Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle

Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1 and 8-isoprostaglandin E2 (8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2 and PGE2 implicated isoprostane activity at the PGE2 receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2 did not reverse KCl tone, suggesting that voltage-dependent Ca2+ influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+ currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2 receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+ channel activation or the direct inhibition of voltage-operated Ca2+ influx or myosin light chain kinase.     

槲皮素填充的脂质体对电离辐射处理的大鼠胸主动脉平滑肌细胞上BK_(Ca)的作用(英文)

本文旨在研究槲皮素填充的卵磷脂脂质体(quercetin-filled phosphatidylcholine liposomes,PCL-Q)对非致死性全身电离辐射处理的大鼠胸主动脉平滑肌细胞(smooth muscle cells,SMCs)上的大电导钙离子依赖性钾通道(large conductance Ca2+-dependent K+channels,BKCa)的作用。我们使用膜片钳技术,观察到去极化阶跃脉冲刺激的SMCs上出现外向K+电流,该电流对paxilline(BKCa的一种特异性抑制剂)敏感,因此认为该电流主体为BKCa电流。相对正常对照大鼠,辐照处理大鼠SMCs的BKCa电流幅度明显减小。这种通道抑制效应在辐照后第9天表现显著,且在整个30天的实验期间是逐渐增强的。辐照处理大鼠的SMCs的血管舒张能力可能因此而减弱。在本实验中,PCL-Q有效地恢复了辐照后SMCs的BKCa功能。而PCL-Q的组分,单一的槲皮素或空白的脂质体(PCL)恢复BKCa功能的能力较二者的组合体有明显的减弱。上述结果表明,PCL-Q能恢复辐照处理后SMCs的BKCa正常功能。PCL-Q的保护能力可能来源于其抗氧化...     

Proteasomal degradation of Kir6.2 channel protein and its inhibition by a Na+ channel blocker aprindine

ATP-sensitive K+ channels (K(ATP):SUR2A+Kir6.2) play a pivotal role in cardiac protection against ischemia and reperfusion injury. When expressed in COS cells, Kir6.2 was short-lived with a half-life time of 1.9 h. The half-life time of Kir6.2 was prolonged by proteasome inhibitors MG132, ALLN, proteasome inhibitor 1, and lactacystine, but not at all by a lysosomal inhibitor chloroquine. MG132 also increased the level of ubiquitinated Kir6.2 without affecting its localization in the endoplasmic reticulum and Golgi apparatus. In electrophysiological recordings, MG132 augmented nicorandil-activated K(ATP) currents in COS cells expressing SUR2A and Kir6.2 as well as the same currents in neonatal rat cardiomyocytes. Like MG132, a Na+ channel blocker aprindine prolonged the half-life time of Kir6.2 and augmented K(ATP). Finally, both aprindine and MG132 inhibited the 20S proteasome activity in vitro. These results suggest a novel activity of aprindine to enhance K(ATP) currents by inhibiting proteasomal degradation of Kir 6.2 channels, which may be beneficial in the setting of cardiac ischemia.     

The protein kinase A inhibitor, H-89, directly inhibits KATP and Kir channels in rabbit coronary arterial smooth muscle cells

The effects of the protein kinase A (PKA) inhibitor H-89 on ATP-sensitive K+ (KATP) and inward rectifier K+ (Kir) currents were examined in rabbit coronary arterial smooth muscle cells using the patch clamp technique. The H-89, in a dose-dependent manner, inhibited KATP and Kir currents with apparent Kd values of 1.19+/-0.18 and 3.78+/-0.37 microM, respectively. H-85, which is considered as an inactive form of H-89, inhibited KATP and Kir currents, similar to the result of H-89. KATP and Kir currents were not affected by either Rp-8-CPT-cAMPs, which is a membrane-permeable selective PKA inhibitor, or KT 5720, which is also known as a PKA inhibitor. Also, these two drugs did not significantly alter the effects of H-89 on the KATP and Kir currents. These results suggest that H-89 directly inhibits the KATP and Kir currents of rabbit coronary arterial smooth muscle cells independently of PKA inhibition.     

Diverse intracellular signalling systems used by growth hormone-releasing hormone in regulating voltage-gated Ca2+ or K channels in pituitary somatotropes

Influx of Ca2+ via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+, are regulated by GH-releasing hormone (GHRH) through G-protein-coupled intracellular signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured ovine and human somatotropes were recorded. Growth hormone-releasing hormone (10 nmol/L) increased both L- and T-type voltage-gated Ca2+ currents. Inhibition of the cAMP/protein kinase A (PKA) pathway by either Rp-cAMP or H89 blocked this increase in both L- and T-type Ca2+ currents. Growth hormone-releasing hormone also decreased voltage-gated transient (IA) and delayed rectified (IK) K+ currents. Protein kinase C (PKC) inhibitors, such as calphostin C, chelerythrine or downregulation of PKC, blocked the effect of GHRH on K+ currents, whereas an acute activation of PKC by phorbol 12, 13-dibutyrate (1 micromol/L) mimicked the effect of GHRH. Intracellular dialysis of a specific PKC inhibitor (PKC19-36) also prevented the reduction in K+ currents by GHRH. It is therefore concluded that GHRH increases voltage-gated Ca2+ currents via cAMP/PKA, but decreases voltage-gated K+ currents via the PKC signalling system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+, leading to an increase in [Ca2+]i and the GH secretion.     

Is ATP Required for K+ Channel Activation in Vicia Guard Cells?

In vivo, K+ entry into guard cells via inward-rectifying K+ channels is indirectly driven by ATP via an H+-ATPase that hyperpolarizes the membrane potential. However, whether activation of the K+ channels of guard cells requires ATP remains unknown. In the present study, both whole-cell and single-channel patch-clamp techniques were used to address this question. Exogenous ATP, ADP, and adenosine-5[prime]-O-(3-thiotriphosphate) applied to the cytoplasm had no effect on whole-cell K+ currents of Vicia faba L. guard cells. Azide, an inhibitor of oxidative phosphorylation, also had no effect. However, an ATP-scavenging system, glucose plus hexokinase, inhibited whole-cell inward K+ currents by 30 to 40%. Single-channel results acquired from cytoplasm-free inside-out membrane patches showed definite activation of inward K+ channels by ATP. Other nucleotides, such as ADP, adenosine-5[prime]-O(3-thiotriphosphate), and GTP, did not increase channel activity in the membrane patches. Inward K+ channel activity in membrane patches preactivated by exogenous ATP was inhibited by glucose plus hexokinase. These results suggest that a low concentration of ATP is required for activation of the inward K+ channels of the guard-cell plasma membrane. The issue of how ATP as a signal regulates these K+ channels is discussed.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes

Single-channel currents were recorded from ATP-sensitive K+ channels in inside-out membrane patches excised from isolated rat ventricular myocytes. Perfusion of the internal surface of excised membrane patches with solutions which contained between 5 and 100 microM free calcium caused the loss of K+ATP channel activity which was not reversed when the membranes were washed with Ca-free solution. K+ATP channel activity could be recovered by bathing the patches in Mg.ATP. The loss of K+ATP channel activity provoked by internal calcium was a process which occurred over a time scale of seconds. Channel closure evoked by internal ATP was essentially instantaneous. The speed of K+ATP channel inactivation increased with the concentration of calcium. Neither a phosphatase inhibitor (fluoride ions) nor a proteinase inhibitor (leupeptin) had any effect upon the loss of K+ channel activity stimulated by internal calcium.     

Properties of Two Outward-Rectifying Channels in Root Xylem Parenchyma Cells Suggest a Role in K+ Homeostasis and Long-Distance Signaling

Xylem parenchyma cells (XPCs) control the composition of the transpiration stream in plants and are thought to play a role in long-distance signaling as well. We addressed the regulation, selectivity, and dependence on the apoplastic ion concentrations of two types of outward rectifiers in the plasma membrane of XPCs, to assess the physiological role of these conductances. In whole-cell recordings, the membrane conductance at depolarization was under the control of cytosolic Ca2+: at physiological Ca2+ levels (150 nM) the K+ outward-rectifying conductance (KORC) predominated, whereas at elevated Ca2+ levels (5 [mu]M), only the nonselective outward-rectifying conductance (NORC) was active. No such regulatory effect of Ca2+ was observed in inside-out experiments. The voltage dependence of whole-cell KORC currents strongly depended on apoplastic K+ concentration: an increase in apoplastic K+ resulted in a positive shift of the current-voltage curve, roughly following the shift in Nernst potential of K+. KORC is impermeable to Na+, but does translocate Ca2+ in addition to K+. In contrast to KORC, NORC selected poorly among monovalent cations and anions, the relative permeability PC+/PA- being about 1.9. Gating of NORC was largely unaffected by the level of K+ in the bath. Under all ionic conditions tested, NORC tail currents or single-channel currents reversed close to 0 mV. Using an in vivo xylem-perfusion technique, tetraethylammonium (an inhibitor of KORC) was shown to block K+ transport to the shoot. These data support the hypothesis that release of K+ to the xylem sap is mediated by KORC. The molecular properties of these two conductances are discussed in the light of the distinct physiological role of XPCs.     

Effects of pine needle extract on pacemaker currents in interstitial cells of Cajal from the murine small intestine

Extracts of pine needles (Pinus densiflora Sieb. et Zucc.) have diverse physiological and pharmacological actions. In this study we show that pine needle extract alters pacemaker currents in interstitial cells of Cajal (ICC) by modulating ATP-sensitive K+ channels and that this effect is mediated by prostaglandins. In whole cell patches at 30 degrees , ICC generated spontaneous pacemaker potentials in the current clamp mode (I = 0), and inward currents (pacemaker currents) in the voltage clamp mode at a holding potential of -70 mV. Pine needle extract hyperpolarized the membrane potential, and in voltage clamp mode decreased both the frequency and amplitude of the pacemaker currents, and increased the resting currents in the outward direction. It also inhibited the pacemaker currents in a dose-dependent manner. Because the effects of pine needle extract on pacemaker currents were the same as those of pinacidil (an ATP-sensitive K+ channel opener) we tested the effect of glibenclamide (an ATP-sensitive K+ channels blocker) on ICC exposed to pine needle extract. The effects of pine needle extract on pacemaker currents were blocked by glibenclamide. To see whether production of prostaglandins (PGs) is involved in the inhibitory effect of pine needle extract on pacemaker currents, we tested the effects of naproxen, a non-selective cyclooxygenase (COX-1 and COX-2) inhibitor, and AH6809, a prostaglandin EP1 and EP2 receptor antagonist. Naproxen and AH6809 blocked the inhibitory effects of pine needle extract on ICC. These results indicate that pine needle extract inhibits the pacemaker currents of ICC by activating ATP-sensitive K+ channels via the production of PGs.     

K+ conductance activated during regulatory volume decrease. The channels in Ehrlich cells and their possible molecular counterpart

K+ currents activated by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the whole-cell recording mode of the patch-clamp technique. K+ together with Cl- currents developed in the absence of added intracellular Ca2+ and with strong buffering of internal Ca2+ in experiments conducted at 37 degrees C. Manipulation of the extracellular medium with other cations suggests a selectivity sequence of K+ > Rb+ > NH4+ > or = Na+ approximately equals Li+ approximately equals Cs+. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and 20 mV at both physiological and high K+ extracellular solutions. The class III antiarrhytmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease (RVD) response of Ehrlich cells. The leukotriene D4 (LTD4) can activate the same current in isotonicity, consistent with a role for this compound in the signalling process of volume regulation. It is suggested that K+ channels activated by cell swelling belong to the so-called background K+ channel group. These are voltage-independent channels which underlie the resting potential of many cells and have recently been identified as belonging to a family of K+ channels with two pore domains in tandem (2P-4TM). Preliminary experiments show the presence of the TASK-2 channel, a member of the 2P-4TM family inhibited by acid extracellular pH, in Ehrlich cells and suggest that it might underlie the swelling-induced K+ current.     

On the block of outward potassium current in rabbit Schwann cells by internal sodium ions.

Currents through delayed rectifier-type K+ channels in Schwann cells cultured from rabbit sciatic nerve were studied with patch-clamp techniques. When the internal and external solutions contained physiological concentrations of sodium, the amplitude of these outward currents declined as the cell was depolarized to potentials above about +40 mV, despite the increased driving force. This reduction in the amplitude of outward K+ currents was observed in many cells before the subtraction of leakage currents; it was also observed for ensemble currents recorded in outside-out patches. It was therefore not the result of a leak-subtraction artefact nor of inadequate voltage-clamp control. Several lines of evidence also suggested that it was not the result of the extracellular accumulation of K+. By contrast, when the Na+ ion concentration of the internal solution was nominally zero, the reduction in the amplitude of outward K+ currents at positive membrane potentials was not observed. The apparent amplitude of single-channel currents through two types of K+ channel was reduced by 30 mM internal Na+, apparently as the result of a rapid 'flickery' block. The results suggest that channel block by internal Na+ is largely responsible for the negative slope conductance seen in current-voltage plots of whole-cell K+ currents at positive membrane potentials. In addition, our analysis of single-channel currents suggests that the current-voltage curve for a delayed rectifier channel in rabbit Schwann cells (in the absence of internal Na+) is roughly linear with internal and external K+ concentrations of 140 mM and 5.6 mM, respectively.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Membrane ionic currents in Rhodobacter capsulatus. Evidence for electrophoretic transport of K+, Rb+ and NH4+

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isoprostanes and PGE2 production in human isolated pulmonary artery smooth muscle cells: concomitant and differential release.

The isoprostanes are a group of biologically active arachidonic acid metabolites initially thought to be formed under conditions of oxidative stress and independently of cyclooxygenase. However, recent studies have demonstrated isoprostane production under conditions in which cyclooxygenase is intentionally activated/induced. Here we describe for the first time formation of isoprostanes by human vascular cells via independent pathways of oxidative stress and cyclooxygenase induction. We compared the release of the isoprostane with that of the traditional prostaglandin, prostaglandin E2. Cyclooxygenase-2 induction was confirmed by Western blot. When cells were stimulated with cytokines, the release of isoprostanes was inhibited by the cyclooxygenase-1 and -2 inhibitor indomethacin as well by as the cyclooxygenase-2 selective inhibitor L-745,337. However, treatment of cells with the superoxide-producing enzyme xanthine oxidase also resulted in isoprostane release, which was not affected by cyclooxygenase inhibition, unlike PGE2 release under the same condition. Thus, two independent pathways relating to oxidative stress and cyclooxygenase-2 induction form isoprostanes. These findings may have particular importance in diseases such as sepsis and ARDS in which oxidant stress occurs and cyclooxygenase is induced.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

Voltage-gated potassium channels in brown fat cells

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.     

Characterization of K+ currents in rat malignant lymphocytes (Nb2 Cells)

Membrane K+ currents of malignant lymphocytes (Nb2 cells) were studied with the whole-cell patch-clamp method. Upon depolarization, K+ currents activate with a delay and follow a sigmoid time course, resembling other delayed rectifier K+ currents present in nerve and muscle cells. The activation time constant of these currents is voltage dependent, increasing from 1 msec at +90 mV to approximately 37 msec at -30 mV. The fractional number of open channels has a sigmoid voltage dependence with a midpoint near -25 mV. Deactivation of K+ currents in Nb2 cells is voltage dependent and follows a simple exponential time course. Time constant of this process increases from 5 msec at -115 mV to almost 80 msec at -40 mV. The relative permeability of K+ channels to different monovalent cations follows the sequence: K+ (1) greater than Rb+ (0.75) greater than NH4+ (0.11) greater than Cs+ (0.07) greater than Na+ (0.05). Inactivation of K+ currents is a biexponential process with time constants of approximately 600 and 7,000 msec. Inactivation of K+ currents in Nb2 cells is not a voltage-dependent process. The steady-state inactivation curve of K+ currents has a midpoint near -40 mV. Following a 500-msec voltage pulse, inactivation of K+ currents recovers with a simple exponential process with a time constant of 9 sec. Short duration (approximately 50 msec) voltage-clamp pulses do not induce significant inactivation of these currents. K+ currents in malignant lymphocytes do not display the phenomenon of cumulative inactivation as described for other delayed rectifier-type K+ channels. Application of a train of voltage pulses to positive potentials at different frequencies induces a moderate decrease in peak outward currents. The use of substances (N-bromoacetamide, trypsin, chloramine-T, and papain) that remove the inactivation of Na+ and K+ currents in other cells are not effective in removing the inactivation of K+ currents present in this lymphoma cell line. Significant differences were found between the characteristics of K+ currents in this malignant cell line and those present in normal lymphocytes. Possible physiological implications for these differences and for the role of K+ currents in the proliferation of normal and malignant lymphocytes are discussed.     

NADPH oxidase does not account fully for O2-sensing in model airway chemoreceptor cells

A key feature of O2 sensing by chemoreceptor tissues is the hypoxic inhibition of K+ channels. However, mechanisms coupling a fall of pO2 to channel closure differ between tissues: O2 regulation of K+ channels in chemoreceptive neuroepithelial bodies and their immortal counterparts, H146 cells, involves altered reactive oxygen species generation by NADPH oxidase. In contrast, this enzyme complex is not involved in O2 sensing by the carotid body and pulmonary vasculature. Here, we provide pharmacological evidence to support a role for NADPH oxidase in hypoxic inhibition of K+ currents in H146 cells. Two structurally unrelated NADPH oxidase inhibitors, diphenylene iodonium and phenylarsine oxide, suppressed hypoxic inhibition of K+ currents recorded using the patch-clamp technique. Most importantly, however, neither inhibitor fully blocked this response. Our findings provide the first evidence that multiple mechanisms may coexist within a specific cell type to account for hypoxic suppression of K+ channel activity.     

Analysis of the K+ Current Profile of Mature Rat Oligodendrocytes in situ

Previous studies have reported that mature oligodendrocytes (OLGs) in vitro display various voltage-dependent K+ currents while in situ OLGs show only voltage-independent K+ currents. Given this discrepancy and the lack of information on myelinating OLG ion channel expression in situ, we characterized mature OLG currents in myelinating corpus callosum slices from 17 to 36-day old rats. OLGs were recorded in cell-attached and whole-cell patch-clamp configurations, displayed morphology typical of mature OLGs, and stained positive for myelin basic protein. OLGs displayed large voltage-independent currents that decayed during the voltage pulse and small voltage-activated outward currents. The latter were blocked by TEA, activated between -40 and -50 mV, and decayed slowly. The former were composed of large voltage-independent, time-dependent Ba2+ (1 mM)-sensitive currents, and voltage-dependent Cs+ (5 mM) and Ba2+ (100 mM)-sensitive currents that reversed near the K+ equilibrium potential and inactivated at hyperpolarized potentials, identifying them as inwardly rectifying K+ currents. Inwardly rectifying single-channel K+currents could be recorded in the cell-attached configuration. The estimated single-channel slope conductance was 30 pS. The steady-state open probability was voltage-dependent and declined from 0.9 to 0.5 between -80 and -150 mV. Overall, mature OLGs in situ possess time- and also voltage-dependent K+ currents, which may facilitate clearance of K+ released during axonal firing.