5
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4666-4670, May 1995 Pharmacology Essential role of adenosine, adenosine Al receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning (forebrain ischemia/gene expression) CATHERINE HEURTEAUX, INGER LAURITZEN, CATHERINE WIDMANN, AND MICHEL LAZDUNSKI* Institut de Pharmacologie Moleculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Communicated by Josef Fried, The University of Chicago, Chicago, IL, February 11, 1995 ABSTRACT Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine Al receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and y-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine Al receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP- sensitive K+ channels. Ischemic preconditioning is an endogenous protective mech- anism in which brief periods of ischemia and reperfusion render the brain more resistant to a subsequent more sustained ischemic insult. Rats and gerbils subjected to sublethal forebrain ischemia induced several days before a secondary lethal ischemic episode do not develop neuronal damage to the hippocampal CA1 subfield, although a single ischemic insult always leads to severe CA1 neuronal loss (1, 2). K+ channel openers (KCOs) are compounds that activate ATP-sensitive K+ (KATP) channels (3-6). KATP channels are present in the brain (7-11), particularly in hippocampus, and have been associated with the control of neurotransmitter release (12). KCOs prevent ischemia-induced neuronal death and ischemia-induced expression of a variety of genes such as immediate early genes and genes for heat shock protein HSP70, amyloid P-protein precursor, and growth factors [nerve growth factor (NGF) and brain-derived neuro- trophic factor (BDNF)] (13, 14). Extracellular levels of adenosine increase during anoxic or ischemic conditions (15). This increase is probably due to the breakdown of intracellular ATP (16, 17). Adenosine inhibits synaptic transmission, decreases K+-stimulated glutamate re- lease, and inhibits presynaptic Ca2+ fluxes via adenosine A1 receptors (for a review, see ref. 18). It has been suggested that adenosine activates presynaptic K+ channels and decreases evoked neurotransmitter release by hyperpolarization of the presynaptic membrane (for review, see ref. 19). The aim of this paper is to study the mechanism of ischemic tolerance by testing the hypothesis that adenosine and KATP channels are involved in the cerebral preconditioning. MATERIALS AND METHODS Male Wistar rats (Charles River Breeding Laboratories), aged 10-12 weeks and weighing 250-300 g, were anesthetized by inhalation of 2% halothane mixed with 30% oxygen and 70% nitrous oxide. Forebrain ischemia was induced by four-vessel The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. occlusion (13). The vertebral arteries were electrocauterized. On the following day, bilateral common carotid arteries were oc- cluded with surgical clips for 3 min. Secondary occlusion of carotid arteries for 6 min was then induced 1 h or 3 days later. A single 6-min period of ischemia 1 h or 3 days after a sham operation was administered in other animals. Body temperature was maintained at 37°C. Rats recovered for 1 h or 1 or 7 days (n = 4 at each time point). Sham control animals were sacrificed just after exposing the carotid arteries without clamping the vessels. Rats after the single 6-min ischemia recovered for 1 h or 1 or 7 days (n = 5). Animals were killed by a transcardial perfusion with 0.9% NaCI followed by ice-cold 1% paraformaldehyde/ phosphate-buffered saline (0.15 M NaCl/0.01 M sodium phos- phate, pH 7.4) (PBS). Brains were postfixed in the same solution for 2 h and then immersed overnight at 4°C in 20% (wt/vol) sucrose/PBS. Coronal frozen sections (10 ,um) at the level of the dorsal hippocampus were cut on a cryostat (Microm, France) at -25°C, collected on 3-aminopropylethoxysilane-coated slides and stored at -70°C until use. In situ hybridization was performed by our method (13, 20) with 33P-radiolabeled oligonucleotide probes complemen- tary to mRNAs for c-fos, c-jun, HSP70, BDNF, NGF, N-methyl-D-aspartate R1 receptor, GluRB receptor (flip and flop variants), and type A y-aminobutyric acid receptor al and ,32 subunits. Sections for each probe were hybridized overnight in 50% (vol/vol) deionized formamide/10% (wt/vol) dextran sulfate/denatured salmon sperm DNA (500 ,tg/ml)/1% Den- hardt's solution/5% (vol/vol) sarcosyl/yeast tRNA (250 ,tg/ ml)/20 mM dithiothreitol/20 mM sodium phosphate/2x SSC. Sections were then washed and apposed to Hyperfilm-3,max (Amersham) for 4 days. The best sections were then dipped in Amersham LM1 emulsion, developed after 12 days, and counterstained with Cresyl violet. The neuronal density (d) of the hippocampal CA1 subfield (i.e., the number of intact pyramidal cells per 1 mm linear length of CA1) was deter- mined by the method of Kirino et al (21). A mean value in each hippocampal CA1 substructure was calculated from 20 bilat- eral measurements on four sections per animal in the five rats of each experimental group. Data correspond to the mean ± SEM of number of intact pyramidal cells in each group of rats. Variations of the neuronal density were assessed by using a Tukey's w test for multiple comparisons. Drug treatments were as follows. Levcromakalim was from Beecham Pharmaceuticals, pinacidil was from Leo-Phar- maceuticals (Denmark), and nicorandil was from Rh6ne- Poulenc Rorer (France). KCOs (10 nmol/5 ,tl) were admin- istered intracerebroventricularly (i.c.v.) 30 min before the induction of cerebral ischemia and once each day during the recirculation. Glipizide (1 ,umol/5 1ul; Pfizer) or tolbu- Abbreviations: KATP channel, ATP-sensitive K+ channel; KCO, K+ channel opener; NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; CPA, N6-cyclopentyladenosine; DPCPX, 8-cyclo- pentyl-1,3-dipropylxanthine; i.c.v., intracerebroventricularly. *To whom reprint requests should be addressed. 4666 Downloaded by guest on January 18, 2021

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Page 1: adenosine, receptors, K+ preconditioning · ficial effects of ischemic preconditioning, whereas CPA(1 mg/kg)protected the brainbutnotaswell as precondition-ing (Figs. 1 and 2EandF)

Proc. Natl. Acad. Sci. USAVol. 92, pp. 4666-4670, May 1995Pharmacology

Essential role of adenosine, adenosine Al receptors, andATP-sensitive K+ channels in cerebral ischemic preconditioning

(forebrain ischemia/gene expression)

CATHERINE HEURTEAUX, INGER LAURITZEN, CATHERINE WIDMANN, AND MICHEL LAZDUNSKI*Institut de Pharmacologie Moleculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

Communicated by Josef Fried, The University of Chicago, Chicago, IL, February 11, 1995

ABSTRACT Preconditioning with sublethal ischemiaprotects against neuronal damage after subsequent lethalischemic insults in hippocampal neurons. A pharmacologicalapproach using agonists and antagonists at the adenosine Alreceptor as well as openers and blockers of ATP-sensitive K+channels has been combined with an analysis of neuronaldeath and gene expression of subunits of glutamate andy-aminobutyric acid receptors, HSP70, c-fos, c-jun, andgrowth factors. It indicates that the mechanism of ischemictolerance involves a cascade of events including liberation ofadenosine, stimulation of adenosine Al receptors, and, viathese receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels.

Ischemic preconditioning is an endogenous protective mech-anism in which brief periods of ischemia and reperfusionrender the brain more resistant to a subsequent moresustained ischemic insult. Rats and gerbils subjected tosublethal forebrain ischemia induced several days before asecondary lethal ischemic episode do not develop neuronaldamage to the hippocampal CA1 subfield, although a singleischemic insult always leads to severe CA1 neuronal loss (1,2). K+ channel openers (KCOs) are compounds that activateATP-sensitive K+ (KATP) channels (3-6). KATP channels arepresent in the brain (7-11), particularly in hippocampus, andhave been associated with the control of neurotransmitterrelease (12). KCOs prevent ischemia-induced neuronaldeath and ischemia-induced expression of a variety of genessuch as immediate early genes and genes for heat shockprotein HSP70, amyloid P-protein precursor, and growthfactors [nerve growth factor (NGF) and brain-derived neuro-trophic factor (BDNF)] (13, 14).

Extracellular levels of adenosine increase during anoxic orischemic conditions (15). This increase is probably due to thebreakdown of intracellular ATP (16, 17). Adenosine inhibitssynaptic transmission, decreases K+-stimulated glutamate re-lease, and inhibits presynaptic Ca2+ fluxes via adenosine A1receptors (for a review, see ref. 18). It has been suggested thatadenosine activates presynaptic K+ channels and decreasesevoked neurotransmitter release by hyperpolarization of thepresynaptic membrane (for review, see ref. 19).The aim of this paper is to study the mechanism of ischemic

tolerance by testing the hypothesis that adenosine and KATPchannels are involved in the cerebral preconditioning.

MATERIALS AND METHODS

Male Wistar rats (Charles River Breeding Laboratories), aged10-12 weeks and weighing 250-300 g, were anesthetized byinhalation of 2% halothane mixed with 30% oxygen and 70%nitrous oxide. Forebrain ischemia was induced by four-vessel

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

occlusion (13). The vertebral arteries were electrocauterized. Onthe following day, bilateral common carotid arteries were oc-cluded with surgical clips for 3 min. Secondary occlusion ofcarotid arteries for 6 min was then induced 1 h or 3 days later. Asingle 6-min period of ischemia 1 h or 3 days after a shamoperation was administered in other animals. Body temperaturewas maintained at 37°C. Rats recovered for 1 h or 1 or 7 days (n= 4 at each time point). Sham control animals were sacrificed justafter exposing the carotid arteries without clamping the vessels.Rats after the single 6-min ischemia recovered for 1 h or 1 or 7days (n = 5). Animals were killed by a transcardial perfusion with0.9% NaCI followed by ice-cold 1% paraformaldehyde/phosphate-buffered saline (0.15 M NaCl/0.01 M sodium phos-phate, pH 7.4) (PBS). Brains were postfixed in the same solutionfor 2 h and then immersed overnight at 4°C in 20% (wt/vol)sucrose/PBS. Coronal frozen sections (10 ,um) at the level of thedorsal hippocampus were cut on a cryostat (Microm, France) at-25°C, collected on 3-aminopropylethoxysilane-coated slidesand stored at -70°C until use.

In situ hybridization was performed by our method (13, 20)with 33P-radiolabeled oligonucleotide probes complemen-tary to mRNAs for c-fos, c-jun, HSP70, BDNF, NGF,N-methyl-D-aspartate R1 receptor, GluRB receptor (flip andflop variants), and typeA y-aminobutyric acid receptor al and,32 subunits. Sections for each probe were hybridized overnightin 50% (vol/vol) deionized formamide/10% (wt/vol) dextransulfate/denatured salmon sperm DNA (500 ,tg/ml)/1% Den-hardt's solution/5% (vol/vol) sarcosyl/yeast tRNA (250 ,tg/ml)/20mM dithiothreitol/20mM sodium phosphate/2x SSC.Sections were then washed and apposed to Hyperfilm-3,max(Amersham) for 4 days. The best sections were then dipped inAmersham LM1 emulsion, developed after 12 days, andcounterstained with Cresyl violet. The neuronal density (d) ofthe hippocampal CA1 subfield (i.e., the number of intactpyramidal cells per 1 mm linear length of CA1) was deter-mined by the method of Kirino et al (21). A mean value in eachhippocampal CA1 substructure was calculated from 20 bilat-eral measurements on four sections per animal in the five ratsof each experimental group. Data correspond to the mean ±SEM of number of intact pyramidal cells in each group of rats.Variations of the neuronal density were assessed by using aTukey's w test for multiple comparisons.Drug treatments were as follows. Levcromakalim was from

Beecham Pharmaceuticals, pinacidil was from Leo-Phar-maceuticals (Denmark), and nicorandil was from Rh6ne-Poulenc Rorer (France). KCOs (10 nmol/5 ,tl) were admin-istered intracerebroventricularly (i.c.v.) 30 min before theinduction of cerebral ischemia and once each day during therecirculation. Glipizide (1 ,umol/5 1ul; Pfizer) or tolbu-

Abbreviations: KATP channel, ATP-sensitive K+ channel; KCO, K+channel opener; NGF, nerve growth factor; BDNF, brain-derivedneurotrophic factor; CPA, N6-cyclopentyladenosine; DPCPX, 8-cyclo-pentyl-1,3-dipropylxanthine; i.c.v., intracerebroventricularly.*To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci USA 92 (1995) 4667

tamide (2 ,mol/5 gl or 2 mmol/5 pl; Pfizer) was injectedi.c.v. 20 min prior to first ischemia. N6-Cyclopentyladenosine(CPA, 1.0 mg/kg) and 8-cyclopentyl-l,3-dipropylxanthine(DPCPX, 1 mg/kg) were purchased from Research Biochemicals(Natick, MA). They were dissolved in ethanol and injected i.p. 15min prior to the sublethal ischemia. None of these drugs weretoxic at the concentrations used. Control experiments wereperformed with i.c.v. injection of 0.9% NaCl under conditionsused for KCOs.

RESULTS AND DISCUSSION

Pretreatment with a 3-min sublethal ischemia protects rathippocampus against subsequent lethal ischemic neuronaldamage induced by a 6-min ischemia. Fig. 1 shows that a single3-min period of ischemia causes no reduction in neuronaldensity (d = 192 ± 10 pyramidal cells per mm, n = 10),whereas 83% of CA1 pyramidal cells are destroyed by 6-minischemia (d = 33 ± 4, n = 10). The interval between ischemicinsults is a critical factor. A 3-min ischemia followed by a 6-minischemia at a 1-h interval causes destruction of almost all CA1pyramidal cells (d = 10 ± 2, n = 10). In contrast, neuronaldamage in the CA1 subfield is prevented when the intervalbetween the two ischemic insults is 3 days (d = 187 + 9, n =

10). Histological investigations on cresyl violet-stained sectionsconfirm the protective effect of preconditioning hippocampalneurons (Fig. 2 A-D).

Since adenosine is massively released during ischemia andsince selective adenosine Al receptor agonists are known tobe neuroprotective (18, 22-25), it appeared to be importantto check. whether the endogenous protective mechanismduring repetitive brain ischemia could be triggered by acti-

1: Sham 6: Glib/3min/3d/6min2: Sham/3min 7 : Levcrom/6min3: Sham/6min 8 : CPA/6min4: 3min/lh/6min 9 : DPCPX/3min/3d/6min5: 3min/3d//6min 10 : Glib+CPA/6min

11: Glib+Levcrom/6min

; 200

150.

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100

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FIG. 1. Neuronal density (d = number of intact pyramidal cellsper 1 mm linear length of CA1) in the CA1 subfield as a function ofthe type of ischemia induced in the rat. Barsil (Sham), control; 2 and3 (Sham/3-min and Sham/6-min), single 3-min or 6-min ischemia;4 and 5 (3-min/lh/6-min and 3-min/3d/6-min), 6-min ischemia withpreconditioning with 3-min ischemia at 1-h or 3-day interval; 6,(Glib/3-min/3d/6-min), glibenclamide (1 /jmol/5 jul) injected i.c.v.20 min prior to first ischemia; 7 (Levcrom/6-min), levcromakalim(10 nmol/5 ,ul) administered 30 min prior to ischemia and once eachday during recirculation; 8 (CPA/6 min), CPA (1 mg/kg) injectedi.p. 15 min prior to ischemia; 9 (DPCPX/3-min/3d/6-min), DPCPX(1 mg/kg, i.p.) 15 min prior to first ischemia; 10 and 11(Glib+CPA/6 min and Glib+Levcrom/6 min), glibenclamide (1/Lmol/5 tIl) 20 min prior to CPA (1 mg/kg) or levcromakalim (10nmol/5 tIl) administration. Neuronal density was analyzed 7 daysafter ischemia. Data were combined from four experiments (n = 5animals per group in each experiment). Statistical analysis was

performed by analysis of variance and Tukey's w test for multiplecomparisons. *, P < 0.05 vs. Sham/6-min; e, P < 0.05 vs. Levcrom/6-min or CPA/6-min.

vation of adenosine Al receptors. Evidence that this isindeed the case was obtained with DPCPX, a selectivehigh-affinity Al receptor antagonist, and CPA, a selectiveAl receptor agonist. DPCPX (1 mg/kg) blocked the bene-ficial effects of ischemic preconditioning, whereas CPA (1mg/kg) protected the brain but not as well as precondition-ing (Figs. 1 and 2 E and F).Opening of KATP channels with KCOs, such as levcrom-

akalim (the active enantiomer of cromakalim), nicorandil,and pinacidil, was recently shown to protect the heart(26-29) and the brain (13) against the deleterious effects ofischemia. This effect was suppressed by glibenclamide, aspecific blocker of KATP channels (12, 30). In the brain, thisprotective effect is thought to be due at least in part toinhibition of synaptic glutamate release during ischemia (13,31). It might also be due to a postsynaptic hyperpolarizationinduced by KCOs (13) that could also lead to a decrease ofglutamate excitotoxicity.

Figs. 1 and 2 compare the protective effects of precondi-tioning, the KCO levcromakalim, and CPA, a selective aden-osine Al receptor agonist. These three treatments provideimpressive protection. Ischemic preconditioning leads tonearly 100% protection in CA1, and treatment with levcro-makalim or CPA results in 70% protection (P < 0.05). In allcases, glibenclamide drastically decreased the protective effects.These results thus suggest that the first short period of ischemiacauses the release of adenosine and that the resultant activationof adenosine Al receptors then activates KATP channels, therebyconferring protection. This would be the major mechanism forprotection (70%). However, preconditioning with a mild ischemicinsult should also activate additional protective mechanisms toexplain the 30% difference between preconditioning and levcro-makalim and CPA treatments.

Global ischemia is associated with changes of expression ofa large number of genes (13, 14, 20, 32, 33). Three main typesof behaviors have been observed: (i) an all-or-nothing stimu-lating effect of ischemia on mRNA levels of the immediateearly genes c-fos and c-jun and heat shock protein HSP70 (13);(ii) a nearly all-or-nothing decrease in mRNA levels for themajor subunit (GluR-2) of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor (32) and for typeAy-aminobutyric acid receptor subunits (33); (iii) an increasedexpression of mRNAs such as subunits of the N-methyl-D-aspartate receptor (20) and growth factors such as NGF andBDNF (14).The effect of ischemic preconditioning was systematically

assayed for these three types of situations. Fig. 3 A-F showsa typical example of in situ hybridizations for HSP70, ana-lyzed 24 h after the reperfusion. As described (34), weobserved that preconditioning accelerated the expression ofHSP70 mRNA after the second longer ischemia. Underthese conditions, the levels of HSP70 mRNA reached a peakat 3 h in CA1 cells (data not shown) and then graduallydeclined to disappear at 24 h (Fig. 3B). The Al adenosinereceptor agonist CPA, and the KCO levcromakalim inhibitedexpression of the HSP70 mRNA in the CA1 field. Gliben-clamide prevented this inhibitory effect in all cases. DPCPXor glibenclamide administration in the absence of ischemiadid not elicit a heat shock response (data not shown). Similarresults were obtained with c-fos and c-jun (data not shown).Fig. 3 G-L shows a typical illustration of case ii with theGluR-2 flip subunit. Ischemic preconditioning, CPA, andthe KCO levcromakalim prevented the decrease of mRNAexpression in CA1. Again, glibenclamide inhibited all ofthese protective effects. Comparable results have been ob-tained with the al and 12 subunits of the type A y-aminobu-tyric acid receptor (data not shown). Ischemia provokes anincrease in mRNA levels for different kinds of growth factorsin CA1, CA3, and the dentate gyrus (case iii). All of thesechanges are prevented by a 3-min ischemic preconditioning, by

Pharmacology: Heurteaux et atl

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4668 Pharmacology: Heurteaux et al

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# AFIG. 2. Rat hippocampus observed 7 days after ischemia. Treatments are as explained in Fig. 1. Treatments: A and B, Sham/6-min; C and D,

3-min/3d/6-min; E and F, CPA/6-min; G and H, Levcrom/6-min; I and J, Glib/3-min/3d/6-min; K and L, Glib+CPA/6-min. (B, D, F, H, J, andL) High-magnification photograph of the CA1 subfield boxed of the hippocampus in A, C, E, G, I, and K, respectively. (Cresyl violet staining. A,C, E, G, I, and K, x5; B, D, F, H, J, and L, x150.)

CPA, and by levcromakalim. The effects of all these treatmentsare abolished by glibenclamide (see Table 1).

* ?*"'' "4"'~:':' ,

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Results, presented in Fig. 3 and in Table 1, completelyconfirm the view that ischemic preconditioning probably ex-

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FIG. 3. (A-F) Distribution of HSP70 mRNAs in rat hippocampus. (G-L) Changes in in situ hybridization of the GluR-2 flip subunit probe toa-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor subunit mRNA in rat hippocampus. Treatments: A and G, single 6-minischemia; B and H, sublethal 3-min ischemia followed by 6-min ischemia at a 3-day interval; C and I, pretreatment with CPA prior to single 6-minischemia; D and J, pretreatment with levcromakalim prior to single 6-min ischemia; E and K, pretreatment with glibenclamide prior to sublethal3-min ischemia followed by 6-min ischemia 3 days later; F and L, pretreatment with glibenclamide prior to CPA administration and followed bysingle 6-min ischemia. Brains were analyzed 24 h after the last ischemia. (x9.)

Proc. Natl, Acad. Sci. USA 92 (1995)

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Proc. Natl. Acad. Sci. USA 92 (1995) 4669

Table 1. Changes in mRNA expression of NGF and BDNF genes in rat hippocampus after preconditioning and ischemia

RNA expressionGlib/ DPCPX/

Brain Sham/ 3min/lh/ 3min/3d/ CPA/ 3min/3d/ Levcrom/ Glib+CPA/ 3min/3d/Probe structure Sham 6min 6min 6min 6min 6min 6min 6min 6min

NGF CA1 + ++++ ++++ + + ++++ + ++++ ++++CA3 + ++++ ++++ + + +++ + +++ +++DG + ++++ ++++ + + +++ + ++++ ++++

BDNF CA1 + ++ ++ + + +++ + +++ +++CA3 + +++ +++ + +++ + +++ +++DG + +++ + +++++ +++ +++

Effect of pretreatment with levcromakalim and glibenclamide (Glib) on preconditioning plus ischemia-induced expression and singleischemia-induced expression of-the two probes, effects of CPA and DPCPX on the mRNA expression of the two probes after preconditioning andischemia, and effect of glibenclamide on single ischemia-induced expression of the two probes in CPA-pretreated rats are shown. Plus signscorrespond to relative intensities of hybridization in hippocampal cells. +, 10-30%; ++, 40-60%; +++, 60-80%; ++++, 80-100% geneinduction; -, no induction (for details, see Fig. 3). Levcrom, levcromakalim; DG, dentate gyrus.erts its protective effects via release of adenosine and activa-tion of KATP channels via A1 receptors. The level of expressionof mRNAs for these different potential protectors remainsunchanged in CA1 cells where they are protected againstneuronal death by the ischemic preconditioning treatment andby levcromakalim and CPA (Fig. 3 and Table 1). The protec-tive mechanism described here associated with cerebral pre-conditioning is a very important addition to the previouslypostulated protection associated with the induction of heatshock proteins in ischemic tolerance and in response to anumber of other brain stresses (refs. 1, 2, 34, and 35; for areview, see ref. 36).

Ischemic preconditioning is not specific to the brain. It hasalso bee.l observed in the heart. In this organ, ischemicpreconditioning prevents arrythmias and cardiac cell deathproduced by a second ischemia (37). In this organ, there isevidence that the protective effect of ischemic preconditioninginvolves KATP channels and adenosine (26, 38, 39).KATP channels are normally activated when the internal

ATP concentration decreases and when the intracellular con-centration of ADP increases (40-42). However, these KATPchannels can also be directly activated by a variety of hormonesand neurotransmitters (43-45), via direct G-protein interac-tions (44, 46), or via cAMP and protein kinase A (46). Probably,during the first short ischemia, the G-protein system liberatesadenosine and, by acting on Al type receptors, adenosine willactivate brain KATP channels, which leads to extensive protectionagainst the deleterious effect of a second longer ischemia. Inaddition to adenosine, other neurotransmitters or peptides couldalso be liberated that might be capable of KATp-channel activationand, therefore, of a protective effect.

We are grateful to Dr. T. C. Hamilton (Beecham Pharmaceuticals)for a gift of levcromakalim. We thank G. Jarretou, C. Roulinat, andF. Aguila for skillful technical assistance. This work was supported bythe Association Francaise contre les Myopathies (AFM) and theCentre National de la Recherche Scientifique. Thanks are due toBristol-Myers Squibb Company for an "Unrestricted Award."

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