Depolarization Of Em Research Articles

Pharmacological inhibition of KSper impairs flagellar pH homeostasis of human spermatozoa.

Sperm-specific potassium channel (KSper) comprised of pore-forming subunit SLO3 and auxiliary subunit LRRC52 is of importance for sperm fertility. The deficiency of KSper in both mice and humans resulted in severe impairments of sperm functions including sperm hyperactivity and acrosome reaction. Previous reports suggested that mouse KSper modulated sperm function possibly by affecting sperm intracellular pH (pHi). However, the precise signaling mechanism of human KSper (hKSper) on the regulation of sperm functions was largely unclear. To explore the regulatory role of hKSper on sperm flagellar pHi. More than 50 sperm donors were recruited during a period of 1 year. As reported in our previous work, we quantitatively measured flagellar pHi by employing a single-cell pH fluorescent recording on human spermatozoa loaded with pH indicator pHrodo Red. Three different hKSper antagonists including clofilium, quinidine, and a polyclonal antibody of LRRC52 (LID1) were utilized to evaluate the effect of hKSper inhibition on sperm flagellar pHi. Given the predominant role of hKSper on the regulation of membrane potential (Em), we first detected a considerable depolarization (about 25-30mV) of Em evoked by clofilium and quinidine. Subsequently, it was shown that flagellar pHi values of human spermatozoa were significantly decreased by the treatment of clofilium (50 µM, from 7.13±0.11 to 6.43±0.12), quinidine (500 µM, from 7.00±0.11 to 6.64±0.08) and LID1 (20 µg/mL, from 6.98±0.16 to 6.67±0.22). Moreover, we found that when human spermatozoa were pre-incubated with a high K+ solution (135mM), both the depolarization of Em and the acidification of flagellar pHi evoked by clofilium and quinidine were abolished. In addition, we found that extracellular substitution of N-methyl-D-glucamine for Na+ abolished pHi acidification induced by hKSper inhibition. Our results demonstrate that hKSper inhibition evokes flagellar pHi acidification of human spermatozoa, suggesting that flagellar pHi maintenance is an important signaling mechanism of hKSper on the regulation of sperm functions.

Acid-sensing ion channel 1 contributes to pulmonary arterial smooth muscle cell depolarization following hypoxic pulmonary hypertension.

Pulmonary hypertension is characterized by sustained vasoconstriction and remodelling of the small pulmonary arteries, which is associated with persistent depolarization of the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs). It is well-known that the underlying mechanism of this depolarization includes inhibition of K+ channels; however, whether other ion channels contribute to this depolarization is unknown. We previously reported that acid-sensing ion channel 1 (ASIC1), a non-selective cation channel (NSCC) that conducts both Na+ and Ca2+ , is present in PASMCs and contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension. Therefore, we tested the hypothesis that ASIC1-mediated Na+ influx contributes to PASMC Em regulation following CH-induced pulmonary hypertension. Using sharp electrode intracellular recordings in isolated, pressurized small pulmonary arteries from rats and mice, we show that exposure to CH leads to PASMC membrane depolarization compared with control animals, and this is independent of intraluminal pressure-induced depolarization. In addition to a decrease in PASMC whole-cell K+ currents following CH, we demonstrate that whole-cell NSCC currents are increased and essential to the persistent CH-induced Em depolarization in PASMCs. Both the specific inhibitor of ASIC1, psalmotoxin 1, and global knockout of ASIC1 (Asic1-/- ) prevents CH-induced Em depolarization and largely inhibits whole-cell NSCC currents, without affecting whole-cell K+ currents. Our results show a combination of factors, including inhibition of K+ efflux and augmented Na+ influx, mediate CH-induced PASMC depolarization. Furthermore, this study demonstrates a novel role for ASIC1 in the regulation of Em in PASMCs during CH-induced pulmonary hypertension. KEY POINTS: In pulmonary hypertensive patients and animal models of pulmonary hypertension, the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs) is persistently depolarized. In addition to the well-established reduction of K+ conductance, we show that non-selective cation channel currents are increased and essential to the persistent Em depolarization in PASMCs following chronic hypoxia (CH)-induced pulmonary hypertension. The current study provides novel evidence that acid-sensing ion channel 1 (ASIC1)-mediated Na+ influx induces membrane depolarization and regulates Em in PASMCs following CH exposure. Although fairly quiescent under control conditions, our findings demonstrate a pathological function of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension.

Electrophysiology of Hypokalemia and Hyperkalemia.

Electrophysiology of Hypokalemia and Hyperkalemia.

Dissimilar responses of membrane potential (EM), permeability properties and respiration to cadmium and nickel in maize root cells

The short-term treatment with Cd2+ and Ni2+ triggered transient depolarization of transplasma membrane potential (EM) in the outer cortical root cells of two maize cultivars (cv. Premia and cv. Blitz), however, both metals changed the EM in a quantitatively different way. The magnitude and duration of EM depolarization were concentration dependent and were greater in the metal susceptible cv. Blitz. The highest EM depolarization was recorded with simultaneous application of Cd2+ + Ni2+ in both maize cultivars. The EM depolarization induced by Cd2+ or Cd2+ + Ni2+ but not Ni2+ alone was accompanied with a tremendous increase of membrane conductivity, but it was not accompanied with the effect of heavy metals (HM) on respiration. Simultaneous application of fusiccocin (FC) with Cd2+ or Cd2+ + Ni2+ during the EM depolarization, inability of FC to stop the depolarization by FC-enhanced proton extrusion and rapid restoration of EM, suggested a transient inhibition of the plasma membrane H+-ATPase by these toxic...

Responses of maize root cells to zearalenone and its derivatives α-zearalenol and β-zearalenol

The extent of mycotoxin-induced responses in living roots of two maize (Zea mays L.) cultivars differing in their susceptibility to Fusarium was studied. Application of mycotoxin zearalenone (ZEN) or its derivates α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) caused a rapid depolarization of plasma membrane potential (EM). The extent of EM depolarization was dependent on the type of mycotoxin that have been used and showed concentration dependence. Interestingly, ZEN, but not its derivatives α-ZEL and β-ZEL, significantly decreased respiration of maize root cells. Electrolyte leakage increased with the duration of toxins treatment and was significantly higher in susceptible cv. Pavla than in resistant cv. Lucia. A strong superoxide dismutase insensitive nitro blue tetrazolium (NBT) reduction activity was identified in the root tips of control plants. This activity was rapidly inhibited by mycotoxin application in the meristematic/distal transition zones of roots in both cultivars examined. The level of recovery was a function of the mycotoxin concentration. Moreover, mycotoxin treatment also resulted in the onset and the progression of root cell death which was dependent on both, the type and concentration of mycotoxin.

Impact of fusaproliferin on primary roots of maize cultivars differing in their susceptibility to Fusarium

Effects of fusaproliferin (FUS) on membrane potential (EM), electrolyte leakage, enzymes activity and respiration of roots, were studied in two maize cultivars (Zea mays L.), differing in their susceptibility to this toxin. In short-term experiments (≤ 6 h), EM has been rapidly and significantly depolarized by FUS. The rapidity of EM depolarization in tolerant cv. Lucia was more expressive in comparison with susceptible cv. Pavla, but the extent of EM depolarization was higher in cv. Pavla. In both maize cultivars, higher depolarization of EM was registered in cells of root zone I. In long-term experiments after the first EM depolarization, which occurred during the first 6 h of FUS treatment, gradual depolarization continued up to 24 h and was represented not only by the active component (EP) but also by the passive component (ED) of EM. The decrease in EM and ED was followed by a loss of K+ ions from FUS treated roots of both cultivars. The leak of K+ ions from the root cells of both root zones as well as both maize cultivars increased with the time of FUS treatment and was significantly higher in susceptible cv. Pavla than in tolerant cv. Lucia. FUS treatment of maize roots resulted in a significant decrease of root respiration which was higher in susceptible cv. Pavla than in tolerant cv. Lucia.

Aluminium-induced ion transport in Arabidopsis: the relationship between Al tolerance and root ion flux.

Aluminium (Al) rhizotoxicity coincides with low pH; however, it is unclear whether plant tolerance to these two factors is controlled by the same mechanism. To address this question, the Al-resistant alr104 mutant, two Al-sensitive mutants (als3 and als5), and wild-type Arabidopsis thaliana were compared in long-term exposure (solution culture) and in short-term exposure experiments (H+ and K+ fluxes, rhizosphere pH, and plasma membrane potential, Em). Based on biomass accumulation, als5 and alr104 showed tolerance to low pH, whereas alr104 was tolerant to the combined low-pH/Al treatment. The sensitivity of the als5 and als3 mutants to the Al stress was similar. The Al-induced decrease in H+ influx at the distal elongation zone (DEZ) and Al-induced H+ efflux at the mature zone (MZ) were higher in the Al-sensitive mutants (als3 and als5) than in the wild type and the alr104 mutant. Under combined low-pH/Al treatment, alr104 and the wild type had depolarized plasma membranes for the entire 30 min measurement period, whereas in the Al-sensitive mutants (als3 and als5), initial depolarization to around –60 mV became hyperpolarization at –110 mV after 20 min. At the DEZ, the Em changes corresponded to the changes in K+ flux: K+ efflux was higher in alr104 and the wild type than in the als3 and als5 mutants. In conclusion, Al tolerance in the alr104 mutant correlated with Em depolarization, higher K+ efflux, and higher H+ influx, which led to a more alkaline rhizosphere under the combined low-pH/Al stress. Low-pH tolerance (als5) was linked to higher H+ uptake under low-pH stress, which was abolished by Al exposure.

δ-Opioid receptor antagonism induces NMDA receptor-dependent excitotoxicity in anoxic turtle cortex

delta-Opioid receptor (DOR) activation is neuroprotective against short-term anoxic insults in the mammalian brain. This protection may be conferred by inhibition of N-methyl-d-aspartate receptors (NMDARs), whose over-activation during anoxia otherwise leads to a deleterious accumulation of cytosolic calcium ([Ca(2+)](c)), severe membrane potential (E(m)) depolarization and excitotoxic cell death (ECD). Conversely, NMDAR activity is decreased by approximately 50% with anoxia in the cortex of the painted turtle, and large elevations in [Ca(2+)](c), severe E(m) depolarization and ECD are avoided. DORs are expressed in high quantity throughout the turtle brain relative to the mammalian brain; however, the role of DORs in anoxic NMDAR regulation has not been investigated in turtles. We examined the effect of DOR blockade with naltrindole (1-10 micromol l(-1)) on E(m), NMDAR activity and [Ca(2+)](c) homeostasis in turtle cortical neurons during normoxia and the transition to anoxia. Naltrindole potentiated normoxic NMDAR currents by 78+/-5% and increased [Ca(2+)](c) by 13+/-4%. Anoxic neurons treated with naltrindole were strongly depolarized, NMDAR currents were potentiated by 70+/-15%, and [Ca(2+)](c) increased 5-fold compared with anoxic controls. Following naltrindole washout, E(m) remained depolarized and [Ca(2+)](c) became further elevated in all neurons. The naltrindole-mediated depolarization and increased [Ca(2+)](c) were prevented by NMDAR antagonism or by perfusion of the G(i) protein agonist mastoparan-7, which also reversed the naltrindole-mediated potentiation of NMDAR currents. Together, these data suggest that DORs mediate NMDAR activity in a G(i)-dependent manner and prevent deleterious NMDAR-mediated [Ca(2+)](c) influx during anoxic insults in the turtle cortex.

Layer-Specific Generation and Propagation of Seizures in Slices of Developing Neocortex: Role of Excitatory GABAergic Synapses

The neonatal period is critical for seizure susceptibility, and neocortical networks are central in infantile epilepsies. We report that application of 4-aminopyridine (4-AP) to immature (P6-P9) neocortical slices generates layer-specific interictal seizures (IISs) that transform after recurrent seizures to ictal seizures (ISs). During IISs, cell-attached recordings show action potentials in interneurons and pyramidal cells in L5/6 and interneurons but not pyramidal neurons in L2/3. However, L2/3 pyramidal neurons also fire during ISs. Using single N-methyl-d-aspartate (NMDA) channel recordings for measuring the cell resting potential (Em), we show that transition from IISs to ISs is associated with a gradual Em depolarization of L2/3 and L5/6 pyramidal neurons that enhances their excitability. Bumetanide, a NKCC1 co-transporter antagonist, inhibits generation of IISs and prevents their transformation to ISs, indicating the role excitatory GABA in epilepsies. Therefore deep layer neurons are more susceptible to seizures than superficial ones. The initiating phase of seizures is characterized by IISs generated in L5/6 and supported by activation of both L5/6 interneurons and pyramidal cells. IISs propagate to L2/3 via activation of L2/3 interneurons but not pyramidal cells, which are mostly quiescent at this phase. In superficial layers, a persistent increase in excitability of pyramidal neurons caused by Em depolarization is associated with a transition from largely confined GABAergic IIS to ictal events that entrain the entire neocortex.

WC5E REDUCED LOW FREQUENCY OSCILLATORY ACTIVITY AS A VULNERABILITY MARKER FOR SCHIZOPHRENIA

Non-steroidal anti-inflammatory drugs (NSAIDs) contribute to gastrointestinal ulcer formation by inhibiting epithelial cell migration and mucosal restitution; however, the drug-affected signaling pathways are poorly defined. We investigated whether NSAID inhibition of intestinal epithelial migration is associated with depletion of intracellular polyamines, depolarization of membrane potential (Em) and altered surface expression of K+ channels. Epithelial cell migration in response to the wounding of confluent IEC-6 and IEC-Cdx2 monolayers was reduced by indomethacin (100 μM), phenylbutazone (100 μM) and NS-398 (100 μM) but not by SC-560 (1 μM). NSAID-inhibition of intestinal cell migration was not associated with depletion of intracellular polyamines. Treatment of IEC-6 and IEC-Cdx2 cells with indomethacin, phenylbutazone and NS-398 induced significant depolarization of Em, whereas treatment with SC-560 had no effect on Em. The Em of IEC-Cdx2 cells was: −38.5 ± 1.8 mV under control conditions; −35.9 ± 1.6 mV after treatment with SC-560; −18.8 ± 1.2 mV after treatment with indomethacin; and −23.7 ± 1.4 mV after treatment with NS-398. Whereas SC-560 had no significant effects on the total cellular expression of Kv1.4 channel protein, indomethacin and NS-398 decreased not only the total cellular expression of Kv1.4, but also the cell surface expression of both Kv1.4 and Kv1.6 channel subunits in IEC-Cdx2. Both Kv1.4 and Kv1.6 channel proteins were immunoprecipitated by Kv1.4 antibody from IEC-Cdx2 lysates, indicating that these subunits co-assemble to form heteromeric Kv channels. These results suggest that NSAID inhibition of epithelial cell migration is independent of polyamine-depletion, and is associated with depolarization of Em and decreased surface expression of heteromeric Kv1 channels.

Sodium and Epithelial Sodium Channels Participate in the Regulation of the Capacitation-associated Hyperpolarization in Mouse Sperm

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 µM to 1 mM) for several hours to one week were studied. The E m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E D) when the Cd concentration exceeded 100 µM. The values of E D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 µM) or complete repolarization (Cd < 100 µM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E m a continuous concentration-dependent decrease of E m followed at all Cd concentrations. Depolarization of E m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 µM to 1 mM Cd, with the exception of root cells treated with 1 and 10 µM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 µM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 µM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H+ ATPase because fusicoccin, an H+ ATPase activator diminished the depolarizing effect of Cd on the E m. The depolarization of E m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H+ ATPase.

Slow volume transients in amphibian skeletal muscle fibres studied in hypotonic solutions.

The influence of extracellular hypotonicity on the relationship between cell volume (V(c)) and resting membrane potential (E(m)) was investigated in Rana temporaria skeletal muscle. V(c) was measured by confocal microscope imaging of fibres through their transverse (xz) planes, and E(m) was determined using standard microelectrode techniques. Hypotonic solutions first elicited a rapid increase in fibre volume, DeltaV(R+) that fulfilled expectations of simple osmotic behaviour described in earlier reports. However, this was consistently followed by a slow increase in V(c) (DeltaV(S+)) to 10-15% above osmotic predictions. Longer (>1 h) exposures to hypotonic solutions permitted a subsequent slow decrease in V(c) (DeltaV(S-)), the eventual magnitude of which exceeded that of the preceding DeltaV(S+). Restoration of isotonic conditions elicited a prompt recovery in V(c) that matched simple osmotic predictions and thus left a net change in V(c). Such alterations in V(c) attributable to DeltaV(S+) then gradually reversed, while those due to DeltaV(S-) persisted. Both DeltaV(S+) and DeltaV(S-) persisted under conditions of Cl- deprivation. The depolarization of E(m) that accompanied DeltaV(R+) was consistent with dilution of intracellular [K(+)]. E(m) did not significantly alter during the subsequent DeltaV(S) transients. These empirical features of DeltaV(S+) and DeltaV(S-) were analysed using the quantitative charge-difference model of Fraser and Huang, published in 2004. This attributed the DeltaV(S+) to an electroneutral increase in the effective osmotic activity of normally membrane-impermeant intracellular anions. In contrast, the DeltaV(S-) could only be explained by an efflux of such anions and was accordingly comparable to organic anion-dependent regulatory volume decreases reported in other cell types.

Effect of NO3- transport and reduction on intracellular pH: an in vivo NMR study in maize roots.

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.

Electrical properties and conduction in reperfused papillary muscle.

The reversibility of ischemia-induced changes of extracellular K(+) concentration ([K(+)](o)), resting membrane potential (E(M)), and passive cable-like properties, ie, extracellular resistance and cell-to-cell electrical coupling, and their relationship to recovery of conduction and contraction is described in 25 reperfused rabbit papillary muscles. No-flow ischemia caused extracellular K(+) accumulation, depolarization of E(M), an increase in whole-tissue (r(t)), external (r(o)), and internal (r(i)) longitudinal resistances, and failure of conduction and contraction. Muscles were reperfused 10 minutes after the onset of ischemia related cell-to-cell electrical uncoupling, ie, 26+/-1 minutes after arrest of perfusion. In 11 muscles, incomplete reflow occurred with only partial recovery of [K(+)](o) and r(t). In the remaining 14 muscles, reperfusion caused a rapid and parallel decrease in [K(+)](o), r(t), and r(o). When complete tissue reperfusion occurred, cell-to-cell electrical uncoupling was largely reversible. Thus, cell-to-cell electrical uncoupling did not indicate irreversible injury. Reperfusion induced a depolarizing current widening the difference between the K(+) equilibrium potential and the E(M). This difference decreased after longer periods of reperfusion. Conduction was restored and conduction velocity approached preischemic values as cell-to-cell electrical interaction was reestablished and E(M) recovered. The recovery of r(o) preceded r(i), decreasing the ratio of the extracellular to intracellular resistance early in reperfusion, an effect predicted to influence the amplitude of the extracellular voltage field and electrocardiographic ST segments during reperfusion.

Stimulation of intracellular chloride accumulation by noradrenaline and hence potentiation of its depolarization of rat arterial smooth muscle in vitro.

1. Double-barrelled ion-selective microelectrodes were used to examine the effects of exogenous noradrenaline upon the membrane potential (Em) and intracellular chloride concentration ([Cl]i) of arterial smooth muscle from the saphenous branch of the femoral artery of the rat. 2. After treatment with 0.6 mM 6-hydroxydopamine (to functionally denervate the tissue), exogenous noradrenaline (5 nM) caused repeatable depolarization of Em from -63.7 +/- 2.4 mV (s.d., n = 18) to -53.8 +/- 3.4 mV (P < 0.0001) and increases in [Cl]i from 31.0 +/- 0.5 mM to 42.5 +/- 2.2 mM (P < 0.0001). 3. In the presence of 10 microM bumetanide (an inhibitor of (Na-K-Cl) cotransport), 5 nM noradrenaline caused a depolarization of Em of 3.0 +/- 3.2 mV, and a rise in [Cl]i of 4.5 +/- 2.5 mM. 4. In the presence of bumetanide and 1 mM acetazolamide (used as an inhibitor of a Na-independent inward Cl pump), noradrenaline had no effect on Em or [Cl]i. 5. In the absence of extracellular chloride, the rise in apparent [Cl]i in response to 5 nM noradrenaline was abolished but there was a depolarization of 2.0 +/- 3.9 mV. 6. These results are consistent with the stimulation of (Na-K-Cl) cotransport and a Na-independent Cl pump by exogenous noradrenaline and with the consequent increase in [Cl]i and shift in ECl potentiating the depolarization caused by noradrenaline. The possibility that modulation of [Cl]i may be a general mechanism of Em regulation is discussed.

Microvillous membrane potential (Em) in villi from first trimester human placenta: comparison toEmat term

The microvillous membrane (MVM) potential (Em) of first trimester human placental villi was measured and compared with that in villi from term human placentas. The median Em in first trimester villi (-28 mV) was significantly more negative than that at term (-21 mV; P < 0.001). The median Em measured in villi from early (weeks 6-11) first trimester (-32 mV) was significantly more negative than that in late (weeks 12 and 13) first trimester villi (-24 mV; P < 0.001). Elevating extracellular KCl concentration induced a significant depolarization of Em in both first trimester and term villi (P < 0.05 and P < 0.001, respectively). The magnitude of this depolarization was greater in first trimester than at term, indicating that the ion conductance of the MVM changes with gestation. Exposure to ouabain induced a significant depolarization of Em (3 mV: P < 0.05) in first trimester villi but had little effect at term. These results suggest that microvillous membrane electrophysiology changes with placental development. An alteration in the relative K+:Cl- conductance of the MVM is likely to be a major contributor to the change in the magnitude of Em.

Differential depolarization-activated calcium responses in fetal and neonatal rat osteoblast-like cells

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

Membrane Response to Diclofop Acid Is pH Dependent and Is Regulated by the Protonated Form of the Herbicide in Roots of Pea and Resistant and Susceptible Rigid Ryegrass.

Electrophysiological studies in roots of pea (Pisum sativum L.) and rigid ryegrass (Lolium rigidum Gaud.) seedlings were conducted to elucidate the mechanism involved in the membrane response to the herbicide diclofop. In pea, a dicotyledonous plant insensitive to diclofop, membrane depolarization at varying pH values and herbicide concentrations increased at higher concentrations of the protonated form of diclofop acid (pKa 3.57). In unbuffered nutrient solution (pH 5.7), diclofop acid (50 [mu]M) depolarized the membrane potential (Em) in roots of both resistant and susceptible biotypes of rigid ryegrass, whereas recovery of Em occurred only in the resistant biotype following removal of the herbicide. This differential response was correlated with an increase (450%) in the rate of acidification of the external solution by the susceptible biotype, and the Em differences between biotypes were eliminated in solutions buffered at pH 5.0 or 6.0. In addition, p-chloromercuribenzene-sulfonic acid did not prevent the depolarization of Em by 50 [mu]M diclofop acid. It is concluded that the differential membrane response to diclofop acid in herbicide-resistant and -susceptible biotypes of rigid ryegrass is due to pH differences at the cell wall/plasmalemma interface. Although the membrane response is probably not involved in the primary inhibitory effect of diclofop on plant growth, it could reduce the concentration of the permeant protonated form of the herbicide and possibly could contribute to increased tolerance to diclofop and other weak acid herbicides.

Electrochemical potentials of potassium in skeletal muscle under different metabolic states.

Intracellular potassium and membrane potential were measured simultaneously by means of double-barrelled liquid ion-exchange microelectrodes in single fibers of rat thigh muscle in vivo in rats maintained in seven different metabolic states. The K+ equilibrium potential (EK) was more negative than the simultaneously measured membrane potential (Em) in the normal state by 18.4 mV. K+ loading, acute and chronic, resulted in depolarization of Em due to increased serum K+ (hyperkalemia) with no increase in intracellular K+. K+ depletion resulted in hyperpolarization of Em as plasma K+ decreased proportionately more than intracellular K+. Low Na+ diet had no effect. Intracellular K+ was decreased in acute acidosis but not in the chronic state. Thus K+ depletion and acute acidosis are associated with intracellular K+ decrease. The fact that hyperpolarization exists in the former and not the latter is a reflection that hypokalemia accompanies the former condition. The hyperpolarizing states of K+ depletion and chronic acidosis are accompanied by decreased excitability and muscle weakness.