Brain Research, 586 (1992) 61-66 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
61
BRES 17843
Inhibition of energy metabolism by 3-nitropropionic acid activates ATP-sensitive potassium channels M a t t h i a s R i e p e ~, Nobuaki H o r i b, Albert C. L u d o l p h a, David O. C a r p e n t e r b, P e t e r S. S p e n c e r a and Charles N. Allen a "Center for Research on Occupational and Em,ironmental Toxicology, Oregon Health Science Unicersity, Portland, OR (USA) and t, Wadsworth Center for Laboratories and Research, Albany, NY (USA) (Accepted 29 January 1992)
Key words: 3-Nitropropionic acid (3-NPA); Histotoxic hypoxia; Oxidative phosphorylation: ATP; Potassium channel; Glibenclamide; Diazoxide; Glutamate antagonists
3-Nitropropionic acid (I raM), which inhibits succinate dehydrogenase activity and reduces cellular energy, produces in the pyramidal cell layer of the hippocampal region CAI a hyperpolarization for variable lengths of time before evoking an irreversible depolarization. Hyperpolarization is caused by an increased potassium conductance that is attenuated by glibenclamide (I-10 ~M), a selective antagonist of ATP-sensitive potassium channels: in contrast, diazoxide ((I.5 raM), an agonist at this channel, induces a hyperpolarization in CA! neurons of rat hippoeampal slices. The transient hyperpolarization after prolonged (ca. I h) application of 3-NPA is followed by a depolarization that is incompletely reversed by brief application of the glutamate antagonists (D-2-amino-5-phosphonopentanoic acid (APV), 6,7-dichloroquinoxaline-2,3.dione (CNQX), 3-(+)-2carbox'ypiperazin-4-yl)propyl-l-phosphonic acid (CPP), 7-chloro-kynurenic acid (7CI-KYN)). Early application of glibenclamide (within the initial 5 rain) blocked or reduced hyperpolarization and accelerated the depolarization. These data suggest that metabolic inhibition by 3-NPA initially activates ATP-sensitive potassium channels. Events other than activation of glutamate receptors participate in the final depolarization resulting from uncoupling of oxidative phosphoryhttion.
INTRODUCTION 3-Nitropropionic acid (3-NPA), a widespread fungal and plant toxin and suicide inhibitor of succinate dehydrogenase, uncouples oxidative phosphorylation2'12 and has been shown to be of pathogenetic significance in certain human and animal neurological diseases 24'2t''33. Children in China who have consumed mildewed sugar cane containing 3-NPA developed an acute illness culminating in encephalopathy 24. After recovery, 2-10% developed a delayed non-regressive dystonia. Rats and mice treated with 3-NPA developed neuropathological damage in basal ganglia and hippocampus, with cytological features reminiscent of excitotoxic lesions 21'24. Similarly, mouse cortical explants treated with 3-NPA developed an excitotoxic pattern of damage that was attenuated by concurrent treatment with glutamate antagonists 34. Detectable tissue damage followed a reduction of cellular energy in the explants 34.
Impairment of cellular energy metabolism by ischemia or metabolic inhibition increases potassium conductance and hyperpolarizes the membrane potential 17'~'22'z~. Ca2*-activated or ATP-regulated potassium channels most likely mediate this conductance increase 17'22'23''~°'3~.Membrane hyperpolarization is followed by a late depolarization, which is believed to be mediated by a failure of ion pumps 15'17'32and/or activation of ionotropic glutamate receptors H~'43'44. The initial events in the toxicity of excitatory amino acids have been shown to be enhanced under conditions of inhibited energy metabolism 25'4~, assumedly because of an increased susceptibility of the cell to NMDA-receptor activation by membrane depolarization 25. It has been demonstrated in tissue cultures that not only glutamate antagonists but also activators of potassium channels can reduce excitotoxic damage t. This study seeks to characterize events mediating membrane potential changes after uncoupling of oxida-
Correspondence: M. Riepe, Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, OR 97201, USA. Fax: (1) (503) 494-4278.
62 Diazoxide and 3-NPA were purchased from Sigma. Glibenclamide was a gift of Hoechst AG.
tive phosphorylation by the exogenous neurotoxin 3NPA,
RESULTS
MATERIALS AND METHODS
lm'estigations with hippocampalslice preparations Slices and media. Male Wistar rats (200-250 g) were killed by cervical dislocation, the brains quickly removed and placed in cold KrebsRinger solution. After trimming, slices (450 #m thickness) were cut on a simple vibrato.me (Nomiyama) perpendicular to the long axis of the hippocampus. Prior to ¢lectrophysiological recording, the slices were incubated for at least 2 h at 35°C in oxygenated (95% O z, 5% CO,,) Krebs-Ringer solution (NaCI 126 raM. KCI, 5 raM: KH:PO 4, 1.26 raM; MgSO4, i.3 raM: CaCl,, 2.4 raM: NaHCO.~. 26 raM; glucose, l0 raM). The glucose concentration during recording was lowered to 4 mM in some experiments. During recording, oxygenated Krebs-Ringer solution with or without 3-NPA or other drugs was perfused ever the submerged slice at 3-5 ml/min. The electrodes for intracellular recording were filled with 4 M potassium acetate (resistances, 60-120 Mll). Data recordingam/analysis. Cells with an initial membrane potential less negative than - 55 mV or without spontaneous action potentials were excluded. Only slices yielding stable recordings for at least 25 rain (3-NPA) or 20 rain (additk)nal other drugs) were accepted for analysis. Stimulation pulses (50 ps. 15V) were applied via a morn)pp. lar electrode placed on the Schaffer collaterals. Recordings were made from the pyramidal cell body in ('AI. Data were printed on a Gould chart recorder and additionally stored on videotape fi)r further digital processing. hw('.~tlgation.~ with priman.' hippocanq)al cuhun,.~' ('ldturt'.~ and m('dhL Primary hippocampal cultures were prepared from newl)orn Sprague~Dawley rats ()it the first day post.partum and grown on colhlgen.coatcd coverslips at 34=35°(' fl)r 8-15 days prior to recording. The medium (Eagle's Minimum Essential Medium with 5r; hor:~e serum) was changed twice w~:ekly. The l)ath solutiot) contained (in raM) Na' 140; K ' 5; Ca ='° 3; M g : ' 2', ( ' I I.~.~', IIEPES l(i', glucose 2,'; and the intrac~llular solulion ('s' 140, Ca:' I', M~ J' 2: ( ) I I 140, ( I o: BAPTA II; IIEI)FS I(I,, ATI ) 2, Data r('cordmg (rod mm/y,~'i,~, Voltage.clamp data were recorded with a List amplifi~:r (EPC 7), Data were filtered with at1 l,i,pole Bessel tilter, ~tored on a vidcotapc, and analysed either after printing on a chart recorder (Gould) or after transfer tO a microcomputer, Elec. trode,~, pulled in two .~teps on u Narishise electrode puller, had a re,,~istance of 5.. 12 M.q whet) filled with any of the pipette solutions. Dru~s were applied externally via a gravity-driven system through barrels having an outer diameter of approximately 400 # M.
To e x a m i n e w h e t h e r 3 - N P A directly induces transm e m b r a n e o u s currents by r e c e p t o r activation, 3 - N P A was a p p l i e d (up to 1 m i n t to cultured h i p p o c a m p a l n e u r o n s using the whole-cell recording t e c h n i q u e in voltage-clamp mode. However no currents were detected over a voltage range from - 120 to + 30 m V for 3 - N P A concentrations of 0 . 1 - 1 0 mM. Since it was known that 3 - N P A time- and concentrat i o n - d e p e n d e n t l y reduces the activity of succinate dehydrogenase and the tissue concentration of A T P in mouse cortical explant cultures "~4, long-term c h a n g e s m e d i a t e d by ion c h a n n e l s r e g u l a t e d by the cellular energy level and metabolites of energy m e t a b o l i s m were investigated.
Changes of membrane potaltial after long-term application of 3.NPA in the slice preparation l n t r a c e l l u l a r recordings were m a d e from the C A I pyramidal cell body, with cells exposed to c o n t i n u o u s perfusion with I m M 3 - N P A (Fig. IA). In 5 of 12 cells, there was an initial maximal depolarization b e t w e e n 2 and 5 m V (3 + 1 mV ( m e a n + S.D.)) which lasted for 7-21 rain (12:1:6 rain ( m e a n + $,D,)), Subsequently, in the c o n t i n u e d presence of 3 - N P A (I raM), all but one (which gradually depolarized) C A I neurons hyperpolarized by a m e a n of - 4 + 7 m V ( m e a n + S.D., n - 12, P > 0,05 in u n p a i r e d t-test c o m p a r e d to initial m e m brane potential) after 20 rain, in those neurons that received no further e x p e r i m e n t a l m a n i p u l a t i o n , hyperpolarization gradually increased to - 1 0 + 7 m V after 40 rain ( m e a n 3: S,D., n = 5, P < 0,03) . Between 46 and 58 rain, the average hyperpolarization was larger
B
t mN 3=NPk
--
-
300 s
llm
I
I mN 3 - N P A
18mY L 60
1OO ~ APV 1 0 IdVl C P P
18mV L a
150
"I
Fig. I. A: intracellular xccording in the cell layer oi" the CAI region in a submerged rat hippocampal slice under continued perfusion of 3-NPA in IO mM extracellular glucose (0.5 nA hyperl~)larizing c~rrent pulses. 50 ms duration. 0.3 Hz). B: same conditions as in (A, but with ~duced extracellular glucose (4 raM).
63 than - 1 4 mV. Two neurons were still hyperpolarized by - 1 7 mV and - 2 2 mV after 100 rain of 3-NPA treatment, The membrane hyperpolarization was accompanied by an increase in the membrane conductance, The initial hyperpolarization was accompanied by a reduction of input resistance to 79 =l=19% at 20 rain (mean + S.D., P < 0.01 in unpaired t-test compared to initial membrane conductance) which increased to 54 + 23% after 40 rain (mean + S.D., P < 0.02). Neurons maintained without any further application of drugs (n = 3) started to depolarize after 65-140 min of 3-NPA treatment. This depolarization was rapid (3-7 m V / m i n for the first 10 mV of depolarization from the initial resting membrane potential) and irreversible for the duration of the recordings. Neurons recorded from slices perfused with Krebs-Ringer solution with the glucose concentration reduced from 10 mM to 4 mM showed no hyperpolarization (Fig. I B). These neurons were already depolarized by 6 4- 10 mV after 20 rain (n = 4) and by 18 :t: 13 mV after 30 min (n - 4) of perfusion with 1 mM 3-NPA, in contrast to the late onset of the depolarization in extracellular solution containing 10 mM glucose.
Actions of agonists and antagonists of the A TP.sensitive potassium channel in the slice preparation Glibenclamide (1-10 ttM), a specific antagonist of ATP-scnsitivc potassium channels, induced spiking, depolarization and conductance decrease in recordings from the cell layer of CAI, suggesting the closure of potassium channels (Fig. 2A, B). The average depolarization amounted to 2 4- 2 mV (n = 7) after 5 rain and 6 4- 10 mV (n = 6) after 8 rain. Fifteen rain after the initial glibenclamide application, the cells remained depolarized by 5 4- 8 mV (n = 6). Concomitant with the depolarization, an increase in membrane resistance to 117 + 24% and 137 4- 43% was observed 5 and 8 rain after application of glibenclamide. In two cells, the depolarization induced by glibenclamide could be reversed by diazoxide (Fig. 3); in these cells, a second application of glibenclamide after repolarization produced less than half the first response to glibenclamide. When glibenclamide was applied during the first 5 min of metabolic inhibition with 1 mM 3-NPA, the mean hyperpolarization was reduced (Fig. 2C); in fact only 3 of 5 cells showed a hyperpolarization. The average hyperpolarization was smaller with this treatment ( - 2 + 10 mV after 20 rain (n = 5) and - 2 + 7 mV after 40 min (n = 3)) than under perfusion with ! mM 3-NPA alone.
18 mV~..4 b 300
s
10 Id4 G l l b e n c X a m X d o
R 18 mY 60
s
60
:,
B
21d4 G l t b e n c l a m t d o
18 mV
2 |IH O l l b o n c l a m l d o O, 5
mH Dt~w.nxlO(~
L a ~ 3-NPA
¢ ',J6 InV I . . ~ 17,0 II
Fig. 2. A: I0 ~M glibenclamide in rat hippocampal slice induces spiking and and irreversible depolarization (0.5 nA hyperpolarizing current pulses, 50 ms duration, 0.3 Hz, application of 3-NPA started 5 rain prior to beginningof trace). B: 2 ~M glibenclamideinduces it reversible depolarization (0.5 nA hyperpolarizingcurrent pulses, 50 ms duration, 0.3 Flz, application of 3.NPA started 27 rain prior to beginning of trace). C: early application of 2 ~M glibenclamide prevents hyperpolarization induced by 3-NPA hut ATP.regulat¢d potassium channels can still be activated by 0.5 mM diazoxide (0.5 nA hyperpolarizingcurrent pulses, 50 ms duration, 0.3 Hz).
U
2 tdd G 1 i b o n c l a m I d o
L__I O. 5 ~
D~azoxldo
36 mY 120
Fig. 3. Diazoxide (0.5 mM) reverses depolarization induced by glibenclamide(2 ~M) under conditions of continued applicationof I mM 3-NPA. (0.5 nA hyperpolarizingcurrent pulses, 5(}ms duration, 0.3 Hz, application of 3-NPA started 25 rain prior to beginning of trace).
64 in a total of 4 of 11 applications in 9 cells, diazoxide (0.5 raM), an agonist at the ATP-regulated potassium channel, evoked a marked hyperpolarization of more than - 4 mV for several min. However, when applied after 80 rain of recording (n = 3 in 3 cells), diazoxide failed to induce hyperpolarization.
Actions of glutamate antagonists during the late hyperpolarization after metabolic inhibition in the slice preparalion Application of excitatory amino acid receptor antagonists, either individually or in combination, to 3NPA-treated CAI neurons was used to determine if membrane depolarization was mediated by extracellular glutamate. A total of 8 different combinations and concentrations of glutamate antagonists (APV 100/~M (n = 2), CNQX 10 pM (n = 1), 7CI-KYN 10/~M (n = 2), APV (50 pM) and CNQX (10/~M) (n = 1), APV 100 ,l~M and CPP 10/~M (n = 1)) was applied for up to 9 rain to 6 different cells at membrane potentials more positive than - 3 5 mV (time after commencement of 3-NPA application about 25 rain in 4 mM glucose and 90 rain in 10 mM glucose). These treatments induced a little slowing of the ongoing depolarization or a minor repolarization of the membrane potential (examples shown in Fig. 4 ) T h e largest effect observed was a conductance decrease of about 25%. No cell completely repolarized under this treatment. DISCUSSION
Hyperpolarization due to actit'ation of A TP.sensitice potassium chamaels after uacoupling of oxMatice phos. phorylation The initial hyperpolarization occurring after application of 3-NPA was accompanied by a reduction in input resistance. Similar changes of membrane poten-
18 mY
tial and input resistance are well known under hypoxic conditions 23 and after impairment of energy metabolism by different metabolic toxins .8. It is established that these changes result from an increased potassium conductance 23. However, the specific channel mediating the potassium current is unclear ee'e3. An early hypothesis indicating calcium-activated channels 3°'3. was challenged recently by reports suggesting involvement of the ATP-regulated potassium channel ~7'n9. The existence of this channel in different neuronal tissues has been substantiated with a variety of methods3- ~,.y~-39.46. The results obtained here support the conclusion that ATP-regulated channels are present in the CAI layer of rat hippocampal slices. This is consistent with the autoradiographic finding of ATP-regulated channels obtained by demonstrating glibenclamide binding to membranes of the basal and proximal regions of apical dendrites of pyramidal cells in the CA1 region as. We cannot exclude the possibility that part of the hyperpolarization is caused by currents associated with calcium-activated potassium channels. The responses to agonists and antagonists of the ATP-regulated channels were more pronounced than reported from some investigations where energy metabolism was impaired by anoxic challenge '~°,'~*. Possibly, this difference is related to the compressed time-course of eleetrophysiologic changes observed in the anoxic situation as opposed to the prolonged time-course with the chemical uncoupling of oxidative phosphorylation by 3-NPA. The latter condition may be more favorable to identify those channols regulated directly by the intracellular energy content because of the more gradual decline of cellular energy levels induced by 3-NPA treatment of CNS tissue in culture ~4. This may be particularly so since the block of the potassium current by the sulfonylureas proved to have a delayed onset of 2-3 min as
~---.4b 300
s
18 lay L . . 4 ~ 300 S
50 ~q
R
10 ~
APY
?CI-KYH
10 f i n CNQX
I~_Jl
II ,
,,,
,m
36D¥~ 120 a
I
l
I
I
I
'
36mV~ 120
s
Fig. 4. A: APV (50 ~M) and CNQX (I0 #M) (same cell as in Fig. IA), and B: 7CI.KYN (I0/~M) (same cell as in Fig. 2A) during depolarized membrane potentials (MP less negative than -35 mY) do not completely reverse the depolarization induced by prolonged inhibition of enerw metabolism by I mM 3-NPA in the in vitro slice preparation of the cell layer in the CAI region. (0.5 nA hypcrpolarizing current pulses, 50 ms duration, 0,3 Hzo)
65 shown previously in cardiac muscle 7 and pancreas 47. The actions of agonists and antagonists themselves depend on a variety of intraceilular modulators 14"!6"28"45"49"50. Among others, the cellular ATP level impacts currents induced by diazoxide; very high and very low concentrations of ATP prevent a response to diazoxide 9'4~'5°. This finding may explain the observation that after a prolonged metabolic inhibition of more than 80 rain, no currents could be evoked by diazoxide. The variety of modulating factors and the very long lasting effects of diazoxide and glibenclamide 7'4~ may account for the observation that currents induced by glibenclamide after the second application were much smaller than after the first application. As with anoxia, when perfusion with sulfonylureas starting prior to the anoxic challenge blocks the hyperpolarization in CA1 ~9 and CA38, the hyperpolarization caused by 3-NPA in some cells could be blocked by short-term application of glibenclamide (I min, 2-10 /zM glibenclamide) in the first five min after starting the perfusion with 3-NPA. Due to the variety of conditions modulating the ATP-regulated potassium channel 13'27'40A~, chemical uncoupling of oxidative phosphorylation may generate a set of conditions different from that triggered by anoxia. Further investigations with these compounds may illuminate the degree of similarity between anoxia and the chemically induced equivalent of hypoxia in vivo and in vitro. This question has been impossible to evaluate with classical inhibitors such as cyanide because of major respiratory side effects associated with this compound in vivo st. Depolarization after prolonged periods o f metabolic inhi. bition Prolonged inhibition of energy metabolism is known to induce depolarization of the membrane potential. The depolarization possibly gates the toxicity of endogenous excitatory compounds. However, after prolonged impairment of mitochondrial oxidative phosphorylation by 3-NPA, the depolarization could only partly be influenced by various combinations of glutamate antagonists. This agrees well with the observation that anoxia suppresses NMDA currents in submerged hippocampal slice preparations zg, probably reflecting the fact that the currents through the NMDA-channei are reduced when the receptor is dephosphorylated as observed in whole-cell recordings in culture systems 3s'36. Moreover, extracellular potassium is known to increase under anoxic conditions or after metabolic inhibition 23 and this is likely to reduce the NMDA response 4z. In contrast, it has been reported that perfusion with glutamate antagonists can prevent anoxic depolarization in CA1 e° and CA3 l°. However, these
experiments differ from ours in that the glutamate antagonists were applied for some min prior to the perfusion with the oxygen-deprived Krebs-Ringer solution. The unchanged slope of the membrane depolarization beyond - 5 0 to - 3 0 mV, and the observation that glutamate antagonists in different concentrations and combinations had only minor impact on the overall ongoing depolarization, might therefore indicate that events other than glutamate-mediated depolarization are important in the final stages of metabolic inhibition and that the toxic events induced by endogenous excitatory agents go beyond a mere depolarization. CONCLUSIONS The present results indicate that the uncoupling of oxidative phosphorylation by the exogenous neurotoxin 3-NPA can activate ATP-regulated potassium channels in preparations of hippocampal slices. Events other than depolarization alone may possibly be involved in gating lesions by excitatory compounds under conditions of prolonged metabolic inhibition by 3-NPA. Acknowk,dgements. This work was supported by grants from the Deutsche Forschungsgcmeinschaft (MR), NIH Grant NS-23807 (DOC), The Medical Research Council of Oregon (CNA), and NIH grant NS-19611 (PSS). REFERENCES I Abele, A.E. lind Miller, R.J., Potassium channel activators abolish excitotoxicity in cultured hippocampal pyramidal neurons, Neurosci. l,ett., I15 (1990) 195-200. 2 Alston, T.A., Mcla, N. and Bright, FIJ,, 3-Nitropropionale, the toxic substance of huliRofera, is a suicide inhibitor of succinale dehydrogenase, Proc, NatL Acad, Sci, USA, 74 (1977) 3767-3771. 3 Amoroso, S., Schmid-Antomarchi,H,, Fosset, M. and Lazdunski, M., Glucose, sull'onylureas,and neurotransmittcrrelease: role of ATP-sensitive K+ channels,Science, 247 (1990) 852-854. 4 Angel,!. and Bidet, S., The binding site for ['~H]glibenclamidein the rat cerebral cortex does not recognize K-channel agonistsor antagonists other than sulphonylureas, Fund. Clin. PharmacoL, 5 (1991) 107-115. 5 Ashford, M.LJ., Boden, P.R. and Treherne, J.M., Glucose-induced excitation of hypothalamic neurons is mediated by ATPsensitive K'~" channels, PfliigersArch., 415 (1990) 479-483. 6 Ashford, M.L.J., Sturgess, N.C., Trout, N.J., Gardner, N.J. and Hales, C.N., Adenosine-5'-triphosphate-sensitiveion channels in neonatal rat cultured central neurons, PfliigersArch., 412 (1988) 297-304, 7 Belles, B., Hescheler, J. and Trube, G., Changes of membrane currents in cardiac cells induced by long whole cell recordings and tolbutamide, PfliigersArch., 409 (1987) 582-588. 8 Ben-Ari, Y., Effect of glibenclamide, a selective blocker of an ATP-K+ channel, on the anoxic response of hippocampal neurones, PfliigersArch., 414 (Suppl 1) (1989) SI 11-S114. 9 Ben-Ari,Y., Krnjevic, K. and Crepel, V., Activatorsof ATP-sensitive K+ channels reduce anoxic depolarization in CA3 hippocampal neurons, Neuroscience, 37 (1990) 55-60. 10 Ben-Ari, Y., Modulation of ATP sensitive K+ channels: a novel strategy to reduce the deleterious effects of anoxia, in Y. Ben-Ari (Ed.), Excitatory Amino Acids and Neuronal Plasticity, Plenum, New York, 1990, pp. 481-489.
66 ! ! Brierley, J.B., Brown, A.W. and Calverley, J., Cyanide intoxication in the rat: physiological and neuropathological aspects, J. Neural. Neumsurg. Psych., 39 11976) 129-140. 12 Colas, C.J., Edmondson, D.E. and Singer, T.P., Inactivation of succinate dehydrogenase by 3-Nitropropionate, J. Biol. Chem., 254 11979) 5161-5167. 13 Davies, N.W., Modulation of ATP-sensitive K ÷ channels in skeletal muscle by intracellular protons, Nature, 343 11990) 375377. 14 Dunne, M.J., Protein phosphorylation is required for diazoxide to open ATP-sensitive potassium channels in insulin (RINm5F) secreting cells, FEBS Lett., 250 11989) 262-266. 15 Erecinska, M. and Dagani, F., Relationships between the neuronal sodium/potassium pump and energy metabolism, J. Gun. Physiol., 95 11990) 591-615. 16 French, J.F., Riera, L.C., Mullins, U.L. and Sarmiento, J.G., Modulation of [3H]glibenclamide binding to cardiac and insulinoma membranes, Eur. J. PharmacoL - Mol. Plmrm. Section, 207 ( 1991) 23-28. 17 Fujiwara. N., Higashi, H., Shimoji, K. and Yoshimura, M., Effects of hypoxia on hippocampal neurons in vitro, J. Physiol., 384 11987) 131-151. '.:, Godfraind, J.M., Kawamura, H.. Krnjevic, K. and Pumain, R., Actions of dinitrophenol and some other metabolic inhibitors on .ortlral neurons, J. Physiol., 215 (1971) 199-222. 19 eh~,'t~,J.J. and Anderson, G.E., Glucose and sulfonylureas modtb lifferent phases of the membrane potential change during hypoxia in rat hippocampal slices, Brain Res., 489 (1989)302-310. 20 Grtgg, JJ. and Anderson, E.G., Competitive and noncompetitive N-methyI-D-aspartate antagonists modify hypoxia-induced membrane potential changes and prtotect rat hippocampal slices from functional failure: a quantitative comparison, J. Phann. Exp. Ther., 253 (1990) 130-135. 21 tlamilton, B.F. and Gould, D.H., Nature and distribution of brain le.~ionsin rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage, Acta Neuropathol., 72 (1987) 286-297. 22 tlansen, A.J., Hounsgaard, J. and Jahnsen, H., Anoxia increases potassium conductance in hippocampal nerve cells, Acta PhysioL Scand., I15 (1982) 301=311), 23 Hansen, A.J., Effect of ,noxi, on ion distribution in the brain, Physiol. Rt,t'., 65 (1985) 101+ 148. 24 lie, F., Zhanll, S,, Zhang, C. el al., Mycotoxin induced an. cephalopalhy and dystonia in children. In G,N. Volans el al, (Eds.), Basic Science #l Texvtcolo~,, Taylor & Francis, London, 1990, pp. 596-604. 25 Henneber~, R.C., Novelli, A., Cox, J.A, and Lysko, P,G., Nauru, toxicity at the N-methyl,t~-aspartate receptor in energy.comprom. ised neurons, An hypothesis for cell death in aging and disease, Attn. NV Acad. Sci., 568 (1989) 225-233. 26 James, L.F., Hartley, J., Williams, M,C. and Van Kampen, K,R,, Field, and experimental studies in cattle and sheep poisoned by nitro-bearing Astralagus or their toxins, J. Am. Vet. Res,, 41 (1980) 377-382, 27 Kakei, M., Kelly, R.P., Ashcmft, SJ.H. and Ashcroft, F.M., The ATP-sensitivity of K + channels in rat pancreatic B-cells is modulated by ADP, FEBS Lett., 208 (1986) 63-66. 28 Kozlowski, R.Z., Hales, C.N. and Ashford, M.LJ., Dual effects of diazoxide on ATP-K + currents recorded from an insulin-secreting cell line, Br. J. Pharmacol., 97 (1989) 1039-1050. 29 Krnjevic, K., Anoxia and NMDA receptors, in Y. Ben-Aft (Ed.), Excitatory Amino Acids and Neuronal Plastic'icy, Plenum, New York, 1990, pp. 475-479. 30 Krnjevic, K., Adenosine triphosphate-sensitive potassium channels in anoxia, Stroke, 21 (Svppl. l i d 11990) !i!-!90-111-193. 31 Leblond, J. and Krnjevic, Hypoxic changes in hippocampal neurons, J. Neurophysiol., 62 11989) i-14. 32 Lipton, P. and Whittingham, T.S., The effect of hypoxia on evoked potentials in the in vitro hippocampus, J. Physiol., 287 (1979) 427-438.
33 Ludolph, A.C., He, F., Spencer, P.S., Hammerstad, J. and Sabri, M., 3-nitropropionic acid - exogenous animal neurotoxin and possible human striatal toxin, Can. J. Neurol. Sci., 18 11992) 492 -498. 34 Ludolph, A.C., Seelig, M., Ludolph, A., Novitt, P., Spencer, P.S. and Sabri, M.I., Cellular energy deficits and excitotoxic pattern of neuronal degeneration induced by 3-nitropropionic acid in vitro, in preparation. 35 MacDonald, J.F., Mody, I. and Salter. M.W., Regulation of N-methyI-D-aspartate receptors reveale by intracellular dialysis of routine neurons in culture, J. Physiol., 414 11989) 17-34. 36 Mody, I., Salter, M.W. and MacDonald, J.F., Requirement of the NMDA receptor/channel for intracellular high-energy phosphates and the extent of intraneuronal calcium buffering in cultured mouse hippocampal neurons, Neurosci. Lett., 93 11988) 73-78. 37 Mourre, C., Ben-Aft, Y., Bernardi, H., Fosset, M. and Lazdunski, M., Antidiabetic sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices, Brain Res., 486 11989) 159-164. 38 Mourre, C., Widmann, C. and Lazdunski, M., Specific hippocam. pal lesions indicate the presence of sulfonylurea binding sites associated to ATP-sensitive K + channels both post-synaptically and on mossy fibers, Brain Res., 540 11991) 340-344. 39 Murphy, K.P.S.J. and Greenfield, S.A., ATP-sensitive potassium channels counteract anoxia in neurones of the substantia nigra, Exp. Brain Res., 84 11991) 355-358. 40 Noma, A. and Shibasaki, T., Membrane current through adenosine-triphosphate-regulated potassium channels in guinea-pig ventricular cells, J. PhysioL, 363 11985) 463-480. 41 Novelli, A., Re;!ly, J.A., Lysko, P.G. and Henneberry, R.C., Glutamate becomes neurotoxic via the N-methyl-o-aspartate receptor when intracellular energy levels are reduced, Brain Res., 4+;I (1988) 205-212. 42 Ozawa, S,, line, M. and Tsuzuki, K., Suppression by extracellular K +x of N-methyI-D-aspartate responses in cultured rat hippocampal neurons, J. Neurophysiol., 64 (1990) 1361-1367, 43 Radar, R.K. and Lanthorn, T.lt,, Experimental ischemia induces a persistent depolarization blocked by decreased calcium and NMDA antagonists, Neur~ci, Lull,, 99 (1989) 125-130, 44 Rosen, A.S. and Morris, M,E,, Depolarizing effects of anoxia on pyramidal cells of rat neocortex, Neur+~'('i, I,ett,, 124 (1991) 169173, 45 Schwanstecher, M,, L6ser, S., Rietze, !. and Panten, U,, Phosphate and thiophosphate group donating adenine and guanine mucleotides inhibit glibendamide binding 1o membranes from pancreatic islets, Naunyn.SchmicdcberR's Arch, Phannacol., 343 (1991) 83-89, 46 Treherne, J.M. and Ashford, M.LJ,, The re8ional distribution of sulphonylurea binding sites in rat brain, Neuroscience, 40 11991) 523-531. 47 Trube, G,, Rorsman, P. and Ohnu-Shosaku, T,, Opposite effects of tolbutamide and diazoxide on the ATP-dependent K + channel in mouse pancreatic ~-cells, Pfliigers Arc'h., 407 (1986) 493-499, 48 Well, K,H., L6nnendonker, U, and Neumcke, B., ATP-sensitive potassium channels in adult mouse skeletal muscle: different modes of blockage by internal cations, ATP and tolbutamide, Pfli~ers Arch., 414 (1989) 622-628, 49 Ztinkler, BJ,, Lins, S., Ohno-Shosaku, T., Trube, G. and Panten, U., Cytosolic ADP enhances the sensitivity to tolbutamide of ATP-dependent K + channels from pancreatic B-cells, FEBS Lute., 239 (I 988) 241-244, 50 Ziinkler, BJ,, Lenzen, S,, Mtinner, K,, Panten, U. and Trube, G., Concentration.dependendent effects of tolbutamide, meglitinide, glipizide, glibendamide and diazoxide on ATP-regulated K + currents in pancreatic B-cells, Naunyn Schmiedeberg's Arch. Phar. macol., 337 (1988) 225-230.
61
BRES 17843
Inhibition of energy metabolism by 3-nitropropionic acid activates ATP-sensitive potassium channels M a t t h i a s R i e p e ~, Nobuaki H o r i b, Albert C. L u d o l p h a, David O. C a r p e n t e r b, P e t e r S. S p e n c e r a and Charles N. Allen a "Center for Research on Occupational and Em,ironmental Toxicology, Oregon Health Science Unicersity, Portland, OR (USA) and t, Wadsworth Center for Laboratories and Research, Albany, NY (USA) (Accepted 29 January 1992)
Key words: 3-Nitropropionic acid (3-NPA); Histotoxic hypoxia; Oxidative phosphorylation: ATP; Potassium channel; Glibenclamide; Diazoxide; Glutamate antagonists
3-Nitropropionic acid (I raM), which inhibits succinate dehydrogenase activity and reduces cellular energy, produces in the pyramidal cell layer of the hippocampal region CAI a hyperpolarization for variable lengths of time before evoking an irreversible depolarization. Hyperpolarization is caused by an increased potassium conductance that is attenuated by glibenclamide (I-10 ~M), a selective antagonist of ATP-sensitive potassium channels: in contrast, diazoxide ((I.5 raM), an agonist at this channel, induces a hyperpolarization in CA! neurons of rat hippoeampal slices. The transient hyperpolarization after prolonged (ca. I h) application of 3-NPA is followed by a depolarization that is incompletely reversed by brief application of the glutamate antagonists (D-2-amino-5-phosphonopentanoic acid (APV), 6,7-dichloroquinoxaline-2,3.dione (CNQX), 3-(+)-2carbox'ypiperazin-4-yl)propyl-l-phosphonic acid (CPP), 7-chloro-kynurenic acid (7CI-KYN)). Early application of glibenclamide (within the initial 5 rain) blocked or reduced hyperpolarization and accelerated the depolarization. These data suggest that metabolic inhibition by 3-NPA initially activates ATP-sensitive potassium channels. Events other than activation of glutamate receptors participate in the final depolarization resulting from uncoupling of oxidative phosphoryhttion.
INTRODUCTION 3-Nitropropionic acid (3-NPA), a widespread fungal and plant toxin and suicide inhibitor of succinate dehydrogenase, uncouples oxidative phosphorylation2'12 and has been shown to be of pathogenetic significance in certain human and animal neurological diseases 24'2t''33. Children in China who have consumed mildewed sugar cane containing 3-NPA developed an acute illness culminating in encephalopathy 24. After recovery, 2-10% developed a delayed non-regressive dystonia. Rats and mice treated with 3-NPA developed neuropathological damage in basal ganglia and hippocampus, with cytological features reminiscent of excitotoxic lesions 21'24. Similarly, mouse cortical explants treated with 3-NPA developed an excitotoxic pattern of damage that was attenuated by concurrent treatment with glutamate antagonists 34. Detectable tissue damage followed a reduction of cellular energy in the explants 34.
Impairment of cellular energy metabolism by ischemia or metabolic inhibition increases potassium conductance and hyperpolarizes the membrane potential 17'~'22'z~. Ca2*-activated or ATP-regulated potassium channels most likely mediate this conductance increase 17'22'23''~°'3~.Membrane hyperpolarization is followed by a late depolarization, which is believed to be mediated by a failure of ion pumps 15'17'32and/or activation of ionotropic glutamate receptors H~'43'44. The initial events in the toxicity of excitatory amino acids have been shown to be enhanced under conditions of inhibited energy metabolism 25'4~, assumedly because of an increased susceptibility of the cell to NMDA-receptor activation by membrane depolarization 25. It has been demonstrated in tissue cultures that not only glutamate antagonists but also activators of potassium channels can reduce excitotoxic damage t. This study seeks to characterize events mediating membrane potential changes after uncoupling of oxida-
Correspondence: M. Riepe, Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, OR 97201, USA. Fax: (1) (503) 494-4278.
62 Diazoxide and 3-NPA were purchased from Sigma. Glibenclamide was a gift of Hoechst AG.
tive phosphorylation by the exogenous neurotoxin 3NPA,
RESULTS
MATERIALS AND METHODS
lm'estigations with hippocampalslice preparations Slices and media. Male Wistar rats (200-250 g) were killed by cervical dislocation, the brains quickly removed and placed in cold KrebsRinger solution. After trimming, slices (450 #m thickness) were cut on a simple vibrato.me (Nomiyama) perpendicular to the long axis of the hippocampus. Prior to ¢lectrophysiological recording, the slices were incubated for at least 2 h at 35°C in oxygenated (95% O z, 5% CO,,) Krebs-Ringer solution (NaCI 126 raM. KCI, 5 raM: KH:PO 4, 1.26 raM; MgSO4, i.3 raM: CaCl,, 2.4 raM: NaHCO.~. 26 raM; glucose, l0 raM). The glucose concentration during recording was lowered to 4 mM in some experiments. During recording, oxygenated Krebs-Ringer solution with or without 3-NPA or other drugs was perfused ever the submerged slice at 3-5 ml/min. The electrodes for intracellular recording were filled with 4 M potassium acetate (resistances, 60-120 Mll). Data recordingam/analysis. Cells with an initial membrane potential less negative than - 55 mV or without spontaneous action potentials were excluded. Only slices yielding stable recordings for at least 25 rain (3-NPA) or 20 rain (additk)nal other drugs) were accepted for analysis. Stimulation pulses (50 ps. 15V) were applied via a morn)pp. lar electrode placed on the Schaffer collaterals. Recordings were made from the pyramidal cell body in ('AI. Data were printed on a Gould chart recorder and additionally stored on videotape fi)r further digital processing. hw('.~tlgation.~ with priman.' hippocanq)al cuhun,.~' ('ldturt'.~ and m('dhL Primary hippocampal cultures were prepared from newl)orn Sprague~Dawley rats ()it the first day post.partum and grown on colhlgen.coatcd coverslips at 34=35°(' fl)r 8-15 days prior to recording. The medium (Eagle's Minimum Essential Medium with 5r; hor:~e serum) was changed twice w~:ekly. The l)ath solutiot) contained (in raM) Na' 140; K ' 5; Ca ='° 3; M g : ' 2', ( ' I I.~.~', IIEPES l(i', glucose 2,'; and the intrac~llular solulion ('s' 140, Ca:' I', M~ J' 2: ( ) I I 140, ( I o: BAPTA II; IIEI)FS I(I,, ATI ) 2, Data r('cordmg (rod mm/y,~'i,~, Voltage.clamp data were recorded with a List amplifi~:r (EPC 7), Data were filtered with at1 l,i,pole Bessel tilter, ~tored on a vidcotapc, and analysed either after printing on a chart recorder (Gould) or after transfer tO a microcomputer, Elec. trode,~, pulled in two .~teps on u Narishise electrode puller, had a re,,~istance of 5.. 12 M.q whet) filled with any of the pipette solutions. Dru~s were applied externally via a gravity-driven system through barrels having an outer diameter of approximately 400 # M.
To e x a m i n e w h e t h e r 3 - N P A directly induces transm e m b r a n e o u s currents by r e c e p t o r activation, 3 - N P A was a p p l i e d (up to 1 m i n t to cultured h i p p o c a m p a l n e u r o n s using the whole-cell recording t e c h n i q u e in voltage-clamp mode. However no currents were detected over a voltage range from - 120 to + 30 m V for 3 - N P A concentrations of 0 . 1 - 1 0 mM. Since it was known that 3 - N P A time- and concentrat i o n - d e p e n d e n t l y reduces the activity of succinate dehydrogenase and the tissue concentration of A T P in mouse cortical explant cultures "~4, long-term c h a n g e s m e d i a t e d by ion c h a n n e l s r e g u l a t e d by the cellular energy level and metabolites of energy m e t a b o l i s m were investigated.
Changes of membrane potaltial after long-term application of 3.NPA in the slice preparation l n t r a c e l l u l a r recordings were m a d e from the C A I pyramidal cell body, with cells exposed to c o n t i n u o u s perfusion with I m M 3 - N P A (Fig. IA). In 5 of 12 cells, there was an initial maximal depolarization b e t w e e n 2 and 5 m V (3 + 1 mV ( m e a n + S.D.)) which lasted for 7-21 rain (12:1:6 rain ( m e a n + $,D,)), Subsequently, in the c o n t i n u e d presence of 3 - N P A (I raM), all but one (which gradually depolarized) C A I neurons hyperpolarized by a m e a n of - 4 + 7 m V ( m e a n + S.D., n - 12, P > 0,05 in u n p a i r e d t-test c o m p a r e d to initial m e m brane potential) after 20 rain, in those neurons that received no further e x p e r i m e n t a l m a n i p u l a t i o n , hyperpolarization gradually increased to - 1 0 + 7 m V after 40 rain ( m e a n 3: S,D., n = 5, P < 0,03) . Between 46 and 58 rain, the average hyperpolarization was larger
B
t mN 3=NPk
--
-
300 s
llm
I
I mN 3 - N P A
18mY L 60
1OO ~ APV 1 0 IdVl C P P
18mV L a
150
"I
Fig. I. A: intracellular xccording in the cell layer oi" the CAI region in a submerged rat hippocampal slice under continued perfusion of 3-NPA in IO mM extracellular glucose (0.5 nA hyperl~)larizing c~rrent pulses. 50 ms duration. 0.3 Hz). B: same conditions as in (A, but with ~duced extracellular glucose (4 raM).
63 than - 1 4 mV. Two neurons were still hyperpolarized by - 1 7 mV and - 2 2 mV after 100 rain of 3-NPA treatment, The membrane hyperpolarization was accompanied by an increase in the membrane conductance, The initial hyperpolarization was accompanied by a reduction of input resistance to 79 =l=19% at 20 rain (mean + S.D., P < 0.01 in unpaired t-test compared to initial membrane conductance) which increased to 54 + 23% after 40 rain (mean + S.D., P < 0.02). Neurons maintained without any further application of drugs (n = 3) started to depolarize after 65-140 min of 3-NPA treatment. This depolarization was rapid (3-7 m V / m i n for the first 10 mV of depolarization from the initial resting membrane potential) and irreversible for the duration of the recordings. Neurons recorded from slices perfused with Krebs-Ringer solution with the glucose concentration reduced from 10 mM to 4 mM showed no hyperpolarization (Fig. I B). These neurons were already depolarized by 6 4- 10 mV after 20 rain (n = 4) and by 18 :t: 13 mV after 30 min (n - 4) of perfusion with 1 mM 3-NPA, in contrast to the late onset of the depolarization in extracellular solution containing 10 mM glucose.
Actions of agonists and antagonists of the A TP.sensitive potassium channel in the slice preparation Glibenclamide (1-10 ttM), a specific antagonist of ATP-scnsitivc potassium channels, induced spiking, depolarization and conductance decrease in recordings from the cell layer of CAI, suggesting the closure of potassium channels (Fig. 2A, B). The average depolarization amounted to 2 4- 2 mV (n = 7) after 5 rain and 6 4- 10 mV (n = 6) after 8 rain. Fifteen rain after the initial glibenclamide application, the cells remained depolarized by 5 4- 8 mV (n = 6). Concomitant with the depolarization, an increase in membrane resistance to 117 + 24% and 137 4- 43% was observed 5 and 8 rain after application of glibenclamide. In two cells, the depolarization induced by glibenclamide could be reversed by diazoxide (Fig. 3); in these cells, a second application of glibenclamide after repolarization produced less than half the first response to glibenclamide. When glibenclamide was applied during the first 5 min of metabolic inhibition with 1 mM 3-NPA, the mean hyperpolarization was reduced (Fig. 2C); in fact only 3 of 5 cells showed a hyperpolarization. The average hyperpolarization was smaller with this treatment ( - 2 + 10 mV after 20 rain (n = 5) and - 2 + 7 mV after 40 min (n = 3)) than under perfusion with ! mM 3-NPA alone.
18 mV~..4 b 300
s
10 Id4 G l l b e n c X a m X d o
R 18 mY 60
s
60
:,
B
21d4 G l t b e n c l a m t d o
18 mV
2 |IH O l l b o n c l a m l d o O, 5
mH Dt~w.nxlO(~
L a ~ 3-NPA
¢ ',J6 InV I . . ~ 17,0 II
Fig. 2. A: I0 ~M glibenclamide in rat hippocampal slice induces spiking and and irreversible depolarization (0.5 nA hyperpolarizing current pulses, 50 ms duration, 0.3 Hz, application of 3-NPA started 5 rain prior to beginningof trace). B: 2 ~M glibenclamideinduces it reversible depolarization (0.5 nA hyperpolarizingcurrent pulses, 50 ms duration, 0.3 Flz, application of 3.NPA started 27 rain prior to beginning of trace). C: early application of 2 ~M glibenclamide prevents hyperpolarization induced by 3-NPA hut ATP.regulat¢d potassium channels can still be activated by 0.5 mM diazoxide (0.5 nA hyperpolarizingcurrent pulses, 50 ms duration, 0.3 Hz).
U
2 tdd G 1 i b o n c l a m I d o
L__I O. 5 ~
D~azoxldo
36 mY 120
Fig. 3. Diazoxide (0.5 mM) reverses depolarization induced by glibenclamide(2 ~M) under conditions of continued applicationof I mM 3-NPA. (0.5 nA hyperpolarizingcurrent pulses, 5(}ms duration, 0.3 Hz, application of 3-NPA started 25 rain prior to beginning of trace).
64 in a total of 4 of 11 applications in 9 cells, diazoxide (0.5 raM), an agonist at the ATP-regulated potassium channel, evoked a marked hyperpolarization of more than - 4 mV for several min. However, when applied after 80 rain of recording (n = 3 in 3 cells), diazoxide failed to induce hyperpolarization.
Actions of glutamate antagonists during the late hyperpolarization after metabolic inhibition in the slice preparalion Application of excitatory amino acid receptor antagonists, either individually or in combination, to 3NPA-treated CAI neurons was used to determine if membrane depolarization was mediated by extracellular glutamate. A total of 8 different combinations and concentrations of glutamate antagonists (APV 100/~M (n = 2), CNQX 10 pM (n = 1), 7CI-KYN 10/~M (n = 2), APV (50 pM) and CNQX (10/~M) (n = 1), APV 100 ,l~M and CPP 10/~M (n = 1)) was applied for up to 9 rain to 6 different cells at membrane potentials more positive than - 3 5 mV (time after commencement of 3-NPA application about 25 rain in 4 mM glucose and 90 rain in 10 mM glucose). These treatments induced a little slowing of the ongoing depolarization or a minor repolarization of the membrane potential (examples shown in Fig. 4 ) T h e largest effect observed was a conductance decrease of about 25%. No cell completely repolarized under this treatment. DISCUSSION
Hyperpolarization due to actit'ation of A TP.sensitice potassium chamaels after uacoupling of oxMatice phos. phorylation The initial hyperpolarization occurring after application of 3-NPA was accompanied by a reduction in input resistance. Similar changes of membrane poten-
18 mY
tial and input resistance are well known under hypoxic conditions 23 and after impairment of energy metabolism by different metabolic toxins .8. It is established that these changes result from an increased potassium conductance 23. However, the specific channel mediating the potassium current is unclear ee'e3. An early hypothesis indicating calcium-activated channels 3°'3. was challenged recently by reports suggesting involvement of the ATP-regulated potassium channel ~7'n9. The existence of this channel in different neuronal tissues has been substantiated with a variety of methods3- ~,.y~-39.46. The results obtained here support the conclusion that ATP-regulated channels are present in the CAI layer of rat hippocampal slices. This is consistent with the autoradiographic finding of ATP-regulated channels obtained by demonstrating glibenclamide binding to membranes of the basal and proximal regions of apical dendrites of pyramidal cells in the CA1 region as. We cannot exclude the possibility that part of the hyperpolarization is caused by currents associated with calcium-activated potassium channels. The responses to agonists and antagonists of the ATP-regulated channels were more pronounced than reported from some investigations where energy metabolism was impaired by anoxic challenge '~°,'~*. Possibly, this difference is related to the compressed time-course of eleetrophysiologic changes observed in the anoxic situation as opposed to the prolonged time-course with the chemical uncoupling of oxidative phosphorylation by 3-NPA. The latter condition may be more favorable to identify those channols regulated directly by the intracellular energy content because of the more gradual decline of cellular energy levels induced by 3-NPA treatment of CNS tissue in culture ~4. This may be particularly so since the block of the potassium current by the sulfonylureas proved to have a delayed onset of 2-3 min as
~---.4b 300
s
18 lay L . . 4 ~ 300 S
50 ~q
R
10 ~
APY
?CI-KYH
10 f i n CNQX
I~_Jl
II ,
,,,
,m
36D¥~ 120 a
I
l
I
I
I
'
36mV~ 120
s
Fig. 4. A: APV (50 ~M) and CNQX (I0 #M) (same cell as in Fig. IA), and B: 7CI.KYN (I0/~M) (same cell as in Fig. 2A) during depolarized membrane potentials (MP less negative than -35 mY) do not completely reverse the depolarization induced by prolonged inhibition of enerw metabolism by I mM 3-NPA in the in vitro slice preparation of the cell layer in the CAI region. (0.5 nA hypcrpolarizing current pulses, 50 ms duration, 0,3 Hzo)
65 shown previously in cardiac muscle 7 and pancreas 47. The actions of agonists and antagonists themselves depend on a variety of intraceilular modulators 14"!6"28"45"49"50. Among others, the cellular ATP level impacts currents induced by diazoxide; very high and very low concentrations of ATP prevent a response to diazoxide 9'4~'5°. This finding may explain the observation that after a prolonged metabolic inhibition of more than 80 rain, no currents could be evoked by diazoxide. The variety of modulating factors and the very long lasting effects of diazoxide and glibenclamide 7'4~ may account for the observation that currents induced by glibenclamide after the second application were much smaller than after the first application. As with anoxia, when perfusion with sulfonylureas starting prior to the anoxic challenge blocks the hyperpolarization in CA1 ~9 and CA38, the hyperpolarization caused by 3-NPA in some cells could be blocked by short-term application of glibenclamide (I min, 2-10 /zM glibenclamide) in the first five min after starting the perfusion with 3-NPA. Due to the variety of conditions modulating the ATP-regulated potassium channel 13'27'40A~, chemical uncoupling of oxidative phosphorylation may generate a set of conditions different from that triggered by anoxia. Further investigations with these compounds may illuminate the degree of similarity between anoxia and the chemically induced equivalent of hypoxia in vivo and in vitro. This question has been impossible to evaluate with classical inhibitors such as cyanide because of major respiratory side effects associated with this compound in vivo st. Depolarization after prolonged periods o f metabolic inhi. bition Prolonged inhibition of energy metabolism is known to induce depolarization of the membrane potential. The depolarization possibly gates the toxicity of endogenous excitatory compounds. However, after prolonged impairment of mitochondrial oxidative phosphorylation by 3-NPA, the depolarization could only partly be influenced by various combinations of glutamate antagonists. This agrees well with the observation that anoxia suppresses NMDA currents in submerged hippocampal slice preparations zg, probably reflecting the fact that the currents through the NMDA-channei are reduced when the receptor is dephosphorylated as observed in whole-cell recordings in culture systems 3s'36. Moreover, extracellular potassium is known to increase under anoxic conditions or after metabolic inhibition 23 and this is likely to reduce the NMDA response 4z. In contrast, it has been reported that perfusion with glutamate antagonists can prevent anoxic depolarization in CA1 e° and CA3 l°. However, these
experiments differ from ours in that the glutamate antagonists were applied for some min prior to the perfusion with the oxygen-deprived Krebs-Ringer solution. The unchanged slope of the membrane depolarization beyond - 5 0 to - 3 0 mV, and the observation that glutamate antagonists in different concentrations and combinations had only minor impact on the overall ongoing depolarization, might therefore indicate that events other than glutamate-mediated depolarization are important in the final stages of metabolic inhibition and that the toxic events induced by endogenous excitatory agents go beyond a mere depolarization. CONCLUSIONS The present results indicate that the uncoupling of oxidative phosphorylation by the exogenous neurotoxin 3-NPA can activate ATP-regulated potassium channels in preparations of hippocampal slices. Events other than depolarization alone may possibly be involved in gating lesions by excitatory compounds under conditions of prolonged metabolic inhibition by 3-NPA. Acknowk,dgements. This work was supported by grants from the Deutsche Forschungsgcmeinschaft (MR), NIH Grant NS-23807 (DOC), The Medical Research Council of Oregon (CNA), and NIH grant NS-19611 (PSS). REFERENCES I Abele, A.E. lind Miller, R.J., Potassium channel activators abolish excitotoxicity in cultured hippocampal pyramidal neurons, Neurosci. l,ett., I15 (1990) 195-200. 2 Alston, T.A., Mcla, N. and Bright, FIJ,, 3-Nitropropionale, the toxic substance of huliRofera, is a suicide inhibitor of succinale dehydrogenase, Proc, NatL Acad, Sci, USA, 74 (1977) 3767-3771. 3 Amoroso, S., Schmid-Antomarchi,H,, Fosset, M. and Lazdunski, M., Glucose, sull'onylureas,and neurotransmittcrrelease: role of ATP-sensitive K+ channels,Science, 247 (1990) 852-854. 4 Angel,!. and Bidet, S., The binding site for ['~H]glibenclamidein the rat cerebral cortex does not recognize K-channel agonistsor antagonists other than sulphonylureas, Fund. Clin. PharmacoL, 5 (1991) 107-115. 5 Ashford, M.LJ., Boden, P.R. and Treherne, J.M., Glucose-induced excitation of hypothalamic neurons is mediated by ATPsensitive K'~" channels, PfliigersArch., 415 (1990) 479-483. 6 Ashford, M.L.J., Sturgess, N.C., Trout, N.J., Gardner, N.J. and Hales, C.N., Adenosine-5'-triphosphate-sensitiveion channels in neonatal rat cultured central neurons, PfliigersArch., 412 (1988) 297-304, 7 Belles, B., Hescheler, J. and Trube, G., Changes of membrane currents in cardiac cells induced by long whole cell recordings and tolbutamide, PfliigersArch., 409 (1987) 582-588. 8 Ben-Ari, Y., Effect of glibenclamide, a selective blocker of an ATP-K+ channel, on the anoxic response of hippocampal neurones, PfliigersArch., 414 (Suppl 1) (1989) SI 11-S114. 9 Ben-Ari,Y., Krnjevic, K. and Crepel, V., Activatorsof ATP-sensitive K+ channels reduce anoxic depolarization in CA3 hippocampal neurons, Neuroscience, 37 (1990) 55-60. 10 Ben-Ari, Y., Modulation of ATP sensitive K+ channels: a novel strategy to reduce the deleterious effects of anoxia, in Y. Ben-Ari (Ed.), Excitatory Amino Acids and Neuronal Plasticity, Plenum, New York, 1990, pp. 481-489.
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