114
Brain Research, 593 (1992) 114-116 •"~ 1t~92Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25400
Short Communications
Potentiation of cyanide neurotoxicity by blockade of ATP-sensitive potassium channels Manisha N. Patel *, G e o r g e K.W. Yim and Gary E. Isom Department of Pharmatology and Toxicology, Purdue Unwersity, West Lafayette, IN 47907 (USA) (Accepted 21 July 1992)
Key wonl~" ATP-sensitive pota,,slum channel; Primary hippocampal neuron; Cyanide; Histotoxic hypoxia
Exposure of primary, hzppocampai cultures to NaCN (2 mM) or glybunde (5 p.M) alone for 3 h did not produce a rise in extracellular lactic dehydrogenase (LDH) activity. Coincubation with NaCN and glyburide produced a significant efflux of LDH from the neurons. Diazoxide or D-2-amino-5-phosphovalerate (APV) partially reversed the release of LDI.-I by the combination of NaCN and glyburide. These observations indicate ATP-sensitive potassium channels (KAle) are activated by nonlethal concentrations of cyanide and their blockade with glyburide unmasks cyanide's toxicity. The cytotoxicity of cyanide appears to result from a combination of processes resulting in altered ion handling and excitotoxicity.
ATP-sensitive potassium channels (K^TP) belong to a class of potassium channels that were first described in cardiac cellst'~. Subsequently, these channels have been identified and characterized in muscle, pancreas and brain a Hyperpolarization mediated via KAI t, is inhibited by normal intracellular ATP concentrations. Sulfonylureas such as glyburide block these channels whereas diazoxide, somatostatin and cromakalin activate them 2. Recent evidence suggests that KA.n, modulate anoxic responses of hippocampal neurons ~.4.u. in CA3 hippocampal neurons of the rat, brief anoxic episodes produce depolarization presumably by glutamate release. This depolarization is sensitive to blockade by diazoxide 4. Anoxia-induced early hyperpolarization can be blocked by glyburide t~. Recently, KA.n, were shown to modulate excitotoxic responses in cultured hippocampal neurons !. Ca 2+-fluctuations and cell death were abolished by diazoxide or cromakalin and these effects were reversed by glyburide, Cyanide produces histotoxic hypoxia following inhibition of cytochrome oxidase ¢'.7. Recent studies in our laboratory suggest that excitotoxic mechanisms mediate cyanide-induced neuronal injury. In brain slices,
cyanide induces the release uf endogenous glutamate t4. Cyanide can elevate neuronal Ca 2+ levelst5 and produce cytotoxicity in hippocampal cultures predominantly by activation of the NMDA receptors. Although Ca 2+ changes were observed early (minutes) following cyanide exposure to hippocampal neurons, cell death occurred only after prolonged (several hours) exposure period. Moreover brief exposure to cyanide followed by washing does not result in cell death over an 18-hour period s. This latency for cyanide-induced neuronal degeneration could be a result of cyanide-mediated ATP depletion and activation of KAXP. In order to determine this, the influence of glyburide on cytotoxicity of cyanide was assessed in hippocampai cultures at a time when cyanide alone did not produce significant damage. Hippocampal cultures were grown as previously described by Patel et al. in 12-well dishes coated with collagen and polyq-lysine Is. Mature cultures (12-17 days in vitro) were used for experiments. Cytotoxicity experiments were conducted using minimum ~ssential medium (MEM) with Earle's salts (GIBCO, Grand Island, NY)without added growth factors and serum.
Correspomlence: G.E. Isom, Department of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907, USA. Fax: (1) (317) 494-1414. * Present address: Department of Medicine, Epilepsy Research Laboratory, Duke University, Durham, NC 27710, USA.
115 Three hours after incubation with treatments, the supernatant medium was removed, the cells were lysed using a lysis buffer containing 0.5% v / v Triton X-100 in 0.1 M potassium phosphate, pH 7. The cells were incubated in the lysis buffer for 30 min, at the end of which the buffer was removed and centrifuged at 10,000 rpm for 5 min. Lactate dehydrogenase (LDH) activity was determined in both the supernatant medium and the lysis buffer as described 8'ts. The cultures were exposed to 2 mM NaCN, 5/~M glyburide, or both for 3 h and LDH efflux was measured. Three hours following incubation, NaCN or glyburide alone produced little LDH efflux, but their combination produced significant LDH efflux (Fig. 1). It has been shown that diazoxide abolished excitotoxicity in hippocampal cultures and this was reversed by glyburide t. Hence attempts were made to reverse the effect of glyburide on NaCN-mediated cytotoxicity. Diazoxide did not completely reverse the effect of glyburide on NaCN-mediated LDH efflux (Fig. 2). It is thought tha~ glutamate release mediates part of the anoxic response of hippocampal CA3 neurons 4'9. A similar mechanism may also be involved in the response of hippocampal neurons to cyanide and glyburide. For this reason the ability of APV, a competitive NMDA receptor antagonist, to protect cultures treated with cyanide and glyburide was tested. The results of this study indicate that APV did not provide complete protection against the neurotoxicity of cyanide and glyburide. The present study indicates that KATP are activated early during cyanide exposure. This is in agreement with the demonstration that KATe are activated during
J W v
CON
OLY
NoON
NaCN +
OLY Fig. 1. The effect of glyburide on cyanide neurotoxicity. Hippocampal cultures were exposed to vehicle (control), 5 p,M glyburide, 2 mM NaCN alm~e or together for 3 h and LDH release was determined. Results are expressed as percent of LDH released as compared to control cells. Control cultures released 3.] +0.6% of the total LDH (cell + media content). Bars represent LDH at the end of the exposure period (mean+ S.E.M., n = 12-19). * Significant difference (P < 0.05) from control.
380 3O0
250 0 (3 200 ILl 150 n, 'I"
100
q
50 0
~14
OI.Y 14o01 OI~Z 14o04 I¢o~I NIV MII04 4. + OLY OLY OIlY + +
NN
Fig. 2. The effect of diazoxide on the neurotoxicity mediated by cyanide and glyburide. Hippocampal cultures were exposed to 500 /~M diazoxide, 2 mM NaCN, 5/~M glyburide, 1 mM APV alone or in combination for 3 h and LDH release was determined. Results are expressed as percent of LDH released compared to control cells. Control cultures released 3.1 + 0.6% of the total LDH (cell + media content). Bars represent LDH at the end of the exposure period (mean+S.E.M., n = 4-19). * Significant difference (P < 0.05) from control.
nitrogen anoxia3 or cyanide t2. In the present studies, LDH release was used as an indirect means to examine the role of KAT P during cyanide exposure. The results indicate that when glyburide, an antagonist of the KATe, was added at a time when cyanide alone produced no detectable damage, the toxicity of cyanide was unmasked It is likely that during cyanide exposure, a fall in ATP levels activates the KAT p which reduces neuronal excitability. After several hours of exposure to cyanide, ionic homeostasis fails and cell viability is threatened. A direct demonstration that cyanide activates the KATp has been shown to occur in substantia nigra neurons 12. The present studies do not exclude the possibility that other potassium channels such as the Cae÷-dependent potassium channel may also be activated during by cyanide. Cyanide-induced Ca 2+ influx may in fact activate Ca2÷-dependent potassium channels as seen with anoxiat°. The demonstration that either diazoxide or APV provided partial protection may reflect that multiple mechanisms are activated following exposure to c y a n i d e . KATP are activated and their blockade accentuates the cyanide-mediated cell injury. Cyanide may also stimulate release of glutamate which activates NMDA receptors, resulting in an excitotoxic response. This study was supported in part by NIH Grant ES04140. M.N.P. was supported by a David Ross Foundation Fellowship. 1 Abele, A.E. and Miller, R.J., Potassium channel activators abolish excitotoxicity in cultured hippocampal pyramidal neurons, Neurosci. Lett., 115 (1990) 195-200.
116 2 Cook, N.S., The pharmaco~'~gy of potassium channels and their therapeutic potential, Trends Pharmacol. Sci., 9 (1988) 21-28. 3 Ben Ari, Y., Effect of glibenclamide, ,~ selective blocker of an ATP-K + channel, on the anoxic response of hippocampal neurons, Pfli~gersArch., 414 (1989) SI 1I-S114. 4 Ben Ari, Y., Krnjevic, K. and Crepel, V., Activators of ATP-sensRive K + channels reduce anoxic depolarization m CA3 hippocampal neurons, Neuroscience, 37 (1990) 55-60. 5 DubinskT, J.M. and Rothman, S.M., Intraceilular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury, J. Neurosci., 11 (1991) 2545-2551. 6 Isom, G.E. and Way, J.L., Lethality of cyanide in the absence of inhibition of liver cytochrome oxidase, Biochem. Pharmacol., 25 (1976) 605-608. 7 Keilin, D., Cytochrome and intracellular oxidase, Proc. R. Soc. Lond. Ser. B, 106 (1930)418-444. 8 Koh, J. and Choi, D.W., Quantitative determination of glu:amate mediated cortical neuronal injury in cell culture by lactate dehydrogenase effiux assay, J. Neuroscl. Methods, 20 (1987) 83-90. 9 Krnjevic, K. and Ben Ari, Y., Anoxic changes in dentate granule cells, Neurosci. Lett., 107 (1989)89-93.
10 Leblond, J. and Krnjevic, K., Hypoxic changes in hlppocampal neuron~, J. Neurophyszol., 62 (1989) 1-14. ! 1 Mourre, C., Ben Ari, Y., Bernardi, H., Fosset, M. and Lazdunskl, M., Antidiabetlc sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices, Brain Res., 486 (1989) 159-164. 12 Murphy, K.P.S. and Greenfield, S.A., ATP-sensitive potassmm channels counteract anoxia in neurons of the substantia nigra, Exp. Brain Res., 84 (1991) 355-358. 13 Noma, A., ATP-regulated K + channels m cardiac muscle, Nature, 305 (1983) 105-111. 14 Patel, M.N., Ardelt, B.K., Yim, G.K.W. and Isom, G.E., Cyanide induces Ca :+ dependent and independent release of glutamate from mouse brain slices, NeuroscL Lett., 131 (1991) 42-44. 15 Patel, M.N., Yim, G.K.W. and Isom, G.E., Blockade of Nmethyl-D-aspartate receptors prevents cyanide-induced neuronal injury in primary hippocampal culture, Toxicol. Appl. Pharmacol., 115 (1992) 124-129.
Brain Research, 593 (1992) 114-116 •"~ 1t~92Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25400
Short Communications
Potentiation of cyanide neurotoxicity by blockade of ATP-sensitive potassium channels Manisha N. Patel *, G e o r g e K.W. Yim and Gary E. Isom Department of Pharmatology and Toxicology, Purdue Unwersity, West Lafayette, IN 47907 (USA) (Accepted 21 July 1992)
Key wonl~" ATP-sensitive pota,,slum channel; Primary hippocampal neuron; Cyanide; Histotoxic hypoxia
Exposure of primary, hzppocampai cultures to NaCN (2 mM) or glybunde (5 p.M) alone for 3 h did not produce a rise in extracellular lactic dehydrogenase (LDH) activity. Coincubation with NaCN and glyburide produced a significant efflux of LDH from the neurons. Diazoxide or D-2-amino-5-phosphovalerate (APV) partially reversed the release of LDI.-I by the combination of NaCN and glyburide. These observations indicate ATP-sensitive potassium channels (KAle) are activated by nonlethal concentrations of cyanide and their blockade with glyburide unmasks cyanide's toxicity. The cytotoxicity of cyanide appears to result from a combination of processes resulting in altered ion handling and excitotoxicity.
ATP-sensitive potassium channels (K^TP) belong to a class of potassium channels that were first described in cardiac cellst'~. Subsequently, these channels have been identified and characterized in muscle, pancreas and brain a Hyperpolarization mediated via KAI t, is inhibited by normal intracellular ATP concentrations. Sulfonylureas such as glyburide block these channels whereas diazoxide, somatostatin and cromakalin activate them 2. Recent evidence suggests that KA.n, modulate anoxic responses of hippocampal neurons ~.4.u. in CA3 hippocampal neurons of the rat, brief anoxic episodes produce depolarization presumably by glutamate release. This depolarization is sensitive to blockade by diazoxide 4. Anoxia-induced early hyperpolarization can be blocked by glyburide t~. Recently, KA.n, were shown to modulate excitotoxic responses in cultured hippocampal neurons !. Ca 2+-fluctuations and cell death were abolished by diazoxide or cromakalin and these effects were reversed by glyburide, Cyanide produces histotoxic hypoxia following inhibition of cytochrome oxidase ¢'.7. Recent studies in our laboratory suggest that excitotoxic mechanisms mediate cyanide-induced neuronal injury. In brain slices,
cyanide induces the release uf endogenous glutamate t4. Cyanide can elevate neuronal Ca 2+ levelst5 and produce cytotoxicity in hippocampal cultures predominantly by activation of the NMDA receptors. Although Ca 2+ changes were observed early (minutes) following cyanide exposure to hippocampal neurons, cell death occurred only after prolonged (several hours) exposure period. Moreover brief exposure to cyanide followed by washing does not result in cell death over an 18-hour period s. This latency for cyanide-induced neuronal degeneration could be a result of cyanide-mediated ATP depletion and activation of KAXP. In order to determine this, the influence of glyburide on cytotoxicity of cyanide was assessed in hippocampai cultures at a time when cyanide alone did not produce significant damage. Hippocampal cultures were grown as previously described by Patel et al. in 12-well dishes coated with collagen and polyq-lysine Is. Mature cultures (12-17 days in vitro) were used for experiments. Cytotoxicity experiments were conducted using minimum ~ssential medium (MEM) with Earle's salts (GIBCO, Grand Island, NY)without added growth factors and serum.
Correspomlence: G.E. Isom, Department of Pharmacology and Toxicology, Purdue University, West Lafayette, IN 47907, USA. Fax: (1) (317) 494-1414. * Present address: Department of Medicine, Epilepsy Research Laboratory, Duke University, Durham, NC 27710, USA.
115 Three hours after incubation with treatments, the supernatant medium was removed, the cells were lysed using a lysis buffer containing 0.5% v / v Triton X-100 in 0.1 M potassium phosphate, pH 7. The cells were incubated in the lysis buffer for 30 min, at the end of which the buffer was removed and centrifuged at 10,000 rpm for 5 min. Lactate dehydrogenase (LDH) activity was determined in both the supernatant medium and the lysis buffer as described 8'ts. The cultures were exposed to 2 mM NaCN, 5/~M glyburide, or both for 3 h and LDH efflux was measured. Three hours following incubation, NaCN or glyburide alone produced little LDH efflux, but their combination produced significant LDH efflux (Fig. 1). It has been shown that diazoxide abolished excitotoxicity in hippocampal cultures and this was reversed by glyburide t. Hence attempts were made to reverse the effect of glyburide on NaCN-mediated cytotoxicity. Diazoxide did not completely reverse the effect of glyburide on NaCN-mediated LDH efflux (Fig. 2). It is thought tha~ glutamate release mediates part of the anoxic response of hippocampal CA3 neurons 4'9. A similar mechanism may also be involved in the response of hippocampal neurons to cyanide and glyburide. For this reason the ability of APV, a competitive NMDA receptor antagonist, to protect cultures treated with cyanide and glyburide was tested. The results of this study indicate that APV did not provide complete protection against the neurotoxicity of cyanide and glyburide. The present study indicates that KATP are activated early during cyanide exposure. This is in agreement with the demonstration that KATe are activated during
J W v
CON
OLY
NoON
NaCN +
OLY Fig. 1. The effect of glyburide on cyanide neurotoxicity. Hippocampal cultures were exposed to vehicle (control), 5 p,M glyburide, 2 mM NaCN alm~e or together for 3 h and LDH release was determined. Results are expressed as percent of LDH released as compared to control cells. Control cultures released 3.] +0.6% of the total LDH (cell + media content). Bars represent LDH at the end of the exposure period (mean+ S.E.M., n = 12-19). * Significant difference (P < 0.05) from control.
380 3O0
250 0 (3 200 ILl 150 n, 'I"
100
q
50 0
~14
OI.Y 14o01 OI~Z 14o04 I¢o~I NIV MII04 4. + OLY OLY OIlY + +
NN
Fig. 2. The effect of diazoxide on the neurotoxicity mediated by cyanide and glyburide. Hippocampal cultures were exposed to 500 /~M diazoxide, 2 mM NaCN, 5/~M glyburide, 1 mM APV alone or in combination for 3 h and LDH release was determined. Results are expressed as percent of LDH released compared to control cells. Control cultures released 3.1 + 0.6% of the total LDH (cell + media content). Bars represent LDH at the end of the exposure period (mean+S.E.M., n = 4-19). * Significant difference (P < 0.05) from control.
nitrogen anoxia3 or cyanide t2. In the present studies, LDH release was used as an indirect means to examine the role of KAT P during cyanide exposure. The results indicate that when glyburide, an antagonist of the KATe, was added at a time when cyanide alone produced no detectable damage, the toxicity of cyanide was unmasked It is likely that during cyanide exposure, a fall in ATP levels activates the KAT p which reduces neuronal excitability. After several hours of exposure to cyanide, ionic homeostasis fails and cell viability is threatened. A direct demonstration that cyanide activates the KATp has been shown to occur in substantia nigra neurons 12. The present studies do not exclude the possibility that other potassium channels such as the Cae÷-dependent potassium channel may also be activated during by cyanide. Cyanide-induced Ca 2+ influx may in fact activate Ca2÷-dependent potassium channels as seen with anoxiat°. The demonstration that either diazoxide or APV provided partial protection may reflect that multiple mechanisms are activated following exposure to c y a n i d e . KATP are activated and their blockade accentuates the cyanide-mediated cell injury. Cyanide may also stimulate release of glutamate which activates NMDA receptors, resulting in an excitotoxic response. This study was supported in part by NIH Grant ES04140. M.N.P. was supported by a David Ross Foundation Fellowship. 1 Abele, A.E. and Miller, R.J., Potassium channel activators abolish excitotoxicity in cultured hippocampal pyramidal neurons, Neurosci. Lett., 115 (1990) 195-200.
116 2 Cook, N.S., The pharmaco~'~gy of potassium channels and their therapeutic potential, Trends Pharmacol. Sci., 9 (1988) 21-28. 3 Ben Ari, Y., Effect of glibenclamide, ,~ selective blocker of an ATP-K + channel, on the anoxic response of hippocampal neurons, Pfli~gersArch., 414 (1989) SI 1I-S114. 4 Ben Ari, Y., Krnjevic, K. and Crepel, V., Activators of ATP-sensRive K + channels reduce anoxic depolarization m CA3 hippocampal neurons, Neuroscience, 37 (1990) 55-60. 5 DubinskT, J.M. and Rothman, S.M., Intraceilular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury, J. Neurosci., 11 (1991) 2545-2551. 6 Isom, G.E. and Way, J.L., Lethality of cyanide in the absence of inhibition of liver cytochrome oxidase, Biochem. Pharmacol., 25 (1976) 605-608. 7 Keilin, D., Cytochrome and intracellular oxidase, Proc. R. Soc. Lond. Ser. B, 106 (1930)418-444. 8 Koh, J. and Choi, D.W., Quantitative determination of glu:amate mediated cortical neuronal injury in cell culture by lactate dehydrogenase effiux assay, J. Neuroscl. Methods, 20 (1987) 83-90. 9 Krnjevic, K. and Ben Ari, Y., Anoxic changes in dentate granule cells, Neurosci. Lett., 107 (1989)89-93.
10 Leblond, J. and Krnjevic, K., Hypoxic changes in hlppocampal neuron~, J. Neurophyszol., 62 (1989) 1-14. ! 1 Mourre, C., Ben Ari, Y., Bernardi, H., Fosset, M. and Lazdunskl, M., Antidiabetlc sulfonylureas: localization of binding sites in the brain and effects on the hyperpolarization induced by anoxia in hippocampal slices, Brain Res., 486 (1989) 159-164. 12 Murphy, K.P.S. and Greenfield, S.A., ATP-sensitive potassmm channels counteract anoxia in neurons of the substantia nigra, Exp. Brain Res., 84 (1991) 355-358. 13 Noma, A., ATP-regulated K + channels m cardiac muscle, Nature, 305 (1983) 105-111. 14 Patel, M.N., Ardelt, B.K., Yim, G.K.W. and Isom, G.E., Cyanide induces Ca :+ dependent and independent release of glutamate from mouse brain slices, NeuroscL Lett., 131 (1991) 42-44. 15 Patel, M.N., Yim, G.K.W. and Isom, G.E., Blockade of Nmethyl-D-aspartate receptors prevents cyanide-induced neuronal injury in primary hippocampal culture, Toxicol. Appl. Pharmacol., 115 (1992) 124-129.
