Resting Membrane Potential Of Cells Research Articles

Expression of the mechanosensitive 2PK+ channel TREK‐1 in human osteoblasts

TREK-1 is a mechanosensitive member of the two-pore domain potassium channel family (2PK+) that is also sensitive to lipids, free fatty acids (including arachidonic acid), temperature, intracellular pH, and a range of clinically relevant compounds including volatile anaesthetics. TREK-1 is known to be expressed at high levels in excitable tissues, such as the nervous system, the heart and smooth muscle, where it is believed to play a prominent role in controlling resting cell membrane potential and electrical excitability. In this report, we use RT-PCR, Western blotting and immunohistochemistry to confirm that human derived osteoblasts and MG63 cells express TREK-1 mRNA and protein. In addition, we show gene expression of TREK2c and TRAAK channels. Furthermore, whole cell patch clamp electrophysiology demonstrates that these cells express a spontaneously active, outwardly rectifying potassium "background leak" current that shares many similarities to TREK-1. The outward current is largely insensitive to TEA and Ba2+, and is sensitive to application of lysophosphatidylcholine (LPC). In addition, blocking TREK-1 channel activity is shown to upregulate bone cell proliferation. It is concluded that human osteoblasts functionally express TREK-1 and that these channels contribute, at least in part, to the resting membrane potential of human osteoblast cells. We hypothesise a possible role for TREK-1 in mechanotransduction, leading to bone remodelling.

Determination of Resting Membrane Potential of Individual Neuroblastoma Cells (IMR-32) Using a Potentiometric Dye (TMRM) and Confocal Microscopy

The potentiometric dye, Tetramethylrhodamine methyl ester (TMRM) has been extensively used with fluorometry or optical microscopy to evaluate the electric potential across plasma or mitochondrial membranes. We present here a TMRM confocal microscopy-based potential measurement technique. Corrections are introduced to minimize nonspecific dye binding and insensitivity to low background levels. We have used this technique to compare the resting membrane potential of proliferating and differentiated human neuroblastoma cells (IMR-32).

A major role for carbon monoxide as an endogenous hyperpolarizing factor in the gastrointestinal tract.

Carbon monoxide (CO) is proposed as a physiological messenger. CO activates cGMP and has a direct effect on potassium channels. Both actions of CO lead to hyperpolarization of a cell's resting membrane potential, suggesting that CO may function as a hyperpolarizing factor, although direct evidence is still lacking. Here we take advantage of the known membrane potential gradient that exists in the muscle layers of the gastrointestinal tract to determine whether CO is an endogenous hyperpolarizing factor. We find that heme oxygenase-2-null mice have depolarized smooth muscle cells and that the membrane potential gradient in the gut is abolished. Exogenous CO hyperpolarizes the membrane potential. Regions of the canine gastrointestinal tract that are more hyperpolarized generate more CO and have higher heme oxygenase activity than more depolarized regions. Our results suggest that CO is a critical hyperpolarizing factor required for the maintenance of intestinal smooth muscle membrane potential and gradient.

Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia.

The systemic vasculature exhibits attenuated vasoconstriction following chronic hypoxia (CH) that is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization. We hypothesized that increased production of arachidonic acid metabolites such as the cyclooxygenase product prostacyclin or cytochrome p-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contributes to VSM cell hyperpolarization following CH. VSM cell resting membrane potential (Em) was measured in superior mesenteric artery strips isolated from rats with control barometric pressure (Pb, congruent with 630 Torr) and CH (Pb, 380 Torr for 48 h). VSM cell Em was normalized between groups following administration of the CYP inhibitors 17-octadecynoic acid and SKF-525A. VSM cell hyperpolarization after CH was not altered by cyclooxygenase inhibition, whereas the selective CYP2C9 inhibitor sulfaphenazole normalized VSM cell Em between groups. Iberiotoxin also normalized VSM cell Em, which suggests that large-conductance, Ca2+-activated K+ (BKCa) channel activity is increased after CH. Sulfaphenazole administration restored phenylephrine-induced and myogenic vasoconstriction and Ca2+ responses of mesenteric resistance arteries isolated from CH rats to control levels. Western blot experiments demonstrated that CYP2C9 protein levels were greater in mesenteric arteries from CH rats. In addition, 11,12-EET levels were elevated in endothelial cells from CH rats compared with controls. We conclude that enhanced CYP2C9 expression and 11,12-EET production following CH contributes to BKCa channel-dependent VSM cell hyperpolarization and attenuated vasoreactivity.

4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

BackgroundIon channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels.MethodsGranulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size.Results4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production.Conclusion4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Quinine suppresses extracellular potassium transients and ictal epileptiform activity without decreasing neuronal excitability in vitro

The effect of quinine on pyramidal cell intrinsic properties, extracellular potassium transients, and epileptiform activity was studied in vitro using the rat hippocampal slice preparation. Quinine enhanced excitatory post-synaptic potentials and decreased fast- and slow-inhibitory post-synaptic potentials. Quinine reduced the peak potassium rise following tetanic stimulation but did not affect the potassium clearance rate. Epileptiform activity induced by either low-Ca 2+ or high-K + artificial cerebrospinal fluid (ACSF) was suppressed by quinine. The frequency of spontaneous inter-ictal bursting induced by picrotoxin, high-K +, or 4-aminopyridine was significantly increased. In normal ACSF, quinine did not affect CA1 pyramidal cell resting membrane potential, input resistance, threshold for action potentials triggered by intracellular or extracellular stimulation, or the orthodromic and antidromic evoked population spike amplitude. The main effects of quinine on intrinsic cell properties were to increase action potential duration and to reduce firing frequency during sustained membrane depolarizations, but not at normal resting membrane potentials. This attenuation was enhanced at increasingly depolarized membrane potentials. These results suggest that quinine suppresses extracellular potassium transients and ictal activity and modulates inter-ictal activity by limiting the firing rate of cells in a voltage-dependent manner. Because quinine does not affect ‘normal’ neuronal function, it may merit consideration as an anticonvulsant.

48-h Hypoxic exposure results in endothelium-dependent systemic vascular smooth muscle cell hyperpolarization.

Chronic hypoxia (CH) results in reduced sensitivity to vasoconstrictors in conscious rats that persists upon restoration of normoxia. We hypothesized that this effect is due to endothelium-dependent hyperpolarization of vascular smooth muscle (VSM) cells after CH. VSM cell resting membrane potential was determined for superior mesenteric artery strips isolated from CH rats (PB = 380 Torr for 48 h) and normoxic controls. VSM cells from CH rats studied under normoxia were hyperpolarized compared with controls. Resting vessel wall intracellular Ca(2+) concentration ([Ca(2+)](i)) and pressure-induced vasoconstriction were reduced in vessels isolated from CH rats compared with controls. Vasoconstriction and increases in vessel wall [Ca(2+)](i) in response to the alpha(1)-adrenergic agonist phenylephrine (PE) were also blunted in resistance arteries from CH rats. Removal of the endothelium normalized resting membrane potential, resting vessel wall [Ca(2+)](i), pressure-induced vasoconstrictor responses, and PE-induced constrictor and Ca(2+) responses between groups. Whereas VSM cell hyperpolarization persisted in the presence of nitric oxide synthase inhibition, heme oxygenase inhibition restored VSM cell resting membrane potential in vessels from CH rats to control levels. We conclude that endothelial derived CO accounts for persistent VSM cell hyperpolarization and vasoconstrictor hyporeactivity after CH.

Endothelium-dependent blunting of myogenic responsiveness after chronic hypoxia.

Blunted agonist-induced vasoconstriction after chronic hypoxia is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and decreased vessel-wall Ca(2+) concentration ([Ca(2+)]). We hypothesized that myogenic vasoconstriction and pressure-induced Ca(2+) influx would also be attenuated in vessels from chronically hypoxic (CH) rats. Mesenteric resistance arteries isolated from CH [barometric pressure (BP), 380 Torr for 48 h] or normoxic control (BP, 630 Torr) rats were cannulated and pressurized. VSM cell resting membrane potential was recorded at intraluminal pressures of 40-120 Torr under normoxic conditions. VSM cells in vessels from CH rats were hyperpolarized compared with control rats at all pressures. Inner diameter was maintained for vessels from control rats, whereas vessels from CH rats developed less tone as pressure was increased. Pressure-induced increases in vessel-wall [Ca(2+)] were also attenuated for arteries from CH rats. Endothelium removal restored myogenic constriction to vessels from CH rats and normalized VSM cell resting membrane potential and pressure-induced Ca(2+) responses to control levels. Myogenic constriction and pressure-induced vessel-wall [Ca(2+)] increases remained blunted in the presence of nitric oxide (NO) synthase inhibition for arteries from CH rats. We conclude that blunted myogenic reactivity after chronic hypoxia results from a non-NO, endothelium-dependent VSM cell hyperpolarizing influence.

A Calcium Aspect of the Effect of Noradrenaline on the Resting Membrane Potential in Somatic Muscle Cells of the Earthworm

A hyperpolarizing effect of noradrenaline (NA) on muscle cells of the earthworm caused by activation of the membrane ion pumps is eliminated in a Ca-free medium, in the case of replacement of Na+ by Mn2+, or when verapamil or chlorpromazine have been added to the bath solution. A decrease or an increase in the Ca2+ concentration in the solution, as well as caffeine application, do not influence the resting membrane potential (RMP) of muscle cells. It is supposed that signal transmission from the membrane adrenoreceptors to the ion pump of earthworm muscle cells by NA is provided via entry of extracellular Ca2+ ions into the cell with subsequent involving of Ca2+ acceptor proteins similar to calmodulin in vertebrate animals.

PP1 inhibitors depolarize Hermissenda photoreceptors and reduce K+ currents.

Previous research indicates that activation of protein kinase C (PKC) plays a critical role in the induction and maintenance of memory-related changes in neural excitability of Type B photoreceptors in the eyes of nudibranch mollusk Hermissenda crassicornis (H.c.). The enhanced excitability of B cells is due in part to PKC-mediated reduction in somatic K+ currents. Here we examined the effects of protein phosphatase inhibitors on Type B photoreceptor excitability and K+ currents to determine the role(s) of protein phosphatases on memory formation in Hermissenda. Using electrophysiological and pharmacological methods, we found that the PP1 inhibitors calyculin A and inhibitor-2 depolarized Type B photoreceptors by 20-30 mV. A broad-spectrum kinase inhibitor, H7, blocked this effect. The depolarization induced by PP1 inhibition occluded that produced by an in vitro associative conditioning procedure. Calyculin and inhibitor-2 reduced the same B cell K+ currents (I(A) and I(delayed)) that are reduced by in vitro and behavioral conditioning. H7 blocked the reductions. Cantharidic acid (PP2A inhibitor) and cyclosporin (PP2B inhibitor) had negligible effects on B cell resting membrane potential, K+ currents, and in vitro conditioning-produced cumulative depolarization of B cells. These results suggest that the functional activity of K+ channels in B cells is sustained by basal activity of PP1. Inhibiting PP1 appears to allow one or more constitutively active kinase(s) to reduce K+ channel activity and thus mimic the effects of conditioning. Our results suggest that PP1 may oppose and/or constrain the extent of learning-produced changes in B cell excitability.

Ca(2+) influx is increased in 2-kidney, 1-clip hypertensive rat aorta.

Arteries from hypertensive rats show a greater contraction in response to Ca(2+) channel activator and an increased sensitivity to Ca(2+) entry blockers compared with those of normotensive rats. These facts suggest an altered Ca(2+) influx through membrane channels. In this study, this hypothesis was tested by direct activation of voltage-gated Ca(2+) channels using Bay K 8644, a dihydropyridine sensitive large conductance (L-type) Ca(2+) channel opener in aortas from 2-kidney, 1-clip (2K1C) hypertensive rats. Because the membrane potential of smooth muscle cells is an important regulator of the conformational state of L-type Ca(2+) channels and, consequently, dihydropyridine affinity, the effect of 10 mmol/L KCl on the responses to Bay K 8644 was also studied. Maximal contraction (ME) and sensitivity to Bay K 8644 were greater in 2K1C rats than in 2K normotensive rats (ME, 1.77+/-0.15 versus 1.25+/-0.19 g; negative log molar value [pD(2)], 8.27+/-0.07 versus 7.92+/-0.08). When the KCl concentration was increased from 4.7 to 10 mmol/L in the bathing medium, no differences were observed in the contractile effect of Bay K 8644 between 2K1C and 2K (ME, 1.28+/-0.13 versus 1.14+/-0.21 g; pD(2), 8.56+/-0.08 versus 8.38+/-0.07). The cell resting membrane potential of 2K1C aorta vascular smooth muscle cells were less negative than in 2K (-35.19+/-4.91 versus -48.32+/-1.88 mV). Basal intracellular Ca(2+) concentration ([Ca(2+)](i)) was greater in cultured vascular smooth muscle cells from 2K1C than from 2K (293.4+/-25.83 versus 205.40+/-12.83 nmol/L). In 2K1C, Bay K 8644 induced a larger increase in [Ca(2+)](i) than in 2K (190.60+/-45.65 versus 92.57+/-14.67 nmol/L), and in 10 mmol/L KCl, this difference was abolished (134.90+/-45.12 versus 125.20+/-32.17 nmol/L). The main conclusion of the present work is that the increased contractile response to Bay K 8644 in 2K1C aortas is due to an increased Ca(2+) influx through voltage-gated Ca(2+) channels.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells.

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 +/- 1.9 mV) compared to NWR (-69 +/- 1.4 mV), NWR 1K-1C (-60.8 +/- 1.6 mV), WKY (-67.1 +/- 3.2 mV) and WKY 1K-1C (-56.9 +/- 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 +/- 0.86) when compared to arteries of all the other groups (NWR 0.93 +/- 0.65, NWR 1K-1C 1.24 +/- 0.45, WKY 1.22 +/- 0.32, WKY 1K-1C 1.15 +/- 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

PH Sensitivity of non-synaptic field bursts in the dentate gyrus.

Under conditions of low [Ca(2+)](o) and high [K(+)](o), the rat dentate granule cell layer in vitro develops recurrent spontaneous prolonged field bursts that resemble an in vivo phenomenon called maximal dentate activation. To understand how pH changes in vivo might affect this phenomenon, the slices were exposed to different extracellular pH environments in vitro. The field bursts were highly sensitive to extracellular pH over the range 7.0-7.6 and were suppressed at low pH and enhanced at high pH. Granule cell resting membrane potential, action potentials, and postsynaptic potentials were not significantly altered by pH changes within the range that suppressed the bursts. The pH sensitivity of the bursts was not altered by pharmacologic blockade of N-methyl-D-aspartate (NMDA), non-NMDA, and GABA(A) receptors at concentrations of these agents sufficient to eliminate both spontaneous and evoked synaptic potentials. Gap junction patency is known to be sensitive to pH, and agents that block gap junctions, including octanol, oleamide, and carbenoxolone, blocked the prolonged field bursts in a manner similar to low pH. Perfusion with gap junction blockers or acidic pH suppressed field bursts but did not block spontaneous firing of single and multiple units, including burst firing. These data suggest that the pH sensitivity of seizures and epileptiform phenomena in vivo may be mediated in large part through mechanisms other than suppression of NMDA-mediated or other excitatory synaptic transmission. Alterations in electrotonic coupling via gap junctions, affecting field synchronization, may be one such process.

Background Nonselective Cationic Current and the Resting Membrane Potential in Rabbit Aorta Endothelial Cells.

The ion channel conductances that regulate the membrane potential was investigated by using a perforated patch-clamp technique in rabbit aorta endothelial cells (RAECs). The whole-cell current/voltage (I-V) relation showed a slight outward rectification under physiological ionic conditions. The resting membrane potential was -23.3 +/- 1.1 mV (mean +/- SEM, n = 19). The slope conductances at the potentials of -80 and 50 mV were 31.0 +/- 4.0 and 62.8 +/- 7.1 pS pF(-1), respectively (n = 15). Changes in the extracellular and intracellular Cl(-) concentrations did not affect the reversal potential on I-V curves. The background nonselective cationic (NSC) current was isolated after the K(+) current was suppressed. The relative permeabilities calculated from the changes in reversal potentials using the constant-field theory were P(K):P(Cs):P(Na):P(Li) = 1:0.87:0.40:0.27 and P(Cs):P(Ca) = 1:0.21. Increases in the external Ca(2+) decreased the background NSC current in a dose-dependent manner. The concentration for half block by Ca(2+) was 1.1 +/- 0.3 mM (n = 7). Through the continuous recording of the membrane potential in a current-clamp mode, it was found that the background NSC conductance is the major determinant of resting membrane potential. Taken together, it could be concluded that the background NSC channels function as the major determinant for the resting membrane potential and can be responsible for the background Ca(2+) entry pathway in freshly isolated RAECs.

Regulation of Ca2+ homeostasis by atypical Na+ currents in cultured human coronary myocytes.

Primary cultured human coronary myocytes (HCMs) derived from ischemic human hearts express an atypical voltage-gated tetrodotoxin (TTX)-sensitive sodium current (I(Na)). The whole-cell patch-clamp technique was used to study the properties of I(Na) in HCMs. The variations of intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were monitored in non-voltage-clamped cells loaded with Fura-2 or benzofuran isophthalate, respectively, using microspectrofluorimetry. The activation and steady-state inactivation properties of I(Na) determined a "window" current between -50 and -10 mV suggestive of a steady-state Na+ influx at the cell resting membrane potential. Consistent with this hypothesis, the resting [Na+]i was decreased by TTX (1 micromol/L). In contrast, it was increased by Na+ channel agonists that also promoted a large rise in [Ca2+]i. Veratridine (10 micromol/L), toxin V from Anemonia sulcata (0.1 micromol/L), and N-bromoacetamide (300 micromol/L) increased [Ca2+]i by 7- to 15-fold. This increase was prevented by prior application of TTX or lidocaine (10 micromol/L) and by the use of Na(+)-free or Ca(2+)-free external solutions. The Ca(2+)-channel antagonist nicardipine (5 micromol/L) blocked the effect of veratridine on [Ca2+]i only partially. The residual component disappeared when external Na+ was replaced by Li+ known to block the Na+/Ca2+ exchanger. The resting [Ca2+]i was decreased by TTX in some cells. In conclusion, I(Na) regulates [Ca2+]i in primary cultured HCMs. This regulation, effective at baseline, involves a tonic control of Ca2+ influx via depolarization-gated Ca2+ channels and, to a lesser extent, via a Na+/Ca2+ exchanger working in the reverse mode.

Modulatory effects of myomodulin on the excitability and membrane currents in Retzius cells of the leech.

Ion channel modulation by the peptide myomodulin (MM) has been demonstrated in a wide variety of organisms including Aplysia, Lymnaea, and Pleurobranchaea. This neural and muscular modulation has been shown to be important for shaping and modifying behavior. In this paper, we report that MM modulates several distinct ionic channels in another species, the medicinal leech Hirudo medicinalis. Experiments have focused on the Retzius cell (R) because the R cell is a multifunction neuron that has been implicated in a number of behaviors including feeding, swimming, secretion, thermal sensing, and the touch elicited shortening reflex and its plasticity. Previous work had identified a MM-like peptide in the leech and demonstrated that this peptide modulated the excitability of the R cell. Using combined current- and voltage-clamp techniques to examine the effects of MM on the R cell, we found that in response to a step pulse, MM increased the excitability of the R cell such that the cell fires more action potentials with a shorter latency to the first action potential. We found that this effect was mediated by the activation of a Na+-mediated inward current near the cell resting membrane potential. Second, we found that MM differentially modulated the potassium currents IA and IK. No effect of MM was found on IA, whereas MM significantly reduced both the peak and steady-state amplitudes of IK by 49 +/- 2.9% and 43 +/- 7.2%, respectively (means +/- SE). Finally we found that MM reduced the amplitude of the Ca2+ current by approximately 20%. The ionic currents modulated by MM are consistent with the overall effect of MM on the cellular activity of the R cell. An understanding of the cellular mechanisms by which MM modulates the activity of the R cell should help us to better understand the roles of both MM and the R cell in a variety of behaviors in the leech.

Effects of fluoxetine, phentermine, and venlafaxine on pulmonary arterial pressure and electrophysiology.

The anorexic agents dexfenfluramine and fenfluramine plus phentermine have been associated with outbreaks of pulmonary hypertension. The fenfluramines release serotonin and reduce serotonin reuptake in neurons. They also inhibit potassium current (IK), causing membrane potential depolarization in pulmonary arterial smooth muscle cells. The recent withdrawal of the fenfluramines has led to the use of fluoxetine and phentermine as an alternative anorexic combination. Because fluoxetine and venlafaxine reduce serotonin reuptake, we compared the effects of these agents with those of phentermine and dexfenfluramine on pulmonary arterial pressure, IK, and membrane potential. Fluoxetine, venlafaxine, and phentermine caused minimal increases in pulmonary arterial pressure at concentrations < 100 microM but did cause a dose-dependent inhibition of IK. The order of potency for inhibition of IK at +50 mV was fluoxetine > dexfenfluramine = venlafaxine > phentermine. Despite the inhibitory effect on IK at more positive membrane potentials, fluoxetine, venlafaxine, and phentermine, in contrast to dexfenfluramine, had minimal effects on the cell resting membrane potential (all at a concentration of 100 microM). However, application of 100 microM fluoxetine to cells that had been depolarized to -30 mV by current injection elicited a further depolarization of >18 mV. These results suggest that fluoxetine, venlafaxine, and phentermine do not inhibit IK at the resting membrane potential. Consequently, they may present less risk of inducing pulmonary hypertension than the fenfluramines, at least by mechanisms involving membrane depolarization.

Altered Resting Membrane Potential of Aortic Smooth Muscle Cells as a Function of Cellular Proliferation

Altered Resting Membrane Potential of Aortic Smooth Muscle Cells as a Function of Cellular Proliferation

A mixture theory for charged-hydrated soft tissues containing multi-electrolytes: passive transport and swelling behaviors.

A new mixture theory was developed to model the mechano-electrochemical behaviors of charged-hydrated soft tissues containing multi-electrolytes. The mixture is composed of n + 2 constituents (1 charged solid phase, 1 noncharged solvent phase, and n ion species). Results from this theory show that three types of force are involved in the transport of ions and solvent through such materials: (1) a mechanochemical force (including hydraulic and osmotic pressures); (2) an electrochemical force; and (3) an electrical force. Our results also show that three types of material coefficients are required to characterize the transport rates of these ions and solvent: (1) a hydraulic permeability; (2) mechano-electrochemical coupling coefficients; and (3) an ionic conductance matrix. Specifically, we derived the fundamental governing relationships between these forces and material coefficients to describe such mechano-electrochemical transduction effects as streaming potential, streaming current, diffusion (membrane) potential, electro-osmosis, and anomalous (negative) osmosis. As an example, we showed that the well-known formula for the resting cell membrane potential (Hodgkin and Huxley, 1952a, b) could be derived using our new n + 2 mixture model (a generalized triphasic theory). In general, the n + 2 mixture theory is consistent with and subsumes all previous theories pertaining to specific aspects of charged-hydrated tissues. In addition, our results provided the stress, strain, and fluid velocity fields within a tissue of finite thickness during a one-dimensional steady diffusion process. Numerical results were provided for the exchange of Na+ and Ca++ through the tissue. These numerical results support our hypothesis that tissue fixed charge density (CF) plays a significant role in modulating kinetics of ions and solvent transport through charged-hydrated soft tissues.

Influence of Bcl-2 overexpression on Na+/K(+)-ATPase pump activity: correlation with radiation-induced programmed cell death.

Bcl-2 overexpression in transfected PW cells is associated with inhibition of radiation-induced programmed cell death (PCD). We have previously reported that there is a relationship between inhibition of radiation-induced PCD and membrane hyperpolarization in these cells. In this article, we report that Na+/ K(+)-ATPase pump activity, as measured by the uptake of Rubidium-86 (86Rb+), is significantly higher in Bcl-2 overexpressing PW cells than in control PW cells, and that pump activity following irradiation with doses > or = 500 cGy was reduced to a lesser extent in the Bcl-2 transfectants than in the control cells. When PW-Bcl-2 cells were incubated with a dose of ouabain (1 microM) that decreased pump activity significantly, but did not induce PCD, the previously reported protection from radiation-induced PCD associated with overexpression of Bcl-2 no longer existed. In order to demonstrate that reactive oxygen species (ROS) affected Na+/ K(+)-ATPase pump activity, cells were incubated with N-acetyl cysteine (NAC) prior to irradiation, or treated with the ROS generating drug buthionine sulphoxamine (BSO). 86Rb+ uptake was significantly higher in irradiated cells incubated with NAC compared to cells irradiated in the absence of NAC, while BSO resulted in lower levels of 86Rb+ uptake, suggesting that the effects of radiation on the Na+/K(+)-ATPase pump were due to ROS. Furthermore, the resting cell membrane potential of cells exposed to NAC were slightly hyperpolarized compared to control PW cells, whereas cells exposed to BSO were depolarized in comparison to control PW cells. In summary, this data suggests that Bcl-2 affects Na+/K(+)-ATPase pump activity, which is associated with the resting membrane potential and the level of susceptibility to radiation-induced PCD.