Hypotonic Stimulation Research Articles

Transient Receptor Potential Vanilloid 4 Mediates Hypotonicity‐Induced Enhancement of Synaptic Transmission in Hippocampal Slices

Changes in cerebrospinal fluid osmotic pressure modulate brain excitability. Transient receptor potential vanilloid 4 (TRPV4), which is sensitive to hypotonic stimulation, is expressed in hippocampus. The present study investigated the effect of hypotonic stimulation on hippocampal synaptic transmission and the role of TRPV4 in hypotonicity-action using electrophysiological recording and pharmacological technique. Accompanied with the decrease in paired pulse facilitation, field excitatory postsynaptic potential (fEPSP) was enhanced by hypotonicity and TRPV4 agonist 4α-PDD in hippocampal slices, which was sensitive to TRPV4 antagonist HC-067047. Hypotonicity-induced increase in fEPSP was blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, but not N-methyl-d-aspartate receptor or N- or P/Q-type voltage-gated calcium channel antagonist. High voltage-gated calcium current (ICa ) in hippocampal CA3 pyramidal neurons was not affected by hypotonicity. AMPA-activated current (IAMPA ) in hippocampal CA1 pyramidal neurons was increased by hypotonicity and 4α-PDD, which was attenuated by HC-067047. Inhibition of protein kinase C or protein kinase A markedly attenuated hypotonicity-increased IAMPA , whereas antagonism of calcium/calmodulin-dependent protein kinase II had no such effect. TRPV4 is involved in hypotonicity-induced enhancement of hippocampal synaptic transmission, which may be mediated through promoting presynaptic glutamate release and increasing postsynaptic AMPA receptor function.

Mechanisms of regulatory volume decrease of human lens epithelial cells associated with hypotonic stimulation

Background It is widely appreciated that many animal cells rely on the mechanism of regulatory volume decrease (RVD) after swell under the hypotonic environment,which involved in some processes of cellular physiology.But the RVD of lens epithelial cells(LECs) still is being further researched.Objective Present study was to clarify the possible mechanisms and influencing factors in the RVD of LECs.Methods Human LECs line (HLE B-3)were cultured and passaged in DMEM/F12 containing 10％ fetal bovine serum(FBS),and before volume measurement,cells were stuck to the base of a perfusion chamber,Ringer solution osmolality was decreased from 15％Hypo to 45％ Hypo,and the cells stimulated by 45％ Hypo Ringer solution were used as the control group.Some experiments were performed in the presence of high extracellular K+ concentration,chloride or potassium channel inhibitor,experiments were also carried out in the nominal absence of Ca2+,Cl-or HCO-3 to test the effect of a decrease in intracellular concentration of these ions on the cell volume response.The volume changes of living cells were measured by lag-time microphotograph acquisition and analysis system (IPP6.0).Results Time course of cell volume change after hypotonic shock in HLE B-3 cells was observed.The cell swelling was followed by a gradual volume recovery,indicating the presence of RVD was influenced by the hypotonic stress.Under the stimulation of 45 ％Hypo Ringer solution,the rates of RVD were (59.1 ±7.8)％.RVD was correlated positively to the maximum cell volume (r =0.99,P＜0.05)in S shape,and RVD changes were sensitive to alter maximum cell volume in the range of 115％-135％.RVD reduced to (16.5 ± 1.6) ％,(14.7 ± 2.3) ％,respectively after acted by potassium channel inhibitor,TEA(10 mmol/L)and BaCl2(5 mmol/L)as well as chloride channel inhibitor,NPPB(100 μmol/L)and DIDS(100 μmol/L),with significant differences in comparison with the control group(all P＜0.01).RVD decreased by(5.8±1.6)％ and(2.7±0.8)％ in high concentration of K+ in extracellular fluid and the absence of Cl-(P＜0.01).RVD was significantly inhibited under the absence of Ca2+.When the 45％ Ringer solution was pH6.8,the process of RVD delayed.The rate of RVD in the first ten minutes was (0.86±0.24)％/min,showing a significant decline in comparison with (3.24±0.84) ％ / min of pH 7.4 (P ＜0.05).Conclusions HLE B-3 have RVD ability under the hypotonic stress stimulation.A certain intracellular Ca2+ concentration is the premise of RVD activation,and Cl efflux and K+ efflux are the key mechanism of RVD of HLE B-3.Acidic environment of hypotonic solution delays the occurrence of RVD. Key words: Lens epithelial cells; Regulatory volume decrease; Hypotonic stress stimulation; Cellular ion channel

Ion regulation in eurythermic teleost fish Fundulus heteroclitus: Cold shock vs. cold acclimation

Killifish overwinter (0–5°C) by decreasing activity and metabolic rate and yet they retain ability to ion/osmoregulate. Fish were acclimated to seawater (32 ppt) at 5°C and 20°C (8+ weeks); cold shock of 20°C opercular epithelia in vitro reversibly decreased Clsecretion by 90% (as short‐circuit current, SCC) and membranes failed to respond to hypotonic shock, IBMX/db‐cAMP (0.1 mM) or isoproterenol (1.0 μM). Opercular epithelia from cold‐acclimated fish also had low transport rates, but were able to respond normally, i.e. rapid inhibition of SCC by hypotonic shock and SCC stimulation by IBMX/db‐cAMP and isoproterenol. Cold acclimation caused homeoviscous adaptive changes, increased fatty acid unsaturation and increased short chain (e.g. reduced C18:0 and increased C14:0, C18:1) to maintain membrane fluidity. Cooling in vitro to 1°C induced a sharp bend in Arrhenius plots below 8°C for warm (Q10 = 6.9) and cold (Q10 = 4.2) acclimated fish, but these nonlinearities were markedly reduced (to Q10 = 2.9) by anaerobiosis and cyanide (0.5 mM), indicating mitochondrial inner membrane ATP production as source of the transition. In the cold, Fundulus markedly reduce ion transport rates but maintain regulatory control of ion transport through homeoviscous adaptation. Supported by NSERC, CFI and UCR.

Hypotonic-induced Stretching of Plasma Membrane Activates Transient Receptor Potential Vanilloid Channels and Sodium–Calcium Exchangers in Mouse Odontoblasts

IntroductionA number of transient receptor potential (TRP) channels have been identified as membrane-bound sensory proteins in odontoblasts. However, the activation properties of these channels remain to be clarified. The purpose of this study was to investigate hypotonic stimulation–induced Ca2+ entry via TRP vanilloid subfamily member (TRPV) 1, TRPV2, and TRPV4 channels, which are sensitive to osmotic and mechanical stimuli, and their functional coupling with Na+-Ca2+ exchangers (NCXs) in mouse odontoblast lineage cells. MethodsWe examined TRP channel activity by measuring intracellular-free Ca2+ concentration by using fura-2 fluorescence and ionic current recordings with whole-cell patch-clamp methods. Protein localization and messenger RNA expression were characterized using immunofluorescence and reverse-transcription polymerase chain reaction analyses. ResultsExtracellular hypotonic solution–induced stretching of plasma membrane resulted in the activation of Ca2+ influx and inward currents. TRPV1, TRPV2, and TRPV4 channel antagonists inhibited the hypotonic stimulation–induced Ca2+ entry and currents. Their respective agonists activated Ca2+ entry. Although the increase in the intracellular free Ca2+ concentration decayed rapidly after the applications of these TRPV channel agonists, NCX inhibitors significantly prolonged the decay time constant. The messenger RNA expression of TRPV1, TRPV2, and TRPV4 channels; NCX isoforms 2 and 3; and dentin sialophosphoprotein were up-regulated after 24 hours of exposure to the hypotonic culture medium. ConclusionsThese results indicate that stretching of the odontoblast membrane activates TRPV1-, TRPV2-, and TRPV4-mediated Ca2+ entry, and increased intracellular-free Ca2+ concentration is extruded via NCXs. These results suggest that odontoblasts can act as sensors that detect stimuli applied to exposed dentin and drive a number of cellular functions including dentinogenesis and/or sensory transduction.

KCNK5 is Functionally Down-Regulated Upon Long-Term Hypotonicity in Ehrlich Ascites Tumor Cells

Background/Aims: Regulatory volume decrease (RVD) in response to acute cell swelling is well described and KCNK5 (also known as TASK-2 or K_2P5.1) has been shown to be the volume sensitive K⁺ channel in Ehrlich cells. Very little is, on the other hand, known about the effects of long-term hypotonicity on expression and function of KCNK5, thus we have investigated the effect of long-term hypotonicity (24h - 48h) on KCNK5 in Ehrlich cells on the mRNA, protein and physiological levels. Methods: Physiological effects of long-term hypotonicity were measured using patch-clamp and Coulter counter techniques. Expression patterns of KCNK5 on mRNA and protein levels were established using real-time qPCR and western blotting respectively. Results: The maximum swelling-activated current through KCNK5 was significantly decreased upon 48h of hypotonicity and likewise the RVD response was significantly impaired after both 24 and 48h of hypotonic stimulation. No significant differences in the KCNK5 mRNA expression patterns between control and stimulated cells were observed, but a significant decrease in the KCNK5 protein level 48h after stimulation was found. Conclusion: The data suggest that the strong physiological impairment of KCNK5 in Ehrlich cells after long-term hypotonic stimulation is predominantly due to down-regulation of the KCNK5 protein synthesis.

Activation of Transient Receptor Potential Vanilloid 4 Increases NMDA-Activated Current in Hippocampal Pyramidal Neurons

The glutamate excitotoxicity, mediated through N-methyl-d-aspartate receptors (NMDARs), plays an important role in cerebral ischemia injury. Transient receptor potential vanilloid 4 (TRPV4) can be activated by multiple stimuli that may happen during stroke. The present study evaluated the effect of TRPV4 activation on NMDA-activated current (INMDA) and that of blocking TRPV4 on brain injury after focal cerebral ischemia in mice. We herein report that activation of TRPV4 by 4α-PDD and hypotonic stimulation increased INMDA in hippocampal CA1 pyramidal neurons, which was sensitive to TRPV4 antagonist HC-067047 and NMDAR antagonist AP-5, indicating that TRPV4 activation potentiates NMDAR response. In addition, the increase in INMDA by hypotonicity was sensitive to the antagonist of NMDAR NR2B subunit, but not of NR2A subunit. Furthermore, antagonists of calcium/calmodulin-dependent protein kinase II (CaMKII) significantly attenuated hypotonicity-induced increase in INMDA, while antagonists of protein kinase C or casein kinase II had no such effect, indicating that phosphorylation of NR2B subunit by CaMKII is responsible for TRPV4-potentiated NMDAR response. Finally, we found that intracerebroventricular injection of HC-067047 after 60 min middle cerebral artery occlusion reduced the cerebral infarction with at least a 12 h efficacious time-window. These findings indicate that activation of TRPV4 increases NMDAR function, which may facilitate glutamate excitotoxicity. Closing TRPV4 may exert potent neuroprotection against cerebral ischemia injury through many mechanisms at least including the prevention of NMDAR-mediated glutamate excitotoxicity.

Dynamics of cellular response to hypotonic stimulation revealed by quantitative phase microscopy and multi-fractal detrended fluctuation analysis

Hypotonic stimulation is known to cause morphological changes in cells and also leads to modulation of cellular physiology. In order to evaluate the dynamics of cellular response to hypotonic stimulation, we utilized digital holographic microscopy for quantitative phase microscopy, achieved by a common-path interferometry geometry based on extraction of reference beam by spatial-filtering. Results from live cell investigations demonstrate the capability of this method for dynamic quantitative phase imaging. Further, wavelet and multi-fractal detrended fluctuation analysis revealed that the dynamic phase changes, in response to hypotonic stimulation, are multifractal in nature.

Unusual localization and translocation of TRPV4 protein in cultured ventricular myocytes of the neonatal rat

TRPV4 protein forms a Ca2+-permeable channel that is sensitive to osmotic and mechanical stimuli and responds to warm temperatures, and expresses widely in various kinds of tissues. As for cardiac myocytes, TRPV4 has been detected only at the mRNA level and there were few reports about subcel-lular localization of the protein. The purpose of the present study was to investigate the expression profile of TRPV4 protein in cultured neonatal rat ventricular myocytes. Using Western blots, immunofluorescence, confocal microscopy and immuno-electron microscopy, we have shown that TRPV4 protein was predominantly located in the nucleus of cultured neonatal myocytes. Furthermore, cardiac myocytes responded to hypotonic stimulation by translocating TRPV4 protein out of the nucleus. The significance and mechanism concerning the unusual distribution and translocation of TRPV4 protein in cardiac myocytes remain to be clarified.

Involvements of the ABC protein ABCF2 and α‐actinin‐4 in regulation of cell volume and anion channels in human epithelial cells

After osmotic swelling, cell volume is regulated by a process called regulatory volume decrease (RVD). Although actin cytoskeletons are known to play a regulatory role in RVD, it is not clear how actin-binding proteins are involved in the RVD process. In the present study, an involvement of an actin-binding protein, α-actinin-4 (ACTN4), in RVD was examined in human epithelial HEK293T cells. Overexpression of ACTN4 significantly facilitated RVD, whereas siRNA-mediated downregulation of endogenous ACTN4 suppressed RVD. When the cells were subjected to hypotonic stress, the content of ACTN4 increased in a 100,000 × g pellet, which was sensitive to cytochalasin D pretreatment. Protein overlay assays revealed that ABCF2, a cytosolic member of the ABC transporter superfamily, is a binding partner of ACTN4. The ACTN4-ABCF2 interaction was markedly enhanced by hypotonic stimulation and required the NH(2) -terminal region of ABCF2. Overexpression of ABCF2 suppressed RVD, whereas downregulation of ABCF2 facilitated RVD. We then tested whether ABCF2 has a suppressive effect on the activity of volume-sensitive outwardly rectifying anion channel (VSOR), which is known to mediate Cl(-) efflux involved in RVD, because another ABC transporter member, CFTR, was shown to suppress VSOR activity. Whole-cell VSOR currents were largely reduced by overexpression of ABCF2 and markedly enhanced by siRNA-mediated depletion of ABCF2. Thus, the present study indicates that ACTN4 acts as an enhancer of RVD, whereas ABCF2 acts as a suppressor of VSOR and RVD, and suggests that a swelling-induced interaction between ACTN4 and ABCF2 prevents ABCF2 from suppressing VSOR activity in the human epithelial cells.

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.

Characterization of phosphorylation of NCC at serine 124

LC‐MS/MS‐based phosphoproteomic profiling identified phosphorylation of NCC at S124 (Feric M et al. Am J Physiol Cell 300: C755–770, 2011). We previously showed pS124‐NCC to be predominantly associated with the apical plasma membrane of DCT cells, where its abundance was regulated by AVP and ANGII. Semi‐quantitative 36Cl uptake studies of various NCC mutants in X. Laevis oocytes showed decreased activity of NCC when replacing S124 with Ala. SPAK or OSR1 protein kinases were unable to phosphorylate synthetic peptides corresponding to the region surrounding S124. Novel tetracycline inducible MDCKII cell lines expressing WT‐NCC and various mutants were generated and used for characterization of the S124 phosphorylation site. WT‐cells displayed thiazide‐sensitive 36Cl uptake, whereas a NCC‐S124A mutant demonstrated decreased activity. WT cells showed robust phosphorylation of NCC at T53 and T58 and membrane targeting following hypotonic low chloride stimulation. Treatment of WT cells with PMA decreased total NCC in the plasma membrane, but the ratio of pNCC to total NCC at the membrane increased, suggesting that non‐phosphorylated NCC is preferentially internalized. Funded by the Lundbeck Foundation.

ATP released from cardiac fibroblasts via connexin hemichannels activates profibrotic P2Y 2 receptors

Cardiac fibroblasts (CFs) play an essential role in remodeling of the cardiac extracellular matrix. Extracellular nucleotide signaling may provoke a profibrotic response in CFs. We tested the hypothesis that physical perturbations release ATP from CFs and that ATP participates in profibrotic signaling. ATP release was abolished by the channel inhibitor carbenoxolone and inhibited by knockdown of either connexin (Cx)43 or Cx45 (47 and 35%, respectively), implying that hypotonic stimulation induces ATP release via Cx43 and Cx45 hemichannels, although pannexin 1 may also play a role. ATP released by hypotonic stimulation rapidly (<10 min) increased phosphorylated ERK by 5-8 fold, an effect largely eliminated by P2Y(2) receptor knockdown or ATP hydrolysis with apyrase. ATP stimulation of P2Y(2) receptors increased α-smooth muscle actin (α-SMA) production, and in an ERK-dependent manner, ATP increased collagen accumulation by 60% and mRNA expression of profibrotic markers: plasminogen activator inhibitor-1 and monocyte chemotactic protein-1 by 4.5- and 4.0-fold, respectively. Apyrase treatment substantially reduced the basal profibrotic phenotype, decreasing collagen and α-SMA content and increasing matrix metalloproteinase expression. Thus, ATP release activates P2Y(2) receptors to mediate profibrotic responses in CFs, implying that nucleotide release under both basal and activated states is likely an important mechanism for fibroblast homeostasis.

The Polymodal Ion Channel Transient Receptor Potential Vanilloid 4 Modulates Calcium Flux, Spiking Rate, and Apoptosis of Mouse Retinal Ganglion Cells

Sustained increase in intraocular pressure represents a major risk factor for eye disease, yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal transient receptor potential vanilloid 4 (TRPV4) cation channel known to mediate osmotransduction and mechanotransduction. TRPV4 antibodies labeled perikarya, axons, and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Müller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4α-PDD and GSK1016790A elevated [Ca2+]i in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca2+](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca2+](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca2+ -free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca2+]i and augmented excitability. Excessive Ca2+ influx through TRPV4 predisposes RGCs to activation of Ca2+ -dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure.

Hypotonicity Modulates Tetrodotoxin-Sensitive Sodium Current in Trigeminal Ganglion Neurons

Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability. We previously reported that the excitability of nociceptor was increased by hypotonic stimulation. The present study tested the effect of hypotonicity on tetrodotoxin-sensitive sodium current (TTX-S current) in cultured trigeminal ganglion (TG) neurons. Our data show that after hypotonic treatment, TTX-S current was increased. In the presence of hypotonicity, voltage-dependent activation curve shifted to the hyperpolarizing direction, while the voltage-dependent inactivation curve was not affected. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers. We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response. We conclude that hypotonic stimulation enhances TTX-S current, which contributes to hypotonicity-induced nociception. TRPV4 receptor and PKC intracellular pathway are involved in the increase of TTX-S current by hypotonicity.

A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

We examined the mechanisms involved in the [Ca2+]i response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging.Reduction of extracellular osmolality from 305 (control) to 195mosmol/kgH2O (hypotonic) evoked transient increase in [Ca2+]i of principal cells of rat CCDs. The [Ca2+]i increase was markedly attenuated by the removal of extracellular Ca2+. The application of a P2 purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2+]i increase. The [Ca2+]i increase in response to extracellular hypotonicity was not influenced by application of Gd3+ and ruthenium red. On the other hand, a voltage-gated Ca2+ channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca2+]i increase in the principal cells. In order to assess Ca2+ entry during the hypotonic stimulation, we measured the quenching of Fura-2 fluorescence intensity by Mn2+. The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn2+, indicating that a Ca2+-permeable pathway was activated by the hypotonicity. The hypotonicity-mediated enhancement of Mn2+ quenching was significantly inhibited by nicardipine.These results strongly suggested that a nicardipine-sensitive Ca2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+]i elevation of principal cells in rat CCD.

Antibodies against ClC7 inhibit extracellular acidification-induced Cl− currents and bone resorption activity in mouse osteoclasts

The Cl⁻ channel/transporter ClC7 is crucial for osteoclastic bone resorption and might become a therapeutic target for osteoporosis. In this study, we raised anti-ClC7 polyclonal antibodies against three different peptide sequences, including G215, P249, and R286, which are the mutation regions found in autosomal dominant osteopetrosis type II patients and examined the effects of these antibodies on the ClC7 Cl⁻ current induced by extracellular acidification (acid-activated Cl⁻ current) using the whole-cell patch clamp technique and bone resorption activity in mouse osteoclasts. Intracellular dialysis of osteoclasts with antibodies to intracellular G215 (Ab-G215) and extracellular application of antibodies to extracellular P249 (Ab-P249) or R286 (Ab-R286) inhibited the acid-activated Cl⁻ current. These antibodies also suppressed the acid-activated Cl⁻ current in ClC7 overexpressing Raw264.7 cells; however, Cl⁻ currents evoked by hypotonic stimulation and the inherent inwardly rectifying K+ currents in mouse osteoclasts were unaffected by these antibodies. Furthermore, extracellularly applied Ab-P249 and Ab-R286 also reduced bone resorption activity. Our results demonstrate that these antibodies specifically block ClC7 in mouse osteoclasts. Thus, anti-ClC7 antibodies have potential promise for treatment of osteoporosis.

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to −60mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current–voltage relationship (from −80 to +40mV) was almost linear with a reversal potential of 1mV and a slope conductance of 34pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca2+ concentration ([Ca2+]i). This [Ca2+]i increase was markedly inhibited in the absence of external Ca2+ or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca2+]i increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.

A microfluidic chip for real-time studies of the volume of single cells.

We report a microfluidic chip that is capable of measuring volume changes in single cells in real-time. Single eukaryotic cells were immobilized in the sensing area and changes in volume in response to hypotonic challenges and drugs were measured using the electrical impedance method. Experiments on MDCK cells showed that the maximum swelling and the time course of swelling vary between individual cells following hypotonic stimulation. The microfluidic chip allows, rapid and convenient change of solutions, enabling detailed studies of various drugs and chemicals that may play important role in cell physiology at the single cell level.

Contribution of Aquaporins to Cellular Water Transport Observed by a Microfluidic Cell Volume Sensor

Here we demonstrate that an impedance-based microfluidic cell volume sensor can be used to study the roles of aquaporin (AQP) in cellular water permeability and screen AQP-specific drugs. Human embryonic kidney (HEK-293) cells were transiently transfected with AQP3- or AQP4-encoding genes to express AQPs in plasma membranes. The swelling of cells in response to hypotonic stimulation was traced in real time using the sensor. Two time constants were obtained by fitting the swelling curves with a two-exponential function, a fast time constant associated with osmotic water permeability of AQP-expressing cells and a slow phase time constant associated mainly with water diffusion through lipid bilayers in the nontransfected cells. The AQP-expressing cells showed at least 10× faster osmotic water transport than control cells. Using the volume sensor, we examined the effects of Hg2+ and Ni2+ on the water transport via AQPs. Hg2+ inhibited the water flux in AQP3-expressing cells irreversibly, while Ni2+ blocked the AQP3 channels reversibly. Neither of the two ions blocked the AQP4 channels. The microfluidic volume sensor can sense changes in cell volume in real time, which enables perfusion of various reagents sequentially. It provides a convenient tool for studying the effect of reagents on the function and regulation mechanism of AQPs.

Osmotic modulation of stimulus‐evoked responses in the rat supraoptic nucleus

Neural information is conveyed by action potentials along axons to downstream synaptic targets. Synapses permit functionally relevant modulation of the information transmitted by converging inputs. Previous studies have measured the amount of information associated with a given stimulus based either on spike counts or on the relative frequencies of spike sequences represented as binary strings. Here we apply information theory to the phase-interval stimulus histogram (PhISH) to measure the extent of the stimulus-evoked response using the statistical relationship between each interspike interval and its phase within the stimulus cycle. We used the PhISH as a novel approach to investigate how different osmotic states affect the flow of information through the osmoreceptor complex of the hypothalamus. The amount of information conveyed from one (afferent) element of the complex, the anteroventral region of the third ventricle (AV3V), to another (an efferent element), the supraoptic nucleus, was increased by hypertonic stimulation (intravenous mannitol, z = 4.39, P < 0.001) and decreased by hypotonic stimulation (intragastric water, z = -3.37, P < 0.001). Supraoptic responses to AV3V stimulation differed from those that follow stimulation of a hypothalamic element outside the osmoreceptor complex, the suprachiasmatic nucleus (SCN), which also projects to the supraoptic nucleus. Thus osmosensitive gain control mechanisms differentially modulate osmotically dependent and osmotically independent inputs, and enhance the osmoresponsiveness of supraoptic cells within a physiological range. The value of the novel approach is that its use is not limited to the osmoreceptor ensemble but it can be used to investigate the flow of information throughout the central nervous system.