrane stretching stimulation increased SA channel activity, followed by IK channel activity. Membrane stretch-induced SA and IK channel activity were positively correlated. However, the emergence of IK channel activity and its increase in response to membrane mechanical stretch was not observed without Ca2+ in the pipette. These results strongly suggest that IK channels are endogenously expressed in cricket oviduct myocytes and that IK channel activity is regulated by neighboring SA channel activity. In conclusion, functional coupling between SA and IK channels may underlie the molecular basis of spontaneous rhythmic contractions.

Tentonin 3/TMEM150C regulates glucose-stimulated insulin secretion in pancreatic β-cells.

Glucose homeostasis is initially regulated by the pancreatic hormone insulin. Glucose-stimulated insulin secretion in β-cells is composed of two cellular mechanisms: a high glucose concentration not only depolarizes the membrane potential of the β-cells by ATP-sensitive K+ channels but also induces cell inflation, which is sufficient to release insulin granules. However, the molecular identity of the stretch-activated cation channel responsible for the latter pathway remains unknown. Here, we demonstrate that Tentonin 3/TMEM150C (TTN3), a mechanosensitive channel, contributes to glucose-stimulated insulin secretion by mediating cation influx. TTN3 is expressed specifically in β-cells and mediates cation currents to glucose and hypotonic stimulations. The glucose-induced depolarization, firing activity, and Ca2+ influx of β-cells were significantly lower in Ttn3-/- mice. More importantly, Ttn3-/- mice show impaired glucose tolerance with decreased insulin secretion invivo. We propose that TTN3, as a stretch-activated cation channel, contributes to glucose-stimulated insulin secretion.

Aquaporins in platelet function

Structurally, aquaporins (AQPs) are small channel proteins with monomers of ~ 30 kDa that are assembled as tetramers to form pores on cell membranes. Aquaporins mediate the conduction of water but at times also small solutes including glycerol across cell membranes and along osmotic gradients. Thirteen isoforms of AQPs have been reported in mammalian cells, and several of these are likely expressed in platelets. Osmotic swelling mediated by AQP1 sustains the calcium entry required for platelet phosphatidylserine exposure and microvesiculation, through calcium permeable stretch-activated or mechanosensitive cation channels. Notably, deletion of AQP1 diminishes platelet procoagulant membrane dynamics in vitro and arterial thrombosis in vivo, independent of platelet granule secretion and without affecting hemostasis. Water entry into platelets promotes procoagulant activity, and AQPs may also be critical for the initiation and progression of venous thrombosis. Platelet AQPs may therefore represent valuable targets for future development of a new class of antithrombotics, namely, anti-procoagulant antithrombotics, that are mechanistically distinct from current antithrombotics. However, the structure of AQPs does not make for easy targeting of these channels, hence they remain elusive drug targets. Nevertheless, thrombosis data in animal models provide compelling reasons to continue the pursuit of AQP-targeted antithrombotics. In this review, we discuss the role of aquaporins in platelet secretion, aggregation and procoagulation, the challenge of drugging AQPs, and the prospects of targeting AQPs for arterial and venous antithrombosis.

TRPV4 channels mediate the mechanoresponse in retinal microglia.

The physiological and neurological correlates of plummeting brain osmolality during edema, traumatic CNS injury, and severe ischemia are compounded by neuroinflammation. Using multiple approaches, we investigated how retinal microglia respond to challenges mediated by increases in strain, osmotic gradients, and agonists of the stretch-activated cation channel TRPV4. Dissociated and intact microglia were TRPV4-immunoreactive and responded to the selective agonist GSK1016790A and substrate stretch with altered motility and elevations in intracellular calcium ([Ca2+ ]i ). Agonist- and hypotonicity-induced swelling was associated with a nonselective outwardly rectifying cation current, increased [Ca2+ ]i , and retraction of higher-order processes. The antagonist HC067047 reduced the extent of hypotonicity-induced microglial swelling and inhibited the suppressive effects of GSK1016790A and hypotonicity on microglial branching. Microglial TRPV4 signaling required intermediary activation of phospholipase A2 (PLA2), cytochrome P450, and epoxyeicosatrienoic acid production (EETs). The expression pattern of vanilloid thermoTrp genes in retinal microglia was markedly different from retinal neurons, astrocytes, and cortical microglia. These results suggest that TRPV4 represents a primary retinal microglial sensor of osmochallenges under physiological and pathological conditions. Its activation, associated with PLA2, modulates calcium signaling and cell architecture. TRPV4 inhibition might be a useful strategy to suppress microglial overactivation in the swollen and edematous CNS.

Functional Coupling of Ion Channels in the Process of Mechano-Dependent Activation in the Membrane of K562 Cells

Mechanically gated cation channels that are activated upon deformation of the membrane are key participants in the transmission of mechanical signals from the cell surface to cytoplasmic structures. It has remained unclear how mechanically dependent reactions involving ion channels in native cells are realized. In this study, we analyzed the development of the activity of single channels in K562 human myeloid leukemia cells in response to the delivery of a mechanical stimulus, this being stretching of the membrane area. The registration of ion currents using the classic versions of the patch–clamp method allowed revealing the functional clustering and interaction of various types of channels in the plasma membrane during mechanotransduction. In K562 cells, coupled activation of mechanosensitive calcium-conducting channels and calcium-activated potassium channels was detected. Real-time recordings of currents demonstrate that the calcium influx from the extracellular medium into the cytoplasm through mechanically gated channels activates potassium channels that are colocalized with them, which do not have their own mechanosensitivity. In experiments on K562 cells and transformed 3T3-SV40 fibroblasts, the preservation of the functional conjugation of the channels during their mechanically gated activation after the action of the microfilament destructor cytochalasin D was shown. The results suggest that clusters including potassium SK channels and Piezo1/2 proteins forming stretch-activated cation channels function in the plasma membrane of K562 and 3T3-SV40 cells.

Heavy Metal Intoxication: A Key-Player in Chronic Kidney Disease (A Review)

Heavy metals gain entry into biological systems mainly via inhalation and ingestion, and also via radiation or radio-therapeutic measures. The accumulation of these heavy metals in biological systems overtime may cause several deleterious health challenges such as liver, kidney and brain damages amongst others. Intoxication with heavy metals may either be acute or chronic, and because the kidney has the ability to reabsorb and accumulate divalent metals, it happens to be the primary target organ for heavy metal toxicity, inducing renal damage. The extent of this damage depends on the dose, nature, route and duration of exposure to the metal. Chronic kidney disease (CKD) is characterized by a permanent loss of nephrons accompanied by an eventual decline in glomerular filtration rate (GFR); this (to a greater extent) maybe due to heavy metal intoxication and the renal reabsorption of these heavy metals. Although 70 percent of the heavy metals are reabsorbed in the proximal tubule, all segments of the nephrons are involved in the reabsorption of these metals, where several transporters such as the Divalent Metal Transporter (DMT)-1, Na+/amino acid co-transporter, Zinc Transporter (ZnT)-1 and stretch-activated cation channels (SAC) facilitate the reabsorption. In the nephrons of each kidney, heavy metals are primarily reabsorbed via the apical membrane and accumulate at the basolateral membrane; these heavy metals do not readily exit the basolateral membrane, which overtime may result in chronic inflammation of the nephrons, fibrosis and kidney failure. However, the loss of nephrons and decline in GFR in CKD are compensated by certain changes (glomerular and cellular, enhanced renal blood flow, enhanced single nephron glomerular filtration rate and tubular hypertrophy) in the remaining functional nephrons. These changes help to deliver solutes to the remaining functional nephrons for uptake by the epithelial cells of the renal tubules. Also, because the luminal and basolateral surfaces of tubular epithelial cells of the remaining healthy nephrons are also potentially exposed to higher levels of metabolic wastes, xenobiotics, heavy metals and other nephrotoxicants, renal injury, tubular or glomerulosclerosis, and death of these nephrons occur. These compensatory changes become insufficient once about 75 percent of the nephrons are no longer functional and incapable of maintaining homeostasis and renal function. This results in the accumulation of metabolic wastes in the blood, and induction of metabolic disturbances and/or organ intoxication.

Mechanically Defined Microenvironment Promotes Stabilization of Microvasculature, Which Correlates with the Enrichment of a Novel Piezo-1+ Population of Circulating CD11b+ /CD115+ Monocytes.

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long-term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long-term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b+ myeloid and CD11b+ /CD115+ monocytes that express the stretch activated cation channel Piezo-1, which is enriched prominently in the clot-like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.

Brv1 Is Required for Drosophila Larvae to Sense Gentle Touch

How we sense touch is fundamental for many physiological processes. However, the underlying mechanism and molecular identity for touch sensation arelargely unknown. Here, we report on defective gentle-touch behavioral responses in brv1 loss-of-function Drosophila larvae. RNAi and Ca2+ imaging confirmed the involvement of Brv1 in sensing touch and demonstrated that Brv1 mediates the mechanotransduction of class III dendritic arborization neurons. Electrophysiological recordings further revealed that the expression of Brv1 protein in HEK293T cells gives rise to stretch-activated cation channels. Purified Brv1 protein reconstituted into liposomes were found to sense stretch stimuli. In addition, co-expression studies suggested that Brv1 amplifies the response of mechanosensitive ion channel NOMPC (no mechanoreceptor potential C) to touch stimuli. Altogether, these findings demonstrate a molecular entity that mediates the gentle-touch response in Drosophila larvae, providing insights into the molecular mechanisms of touch sensation.

Electrotaxis of tumor-initiating cells of H1975 lung adenocarcinoma cells is associated with both activation of stretch-activated cation channels (SACCs) and internal calcium release

The metastatic potential of cancer cells is related to their migratory ability, which is influenced by in vivo microenvironment possessing specific physiochemical factors including electric properties. In the present study, we isolated two different subsets of lung adenocarcinoma H1975 cells, as side population (SP) and main population (MP). SP cells were demonstrated to have cancer stem cell characteristics. Using a microscale device to provide physiological direct-current electric field (dcEF), we investigated the electrotactic responses of the SP and MP cells. The results showed that both SP and MP cells exhibited enhanced cathodal migration ability with actin reorganization and transient intracellular calcium ions ([Ca2+]i) increase under dcEF stimulation. For SP cells, the treatment of either stretch-activated cation channels (SACCs) inhibitor or the blockage of intracellular Ca2+ release could partially inhibited dcEF-activated [Ca2+]i increase, and the concomitant treatment led to a complete inhibition. For MP cells, SACCs activation was entirely responsible for EF-activated increase of [Ca2+]i. All these results suggested that that intracellular Ca2+ activation may be associated with cancer cell tumorigenicity and metastasis.

Endocardial endothelium as a blood-heart barrier

Introduction. Endocardial endothelium is formed from a single layer of closely related cells with complex interrelationships and extensive overlap at the junctional edges. Morphological characteristics of blood-heart barrier. Endocardium is composed of three layers: endocardial endothelium, subendothelial loose connective tissue and subendocardium. The fibrous component of the subendothelium consists of small amount of collagen and elastic fibers. Several cell types are present in subendocardium: telocytes, fibroblasts and nerve endings. Intercellular bonds between the endocardial endothelial cells. Endocardial endothelial cells are attached to one another via sets of binding proteins forming solid, adherent and communicating connections. Communicating connections form transmembrane channels between the neighboring cells, while solid and adherent connections form pericellular structures like stitches. The maintenance of the presumed transendocardial electrochemical potential difference provides a high gradient for certain ions as well as a selective boundary barrier, basal lamina, preventing ionic leakage. The negatively charged glycocalyx also modulates endothelial permeability. Electrophysiological characteristics of heart-blood barrier. Electrophysiological studies have shown the existence of a large number of membrane ion channels in the endocardial endothelial cells: inward rectifying K+ channels, Ca2+ dependent K+channels, voltage-dependent Cl-channels, volume-activated Cl-channels, stretch-activated cation channels and one carrier mediated transport mechanism - Na+K+adenosine triphosphatase. Conclusion. Numerous diseases of the cardiovascular system may be a consequence, but also the cause of the endocardial endothelium dysfunction. Selective damage to the endocardial endothelium and subendocardium is found in arrhythmia, atrial fibrillation, ischemia/reperfusion injury and heart failure. Typical lesions of endocardial and microvascular endothelium have also been described in sepsis, myocardial infarction, inflammation and thrombosis. The result of endothelial dysfunction is the weakening of the endothelial barrier regulation and electrolyte imbalance of the subendocardial interstitium.

Dysregulation of Intracellular Ca2+ in Dystrophic Cortical and Hippocampal Neurons.

Duchenne muscular dystrophy (DMD) is an inherited X-linked disorder characterized by skeletal muscle wasting, cardiomyopathy, as well as cognitive impairment. Lack of dystrophin in striated muscle produces dyshomeostasis of resting intracellular Ca2+ ([Ca2+]i), Na+ ([Na+]i), and oxidative stress. Here, we test the hypothesis that similar to striated muscle cells, an absence of dystrophin in neurons from mdx mice (a mouse model for DMD) is also associated with dysfunction of [Ca2+]i homeostasis and oxidative stress. [Ca2+]i and [Na+]i in pyramidal cortical and hippocampal neurons from 3 and 6months mdx mice were elevated compared to WT in an age-dependent manner. Removal of extracellular Ca2+ reduced [Ca2+]i in both WT and mdx neurons, but the decrease was greater and age-dependent in the latter. GsMTx-4 (a blocker of stretch-activated cation channels) significantly decreased [Ca2+]i and [Na+]i in an age-dependent manner in all mdx neurons. Blockade of ryanodine receptors (RyR) or inositol triphosphate receptors (IP3R) reduced [Ca2+]i in mdx. Mdx neurons showed elevated and age-dependent reactive oxygen species (ROS) production and an increase in neuronal damage. In addition, mdx mice showed a spatial learning deficit compared to WT. GsMTx-4 intraperitoneal injection reduced neural [Ca2+]i and improved learning deficit in mdx mice. In summary, mdx neurons show an age-dependent dysregulation in [Ca2+]i and [Na+]i which is mediated by plasmalemmal cation influx and by intracellular Ca2+ release through the RyR and IP3R. Also, mdx neurons have elevated ROS production and more extensive cell damage. Finally, a reduction of [Ca2+]i improved cognitive function in mdx mice.

Development and localization of reverse-polarity mechanotransducer channels in cochlear hair cells

Cochlear hair cells normally detect positive deflections of their hair bundles, rotating toward their tallest edge, which opens mechanotransducer (MT) channels by increased tension in interciliary tip links. After tip-link destruction, the normal polarity of MT current is replaced by a mechanically sensitive current evoked by negative bundle deflections. The "reverse-polarity" current was investigated in cochlear hair cells after tip-link destruction with BAPTA, in transmembrane channel-like protein isoforms 1/2 (Tmc1:Tmc2) double mutants, and during perinatal development. This current is a natural adjunct of embryonic development, present in all wild-type hair cells but declining after birth with emergence of the normal-polarity current. Evidence indicated the reverse-polarity current seen developmentally was a manifestation of the same ion channel as that evident under abnormal conditions in Tmc mutants or after tip-link destruction. In all cases, sinusoidal fluid-jet stimuli from different orientations suggested the underlying channels were opened not directly by deflections of the hair bundle but by deformation of the apical plasma membrane. Cell-attached patch recording on the hair-cell apical membrane revealed, after BAPTA treatment or during perinatal development, 90-pS stretch-activated cation channels that could be blocked by Ca(2+) and by FM1-43. High-speed Ca(2+) imaging, using swept-field confocal microscopy, showed the Ca(2+) influx through the reverse-polarity channels was not localized to the hair bundle, but distributed across the apical plasma membrane. These reverse-polarity channels, which we propose to be renamed "unconventional" mechanically sensitive channels, have some properties similar to the normal MT channels, but the relationship between the two types is still not well defined.

Confinement Sensing and Signal Optimization via Piezo1/PKA and Myosin II Pathways.

SUMMARYCells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments.

Cardiovascular-Active Venom Toxins: An Overview.

Animal venoms are a mixture of bioactive compounds produced as weapons and used primarily to immobilize and kill preys. As a result of the high potency and specificity for various physiological targets, many toxins from animal venoms have emerged as possible drugs for the medication of diverse disorders, including cardiovascular diseases. Captopril, which inhibits the angiotensin-converting enzyme (ACE), was the first successful venom-based drug and a notable example of rational drug design. Since captopril was developed, many studies have discovered novel bradykinin-potentiating peptides (BPPs) with actions on the cardiovascular system. Natriuretic peptides (NPs) have also been found in animal venoms and used as template to design new drugs with applications in cardiovascular diseases. Among the anti-arrhythmic peptides, GsMTx-4 was discovered to be a toxin that selectively inhibits the stretch-activated cation channels (SACs), which are involved in atrial fibrillation. The present review describes the main components isolated from animal venoms that act on the cardiovascular system and presents a brief summary of venomous animals and their venom apparatuses.

Wall stretch and thromboxane A₂ activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H₂O₂ and Akt-dependent phosphorylation.

Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 μM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli.

Activation of Ca2+-Dependent Cation Current by Fluid Shear Force in Atrial Myocytes

Atrial myocytes are subjected to fluid shear force (FSF) during each contraction and relaxation. Ionic currents regulated by shear force and their molecular integrity in cardiac myocytes have not been well-understood. We examined whether FSF activates specific current in atrial myocytes and underlying mechanisms for FSF-sensitive ionic current using whole-cell patch-clamp technique. A FSF of ∼16 dyne/cm2 was applied to entire single atrial myocyte using automated micro-puffing apparatus. A FSF-sensitive current (IFSF) was detected in lowly Ca2+-buffered (0.5 mM EGTA) atrial myocytes, but not in highly Ca2+-buffered (≥4 mM EGTA or 10 mM BAPTA) myocytes. The IFSF showed an outward rectification with a reversal potential of about -6 mV. The IFSF was inhibited by high concentrations (20-50 μM) of ryanodine and by replacement of external and internal cation with impermeant NMDG+, suggesting that IFSF is a Ca2+ release-dependent cation current. Application of either transient receptor potential melastatin subfamily 4 (TRPM4) inhibitor 9-phenanthrol or TRPM4-specific antibodies removed most of inward IFSF and ∼80% of outward IFSF. However, stretch-activated cation channel blocker GsMTx-4 did not affect IFSF. Interestingly, IFSF was strongly inhibited by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, 2-APB (2 μM) or xestospongin C. In addition, in atrial myocytes isolated from type 2 IP3R (IP3R2) knock-out mouse, IFSF was not detected, although 9-phenanthrol-sensitive IFSF was recorded in wild-type myocytes. Co-immunostaining of TRPM4 and IP3R2 in rat atrial myocytes revealed peripheral localization of these proteins with some co-localizations. These results suggest that fluid shear stimuli may activate TRPM4 channels in atrial myocytes via Ca2+ releases triggered by the activation of nearby IP3R2.

Fluid Shear Induces Proarrhythmic Ca2+ Wave and Alters Atrial Ca2+ Signaling: A Role of Autocrine Activations of P2-Purinergic/Type 2 Inositol Trisphosphate Receptor Signaling

Cardiac myocytes are subjected to fluid shear force (FSF) during each contraction and relaxation. Under pathological conditions, such as valve disease, heart failure and hypertension, FSF in cardiac chamber increases due to high blood volume and pressure. We have previously shown that FSF induces proarrhythmic longitudinal global Ca2+ waves and premature beat in rat atrial myocytes (Biophys J, 2014; 106 (2): 320a). In the present study, we further explored underlying cellular mechanisms for the FSF-induced longitudinal global Ca2+ waves and their effects on Ca2+ signaling in rat atrial myocytes. A FSF of ∼16 dyn/cm2 was applied onto entire single myocyte using pressurized fluid puffing. Confocal Ca2+ imaging was performed to measure local and global Ca2+ signals. Proarrhythmic longitudinal Ca2+ waves in atrial cells under FSF were resistant to the blockers for stretch-activated cation channel, Na+-Ca2+ exchange or Ca2+-activated cation channel (TRPM4), and to removal of external Ca2+. Interestingly, this FSF-induced atrial Ca2+ wave was eliminated by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, tetracaine, or by phospholipase C inhibitor, and were absent in the type 2 IP3R (IP3R2) knockout cells. Furthermore, the FSF-induced atrial Ca2+ waves were suppressed by blockade of P2-purinergic receptors or by inhibition of gap junction that can secrete ATP. In field-stimulated cells, FSF immediately enlarged Ca2+ transients with larger increase in non-junctional Ca2+ release relative to junctional release. This FSF-induced enhancement in Ca2+ release was soon followed by significant reductions in Ca2+ transient and sarcoplasmic reticulum Ca2+ content. Our data suggest that IP3R2-mediated Ca2+ release, triggered by autocrine activation of P2-purinergic signaling, may generate proarrhythmic Ca2+ wave, thereby altering atrial Ca2+ signaling.

Concerted actions of TXA2 and mechanical stretch stimulate NOS3 (eNOS) in rat pulmonary arterial myocytes putatively via ROS generation and mechanosensitive channel activation (LB673)

Nitric oxide (NO), a potent vasodilator, is produced by type 3 NO synthase (NOS3, eNOS) in endothelium. In endothelium‐denuded pulmonary arteries (PAs) from rat, however, we found that unspecific NOS inhibitor (L‐NAME) induces a weak contraction in the presence of 2 ‐ 5 nM U46619 (thromboxane A2 agonist). With additional increase in wall tension equivalent to pressure elevation to 30 mmHg (pre‐stretch), L‐NAME induced robust contraction of PAs (L‐NAME contraction; 110 % of 80 mM KCl contraction). In endothelium‐denuded systemic arteries (e.g. renal and mesenteric arteries), no such L‐NAME induced contraction was observed. Interestingly, contraction of PAs by 20 mM KCl and 2 nM U46619 was also robustly augmented by L‐NAME, suggesting that the pre‐stretch effect was mimicked by partial depolarization. Pretreatment with 30 µM DIDS, known as an inhibitor of stretch‐activated cation channels (SACs) in arterial myocytes, the L‐NAME contraction was abolished in the PAs with pre‐stretch. The L‐NAME contraction was also diminished by ROS scavengers (Tiron and PEG‐Catalase). Consistently, exogenous hydrogen peroxide (H2O2, 1 µM) effectively augmented the contraction by L‐NAME without pre‐stretch whereas 1 µM H2O2 alone had no effect. Neither NOS1 inhibitor (SMTC) nor NOS2 inhibitor (1400W) mimicked the L‐NAME contraction. Muscular expression of NOS3 was confirmed by RT‐PCR and immunohistochemistry. Taken together, we suggest that PA myocytes express NOS3 that is concertedly activated by TXA2 and mechanical stretch. ROS generation and facilitated SACs by wall stretch might mediate the NOS3 recruitment in PA myocytes, and the resultant decrease in the contractility might contribute to the relatively low peripheral resistance of pulmonary circulation with fluctuating PA pressure.

The effect of pressure on potassium currents in the inner hair cells isolated from guinea-pig cochlea.

An increase in hydrostatic pressure in the endolymphatic system causes hydrops-related inner ear diseases such as Meniere's disease or low tone sensorineural hearing loss. In the present study, we investigated the effects of pressure exerted on potassium currents in acutely isolated inner hair cells of the guinea-pig cochlea using whole-cell voltage-clamp techniques. By applying negative or positive pressure via the patch pipette using a syringe, intracellular hydropressure was changed between -40 cm H2O to +20 cm H2O. Negative pressure potentiated the amplitude of potassium currents, whereas positive pressure suppressed the amplitude of potassium currents. Gadolinium, a blocker of stretch-activated cation channels, did not influence pressure-dependent changes in potassium currents; however, cinnarizine blocked pressure-dependent changes in potassium currents. The current changes were not dependent on the sign of the pressure change, that is, similar increases in negative pressures (between -10 cm H2O and -40 cm H2O) and similar decreases in positive pressures (between +10 cm H2O and +20 cm H2O) were observed.