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Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures
Neuroscience Letters 250 (1998) 111–114
Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures Ayelet Reshef a, Oded Sperling a , b , c ,*, Esther Zoref-Shani a a
Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel b Department of Clinical Biochemistry, Rabin Medical Center (Beilinson Campus), Petah-Tikva, Israel c Felsenstein Medical Research Institute, Rabin Medical Center, Petah-Tikva, Israel Received 11 February 1998; received in revised form 25 May 1998; accepted 25 May 1998
Abstract The effect of opening and of blocking of ATP-sensitive potassium (KATP) channels on the short-term capacity of neurons to resist ischemia–reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to metabolic poisoning by iodoacetic acid (150 mM, 150 min), followed by reperfusion (1 h). The metabolic poisoning resulted in a marked decrease in cellular ATP content (from 65.3 ± 13.4 to 21.6 ± 11.7 nmole/mg protein), simulating an ischemia, or hypoxiainduced condition of energy crisis. The degree of neuronal damage was assessed by the trypan blue exclusion test. Exposure of the neurons to the channel-opener cromakalim (10 mM; 15 min), prior to the insult, induced resistance, which could be abolished by the specific channel blocker glibenclamide (2 mM). Glibenclamide also abolished the protection acquired by preconditioning of the neurons with iodoacetate (IA; 100 mM), the adenosine A1 agonist N6-(R)-phenylisopropyladenosine (RPIA; 100 mM), or with the protein kinase C (PKC) activator 1,2 dioctanoyl-rac-glycerol (DOG; 1 mM). The results indicate that in the neurons, opening of the KATP channels confers protection against an ATP-depleting crisis, and suggest that the protective effects induced by adenosine and by activation of PKC, are mediated by the opening of these channels. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Adenosine; ATP-sensitive potassium channels; Cromakalim; 1,2 Dioctanoyl-rac-glycerol; Glibenclamide; Ischemia– reperfusion damage; Ischemic tolerance; N6-(R)-phenylisopropyladenosine; Neuronal cultures
The brain and the heart can be preconditioned, or protected against ischemic, or a combined ischemia–reperfusion insult, by exposure of the tissue to sublethal ischemia [14,16,18]. The mechanism underlying the classical phenomenon in the heart is activated by adenosine, the ischemia/hypoxia-induced degradation product of ATP [13]. Binding of this endogenously-derived ischemia signal to adenosine A1 receptors, activates a ‘protective’ signaltransduction cascade, resulting in acquisition of ischemic tolerance [16]. The classical adenosine mechanism was demonstrated to induce a rapid appearance (within minutes), of a short-lasting (60–120 min) protection [16]. * Corresponding author. Tel.: +972 3 9376597; fax: +972 3 9376596; e-mail: [email protected]
Recently, this mechanism was demonstrated to induce also a delayed, relatively longer, ‘second window of protection’ [26]. An ‘adenosine protective mechanism’ has also been demonstrated in the neurons [7,11,14,19,22], but it is not yet clarified whether the heart and the neuronal mechanisms are similar or represent different, tissue-specific mechanisms [20]. The short-term heart ‘adenosine protective mechanism’ was suggested to include the participation of G-proteins, inositol phosphates, protein kinase C (PKC), ATP-sensitive potassium (KATP) channels and probably also calcium channels [16]. Three steps in this adenosine-activated signal transduction cascade, the binding of adenosine to its receptors, the activation of PKC and the opening of the KATP channels, were established to be the most important, characteristic markers of this mechanism [15]. Demonstra-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00458- 3
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tion of similar protective effects for these three events in the neurons, may be taken to imply resemblance of the neuronal ‘adenosine protective mechanism’ to that operating in the heart. For this purpose we have established recently an experimental model of cultured rat neurons, subjected to poisoning by iodoacetic acid. Employing this model system, we could demonstrate that activation of the adenosine receptors (by adenosine or by the A1 receptor agonist N6(R)-phenylisopropyladenosine; R-PIA), or activation of PKC (by the PKC activators 1,2 dioctanoyl-rac-glycerol; DOG, or phorbol 12-myristate 13-acetate; PMA), induce in the neurons protection against the chemical ischemic insult [19,20]. The aim of the present study was to further investigate the degree of resemblance between the ‘adenosine protective mechanisms’ operating in the heart and in the neurons. Employing the same model system simulating the ischemic energy crisis, we assessed the effect of opening of the KATP channels on the capacity of the neurons to resist ischemia–reperfusion insult, and attempted to clarify if the protective effects induced by adenosine, and by PKC activators are mediated through opening of these channels. Neuronal cultures were prepared from cerebral hemispheres from embryos at 16–17 days of gestation, according to Yavin and Yavin [25], as described before [19,20]. The experimental design (Fig. 1), was essentially that reported by us before [19,20]. Nine to 13-day-old cultures were employed for the experiments. The cultures were preconditioned against ischemic injury by exposure of the cells to iodoacetic acid (150 mM) for 5 min, simulating sublethal ischemia [20]. For the evaluation of the capacity of the various drugs to confer protection (Fig. 1A), the cultures were exposed first to these drugs for 15 min. The following drugs were studied: the KATP channel opener (±)-cromakalim (10 mM), the PKC activator DOG (1 mM), the PKC blocker chelerythrine (1 mM) (all from Sigma, St. Louis, MO), and the adenosine A1 receptor agonist R-PIA (100 mM) (Research Biochemicals International, Natick, MA). The above concentrations were found by dose–response experiments to exert the maximal effects. Following incubation with IA or the drugs, all media were discarded and the cultures incubated in fresh medium for a washout period of 10 min. The cultures were then exposed to the metabolic poisoning insult, by the addition to the culture medium of iodoacetic acid to a final concentration of 150 mM, for 150 min. Control cultures were left in the regular medium for the same time period. Following the insult, all cultures were washed with fresh medium and incubated for 1 h (reperfusion). For the study of the effect of the KATP channelblocker, glibenclamide, on the capacity of the neurons to acquire protection against the insult, by exposure to sublethal ischemia, or to cromakalim, R-PIA, DOG and chelerythrine (Fig. 1B), the cultures were exposed to glibenclamide (2 mM; Research Biochemicals), starting 15 min prior to the drugs and continued during all stages of the experimental protocol. This concentration of glibenclamide, out of a wide range of concentrations studied (0.2–50 mM),
was found to exert the most effective abolishment of the cromakalim-induced protection. In all protocols, the injury inflicted on the cells was assessed by the trypan-blue exclusion test [5]. ATP in the cultured neurons was determined employing HPLC methodology [23]. P values were calculated according to Student’s t-test. Exposure of the cultured neurons to iodoacetic acid for 150 min, resulted in a marked decrease in neuronal ATP content, from 65.3 ± 13.4 to 21.6 ± 11.7 nmole/mg protein (seven determinations on different cultures; difference between the two groups, P = 0.00003). This finding indicates that the metabolic poisoning employed represents a plausible simulation of an ischemia- or hypoxia-induced energy crisis. Accordingly, exposure of the cultures to the insult of chemical ischemia, followed by reperfusion, resulted in severe damage, reflected by the highly significant, 4-fold increase in the proportion of dead cells (stained by trypan blue) (Fig. 2). No attempt was made to clarify the mode of cell death. Exposure of the cultures to sublethal ischemia (IA for 5 min), cromakalim, R-PIA, DOG and chelerythrine conferred significant protection against the insults (Fig. 2; difference from the ischemic control for all compounds, P , 0.001). Glibenclamide abolished the protection induced by cromakalim, sublethal ischemia, R-PIA and DOG (Fig. 2; difference between the injury in presence of the combination of these compounds with glibenclamide in comparison to the respective compound alone, P , 0.001), but not that induced by chelerythrine. Exposure of the cultures to glibenclamide alone did not affect the degree of damage induced by the insult. The role of the KATP channels in the acquisition of ischemic tolerance was demonstrated first and mainly in various experimental models of the heart [2,3,21]. Recently, evidence was presented for a similar role of these channels also in the brain [9–11,17] and very recently [12] also in neurons in culture. In the present study we demonstrated that the experimental model employed indeed represents a plausible simulation of the ischemia- or hypoxia-induced
Fig. 1. The experimental protocol. (A) The protocol for the evaluation of the capacity of the various drugs to confer protection. (B) The protocol for the study of the effect of the KATP channel blocker, glibenclamide, on the capacity of cromakalim, R-PIA, DOG and chelerythrine to induce protection.
A. Reshef et al. / Neuroscience Letters 250 (1998) 111–114
Fig. 2. Effect of exposure of primary neuronal cultures to modulators of KATP channels on neuronal capacity to resist ischemia–reperfusion insult. Cultures of 9–13 days were subjected to the experimental protocol, as described in the text (Fig. 1). Each bar represents the mean ± SD (n for IA and for Glib + IA was 4; for all other bars n ranged from 8 to 14).
condition of ATP depletion, as indicated by the marked and highly-significant, 67% decrease in cellular ATP content. Employing this neuronal model system, we confirmed that opening of the KATP channels confers protection against ischemia–reperfusion insult. That the cromakalim-induced protective effect is indeed a true result of opening of the KATP channels, is indicated by the finding that glibenclamide, the specific KATP channel blocker, abolished this protection. The demonstrated capacity of cromakalim to induce protection against a condition associated with fast ATP depletion, is in accordance with the suggested mechanism, by which opening of the KATP channels preserve ATP. Opening of the KATP channels in the neurons results in repolarization or hyperpolarization, reducing membrane excitability. This effect downregulates neuronal metabolic and energy-consuming activity, resulting in protection against ischemic or hypoxic damage [24]. The results also furnished evidence implying that opening of the KATP channels is part of the neuronal ‘adenosine protective mechanism’, since glibenclamide abolished the protection induced by sublethal ischemia and by the adenosine A1 receptor agonist, R-PIA, (we have verified before that the protective effect induced by sublethal ischemia or by R-PIA is obtained by activation of the adenosine receptors, by demonstrating that the adenosine receptor antagonist 8-sulphophenyl theophylline (SPT) abolished this effect [20]). The finding that opening of the KATP channels is part of the neuronal ‘adenosine protective mechanism’ conforms to results of studies performed before in whole-brain tissue [11], demonstrating
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the involvement of the adenosine A1 receptors and of the KATP channels in the acquisition of long-term ischemic tolerance in the brain. In our study, the blocking by glibenclamide of the protection conferred by DOG, an established activator of PKC, may be taken to suggest, that also this effect is mediated through opening of the KATP channels. Indeed, it was suggested that opening of the KATP channels, during ischemia, depends on their prior phosphorylation (priming) by activated PKC [1,3,6,8,9,13,15,24]. We could not clarify whether activation of PKC is part of the adenosine-induced mechanism, or whether it is a component of a different protective mechanism. It was impossible in our experimental model, to evaluate the effect of PKC inhibition on the protection effect induced by adenosine or RPIA, since the established PKC inhibitors, chelerythrine and calphostin C, induced protection, rather than abolishing the protection induced by PKC activation [20]. The protective effect of chelerythrine was attributed to inhibition by this compound of the effect of a ‘destructive’ PKC isoform, which probably mediates the ‘destructive’ signal induced by the sustained ischemic insult [20]. The finding in the present study that glibenclamide did not abolish this protective effect, is in accordance with this suggestion, since such a mechanism of protection does not involve opening of the KATP channels. The results of the present study demonstrate, for the first time in pure neuronal cultures, that the neuronal adenosineactivated, short-term mechanism to tolerate ischemic insult, involves opening of the KATP channels. In addition, these results, combined with those of our previous studies concerning the neuronal ‘adenosine protective mechanism’ [19,20], and in agreement with results of other studies [4,7,10–12,22], demonstrate that each of the three important and characteristic steps, operating in the classical heart ‘adenosine protective mechanism,’ i.e. activation of the adenosine receptors, activation of PKC and opening of the KATP channels, operate also in the neurons. At present, only the latter step has been shown to be part of the neuronal ‘adenosine protective mechanism’. Nevertheless, in view of the known dependence of the opening of the channels during ischemic crisis, on prior phosphorylation by activated PKC, it seems probable that in the neurons, as in the heart, PKC activation is also part of the ‘adenosine protective mechanism’. This work partially fulfills the requirements of the Ph.D. thesis of A.R. at the Sackler Faculty of Medicine, Tel Aviv University. [1] Ashcroft, F.M., Adenosine 5′-triphosphate-sensitive potassium channels, Ann. Rev. Neurosci., 11 (1988) 97–118. [2] Cavero, I., Djellas, Y. and Guillon, J.M., Ischemic myocardial cell protection conferred by the opening of ATP-sensitive potassium channels, Cardiovasc. Drugs Ther., 9 (1995) 245–255. [3] Cole, W.C., ATP-sensitive K + channels in cardiac ischemia: an endogenous mechanism for protection of the heart, Cardiovasc. Drugs Ther., 7 (1993) 527–537.
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[4] Davis, J.B. and Maher, P., Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line, Brain Res., 652 (1994) 169–173. [5] Dessi, F., Chariaut-Marlangue, C., Khrestchantiski, M. and Ben-Ari, Y., Glutamate-induced neuronal death is not programmed cell death in cerebellar culture, J. Neurochem., 60 (1993) 1953–1955. [6] Edwards, G. and Weston, A.H., The pharmacology of ATPsensitive potassium channels, Ann. Rev. Pharmacol. Toxicol., 33 (1993) 597–637. [7] Goldberg, M.P., Monyer, H., Eeiss, J.H. and Choi, D.W., Adenosine reduces cortical neuronal injury induced by oxygen or glucose deprivation in vitro, Neurosci. Lett., 89 (1988) 323– 327. [8] Grover, G.J., Role of the ATP-sensitive potassium channel in ischaemic preconditioning. In M.S. Marber and D.M. Yellon (Eds.), Ischemia: Preconditioning and Adaptation, BIOS Scientific, Oxford, 1996, pp. 35–58. [9] Haddad, G.G. and Jiang, C., Mechanisms of neuronal survival during hypoxia: ATP-sensitive K + channels, Biol. Neonate, 65 (1994) 160–165. [10] Heurteaux, C., Bertaina, V., Widmann, C. and Lazdunski, M., K + channel openers prevent global ischemia-induced expression of c-fos, c-jun, heat shock protein, and amyloid b-protein precursor genes and neuronal death in rat hippocampus, Proc. Natl. Acad. Sci. (USA), 90 (1993) 9431–9435. [11] Heurteaux, C., Lauritzen, I., Widmann, C. and Lazdunski, M., Essential role of adenosine A1 receptors, and ATP-sensitive K + channels in cerebral ischaemic preconditioning, Proc. Natl. Acad. Sci. (USA), 92 (1995) 4666–4670. [12] Lauritzen, I., De Weille, J.R. and Lazdunski, M., The potassium channel opener (−)-cromakalim prevents glutamate-induced cell death in hippocampal neurons, J. Neurochem., 69 (1997) 1570–1579. [13] Light, P.E., Sabir, A.A., Allen, B.G., Walsh, M.P. and French, R.J., Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K + channels. A possible mechanistic link to ischemic preconditioning, Circ. Res., 79 (1996) 399–406. [14] Liu, Y., Kato, H., Kato, N. and Kogure, K., Protection of rat
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Opening of ATP-sensitive potassium channels by cromakalim confers tolerance against chemical ischemia in rat neuronal cultures Ayelet Reshef a, Oded Sperling a , b , c ,*, Esther Zoref-Shani a a
Department of Clinical Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel b Department of Clinical Biochemistry, Rabin Medical Center (Beilinson Campus), Petah-Tikva, Israel c Felsenstein Medical Research Institute, Rabin Medical Center, Petah-Tikva, Israel Received 11 February 1998; received in revised form 25 May 1998; accepted 25 May 1998
Abstract The effect of opening and of blocking of ATP-sensitive potassium (KATP) channels on the short-term capacity of neurons to resist ischemia–reperfusion-induced cell injury, was studied in a model of primary rat neuronal cultures, subjected to metabolic poisoning by iodoacetic acid (150 mM, 150 min), followed by reperfusion (1 h). The metabolic poisoning resulted in a marked decrease in cellular ATP content (from 65.3 ± 13.4 to 21.6 ± 11.7 nmole/mg protein), simulating an ischemia, or hypoxiainduced condition of energy crisis. The degree of neuronal damage was assessed by the trypan blue exclusion test. Exposure of the neurons to the channel-opener cromakalim (10 mM; 15 min), prior to the insult, induced resistance, which could be abolished by the specific channel blocker glibenclamide (2 mM). Glibenclamide also abolished the protection acquired by preconditioning of the neurons with iodoacetate (IA; 100 mM), the adenosine A1 agonist N6-(R)-phenylisopropyladenosine (RPIA; 100 mM), or with the protein kinase C (PKC) activator 1,2 dioctanoyl-rac-glycerol (DOG; 1 mM). The results indicate that in the neurons, opening of the KATP channels confers protection against an ATP-depleting crisis, and suggest that the protective effects induced by adenosine and by activation of PKC, are mediated by the opening of these channels. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Adenosine; ATP-sensitive potassium channels; Cromakalim; 1,2 Dioctanoyl-rac-glycerol; Glibenclamide; Ischemia– reperfusion damage; Ischemic tolerance; N6-(R)-phenylisopropyladenosine; Neuronal cultures
The brain and the heart can be preconditioned, or protected against ischemic, or a combined ischemia–reperfusion insult, by exposure of the tissue to sublethal ischemia [14,16,18]. The mechanism underlying the classical phenomenon in the heart is activated by adenosine, the ischemia/hypoxia-induced degradation product of ATP [13]. Binding of this endogenously-derived ischemia signal to adenosine A1 receptors, activates a ‘protective’ signaltransduction cascade, resulting in acquisition of ischemic tolerance [16]. The classical adenosine mechanism was demonstrated to induce a rapid appearance (within minutes), of a short-lasting (60–120 min) protection [16]. * Corresponding author. Tel.: +972 3 9376597; fax: +972 3 9376596; e-mail: [email protected]
Recently, this mechanism was demonstrated to induce also a delayed, relatively longer, ‘second window of protection’ [26]. An ‘adenosine protective mechanism’ has also been demonstrated in the neurons [7,11,14,19,22], but it is not yet clarified whether the heart and the neuronal mechanisms are similar or represent different, tissue-specific mechanisms [20]. The short-term heart ‘adenosine protective mechanism’ was suggested to include the participation of G-proteins, inositol phosphates, protein kinase C (PKC), ATP-sensitive potassium (KATP) channels and probably also calcium channels [16]. Three steps in this adenosine-activated signal transduction cascade, the binding of adenosine to its receptors, the activation of PKC and the opening of the KATP channels, were established to be the most important, characteristic markers of this mechanism [15]. Demonstra-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00458- 3
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tion of similar protective effects for these three events in the neurons, may be taken to imply resemblance of the neuronal ‘adenosine protective mechanism’ to that operating in the heart. For this purpose we have established recently an experimental model of cultured rat neurons, subjected to poisoning by iodoacetic acid. Employing this model system, we could demonstrate that activation of the adenosine receptors (by adenosine or by the A1 receptor agonist N6(R)-phenylisopropyladenosine; R-PIA), or activation of PKC (by the PKC activators 1,2 dioctanoyl-rac-glycerol; DOG, or phorbol 12-myristate 13-acetate; PMA), induce in the neurons protection against the chemical ischemic insult [19,20]. The aim of the present study was to further investigate the degree of resemblance between the ‘adenosine protective mechanisms’ operating in the heart and in the neurons. Employing the same model system simulating the ischemic energy crisis, we assessed the effect of opening of the KATP channels on the capacity of the neurons to resist ischemia–reperfusion insult, and attempted to clarify if the protective effects induced by adenosine, and by PKC activators are mediated through opening of these channels. Neuronal cultures were prepared from cerebral hemispheres from embryos at 16–17 days of gestation, according to Yavin and Yavin [25], as described before [19,20]. The experimental design (Fig. 1), was essentially that reported by us before [19,20]. Nine to 13-day-old cultures were employed for the experiments. The cultures were preconditioned against ischemic injury by exposure of the cells to iodoacetic acid (150 mM) for 5 min, simulating sublethal ischemia [20]. For the evaluation of the capacity of the various drugs to confer protection (Fig. 1A), the cultures were exposed first to these drugs for 15 min. The following drugs were studied: the KATP channel opener (±)-cromakalim (10 mM), the PKC activator DOG (1 mM), the PKC blocker chelerythrine (1 mM) (all from Sigma, St. Louis, MO), and the adenosine A1 receptor agonist R-PIA (100 mM) (Research Biochemicals International, Natick, MA). The above concentrations were found by dose–response experiments to exert the maximal effects. Following incubation with IA or the drugs, all media were discarded and the cultures incubated in fresh medium for a washout period of 10 min. The cultures were then exposed to the metabolic poisoning insult, by the addition to the culture medium of iodoacetic acid to a final concentration of 150 mM, for 150 min. Control cultures were left in the regular medium for the same time period. Following the insult, all cultures were washed with fresh medium and incubated for 1 h (reperfusion). For the study of the effect of the KATP channelblocker, glibenclamide, on the capacity of the neurons to acquire protection against the insult, by exposure to sublethal ischemia, or to cromakalim, R-PIA, DOG and chelerythrine (Fig. 1B), the cultures were exposed to glibenclamide (2 mM; Research Biochemicals), starting 15 min prior to the drugs and continued during all stages of the experimental protocol. This concentration of glibenclamide, out of a wide range of concentrations studied (0.2–50 mM),
was found to exert the most effective abolishment of the cromakalim-induced protection. In all protocols, the injury inflicted on the cells was assessed by the trypan-blue exclusion test [5]. ATP in the cultured neurons was determined employing HPLC methodology [23]. P values were calculated according to Student’s t-test. Exposure of the cultured neurons to iodoacetic acid for 150 min, resulted in a marked decrease in neuronal ATP content, from 65.3 ± 13.4 to 21.6 ± 11.7 nmole/mg protein (seven determinations on different cultures; difference between the two groups, P = 0.00003). This finding indicates that the metabolic poisoning employed represents a plausible simulation of an ischemia- or hypoxia-induced energy crisis. Accordingly, exposure of the cultures to the insult of chemical ischemia, followed by reperfusion, resulted in severe damage, reflected by the highly significant, 4-fold increase in the proportion of dead cells (stained by trypan blue) (Fig. 2). No attempt was made to clarify the mode of cell death. Exposure of the cultures to sublethal ischemia (IA for 5 min), cromakalim, R-PIA, DOG and chelerythrine conferred significant protection against the insults (Fig. 2; difference from the ischemic control for all compounds, P , 0.001). Glibenclamide abolished the protection induced by cromakalim, sublethal ischemia, R-PIA and DOG (Fig. 2; difference between the injury in presence of the combination of these compounds with glibenclamide in comparison to the respective compound alone, P , 0.001), but not that induced by chelerythrine. Exposure of the cultures to glibenclamide alone did not affect the degree of damage induced by the insult. The role of the KATP channels in the acquisition of ischemic tolerance was demonstrated first and mainly in various experimental models of the heart [2,3,21]. Recently, evidence was presented for a similar role of these channels also in the brain [9–11,17] and very recently [12] also in neurons in culture. In the present study we demonstrated that the experimental model employed indeed represents a plausible simulation of the ischemia- or hypoxia-induced
Fig. 1. The experimental protocol. (A) The protocol for the evaluation of the capacity of the various drugs to confer protection. (B) The protocol for the study of the effect of the KATP channel blocker, glibenclamide, on the capacity of cromakalim, R-PIA, DOG and chelerythrine to induce protection.
A. Reshef et al. / Neuroscience Letters 250 (1998) 111–114
Fig. 2. Effect of exposure of primary neuronal cultures to modulators of KATP channels on neuronal capacity to resist ischemia–reperfusion insult. Cultures of 9–13 days were subjected to the experimental protocol, as described in the text (Fig. 1). Each bar represents the mean ± SD (n for IA and for Glib + IA was 4; for all other bars n ranged from 8 to 14).
condition of ATP depletion, as indicated by the marked and highly-significant, 67% decrease in cellular ATP content. Employing this neuronal model system, we confirmed that opening of the KATP channels confers protection against ischemia–reperfusion insult. That the cromakalim-induced protective effect is indeed a true result of opening of the KATP channels, is indicated by the finding that glibenclamide, the specific KATP channel blocker, abolished this protection. The demonstrated capacity of cromakalim to induce protection against a condition associated with fast ATP depletion, is in accordance with the suggested mechanism, by which opening of the KATP channels preserve ATP. Opening of the KATP channels in the neurons results in repolarization or hyperpolarization, reducing membrane excitability. This effect downregulates neuronal metabolic and energy-consuming activity, resulting in protection against ischemic or hypoxic damage [24]. The results also furnished evidence implying that opening of the KATP channels is part of the neuronal ‘adenosine protective mechanism’, since glibenclamide abolished the protection induced by sublethal ischemia and by the adenosine A1 receptor agonist, R-PIA, (we have verified before that the protective effect induced by sublethal ischemia or by R-PIA is obtained by activation of the adenosine receptors, by demonstrating that the adenosine receptor antagonist 8-sulphophenyl theophylline (SPT) abolished this effect [20]). The finding that opening of the KATP channels is part of the neuronal ‘adenosine protective mechanism’ conforms to results of studies performed before in whole-brain tissue [11], demonstrating
113
the involvement of the adenosine A1 receptors and of the KATP channels in the acquisition of long-term ischemic tolerance in the brain. In our study, the blocking by glibenclamide of the protection conferred by DOG, an established activator of PKC, may be taken to suggest, that also this effect is mediated through opening of the KATP channels. Indeed, it was suggested that opening of the KATP channels, during ischemia, depends on their prior phosphorylation (priming) by activated PKC [1,3,6,8,9,13,15,24]. We could not clarify whether activation of PKC is part of the adenosine-induced mechanism, or whether it is a component of a different protective mechanism. It was impossible in our experimental model, to evaluate the effect of PKC inhibition on the protection effect induced by adenosine or RPIA, since the established PKC inhibitors, chelerythrine and calphostin C, induced protection, rather than abolishing the protection induced by PKC activation [20]. The protective effect of chelerythrine was attributed to inhibition by this compound of the effect of a ‘destructive’ PKC isoform, which probably mediates the ‘destructive’ signal induced by the sustained ischemic insult [20]. The finding in the present study that glibenclamide did not abolish this protective effect, is in accordance with this suggestion, since such a mechanism of protection does not involve opening of the KATP channels. The results of the present study demonstrate, for the first time in pure neuronal cultures, that the neuronal adenosineactivated, short-term mechanism to tolerate ischemic insult, involves opening of the KATP channels. In addition, these results, combined with those of our previous studies concerning the neuronal ‘adenosine protective mechanism’ [19,20], and in agreement with results of other studies [4,7,10–12,22], demonstrate that each of the three important and characteristic steps, operating in the classical heart ‘adenosine protective mechanism,’ i.e. activation of the adenosine receptors, activation of PKC and opening of the KATP channels, operate also in the neurons. At present, only the latter step has been shown to be part of the neuronal ‘adenosine protective mechanism’. Nevertheless, in view of the known dependence of the opening of the channels during ischemic crisis, on prior phosphorylation by activated PKC, it seems probable that in the neurons, as in the heart, PKC activation is also part of the ‘adenosine protective mechanism’. This work partially fulfills the requirements of the Ph.D. thesis of A.R. at the Sackler Faculty of Medicine, Tel Aviv University. [1] Ashcroft, F.M., Adenosine 5′-triphosphate-sensitive potassium channels, Ann. Rev. Neurosci., 11 (1988) 97–118. [2] Cavero, I., Djellas, Y. and Guillon, J.M., Ischemic myocardial cell protection conferred by the opening of ATP-sensitive potassium channels, Cardiovasc. Drugs Ther., 9 (1995) 245–255. [3] Cole, W.C., ATP-sensitive K + channels in cardiac ischemia: an endogenous mechanism for protection of the heart, Cardiovasc. Drugs Ther., 7 (1993) 527–537.
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