Nitric oxide activates ATP-dependent K+ channels in human eosinophils

Nitric oxide (NO) affects the function of ion channels in many cell types, but its role in the regulation of eosinophil ion channels is unknown. In this study, we used the perforated patch-clamp method to investigate the effect of endogenous and exogenous NO on eosinophil ion channels. Using the NO synthase inhibitor, N-nitro-L-arginine methyl ester, we showed that endogenous NO did not affect the whole-cell current in eosinophil. However, two NO donors, S-nitroso-glutathione and S-nitroso-N-acetyl penicillamine, activated whole-cell currents via a NO/cGMP-dependent pathway. Ion substitution and pharmacological studies showed that NO-activated currents were carried by K+ ions, likely through ATP-dependent K+ channels (K(ATP)). Although RT-PCR studies showed the expression of several classes of K+ channels in human eosinophils, NO donors affected only K(ATP) channel function. We conclude that NO, at concentrations likely to be encountered in vivo, could prevent eosinophil activation by opening K(ATP) channels.     

Three pharmacologically distinct potassium channels in molluscan neurones.

1. Potassium currents were studied under voltage-clamp conditions in nerve cell bodies of the nudibranch Tritonia diomedia. 2. Potassium currents could be separated into three distinct components on the basis of their sensitivity to 4-aminopyridine (4-AP), tetraethyl-ammonium (TEA) and to Co2+ and Mn2+ ions. 3. A transient potassium current, similar to the fast outward current described by Connor & Stevens (1971b) and Neher (1971), was blocked by externally applied 4-AP but was much less sensitive to TEA or to Co2+ or Mn2+. A single 4-AP ion binds each receptor with an apparent dissociation constant of 1-5 X 10(-3) M. 4-AP decreases the rates of activation and inactivation and reduces the maximum conductance of transient current channels. 4. Delayed outward current was not effected by 4-AP at concentrations which blocked the transient current, but it could be divided into two components by external application of TEA and Co2+ or Mn2+. 5. A voltage-dependent component of delayed current, termed K-current, was blocked by TEA. Each K-current receptor binds a single TEA ion with an apparent dissociation constant of 8 X 10(-3) M. Co2+ and Mn2+ have little or no effect on K-current. 6. A second component of delayed outward current, termed C-current, depends on Ca2+ entry for its activation. It is similar to the Ca2+ dependent potassium current reported by Meech & Stranden (1975) in Helix cells. C-current is essentially blocked by 30 mM external Co2+ or Mn2+. It is little affected by TEA, however, being reduced by about 20% at a TEA concentration of 100 mM. 7. It is concluded that three sets of potassium selective channels contribute to the outward current and that these channels can be separated pharmacologically.     

ATP-sensitive potassium channels counteract anoxia in neurones of the substantia nigra

Summary The ATP-sensitive potassium channel (K_ATP channel) is a unique ionophore in that it appears to reflect cell metabolism. In the brain, the highest density of binding sites for the K_ATP channel is the substantia nigra. To evaluate the role of the K_ATP channel in this key brain area for motor control, we used exposure to cyanide to lower intracellular ATP and thereby mimic anoxia and ischemia. Treatment with cyanide caused the activation of a potassium current in a sub-population of nigral neurones with distinct pharmacological and electrophysiological properties. The response to cyanide was abolished by the sulphonylurea tolbutamide, a potent blocker of the K_ATP channel. These results suggest that in the substantia nigra, the K_ATP channel plays a pivotal role in normal mechanisms of neuronal homeostasis in response to anoxia and ischaemia. The significance of these findings for our understanding of the cellular mechanisms in Parkinsonian degeneration is discussed.     

NO对新生大鼠下丘脑钙激活钾通道的直接激活作用

目的：研究NO对下丘脑神经元钙激活钾通道（K_Ca）的作用及其机制。方法：采用膜片钳技术内面向外式。结果：NO可显著提高通道的开放概率，这种增强作用是通过延长通道开放时间及增加开放频率实现的。结论：下丘脑神经元中NO可直接激活K_Ca，它的病理生理作用还有待于进一步研究。     

Adenosine-5′-triphosphate-sensitive ion channels in neonatal rat cultured central neurones

ATP-sensitive channels were observed in isolated inside-out membrane patches from rat cultured central neurones. Two types of ATP-sensitive K⁺ channels were present in cortical neurones, one which had its open-state probability increased, the other its open-state probability decreased by application of ATP to the cytoplasmic membrane surface. Another, ATP-sensitive channel differing in ion conductance from all previously reported ATP-sensitive channels was also seen in patches from cortical neurones. This channel was noselective with respect to Na⁺, K⁺ and Cl^– ions and ATP produced a flickery type of block. The non-hydrolysable analogue, AMPPNP, did not mimic ATP and prevented ATP action Preliminary experiments indicate that similar, but not, identical ATP-sensitive channels exist in cerebellar neurones     

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.     

Bilateral coordination of inspiratory neurones in the rat

Inspiratory activity on the left and right sides must be coordinated to be effective. We used cross-correlation to examine the hypothesis that the coordination of left and right medullary inspiratory neurones is produced by excitation from common sources and by midline-crossing excitatory connections among these neurones. In adult rats, a total of 185 contralateral pairs of inspiratory neurones ( n=370) were recorded extracellularly, and classified, according to their firing pattern, as augmenting ( n=262), constant ( n=82) or decrementing ( n=26). Of the 262 augmenting inspiratory neurones, 98 were classified as phrenic premotor neurones by cross-correlation with phrenic nerve discharge. The 185 cross-correlograms showed little evidence of common activation, or midline-crossing excitatory connections. Of the 45 cross-correlograms for pairs of augmenting neurones, only 4 (approximately equal to 9%) indicated a common activation, and only one a monosynaptic connection. Of the 45 for pairs of augmenting and phrenic premotor neurones, only 9 (20%) showed a common activation, and only 2 a monosynaptic excitatory connection. Of the 19 pairs of phrenic premotor neurones, 5 from the same rat showed high-frequency oscillations, and 1 a monosynaptic excitatory connection. Cross-correlograms for pair combinations of other types of neurones also exhibited few features. We suggest that, in the adult rat, although both common activation and excitatory cross-connections exist as a means for coordinating left and right ventral group inspiratory neurones to the same respiratory rhythm, they are insufficient to account for it.     

The flickery block of ATP-dependent potassium channels of skeletal muscle by internal 4-aminopyridine

We have examined the effects of 4-aminopyridine (4-AP) on single ATP-dependent potassium channels in patches excised from frog skeletal muscle. 4-AP applied to the internal face of the membrane caused a flickery block. We could not detect any flickery block when 10 mM 4-AP was applied to the external surface of the membrane. The reduction in mean unitary current by internal 4-AP was consistent with 11 binding with a K _d of 3.3 mM at 0 mV. The block was voltage-dependent, increasing with depolarization with an effective valency of 0.57. Rate constants for blocking and unblocking by 4-AP were obtained by fitting functions to the distribution of current amplitudes. Both rate constants were voltage-dependent. At 0 mV they were 17 mM^–1 ms^–1 and 61 ms^–1. Simulation of the block using these rate constants produced a flickery block very similar to that observed experimentally.     

A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ.

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around -55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At -60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01-1 mM). Tetraethylammonium (0.1-30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials.This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.     

Calcium influx through N-methyl-D-aspartate channels activates a potassium current in postnatal rat hippocampal neurons

Calcium-activated potassium conductances play important roles in modulating neuronal excitability. Indeed, the effects of some neurotransmitters such as acetylcholine and norepinephrine are, in part, due to actions on these conductances. We have found that the N-methyl-D-aspartate (NMDA) class of excitatory amino acid receptors also is coupled to a calcium activated potassium current. In voltage-clamped postnatal rat hippocampal neurons, NMDA responses consist of an initial inward cationic current followed by a slowly developing outward current carried by potassium ions. The slow outward current always follows the inward current, is associated with an increase in membrane conductance and is dependent on the influx of calcium ions. Similar responses are produced by other agonists active at NMDA receptors, including aspartate, glutamate and ibotenate, but are not activated by kainate, quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Inhibition of the NMDA gated inward current by a competitive antagonist, 2-amino-5-phosphonovalerate (APV), eliminates the outward current. From these results we conclude that calcium influx through NMDA channels activates a potassium current. The extended time course of this outward current suggests that NMDA receptors may modulate neuronal excitability long after the opening of the NMDA channel.     

ATP-dependent potassium channels and mitochondrial permeability transition pores play roles in the cardioprotection of theaflavin in young rat

Previous studies have confirmed that tea polyphenols possess a broad spectrum of biological functions such as anti-oxidative, anti-bacterial, anti-tumor, anti-inflammatory, anti-viral and cardiovascular protection activities, as well as anti-cerebral ischemia-reperfusion injury properties. But the effect of tea polyphenols on ischemia/reperfusion heart has not been well elucidated. The aim of this study was to investigate the protective effect of theaflavin (TF1) and its underlying mechanism. Young male Sprague-Dawley (SD) rats were randomly divided into five groups: (1) the control group; (2) TF1 group; (3) glibenclamide + TF1 group; (4) 5-hydroxydecanoate (5-HD) + TF1 group; and (5) atractyloside + TF1 group. The Langendorff technique was used to record cardiac function in isolated rat heart before and after 30 min of global ischemia followed by 60 min of reperfusion. The parameters of cardiac function, including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of LVDP (±LVdP/dt _max) and coronary flow (CF), were measured. The results showed: (1) compared with the control group, TF1 (10, 20, 40 μmol/l) displayed a better recovery of cardiac function after ischemia/reperfusion in a concentration-dependent manner. At 60 min of reperfusion, LVDP, ±LVdP/dt _max and CF in the TF1 group were much higher than those in the control group, whereas left ventricular end-diastolic pressure (LVEDP) in the TF1 group was lower than that in the control group (P < 0.01). (2) Pretreatment with glibenclamide (10 μmol/l), a K_ATP antagonist, completely abolished the cardioprotective effects of TF1 (20 μmol/l). Also, most of the effects of TF1 (20 μmol/l) on cardiac function after 60 min of reperfusion were reversed by 5-HD (100 μmol/l), a selective mitochondria K_ATP antagonist. (3) Atractyloside (20 μmol/l), a mitochondrial permeability transition pore (mPTP) opener, administered at the beginning of 15 min of reperfusion completely abolished the cardioprotection of TF1 (20 μmol/l). The results indicate that TF1 protects the rat heart against ischemia/reperfusion injury through the opening of K_ATP channels, particularly on the mitochondrial membrane, and inhibits mPTP opening.     

Activation of mitochondrial ATP-sensitive potassium channels prevents neuronal cell death after ischemia in neonatal rats

Activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) has been shown to protect against cell death following ischemia/reperfusion in the heart but not in brain. We examined whether mK(ATP) activation with diazoxide (DIZ) prevents neuronal cell death following hypoxia-ischemia (HI) in 7-day-old rat pups. Rat pups were subjected to HI (left carotid ligation; 8% O(2); 2.5 h), following administration of vehicle, 1.9 mg/kg DIZ, 3.8 mg/kg DIZ or DIZ plus 10 mg/kg 5-hydroxydecanoic acid (mK(ATP) antagonist). Total infarct volume was reduced from 99.8+/-2.7% in vehicle animals to 80.6+/-4.2% in 3.8 mg/kg DIZ treated animals (n=85, P<0.05). Western blotting showed K(ATP) subunits concentrated in mitochondria. Fluorescent studies indicated DIZ directly depolarized the mitochondria. In conclusion, selective opening of mK(ATP) prior to HI results in neuroprotection in immature rats.     

Dopamine directly activates a ligand-gated channel in snail neurones

Synaptically released dopamine is known to evoke fast as well as slow synaptic potentials in neurones of gastropod molluscs. Here evidence is presented that the fast excitatory response to dopamine is mediated by the direct activation of a ligand-gated channel unitary currents were observed in outside-out patches of neurones exposed to dopamine, and the response persisted in the presence of intracellular guanosine 5-o-(2-thiobiphosphate), GDP[-S], a condition known to block G-protein-coupled responses to dopamine and other agents. In whole-cell recordings, the fast response desensitized very rapidly; it was less desensitized in outside-out patches, suggesting dependence of desensitization on an intracellular factor. The response to dopamine was blocked by D-tubocurarine and strychnine (both probably by channel blockade), by apomorphine, chlorpromazine and relatively high doses of (±)-sulpiride and spiperone. The channel conducts predominantly monovalent cations. Unexpectedly, the fast response to dopamine was also observed in an identified dopaminergic neurone when maintained in isolation in culture. The receptors on the dopaminergic neurone were unevenly distributed, being more abundant on the axon-hillock, axon and neurite terminals.     

Blockades of ATP-sensitive potassium channels and L-type calcium channels improve analgesic effect of morphine in alloxan-induced diabetic mice

In the present study interactions between analgesic effect of morphine with blockade of ATP-sensitive potassium channels and L-type calcium channels were investigated in alloxan-induced diabetic mice. A hot plate test was used to assess analgesic effect of drugs in adult male NMRI mice. All drugs were injected through an intraperitoneal route. A diabetic mouse model was established by injections of alloxan for three consecutive days. Seventy-two hours after the final injection, mice with a blood glucose level higher than 11.1mmol/l were considered as diabetic. The results showed that morphine at doses of 10 and 15mg/kg induced analgesia in both non-diabetic and diabetic mice, but the analgesic effect of morphine at dose of 7.5mg/kg was decreased in diabetic mice. Injections of an ATP-sensitive potassium channel blocker, glibenclamide (4, 8, 12, 20mg/kg) had no effect in non-diabetic mice, while at doses of 12 and 20mg/kg induced analgesia in diabetic mice. Blockade of L-type calcium channels with nimodipine at different doses (2.5, 5, 10 and 20mg/kg) was ineffective in non-diabetic mice, but at dose of 20mg/kg induced analgesia in diabetic mice. Co-administrations of glibenclamide (20mg/kg) or nimodipine (20mg/kg) along with different doses of morphine (5, 7.5 and 10mg/kg) improved the analgesic effect of the later drug in diabetic mice. According to the present results, it is possible that diabetes via affecting the potassium and calcium channels in the pain pathways may alter processing of pain in the peripheral and central nervous system.     

Modulation of ligand-gated dopamine channels in Helix neurones

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca²⁺] by various procedures (external zero Ca²⁺ or 1 mM Co²⁺, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.     

链脲佐菌素诱导的糖尿病早期小鼠胸主动脉钾通道变化

目的:探讨链脲佐菌素诱导的糖尿病早期小鼠胸主动脉钾通道的变化。方法:实验采用离体血管的方法测定糖尿病鼠和正常鼠胸主动脉环对血管收缩剂:60mmol/LKCl和苯肾上腺素(PE)、内皮非依赖性舒张剂:硝普钠(SNP)以及电压依赖性钾通道(K_V通道),钙激活型钾通道(K_Ca通道),ATP敏感钾通道(K_ATP通道)阻断剂的反应。结果:糖尿病鼠胸主动脉环对60mmol/LKCl、PE和SNP的效应都显著大于对照组;K_Ca通道阻断剂四乙铵(TEA)显著降低糖尿病小鼠胸主动脉环在PE的激动下SNP的舒张效应,而且其-logIC₅₀的差值较对照组显著增大;K_V通道阻断剂4-氨基吡啶(4-AP)显著降低糖尿病和正常小鼠胸主动脉环对SNP的舒张效应,但是-logIC₅₀差值无显著差异;K_ATP通道阻断剂格列苯脲(Glibenclamide)显著降低糖尿病小鼠胸主动脉环对SNP的舒张效应,而对照组无显著阻断作用,-logIC₅₀的差值也无显著差异。结论:糖尿病早期小鼠胸主动脉平滑肌细胞K_Ca通道的开放或表达显著增强,也证实了K_ATP通道开放增强。