Pathophysiological and protective roles of mitochondrial ion channels

Mitochondria possess a highly permeable outer membrane and an inner membrane that was originally thought to be relatively impermeable to ions to prevent dissipation of the electrochemical gradient for protons. Although recent evidence has revealed a rich diversity of ion channels in both membranes, the purpose of these channels remains incompletely determined. Pores in the outer membrane are fundamental participants in apoptotic cell death, and this process may also involve permeability transition pores on the inner membrane. Novel functions are now being assigned to other ion channels of the inner membrane. Examples include protection against ischaemic injury by mitochondrial K_ATP channels and the contribution of inner membrane anion channels to spontaneous mitochondrial oscillations in cardiac myocytes. The central role of mitochondria in both the normal function of the cell and in its demise makes these channels prime targets for future research and drug development.     

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.     

Iptakalim protects against hypoxic brain injury through multiple pathways associated with ATP-sensitive potassium channels

The rapid and irreversible brain injury produced by anoxia when stroke occurs is well known. Cumulative evidence suggests that the activation of neuronal ATP-sensitive potassium (KATP) channels may have inherent protective effects during cerebral hypoxia, yet little information regarding the therapeutic effects of KATP channel openers is available. We hypothesized that pretreatment with a KATP channel opener might protect against brain injury induced by cerebral hypoxia. In this study, adult Wistar rats were treated with iptakalim, a new KATP channel opener, which is selective for SUR2 type KATP channels, by intragastric administration at doses of 2, 4, or 8 mg/kg/day for 7 days before being exposed to simulated high altitude equivalent to 8000 m in a decompression chamber for 8 h leading to hypoxic brain injury. By light and electron microscopic images, we observed that hypobaric hypoxia-induced brain injury could be prevented by pretreatment with iptakalim. It was also observed that the permeability of the blood-brain barrier, water content, Na+ and Ca2+ concentration, and activities of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase in rat cerebral cortex were increased and the gene expression of the occludin or aquaporin-4 was down- or upregulated respectively, which could also be prevented by the pretreatment with iptakalim at doses of 2, 4, or 8 mg/kg in a dose-dependent manner. Furthermore, we found that in an oxygen-and-glucose-deprived model in ECV304 cells and rat cortical astrocytes, pretreatment with iptakalim significantly increased survived cell rates and decreased lactate dehydrogenate release, which were significantly antagonized by glibenclamide, a K(ATP) channel blocker. We conclude that iptakalim is a promising drug that may protect against brain injury induced by acute hypobaric hypoxia through multiple pathways associated with SUR2-type K(ATP) channels, suggesting a new therapeutic strategy for stroke treatment.     

Isobolographic analysis of caramiphen and lidocaine on spinal anesthesia in rats

The aims of the study were to evaluate the spinal anesthetic effect of caramiphen and also assess spinal anesthetic interactions of caramiphen with lidocaine. Lidocaine, a common local anesthetic, was used as control. Dose-dependent responses of intrathecal caramiphen on spinal anesthesia were compared with lidocaine in rats. The interactions of caramiphen with lidocaine were evaluated via an isobolographic analysis. Caramiphen and lidocaine produced a dose-dependent local anesthetic effect as spinal anesthesia. On a 50% effective dose (ED₅₀) basis, the spinal anesthetic effect of caramiphen was more potent than lidocaine (P < 0.01 for each comparison). Co-administration of caramiphen with lidocaine produced an additive effect. Caramiphen and lidocaine are known to have local anesthetic effects as spinal anesthesia in rats. The spinal anesthetic effects of adding caramiphen to lidocaine are similar to the combinations of other anesthetics with lidocaine.     

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.     

Diverse roles for auxiliary subunits in phosphorylation-dependent regulation of mammalian brain voltage-gated potassium channels

Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse partly due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here, we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels.     

Intrathecal oxybuprocaine and proxymetacaine produced potent and long-lasting spinal anesthesia in rats

Proxymetacaine and oxybuprocaine were clinically used for topical ocular anesthesia but never for spinal anesthesia, and therefore spinal anesthetic effects of proxymetacaine and oxybuprocaine were performed and compared with bupivacaine and lidocaine. After rats were injected intrathecally with proxymetacaine, oxybuprocaine, bupivacaine, and lidocane, dose–response curves were constructed. We evaluated the potencies (ED₅₀) and durations (time to full recovery) of proxymetacaine and oxybuprocaine on spinal blockades of motor function, proprioception, and nociception and compared with bupivacaine and lidocaine in rats. We found that proxymetacaine and oxybuprocaine acted like bupivacaine or lidocaine and produced dose-related spinal blockades of motor function, proprioception and nociception. On the ED₅₀ basis, the ranks of potencies in motor, proprioception, and nociception were proxymetacaine > oxybuprocaine > bupivacaine > lidocaine (P < 0.01 for the differences). On an equipotent basis (ED₂₀, ED₅₀, ED₈₀), oxybuprocaine and bupivacaine produced similarly longer spinal blockades than did proxymetacaine or lidocaine (P < 0.05 for the differences). Intrathecal proxymetacaine, oxybuprocaine, and bupivacaine also produced longer sensory blockade than motor blockade. These data demonstrated that oxybuprocaine and proxymetacaine produced more potent spinal blockades, when compared with bupivacaine or lidocaine. Oxybuprocaine and bupivacaine with a more sensory-selective action over motor blockade produced longer spinal blockade than did proxymetacaine or lidocaine.     

Effect of anoxic preconditioning on ATP-sensitive potassium channels in guinea-pig ventricular myocytes

Ischemic or hypoxic preconditioning in experimental animals and humans is described. The mechanism of preconditioning may involve several endogenous substances released from ischemic or hypoxic tissues (such as adenosine, noradrenaline and bradykinin) that stimulate protein kinase C (PKC), which then phosphorylates ATP-sensitive potassium channels (K(ATP) channels). However, the effect of hypoxic preconditioning on K(ATP) channels in guinea-pig ventricular myocytes is unclear. The uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) has been shown to activate K(ATP) channels in isolated cardiac cells. In the present study we tested whether anoxic preconditioning (APC) could affect the opening of K(ATP) channels activated by metabolic inhibition (MI) induced by FCCP in cell-attached and inside-out patches from guinea-pig ventricular myocytes. We measured the channel activity as NP(o)i and calculated it using the formula Po=I/(Ni), where Po is open-state probability, I is the mean patch current carried by all K(ATP) channels activated in a particular patch for a certain period of time, N is the number of functioning channels in the patch, and i is the unitary current of the K(ATP) channels. In cell-attached membrane patches, after about 5 min of initiating MI, K(ATP) channels were activated at a holding potential of +40 mV (NP(o)i=3.70+/-0.9 pA); APC pretreatment (3 min of anoxia followed by 7 min of reoxygenation) before MI (APC+MI group) shortened the time to activate K(ATP) channels by MI (2.3+/-0.5 min) and increased the activity of K(ATP)currents (NP(o)i=8.4+/-0.5 pA). This effect of APC was eliminated by administration of a PKC blocker, chelerythrine (5 microM), for 5 min before the APC pretreatment. In the inside-out patches, the IC50 of intracellular ATP against the K(ATP) channels in the APC+MI group was significantly increased to 642 microM compared to that in the MI group (IC50 of intracellular ATP =252 microM). Chelerythrine inhibited the effect of APC on the sensitivity of K(ATP) channels to the intracellular ATP concentration (IC50 of [ATP]i=301 microM). Our results demonstrate that APC can increase and accelerate the opening of K(ATP) channels induced by MI, and decrease the sensitivity of K(ATP) channels to [ATP]i, which is mediated by promoting the activation of PKC induced by APC.     

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 ATP-sensitive potassium channels in follicle-enclosedXenopus oocytes by the epileptogenic agent pentylenetetrazol

For further investigation of the epileptogenic properties of pentylenetetrazol (PTZ), membrane currents elicited by PTZ were analysed in native Xenopus oocytes. PTZ elicited a sequence of membrane currents. Two inward currents have been described to be due to a decrease in potassium permeability and an increase in chloride permeability. Experiments performed up to 3 days after preparation of the oocytes showed that PTZ is also able to activate an outward current. This current is: (1) reversed near the potassium equilibrium potential, (2) associated with a decrease in membrane resistance, (3) reduced by tetraethylammonium and caesium, (4) abolished by defolliculation, and (5) blocked by glibenclamide. Thus, the current can be interpreted to be due to an activation of ATP-sensitive potassium (K_ATP) channels located in the follicle cells. An activation of K_ATP channels by PTZ may contribute to termination and re-initiation of seizure activity. Received: 7 June 1995/Received after revision: 7 September 1995/Accepted: 6 October 1995     

Differential activation of ATP-sensitive potassium channels during energy depletion in CA1 pyramidal cells and interneurones of rat hippocampus

In the hippocampus, pyramidal cells are more vulnerable than granule cells and interneurones to energy depletion during hypoxia and ischaemia. The aim of the present study was to explore whether this difference is related to the lower expression of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channels in pyramidal cells compared to other hippocampal neurones. Hippocampal slices were prepared from 10- to 13-day-old rats, and CAI pyramidal cells and interneurones of the stratum radiatum were visually and electrophysiologically identified. Energy depletion was produced by removing glucose from the bath or by inhibiting mitochondrial metabolism using rotenone. In the perforated-patch configuration, both protocols elicited outward currents in only a minority of the pyramidal cells but in most of the interneurones. The currents started to develop 9-57 min after glucose deprivation and 4-16 min after rotenone application and reversed near the K+ equilibrium potential. Bath-applied diazoxide (0.3 mM), an opener of K(ATP) channels, could activate additional currents. The sulphonylureas tolbutamide (0.5 mM) or glibenclamide (20 microM), two blockers of K(ATP) channels, totally inhibited the currents induced by energy depletion and activated by diazoxide. The results demonstrate the differential activation of K(ATP) channels during energy depletion in pyramidal cells and interneurones, and suggest that channel activation is neuroprotective against the deleterious effects of energy depletion.     

Gating kinetics of ATP-sensitive single potassium channels in myocardial cells depends on electromotive force

ATP-sensitive single-channel potassium currents were studied in the membrane of rat ventricular myocytes. With an internal K⁺ concentration of [K⁺]_i=140 mM, the outwardly directed currents saturated at 1.8 pA in the region of positive potentials independently of the external K⁺ concentration [K⁺]_o, whereas an increase in [K⁺]_i of up to 300 mM caused a positive shift in the region of current saturation (from +40 mV to +100 mV) and an increase in the level of the saturation up to 4 pA. The openings of the channels appeared in bursts. Gating kinetics within the bursts were investigated. It was shown that the channel mean open (_o) and closed (_c) times during a burst depended primarily on the electromotive force (V-V _k) for potassium ions. For different [K⁺]_o, _o was maximal and _c was minimal in the region of reversal potentials (V _rev); _o decreased and _c increased gradually with deviation ofV fromV _rev. Therefore we conclude that the gating properties of the ATP-sensitive K channels depend on the ion flux parameters.     

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.     

Functional roles of TRPC channels in the developing brain

Transient receptor potential canonical (TRPC) channels are Ca²⁺-permeable, nonselective cation channels formed by homomeric or heteromeric complexes of TRPC proteins that contain six transmembrane domains. These channels can be activated through a phospholipase-C-dependent mechanism, making them sensors for environmental cues. Their expression begins early in embryonic days and remains in adulthood. These channels have important roles in the processes of neuronal development, including neural stem cell proliferation, cerebellar granule cell survival, axon path finding, neuronal morphogenesis, and synaptogenesis. In this review, we will discuss functional implications of TRPC channels during brain development.     

Physiological roles of ATP-sensitive K+ channels in smooth muscle

Potassium channels that are inhibited by intracellular ATP (ATP_i) were first identified in ventricular myocytes, and are referred to as ATP-sensitive K⁺ channels (i.e. K_ATP channels). Subsequently, K⁺ channels with similar characteristics have been demonstrated in many other tissues (pancreatic β-cells, skeletal muscle, central neurones, smooth muscle). Approximately one decade ago, K_ATP channels were cloned and were found to be composed of at least two subunits: an inwardly rectifying K⁺ channel six family (Kir6.x) that forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR) that accounts for several pharmacological properties. Various types of native K_ATP channels have been identified in a number of visceral and vascular smooth muscles in single-channel recordings. However, little attention has been paid to the molecular properties of the subunits in K_ATP channels and it is important to determine the relative expression of K_ATP channel components which give rise to native K_ATP channels in smooth muscle. The aim of this review is to briefly discuss the current knowledge available for K_ATP channels with the main interest in the molecular basis of native K_ATP channels, and to discuss their possible linkage with physiological functions in smooth muscle.     

Dexmedetomidine for the prevention of shivering during spinal anesthesia

PURPOSE:

The aim of this study was to evaluate the effect of dexmedetomidine on shivering during spinal anesthesia.

METHODS:

Sixty patients (American Society of Anesthesiologists physical status I or II, aged 18-50 years), scheduled for elective minor surgical operations under spinal anesthesia with hyperbaric bupivacaine, were enrolled. They were administered saline (group C, n = 30) or dexmedetomidine (group D, n = 30). Motor block was assessed using a Modified Bromage Scale. The presence of shivering was assessed by a blinded observer after the completion of subarachnoid drug injection.

RESULTS:

Hypothermia was observed in 21 patients (70%) in group D and in 20 patients (66.7%) in group C (p = 0.781). Three patients (10%) in group D and 17 patients (56.7%) in group C experienced shivering (p = 0.001). The intensity of shivering was lower in group D than in group C (p = 0.001). Time from baseline to onset of shivering was 10 (5-15) min in group D and 15 (5-45) min in group C (p = 0.207).

CONCLUSION:

Dexmedetomidine infusion in the perioperative period significantly reduced shivering associated with spinal anesthesia during minor surgical procedures without any major adverse effect during the perioperative period. Therefore, we conclude that dexmedetomidine infusion is an effective drug for preventing shivering and providing sedation in patients during spinal anesthesia.     

腰麻联合硬膜外麻醉对剖宫产术麻醉效果的影响

目的 分析腰麻联合硬膜外麻醉对剖宫产术临床麻醉效果和并发症的影响。方法 收集2018年2月~2019年2月在我院行剖宫产术的110例产妇临床资料,采用随机数字表法分为对照组和观察组,各55例。对照组采用硬膜外麻醉,观察组在对照组基础上联合腰麻,比较两组临床麻醉效果、肌肉松弛效果、麻醉起效时间、术中出血量、麻药用量、疼痛评分以及不良反应。结果 观察组麻醉效果优于对照组,差异有统计学意义（P＜0.05）;观察组肌肉松弛效果为100.00%,优于对照组的83.63%,差异有统计学意义（P＜0.05）;观察组麻醉起效时间（3.31±0.89）min、术中出血量（130.51±24.70）ml、麻药用量（20.33±8.45）mg、疼痛评分（2.88±0.99）分,均低于对照组的（8.71±0.69）min、（158.88±22.81）ml、（43.27±1.13）mg、（5.02±0.78）分,差异有统计学意义（P＜0.05）;观察组不良反应发生率（9.09%）低于对照组（21.81%）,差异有统计学意义（P＜0.05）。结论 腰麻联合硬膜外麻醉用于剖宫产手术,麻醉效果佳,肌肉松弛理想,麻醉起效快,镇痛效果良好,且并发症少,安全性高,临床应用效果理想。     

ATP-sensitive potassium channels in rat primary afferent neurons: the effect of neuropathic injury and gabapentin

ATP-sensitive potassium (K(ATP)) currents were examined in dorsal root ganglion neurons from neuropathic and control rats using whole-cell voltage clamp recordings. K(ATP) channel openers (diazoxide and pinacidil) enhanced, and the blocker glibenclamide inhibited an outward current in control neurons in a manner dependent on the pipette ATP concentration. Analysis of reversal potentials showed that this current is carried by K(+) ions. Outward current in cells from rats with peripheral nerve injury was not sensitive to modulators of K(ATP) channels. Gabapentin, a putative K(ATP) channel opener, had minimal effect on currents in either group of neurons. We conclude that normal primary afferent neurons express K(ATP) channels that conduct current which is eliminated by peripheral nerve injury. Gabapentin does not affect this current significantly.     

Modulation of muscle contractility during fatigue and recovery by ATP-sensitive potassium channel

The activity of ATP-sensitive potassium channels of skeletal muscle is controlled by changes in the bioenergetic state of the cell. These channels are inactive in unfatigued muscle and become activated during fatigue. It has been postulated that ATP-sensitive potassium channels shorten the action potential duration, increase the potassium efflux and contribute to the decrease in force during fatigue. Although blocking ATP-sensitive potassium channels during fatigue prolongs the action potential duration and decreases the potassium efflux as expected, it does not affect the rate of fatigue development, as observed from the decrease in tetanic force. Even though such results are not consistent with the hypothesis that ATP-sensitive potassium channels contribute to the decrease in force during fatigue, a reduced capacity of skeletal muscles to recover their tetanic force following fatigue is also observed when ATP-sensitive potassium channels are blocked during fatigue, suggesting that these channels have a myoprotective effect. It is thus possible that removing this myoprotection during fatigue results in deleterious effects which counteract the expected slower decrease in force. However, ATP-sensitive potassium channel openers also fail to affect the rate of fatigue development. Therefore, the results obtained so far do not support the hypothesis that ATP-sensitive potassium channels contribute to the decrease in force during fatigue.