Pflügers Arch – Eur J Physiol (2000) 439:808–813 Digital Object Identifier (DOI) 10.1007/s004249900200
O R I G I N A L A RT I C L E
Zhen Zhu · Yu-Long Li · De-Pei Li · Rui-Rong He
Effect of anoxic preconditioning on ATP-sensitive potassium channels in guinea-pig ventricular myocytes
Received: 3 June 1999 / Received after revision: 9 October 1999 / Accepted: 25 October 1999 / Published online: 4 February 2000 © Springer-Verlag 2000
Abstract 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 (KATP channels). However, the effect of hypoxic preconditioning on KATP channels in guinea-pig ventricular myocytes is unclear. The uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) has been shown to activate KATP channels in isolated cardiac cells. In the present study we tested whether anoxic preconditioning (APC) could affect the opening of KATP 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 NPoi and calculated it using the formula Po=I/(Ni), where Po is open-state probability, I is the mean patch current carried by all KATP 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 KATP channels. In cell-attached membrane patches, after about 5 min of initiating MI, KATP channels were activated at a holding potential of +40 mV (NPoi=3.7±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 KATP channels by MI (2.3±0.5 min) and increased the activity of KATP currents (NPoi=8.4±0.5 pA). This effect of APC was eliminated by administration of a PKC blocker, chelerythrine (5 µM), for 5 min before the APC pretreatment. In the inside-out patches, the IC50 of intracellular ATP against the KATP channels in the APC+MI group Z. Zhu · Y.L. Li · D.P. Li · R.R. He (✉) Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, People’s Republic of China e-mail:
[email protected] Fax: +86-311-6048177
was significantly increased to 642 µM compared to that in the MI group (IC50 of intracellular ATP =252 µM). Chelerythrine inhibited the effect of APC on the sensitivity of KATP channels to the intracellular ATP concentration (IC50 of [ATP]i=301 µM). Our results demonstrate that APC can increase and accelerate the opening of KATP channels induced by MI, and decrease the sensitivity of KATP channels to [ATP]i, which is mediated by promoting the activation of PKC induced by APC. Key words Anoxic preconditioning · ATP-sensitive K+ channels · Cardiomyocytes · Metabolic inhibition · Protein kinase C
Introduction Single or multiple brief periods of myocardial ischemia and reperfusion render the heart resistant to injury after a subsequent prolonged period of coronary artery occlusion [20]. This phenomenon, termed ischemic preconditioning, has been described in studies of experimental animals (including dogs [12, 20], rabbits [14], pigs [23, 24] and rats [15]) as well as humans [30]. Walker et al. [26] and Kasamaki et al. [10] also found that hypoxia preconditioning prior to prolonged hypoxia challenge improves the post-hypoxia recovery of developed force in rabbit and guinea-pig papillary muscles. Although a great deal of effort has been made to identify the mechanisms underlying ischemic or hypoxic preconditioning, these remain poorly understood. One recently proposed and popular hypothesis is that several endogenous substances released from ischemic or hypoxic tissues (such as adenosine, noradrenaline and bradykinin) during the preconditioning ischemia or hypoxia activate protein kinase C (PKC), which then phosphorylates ATP-sensitive potassium (KATP) channels. The phosphorylated KATP channels were found [17] to open earlier and/or more intensely during the prolonged ischemia or hypoxia, and shortened the action potential duration, decreased Ca2+ influx and conserved energy, and thus improved the post-
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ischemia or post-hypoxia recovery of developed tension and limited myocardial infarct size [17]. However, the effect of hypoxic preconditioning on KATP channels and the underlying mechanisms in single ventricular myocytes have not yet been elucidated. Metabolic inhibition (MI) induced by the uncoupler carbonyl cyanide p(trifluoromethoxy)phenylhydrazone (FCCP) has been shown to activate KATP channels in isolated cardiac cells [11]. Therefore, we investigated the effect of anoxic preconditioning (APC) on KATP channels opened by MI and also tested whether the effect of APC is mediated by PKC.
Electrophysiologocal measurement Conventional patch-clamp techniques [6] were used to record single KATP channel currents from cell-attached and inside-out membrane patches. The resistance of the patch electrodes ranged from 3 to 5 MΩ. The composition of the pipette solution (extracellular medium) was (in mM): KCl 140, CaCl2 1.8, MgCl2 0.53, glucose 5.5, HEPES 5 (pH 7.4 with KOH). In the cell-attached experiments, Tyrode’s solution was used as the bath solution. In the inside-out experiments, the bath solution was changed to a highK+, ATP-free solution containing (in mM): KCl 140, HEPES 5, EGTA 5, glucose 5.5 (pH 7.4 with KOH). Single-channel currents were recorded with a patch-clamp amplifier (Axopatch 200B, Axon Instruments, USA). Data analysis
Materials and methods The investigations were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No 85–23, revised 1996). All procedures conformed to the guidelines stipulated by the Physiological Society of China and the Animal Ethics Committee of Hebei Medical University. Cell isolation Single ventricular cells were isolated from guinea-pigs (250–350 g body mass) by the enzymatic dissociation technique according to Powell et al. [21]. Briefly, guinea-pigs were stunned by a blow on the neck. After mounting the excised heart on a Langendorff apparatus, it was perfused with Tyrode’s solution. The composition of Tyrode’s solution used here was (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, NaH2PO4 0.3, MgCl2 0.5, glucose 5, HEPES 5, and the pH was adjusted to 7.4. After perfusing with about 100 ml of Ca2+-free Tyrode’s solution, the perfusing solution was switched to this same solution containing collagenase (0.04%, >260 units/mg, Sigma, type I). After a 20-min perfusion, the heart was washed with a KB solution of the following composition (in mM): KCl 30, glutamic acid 70, taurine 20, MgCl2 1.0, KH2PO4 10, HEPES 10, glucose 10, EGTA 0.3, and the pH was adjusted to 7.4. The temperature of these perfusates was kept at 35–36°C. In the KB solution, single myocytes were mechanically dispersed from the left ventricle and placed in the recording chamber (0.8 ml in volume) mounted on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan). The chamber was continuously perfused with Tyrode’s solution at a rate of 2–3 ml/min. Only rod-shaped cells with a clear margin and striation were used for the experiment. Chamber for patch experiments under anoxic conditions Experiments were performed using a chamber as reported by Shigematsu and Arita [25]. A semi-closed airtight chamber was specially designed to permit establishment of an argon layer between the bath solution and air, which sufficiently isolated atmospheric oxygen while providing small access for the patch electrode to the cells. Under the control condition, the cells were perfused with Tyrode’s solution. Anoxia was produced by perfusing glucosefree Tyrode’s solution containing pure nitrogen gas (99.99%). Nitrogen gas was bubbled under positive pressure (>30 cmH2O) for more than 3 h. The PO2 of the anoxic solution was less than 3.8×10–8 mmHg, as determined by redox reaction with resazurin [1]. All experiments were carried out at 36±0.5°C.
The single-channel current signals were filtered at 2 kHz and then stored on videotape. The data were replayed by an IBM PCcompatible computer (486) equipped with a 12-bit Labmaster analog-to-digital converter (Axon Instruments). The current signals were digitized at 5 kHz. The data were analyzed using pCLAMP v 6.0 software (Axon Instruments). We measured the channel activity as NPoi and calculated it using the formula Po=I/(Ni), where Po is open-state probability, I is the mean patch current carried by all KATP 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 KATP channels. The mean current (I) was obtained over 20 s as the time-averaged proportion of total KATP current, measured as a difference current between the baseline (the current level where all channels are in the closed state) and the current levels where some channels are in the open state. The time to activate KATP channels was determined by the time from the beginning of MI to the first KATP channel opening. Protocols MI was achieved by adding 0.1 µM FCCP and omitting glucose from Tyrode’s solution. The cells were divided into four groups. In the first group (the MI group), the cell was exposed to MI after the cell-attached patch was established. In the second group (the APC+MI group), the cell was treated with 3 min of anoxia followed by 7 min of reoxygenation before exposure to MI. In the third group (the chelerythrine+MI group), the cell was treated with chelerythrine (5 µM) for 5 min followed by a 10-min washout before exposure to MI. In the fourth group (the chelerythrine+APC+MI group), the cell was first treated with chelerythrine (5 µM) for 5 min, and then the cell was treated in the same way as the second group. After the experiments in the cell-attached mode ended, the bath solution was changed to a high-K+, ATP-free solution containing 0.1 µM FCCP and the patch was excised into an inside-out patch in order to investigate the sensitivity of KATP channels to the intracellular ATP concentration ([ATP]i) in the above groups. Chemicals All chemicals were purchased from Sigma (St. Louis, Mo., USA). FCCP was dissolved in dimethylsulfoxide (DMSO) as a 1 mM stock solution. Chelerythrine was dissolved in distilled water and kept as a 1 mM stock solution. Each stock solution was added to the perfusate immediately before use to produce the final concentration given in the text. After dilution, the final concentration of DMSO was less than 0.1%, which had no effect on the membrane currents [7]. Statistical methods All results are expressed as the mean ±SEM. Student’s unpaired t-test was used to evaluate statistical significance. Differences with values of P
