An electrophysiological technique to measure change in hepatocyte water volume

We have applied an electrophysiologic technique (Reuss, L. (1985) Proc. Natl. Acad. Sci. USA 82, 6014) to measure changes in steady-state hepatocyte volume during osmotic stress. Hepatocytes in mouse liver slices were loaded with tetramethylammonium ion (TMA+) during transient exposure of cells to nystatin. Intracellular TMA+ activity (alpha 1TMA) was measured with TMA(+)-sensitive, double-barrelled microelectrodes. Loading hepatocytes with TMA+ did not change their membrane potential (Vm), and under steady-state conditions alpha iTMA remained constant over 4 min in a single impalement. Hyperosmotic solutions (50, 100 and 150 mM sucrose added to media) and hyposmotic solutions (sucrose in media reduced by 50 and 100 mM) increased and decreased alpha iTMA, respectively, which demonstrated transmembrane water movements. The slope of the plot of change in steady-state cell water volume, [(alpha iTMA)0/(alpha iTMA)4min] -1, on the relative osmolality of media, (experimental mosmol/control mosmol) -1, was less predicted for a perfect osmometer. Corresponding measurements of Vm showed that its magnitude increased with hyposmolality and decreased with hyperosmolality. When Ba2+ (2 mM) was present during hyposmotic stress of 0.66 X 286 mosmol (control), cell water volume increased by a factor of 1.44 +/- 0.02 compared with that of hyposmotic stress alone, which increased cell water volume by a factor of only 1.12 +/- 0.02, P less than 0.001. Ba2+ also decreased the hyperpolarization of hyposmotic stress from a factor of 1.62 +/- 0.04 to 1.24 +/- 0.09, P less than 0.01. We conclude that hepatocytes partially regulate their steady-state volume during hypo- and hyperosmotic stress. However, volume regulation during hyposmotic stress diminished along with hyperpolarization of Vm in the presence of K(+)-channel blocker, Ba2+. This shows that variation in Vm during osmotic stress provides an intercurrent, electromotive force for hepatocyte volume regulation.     

Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.     

Axonal contribution to subthreshold currents inaplysia bursting pacemaker neurons

The contribution of axonal activity to the ionic currents which generate bursting pacemaker activity was studied by using the two-electrode voltage-clamp technique in Aplysia bursting neuron somata in conjunction with intraaxonal voltage recordings. Depolarizing voltage-clamp pulses applied to bursting cell somata triggered axonal action potentials. The voltage-clamp current recording exhibited transient inward current "notches" corresponding to each of the axonal spikes. The addition of 50 microM tetrodotoxin (TTX) to the bathing medium blocked the fast axonal spikes and current notches, revealing a slower axonal spike which was blocked by the replacement of external Ca2+ with Co2+. The inward current evoked by applying a depolarizing voltage-clamp pulse in the soma is distorted by the occurrence of the axonal Ca2+ spike. Elimination of the axonal spike, by injecting hyperpolarizing current into the axon, changes both the time course and the magnitude of the inward current. The axonal Ca2+ spikes are followed by a series of Ca2+-dependent afterpotentials: a rapid postspike hyperpolarization, a depolarizing afterpotential (DAP) and, finally, a long-lasting postburst hyperpolarization. The long-lasting hyperpolarization is not blocked by 50 mM external tetraethyl ammonium, an effective blocker of Ca2+-activated K+ current [IK(Ca)], and does not appear to reverse at EK. Hence, the axonal long-lasting hyperpolarization may not be due to IK(Ca). Somatic voltage-clamp pulses in bursting neurons are followed by a slow inward tail current, which is sometimes coincident with a DAP in the axon. In some cells, the amplitude of the slow inward tail current is greatly reduced if axonal spikes and DAPs are prevented by hyperpolarization of the axon, while, in other cells, elimination of axonal activity has little effect. Therefore, the slow inward tail current is not necessarily an artifact of poor voltage-clamp control over the axonal membrane potential but probably results from the activation of an ionic conductance mechanism located partly in the axon and partly in the soma.     

Volume changes in cardiac ventricles from Aplysia brasiliana upon exposure to hyposmotic shock

We investigated the possible role of ion channels and transporters in cell volume control using Aplysia brasiliana ventricular tissues exposed to a 26% hyposmotic shock, by assessing changes in wet weight, intracellular water and ionic contents. Thirty minutes after the shock, the wet weight of isolated ventricles increase about 20% above control levels and then attain near original weight within 60 min after the shock. At the time when the wet weight returned to control values, intracellular water and KCl contents are decreased by 22 and 20%, respectively. The K(+) channel blockers, 4-AP and TEA, but not the cotransport blockers, hydrochlorothiazide and furosemide, greatly affect the magnitude of wet weight gain and the time course of weight recovery, indicating that KCl loss occur through conductive pathways. Intracellular recordings performed on ventricular myocytes during exposure to the osmotic shock showed an immediate membrane hyperpolarization and blockade of spontaneous electrical activity; diastolic membrane potential recover over time and spontaneous action potentials are completely restored 60 min after the hyposmotic shock. Because significant weight loss is observed during the exposure of ventricular tissues to 26% hypo-ionic, but isosmotic saline, it is suggested that ventricular volume restoration is accomplished by two distinct but simultaneously occurring processes: a volume-dependent and a volume-independent mechanism. Because wet weight restoration is completely prevented by exposing ventricular tissue to a Ca(2+)-free hyposmotic solution, we postulate that both processes involved in A. brasiliana ventricular weight restoration are Ca(2+)-dependent mechanisms.     

Amino Acids as Osmolytes in the Retina

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.     

Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells

Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.     

Negative surface potential shift and responses of neurons and glial cells during tetanic stimulation of the cortical surface

The negative potential shift in response to tetanic stimulation of the surface of the cortex or thalamic nucleus was recorded from the cortical surface in cats lightly anesthetized with pentobarbital. Parallel intracellular recordings were obtained of activity of neurons and glial cells. Glial cells responded to this stimulation by slow depolarization, which, under certain conditions of stimulation, was followed by slow hyperpolarization; hyperpolarization shifts were observed in neurons. Depolarization and hyperpolarization of glial cells, like hyperpolarization of neurons, did not correlate in time with the development of a negative shift of the surface potential. It is postulated that this shift is a response of complex origin involving the participation not only of glial cells, but also of cortical neurons.I. S. Beritashvili Institute of Physiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 248–253, May–June, 1982.     

Cell Swelling Activates the K+ Conductance and Inhibits the Cl? Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium

Necturus gallbladder epithelial cells bathed in 10 mM HCO₃/1% CO₂ display sizable basolateral membrane conductances for Cl⁻ (G_Cl ^b) and K ⁺ (G_K ^b). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K ⁺/Cl⁻ selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca²⁺]_i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca²⁺ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (V_cs) produced a significant increase in the change in V_cs elicited by lowering basolateral solution [Cl⁻], whereas hyperpolarization of V_cs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases G_K ^b and decreases G _Cl ^b. These two effects appear to be linked, i.e., the increase in G _K ^b produces membrane hyperpolarization, which in turn reduces G _Cl ^b. ( b) Hyperosmotic shrinkage has the opposite effects on G_K ^b and G _Cl ^b. ( c) Cell swelling causes a transient increase in [Ca²⁺]_i, but this response may not be necessary for the increase in G_K ^b during cell swelling.     

微丝在低渗牵张诱导毒蕈碱电流增加中的作用

在急性分离的豚鼠胃窦平滑肌细胞上 ,利用膜片钳技术的传统全细胞模式记录离子电流的方法 ,探讨微丝在低渗牵张诱导毒蕈碱电流增加中的作用。当豚鼠胃窦平滑肌细胞的膜电位钳制在 - 2 0mV时 ,灌流液中 5 0μmol/L 卡巴胆碱 (carbachol,CCh)或电极内液中 0 5mmol/LGTPγS均可引导毒蕈碱电流 (muscariniccurrentICCh) ,低渗牵张 ( 2 0 2mOsmol/L)分别使其增加 145± 2 7%和 183± 3 0 % ;当电极内液中加入 2 0 μmol/L的细胞松弛B (一种微丝骨架的解聚剂 )时 ,低渗牵张使ICCh只增加 70± 6% ;而电极内液中加入 2 0 μmol/L的鬼笔环肽 (一种微丝骨架的稳定剂 )则使ICCh增加了 5 45± 81%。结果表明 ,低渗牵张可增加由卡巴胆碱或GTPγS诱导的毒蕈碱电流 ,微丝参与调节低渗牵张诱导豚鼠胃窦平滑肌细胞ICCh增加的作用     

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P < 0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P < 0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.     

Characterization of the taurine transport pathway in A6 kidney cells

We investigated the role of taurine in cell homeostasis and characterized the taurine transport pathway in cultured kidney cells (A6). The taurine concentration in A6 cells varies with the osmolarity of the culture medium, suggesting that taurine participates in cell osmolarity. Under isosmotic conditions, 14C-taurine efflux through the apical membranes (aJtaur) was 6-7 times lower than that through the basolateral membranes (bJtaur). Under hyposmotic conditions, aJtaur remained almost unchanged. On the contrary, bJtaur increased 8 times in comparison with isosmotic conditions. In hyposmotic conditions, bJtaur was inhibited by 500 microM DIDS, 50 microM NPPB, 10 microM of the two oxonol derivatives DISBAC(2)3 and WW-791, and 100 microM ketoconazole. Conversely, 100 microM 1,9-dideoxyforskolin, 10 microM tamoxifen, 100 microM niflumic acid and 50 microM verapamil had no inhibitory effects. Cell volume regulation upon hyposmotic stress was also found to be inhibited by DISBAC(2)3 (K0.5 of 5+/-1 microM) and by ketoconazole. Nystatin was used to permeabilize the apical membranes with the aim to further characterize bJtaur. 14C-taurine transepithelial fluxes in nystatin-treated cells were found to be linear over taurine concentrations ranging from 3.5 microM to 35 mM. Clamping the transepithelial voltage at positive values (serosal side) slightly stimulated the 14C-taurine transport. Similar time courses of 14C-taurine, 36Cl and 86Rb transepithelial fluxes were found under osmotic stimulation followed by DIDS inhibition in nystatin-treated cells. In whole cell patch-clamp experiments, DISBAC(2)3 application resulted in a strong and reversible decrease of the global Cl- current which was stimulated by hyposmotic stress. Our study indicates that taurine participates in the control of A6 cell osmolarity and that the transporting taurine pathway (efflux) is on the basolateral membranes. In addition to usual chloride channel blockers, oxonol was found to be a potent blocker of the taurine transport and of the swelling-activated chloride current. Using a pharmacological approach, we could not distinguish between a common or different pathway for Cl- and taurine.     

Local anaesthetic block protects against electrically-induced damage in peripheral nerve

This study is one of a series addressing the mechanisms involved in the production of neural damage caused by continuous, prolonged electrical stimulation of peripheral nerve. It has been previously shown that sustained, high frequency electrical stimulation of the cat's peroneal nerve may cause irreversible neural damage in the form of axonal degeneration of the large myelinated fibres. In this study we demonstrate that blocking the action potentials on most of the nerve fibres with local anaesthetics (10% procaine or 2% lidocaine) almost completely prevents the axonal degeneration. The abolition of axonal injury by local anaesthetic block strongly suggests that the electrically-induced damage is due to prolonged electrical excitation of axons. Furthermore, since less than complete suppression of the induced neural activity by local anaesthetic engenders essentially complete sparing of all axons, our results suggest that the damage to individual axons derives, at least in part, from stimulation-induced global changes in the nerve.     

Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K+ from the cells. Hyperpolarization shows a peak at about 3 min and a net K+ efflux at 4-5 min. An additional transient hyperpolarization and net K+ efflux are found after 60-80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K+ efflux. At low pH, neither transient hyperpolarization nor a transient K+ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K+ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K+ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K+, whereas the same amount of Na+ does not reduce the hyperpolarization.     

HDAC1 in axonal degeneration: A matter of subcellular localization

Multiple sclerosis (MS) is a disease characterized by inflammatory demyelination and a strong neurodegenerative component. Axonal damage is characteristically detected in MS brains, although the pathogenic mechanisms are not clearly understood. Here, we discuss the importance of HDAC1 localization as one of the potential mechanisms initiating damage in demyelinating conditions. We suggest the occurrence of a two-stage mechanism of damage. The first event is a calcium-dependent HDAC1 nuclear export in a CRM1-dependent manner and the second event is the interruption of mitochondrial transport resulting from the cytoplasmic localization of HDAC1. In the cytosol of neurons challenged by cytokines and excitatory aminoacids, HDAC1 formed complexes with motor-protein and microtubules and this resulted in blockade of axonal transport and release of cargo from motor proteins. We suggest that these findings might be the framework for future studies and for the development of novel therapeutic targets for axonal damage in demyelinating conditions.Key words: neurodegeneration, histone deacetylase, multiple sclerosis, demyelination, nuclear export     

Role of kinases and G-proteins in the hyposmotic stimulation of cardiac IKs

Exposure of cardiac myocytes to hyposmotic solution stimulates slowly-activating delayed-rectifying K(+) current (I(Ks)) via unknown mechanisms. In the present study, I(Ks) was measured in guinea-pig ventricular myocytes that were pretreated with modulators of cell signaling processes, and then exposed to hyposmotic solution. Pretreatment with compounds that (i) inhibit serine/threonine kinase activity (10-100 microM H89; 200 microM H8; 50 microM H7; 1 microM bisindolylmaleimide I; 10 microM LY294002; 50 microM PD98059), (ii) stimulate serine/threonine kinase activity (1-5 microM forskolin; 0.1 microM phorbol-12-myristate-13-acetate; 10 microM acetylcholine; 0.1 microM angiotensin II; 20 microM ATP), (iii) suppress G-protein activation (10 mM GDPbetaS), or (iv) disrupt the cytoskeleton (10 microM cytochalasin D), had little effect on the stimulation of I(Ks) by hyposmotic solution. In marked contrast, pretreatment with tyrosine kinase inhibitor tyrphostin A25 (20 microM) strongly attenuated both the hyposmotic stimulation of I(Ks) in myocytes and the hyposmotic stimulation of current in BHK cells co-expressing Ks channel subunits KCNQ1 and KCNE1. Since attenuation of hyposmotic stimulation was not observed in myocytes and cells pretreated with inactive tyrphostin A1, we conclude that TK has an important role in the response of cardiac Ks channels to hyposmotic solution.     

Hyposmotic activation of ICl,swell in rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl(-) conductance (I(Cl,swell)), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I(Cl,swell) following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl(-) current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I(Cl,swell). Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I(Cl,swell). Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I(Cl,swell) following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.     

Hepatopancreas gluconeogenesis during hyposmotic stress in crabs Chasmagnathus granulata maintained on high-protein or carbohydrate-rich diets

The incorporation of [14C]-alanine or [14C]-lactate into glucose was measured in hepatopancreas fractions from Chasmagnathus granulata crabs adapted to a high protein or a carbohydrate-rich diet and submitted or not (control group) to hyposmotic stress. Gluconeogenic capacity and phosphoenolpyruvate carboxykinase (PEPCK) activity increased during acclimation to a dilute medium in C. granulata hepatopancreas. In intact animals, high hemolymph urea levels occurred for the high-protein regimen and for crabs fed both diets and submitted to hyposmotic stress. It could be that the amino acids released during hyposmotic stress are deaminated in the hepatopancreas, and that the carbon chains are used as substrate for gluconeogenesis. Hepatopancreas gluconeogenesis seems to be one of the pathways implicated in the metabolic adjustment of the amino acid pool during hyposmotic stress in C. granulata.     

Membrane mechanisms of testosterone action]

The experiments on adult Wistar rats have shown that testosterone administration provoke hyperpolarization of hepatocytes and adrenocorticocytes plasmatic membranes. It was discovered that this hyperpolarization was caused by cell Na, K-ATPase activation. Inhibitors of protein biosynthesis prevent both testosterone-induced hyperpolarization and Na, K-ATPase activation. It is shown that some hyperpolarization factor appeared in the cytosol and blood serum under testosterone influence. It was suggested that this mechanism is mediated by cell genome.     

TRPV4在渗透压对大鼠心肌收缩性影响中的作用

本文旨在探讨低渗和高渗内环境对心肌收缩性的影响及机制.取Sprague-Dawley(SD)大鼠左心室乳头状肌,在电刺激引起兴奋的条件下,分别记录在低渗、等渗和高渗灌流液中肌条的收缩力;同样条件下观察在低渗、等渗和高渗灌流液中加入渗透压敏感蛋白瞬时感受器电位离子通道家族香草素受体亚家族IV型(transient receptor potential vanilloid 4,TRPV4)的拮抗剂和激动剂后肌条收缩力的变化.结果显示:(1)与等渗(310 mOsm/L)时心肌收缩力相比,渗透压为290、270和230 mOsm/L时心肌收缩力分别增加11.5%、21.5%、25.O%(P<0.05);渗透压为350、370、390 mOsm/L时心肌收缩力分别降低16.0%、23.7%、55.2%(P<0.05).(2)在低渗液(270 mOsm/L)中加入TRPV4拮抗剂钌红(ruthenium red,RR),低渗对心肌收缩力的增强作用被抑制36%(P<0.01);在高渗液(390 mOsm/L)中加入RR,高渗对心肌收缩力的抑制作用增加56.1%(P<0.01).(3)在等渗液中(310 mOsm/L)加入TRPV4激动剂4-α-佛波醇-12,13-二癸酸(4-α-phorbol-12,13-didecanoate,4 α-PDD),心肌收缩力没有改变;在高渗液中(390 mOsm/L)加入4α-PDD,高渗对心肌收缩力的抑制作用增加27.1%(P<0.01).以上结果提示,TRPV4参与渗透压引起的心肌收缩力变化.     

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K⁺ channel inhibitors Ba²⁺, quinine, and tetraethylammonium chloride, implicating K⁺ channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K⁺ channel, and also a dominant-negative NH₂-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K⁺ current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K⁺ secretion. potassium channel; volume regulation; mammary gland