Brain Research, 577 (1992) 343-346 ~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
343
BRES 25150
Cyclic GMP modulators and excitotoxic injury in cerebral cortical cultures Heather S. Lustig, Kristine L. von Brauchitsch, Jane Chan and David A. Greenberg Department of Neurology, University of California and San Francisco General Hospital, San Francisco, CA 94110 (USA)
(Accepted 21 January 1992) Key words: Cyclic GMP; Excitotoxicity; Cell culture; N-Methyl-D-aspartate; Sodium nitroprusside; Guanylate cyclase; Protein kinase;
Phosphodiesterase
N-Methyl-D-aspartate (NMDA) receptor activation generates nitric oxide (NO) and cyclic GMP (cGMP) and produces 'excitotoxic' neuronal injury. To examine the possible role of cGMP in excitotoxicity, we evaluated the effects of agents that stimulate or inhibit cGMP activity on the release of lactate dehydrogenase from neuron-enriched cortical cultures, cGMP analogs exhibited no toxicity, and inhibitors of guanylate cyclase or of cGMP-dependent enzymes failed to protect cultures from the toxic effects of NMDA or the NO donor sodium nitroprusside. These findings argue against a role for cGMP in the pathogenesis of excitotoxic neuronal injury. The action of excitatory amino acid neurotransmitters on N M D A receptors contributes to ischemic and other forms of 'excitotoxic' neuronal injury. Evidence for such a contribution includes the toxicity of N M D A in neuronal cultures in vitro 15 and the ability of N M D A antagonists to reduce ischemic cerebral damage in vivo 24. N M D A receptor activation stimulates production of the second messenger nitric oxide (NO), which promotes the synthesis of cGMP by soluble guanylate cyclase (GC) 13. NO appears to have a role in mediating the toxic effect of N-methyl-D-aspartate (NMDA) on cortical neurons because toxicity can be reproduced by the NO donor, sodium nitroprusside (SNP) 5. Like N M D A , SNP also stimulates cGMP production by neurons 5'13, but whether cGMP is involved in the toxicity of N M D A or SNP is uncertain. Little direct evidence supports a neurotoxic role for cGMP, and several studies suggest that cGMP could be neuroprotective. For example, the cGMP analog dibutyryl-cGMP interferes with the naturally occurring death of motor neurons in embyronic chick spinal cord 2s and inhibitors of GC cause death of granule cell neurons in cerebellar slices H. Nevertheless, the possibility that cGMP is involved in excitotoxicity is of interest, partly because it might explain the relative resistance of NOproducing neurons 12. Thus, neurons expressing N M D A receptors and NO synthase might be protected by receptor-stimulated elevation of free intracellular Ca 2÷ (Ca2+i), which at high concentrations inhibits GC 18,
while such protection would not occur in neighboring neurons into which NO diffuses 13. A variety of mechanisms could underlie cGMP toxicity, since cGMP is involved in diverse neurocellular functions, including the gating of ion channels and the regulation of protein kinases and cyclic nucleotide phosphodiesterases (PDEs) 14. To investigate the possible role of cGMP in excitotoxic injury, we examined the effects of cGMP analogs, and inhibitors of GC or of cGMP-dependent enzymes, in neuron-enriched cultures prepared from rat cerebral cortex. Release of lactate dehydrogenase (LDH) was used as an index of neuronal injury 19 induced by N M D A or SNP. Drugs were purchased from Sigma Chemical Co. (St. Louis, MO), except for 6-anilinoquinoline-5, 8-quinone (LY 83583) and N-[2-(methylamino)-ethyl]-5-isoquinoline-sulfonamide dihydrochloride (H-8), which were from Calbiochem Corporation (LaJolla, CA), and N-(2-aminoethyl)-5-isoquinoline-sulfonamide dihydrochloride (H9), quazinone and dipyridamole, which were from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). Cultures were prepared from cerebral cortex of 16-17 day Sprague-Dawley rat embryos as described previously 21. Exposure to cytosine arabinoside on day 5 in vitro resulted in cultures containing - 8 5 % neuronspecific enolase-immunoreactive cells on day 12, when experiments were performed. Cultures were exposed to drugs for 20 min in (mM) NaC1 120, KC1 5.4, CaC12 1.8, glucose 15 and Tris-HC1 25 (exposure buffer; p H 7.4 at
Correspondence: D.A. Greenberg, Department of Neurology, University of California, 4M62 San Francisco General Hospital, San Francisco, CA 94110, USA. Fax: (1) (415) 826-1313.
344 37°C). Toxicity was assessed 24 h after drug e x p o s u r e by m e a s u r i n g L D H released into the m e d i u m t9 using a commercially available s p e c t r o p h o t o m e t r i c assay kit
tion b e t w e e n L D H release a n d loss of the ability of cult u r e d cortical n e u r o n s to exclude T r y p a n blue dye over a wide range of N M D A c o n c e n t r a t i o n s (r = 0.957; unp u b l i s h e d data).
(340-LD, Sigma C h e m i c a l Co., St. Louis, M O ) . L D H m e a s u r e m e n t s were c o n d u c t e d in triplicate or q u a d r u p l i cate a n d r e p e a t e d o n m u l t i p l e platings, a n d L D H release was expressed as a p e r c e n t of the total L D H activity re-
T h e c G M P analogs d i b u t y r y l - c G M P a n d 8 - b r o m o c G M P had n o effect o n L D H release. L D H release was also u n c h a n g e d following exposure to 8 - b r o m o i n o s i n e 3',
leased from cultures by freeze-thawing. D a t a are rep o r t e d as m e a n values + S . E . M . T h e significance of dif-
5'-cyclic m o n o p h o s p h a t e ( 8 - b r o m o - c l M P ) , which mimics some actions of c G M P 7. H y d r a l a z i n e , which has b e e n r e p o r t e d to activate soluble G C 26, increased L D H re-
ferences b e t w e e n m e a n s was assessed b y S t u d e n t ' s t-test for single c o m p a r i s o n s a n d by analysis of variance followed by post hoc S t u d e n t - N e w m a n - K e u l s tests for m u l t i p l e c o m p a r i s o n s , with P < 0.05 c o n s i d e r e d statisti-
lease. H o w e v e r , the m e c h a n i s m u n d e r l y i n g hydralazine toxicity is u n c l e a r since, in some systems, hydralazine has c G M P - i n d e p e n d e n t toxic effects m e d i a t e d t h r o u g h d a m a g e to D N A 7'29. D i p y r i d a m o l e , which inhibits the
cally significant. As shown in Table I, control cultures released a b o u t 20% of their total L D H c o n t e n t over 24 h. L D H release increased m o r e t h a n 2-fold following e x p o s u r e to 1 m M N M D A a n d almost 3-fold following exposure to 1 m M SNP ( P < 0.001); we f o u n d previously that the toxic effects of b o t h N M D A a n d SNP were m a x i m a l at this conc e n t r a t i o n (Lustig et al., s u b m i t t e d for p u b l i c a t i o n ) .
hydrolysis of c G M P by cGMP-specific (type V) P D E 1, did n o t affect L D H release. N e i t h e r the c G M P analogs n o r d i p y r i d a m o l e r e d u c e d the toxicity of N M D A or SNP, while hydralazine decreased SNP toxicity, p e r h a p s due to its ability to b i n d to SNP 2. Several G C inhibitors increased L D H release. These i n c l u d e d M e t h y l e n e blue, h y d r o x y l a m i n e , a n d N - m e t h -
L D H release has b e e n s h o w n to reflect the e x t e n t of m o r p h o l o g i c N M D A toxicity in cortical cultures in o t h e r studies 3, a n d we have f o u n d a similarly strong correla-
y l h y d r o x y l a m i n e , which are also toxic to g r a n u l e cells in cerebellar slices u , a n d LY 835836. H o w e v e r , the toxicity of these agents c a n n o t necessarily be a t t r i b u t e d to inhi-
TABLE I Effects of cGMP modulators on L D H release from cortical cultures
LDH release is expressed as a percent of total LDH released by freeze-thawing. Data shown are means + S.E.M. from the number of cultures indicated in brackets. Treatment
Treatment alone
Treatment + NMDA (1 mM)
Treatment + SNP (1 mM)
None
23 + 1 [141]
54 _+ 3 [39]
65 + 2 [36]
-
29 + 2 [16] 21 + 3 [11] 22 + 2 [11]
52 _+ 6 [7] 43 + 2 [7] 45 + 4 [8]
61 + 3 [12] 61 + 2 [11] 62 + 1 [11]
-
37 + 2 [25]*
43 + 4 [15]
44 + 4 [22]*
-
24 _+ 4 [15]
50 + 5 [8]
63 _+ 5 [12]
-
44 66 57 39 40
54 53 60 48 52
68 82 71 78 61
Activators cGMP analogs Dibutyryl-cGMP (1 mM) 8-Bromo-cGMP (1 mM) 8-Bromo-clMP (1 mM) Guanylate cyclase activator Hydralazine (1 mM) cGMP-specific (type V) PDE inhibitor Dipyridamole (10/tM) Inhibitors Guanylate cyclase inhibitors Methylene blue (10 #M) LY 83583 (1 mM) Hydroxylamine (1 mM) N-Methylhydroxylamine (1 mM) KC1 (50 mM) depolarization cGMP-dependent protein kinase inhibitors H-8 (100 ~M) H-9 (100 ~M) cOMP-inhibited (type III) PDE inhibitor Quazinone (10 ~M) *P < 0.05 relative to 'None' (Student-Newman-Keuls).
+ + + + +
3 4 4 5 2
[221" [161" [20]* [14]* [231"
+ + + + +
8 7 6 5 8
[lll [131 [6] [8] [81
-+ 5 -+ 5 _+ 4 ___6 + 3
Treatment + dibutyryl cGMP (1 mM)
I141 [131 16] [6] [81
49 70 70 44 34 -
26 + 4 [10] 41 _+ 7 [12]*
41 + 5 [10] 67 + 5 [91
59 + 9 [71 74 _+ 4 [12]
28 + 5 [131
56 + 2 [9]
61 ± 4 [121
-
+ + + + +
1 7 9 8 3
[4] [3] [10] [3] [4]
345 bition of GC, since hydroxylamine can be metabolized to N O 25 and LY 83583 generates superoxide radicals 8. Moreover, L D H release was not reduced by adding 1 mM dibutyryl c G M P to cultures treated with G C inhibitors. None of the G C inhibitors reduced the toxicity of N M D A or SNP. A small percentage of cultured cortical neurons express the enzyme N A D P H diaphorase, which has been identified as an N O synthase 17, and are comparatively resistant to N M D A toxicity 2°. Considering the apparent role of N O as a mediator of N M D A toxicity 5, this seems paradoxical. However, N M D A receptor activation elevates [Ca2+]i , which can inhibit soluble G C TM. Accordingly, it has been suggested that N M D A receptor-mediated increases in [Ca2+]i may protect NO-producing neurons from cGMP-dependent toxicity, while N O kills neighboring cells into which it diffuses, and in which [Ca2+]i is not elevated ~2. If this were the case, then increasing [Ca2+]i by an N M D A - i n d e p e n d e n t mechanism might protect against N M D A toxicity in the larger percentage of neurons that do not produce NO. To investigate this possibility, some cultures were depolarized by increasing the concentration of KC1 in the exposure buffer to 50 mM. Microspectrofluorometry on single cultured cortical neurons using the Ca 2+ indicator indo-1 showed that 50 mM KC1 increased [Ca2+]i from a resting value of 145 + 13 nM to 491 + 46 nM (n = 14, P < 0.001), which is within the range of [Ca2+]i reported to inhibit G C TM. However, KCI depolarization failed to protect against N M D A or SNP toxicity. Known intracellular targets for the effects of c G M P include cGMP-dependent protein kinase and cGMP-
stimulated (type II) and cGMP-inhibited (type III) cyclic nucleotide P D E s TM. If cGMP-dependent enzymes were involved in excitotoxicity, then inhibitors of these enzymes might reduce the toxic effects of N M D A and SNP. However, H-8 and H-9, protein kinase inhibitors with preferential high affinity for cGMP-dependent protein kinase 16, had no effect on N M D A or SNP toxicity, and H-9 was itself toxic. Selective inhibitors of cGMPstimulated (type II) P D E have not been described, but the selective type III (cGMP-inhibited) P D E inhibitor quazinone 1 was also ineffective as a neuroprotective agent. The major finding of this study is that the toxic effects of N M D A and SNP on cortical cultures are neither reproduced by cGMP analogs nor prevented by inhibitors of G C or of cGMP-dependent effector enzymes. These observations suggest that although both N M D A and SNP are capable of stimulating c G M P synthesis s, c G M P is unlikely to mediate their neurotoxic effects. The effects of N O on [Ca2+]i , mitogenesis, and proliferation in fibroblasts 9'1°, protein synthesis in hepatocytes 4 and osteoclast function 22 have also been shown to be independent of cGMP. O u r findings do not identify the specific mechanism underlying NO-mediated excitotoxic neural injury but, as proposed for immune complex-induced injury to lung and skin 23, directly toxic actions of N O or its oxidative metabolites may be involved.
1 Beavo, J.A. and Reifsnyder, D.H., Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors, Trends Pharmacol. Sci., 11 (1990) 150-155. 2 Castle, R.N., The Chemistry of Heterocyclic Compounds, Vol. 27, Wiley Interscience, New York, 1973, p. 610 3 Choi, D.W., Koh, J.-Y. and Peters, S., Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists, J. Neurosci., 8 (1988) 185-196. 4 Curran, R.D., Ferrari, F.K., Kispert, P.H., Stadler, J., Stuehr, D.J., Simmons, R.L. and Billiar, T.R., Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis, FASEB J., 5 (1991) 2085-2092. 5 Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 6368-6371. 6 Diamond, J. and Chu, E.B., A novel cyclic GMP-lowering agent, LY83583, blocks carbachol-induced cyclic GMP elevation in rabbit atrial strips without blocking the negative inotropic effects of carbachol, Can. J. Physiol. Pharmacol., 63 (1985) 908911. 7 Doerner, D. and Alger, B.E., Cyclic GMP depresses hippocampal Ca2+ current through a mechanism independent of cGMP-dependent protein kinase, Neuron, 1 (1988) 693-699. 8 Furchgott, R.F. and Jothianandan, D., Endothelium-dependent
and -independent vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light, Blood Vessels, 28 (1991) 52-61. Garg, U.C. and Hassid, A., Nitric oxide-generating vasodilators inhibit mitogenesis and proliferation of BALB/C 3T3 fibroblasts by a cyclic GMP-independent mechanism, Biochern. Biophys. Res. Commun., 171 (1990) 474-479. Garg, U.C. and Hassid, A., Nitric oxide decreases cytosolic free calcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism, J. Biol. Chem., 266 (1991) 9-12. Garthwaite, G. and Garthwaite, J., Cyclic GMP and cell death in rat cerebellar slices, Neuroscience, 26 (1988) 321-326. Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-68. Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. Goy, M.F., cGMP: the wayward child of the cyclic nucleotide family, Trends Neurosci., 14 (1991) 293-299, Hartley, D.M. and Choi, D.W., Delayed rescue of N-methylD-aspartate receptor-mediated neuronal injury in cortical culture, J. Pharmacol. Exp. Ther., 250 (1989) 752-758. Hidaka, H., Inagaki, M., Kawamoto, S. and Sasaki, Y., Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nu-
Supported by grants from the USPHS (AA07032 and NS14543), the Dr. Louis Sklarow Memorial Fund, and the UCSF Academic Senate Committee on Research (D.A.G.), and by fellowships from the American Academy of Neurology and the American Heart Association, California Affiliate and Alameda County Chapter (J.C.).
9
10 11 12 13
14 15 16
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17
18
19
20
21
22
cleotide dependent protein kinase and protein kinase C, Biochemistry, 23 (1984) 5036-41. Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 2811-2814. Knowles, R.G., Palacios, M., Palmer, R.M.J. and Moncada, S., Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 5159-5162. Koh, J.Y. and Choi, D.W., Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay, J. Neurosei. Methods, 20 (1987) 83 -90. Koh, J.Y., Peters, S. and Choi, D.W., Neurons containing NADPH-diaphorase are selectively resistant to quinolinate toxicity, Science, 234 (1986) 73-76. Lustig, H.S., Chan, J. and Greenberg, D.A., Ethanol inhibits excitotoxicity in cerebral cortical cultures, Neurosci. Lett., 135 (1992) 259-261. MacIntyre, I., Zaidi, M., Alam, A.S.M.T., Datta, H.K., Moonga, B.S., Lidbury, ES., Hecker, M. and Vane, J.R., Osteoclastic inhibition: an action of nitric oxide not mediated by cyclic GMP, Proc. Natl. Aead. Sci. U.S.A., 88 (1991) 29362940.
23 Mulligan, M.S., Hevel, J.M., Marietta, M.A. and Ward, P.A., Tissue injury caused by deposition of immune complexes is e-arginine dependent, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 6338-6342. 24 Simon, R.R, Swan, J.H., Griffiths, T. and Meldrum, B.S., Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850-852. 25 Southam, E. and Garthwaite, J., Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices, Neurosci. Lett., 130 (1991) 107-111. 26 Tremblay, J., Gerzer, R. and Hamet, P., Cyclic GMP in cell function. In P. Greengard and G.A. Robison (Eds.), Advances in Second Messenger and Phosphoprotein Research, Vol. 22, Raven, New York, 1988, pp. 319-383. 27 Weglarz, L. and Bartosz, G., Spin label detection and free radical nature of DNA damage by hydralazine, Int. J. Biochem., 23 (1991) 663-667. 28 Weill, C.L. and Greene, D.P., Prevention of natural motoneurone cell death by dibutyryl cyclic GMP, Nature, 308 (1984) 452-454. 29 Yamamoto, K. and Kawanishi, S., Free radical production and site-specific DNA damage induced by hydralazine in the presence of metal ions or peroxidase/hydrogen peroxide, Biochem. Pharmacol., 41 (1991) 905-914.
343
BRES 25150
Cyclic GMP modulators and excitotoxic injury in cerebral cortical cultures Heather S. Lustig, Kristine L. von Brauchitsch, Jane Chan and David A. Greenberg Department of Neurology, University of California and San Francisco General Hospital, San Francisco, CA 94110 (USA)
(Accepted 21 January 1992) Key words: Cyclic GMP; Excitotoxicity; Cell culture; N-Methyl-D-aspartate; Sodium nitroprusside; Guanylate cyclase; Protein kinase;
Phosphodiesterase
N-Methyl-D-aspartate (NMDA) receptor activation generates nitric oxide (NO) and cyclic GMP (cGMP) and produces 'excitotoxic' neuronal injury. To examine the possible role of cGMP in excitotoxicity, we evaluated the effects of agents that stimulate or inhibit cGMP activity on the release of lactate dehydrogenase from neuron-enriched cortical cultures, cGMP analogs exhibited no toxicity, and inhibitors of guanylate cyclase or of cGMP-dependent enzymes failed to protect cultures from the toxic effects of NMDA or the NO donor sodium nitroprusside. These findings argue against a role for cGMP in the pathogenesis of excitotoxic neuronal injury. The action of excitatory amino acid neurotransmitters on N M D A receptors contributes to ischemic and other forms of 'excitotoxic' neuronal injury. Evidence for such a contribution includes the toxicity of N M D A in neuronal cultures in vitro 15 and the ability of N M D A antagonists to reduce ischemic cerebral damage in vivo 24. N M D A receptor activation stimulates production of the second messenger nitric oxide (NO), which promotes the synthesis of cGMP by soluble guanylate cyclase (GC) 13. NO appears to have a role in mediating the toxic effect of N-methyl-D-aspartate (NMDA) on cortical neurons because toxicity can be reproduced by the NO donor, sodium nitroprusside (SNP) 5. Like N M D A , SNP also stimulates cGMP production by neurons 5'13, but whether cGMP is involved in the toxicity of N M D A or SNP is uncertain. Little direct evidence supports a neurotoxic role for cGMP, and several studies suggest that cGMP could be neuroprotective. For example, the cGMP analog dibutyryl-cGMP interferes with the naturally occurring death of motor neurons in embyronic chick spinal cord 2s and inhibitors of GC cause death of granule cell neurons in cerebellar slices H. Nevertheless, the possibility that cGMP is involved in excitotoxicity is of interest, partly because it might explain the relative resistance of NOproducing neurons 12. Thus, neurons expressing N M D A receptors and NO synthase might be protected by receptor-stimulated elevation of free intracellular Ca 2÷ (Ca2+i), which at high concentrations inhibits GC 18,
while such protection would not occur in neighboring neurons into which NO diffuses 13. A variety of mechanisms could underlie cGMP toxicity, since cGMP is involved in diverse neurocellular functions, including the gating of ion channels and the regulation of protein kinases and cyclic nucleotide phosphodiesterases (PDEs) 14. To investigate the possible role of cGMP in excitotoxic injury, we examined the effects of cGMP analogs, and inhibitors of GC or of cGMP-dependent enzymes, in neuron-enriched cultures prepared from rat cerebral cortex. Release of lactate dehydrogenase (LDH) was used as an index of neuronal injury 19 induced by N M D A or SNP. Drugs were purchased from Sigma Chemical Co. (St. Louis, MO), except for 6-anilinoquinoline-5, 8-quinone (LY 83583) and N-[2-(methylamino)-ethyl]-5-isoquinoline-sulfonamide dihydrochloride (H-8), which were from Calbiochem Corporation (LaJolla, CA), and N-(2-aminoethyl)-5-isoquinoline-sulfonamide dihydrochloride (H9), quazinone and dipyridamole, which were from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). Cultures were prepared from cerebral cortex of 16-17 day Sprague-Dawley rat embryos as described previously 21. Exposure to cytosine arabinoside on day 5 in vitro resulted in cultures containing - 8 5 % neuronspecific enolase-immunoreactive cells on day 12, when experiments were performed. Cultures were exposed to drugs for 20 min in (mM) NaC1 120, KC1 5.4, CaC12 1.8, glucose 15 and Tris-HC1 25 (exposure buffer; p H 7.4 at
Correspondence: D.A. Greenberg, Department of Neurology, University of California, 4M62 San Francisco General Hospital, San Francisco, CA 94110, USA. Fax: (1) (415) 826-1313.
344 37°C). Toxicity was assessed 24 h after drug e x p o s u r e by m e a s u r i n g L D H released into the m e d i u m t9 using a commercially available s p e c t r o p h o t o m e t r i c assay kit
tion b e t w e e n L D H release a n d loss of the ability of cult u r e d cortical n e u r o n s to exclude T r y p a n blue dye over a wide range of N M D A c o n c e n t r a t i o n s (r = 0.957; unp u b l i s h e d data).
(340-LD, Sigma C h e m i c a l Co., St. Louis, M O ) . L D H m e a s u r e m e n t s were c o n d u c t e d in triplicate or q u a d r u p l i cate a n d r e p e a t e d o n m u l t i p l e platings, a n d L D H release was expressed as a p e r c e n t of the total L D H activity re-
T h e c G M P analogs d i b u t y r y l - c G M P a n d 8 - b r o m o c G M P had n o effect o n L D H release. L D H release was also u n c h a n g e d following exposure to 8 - b r o m o i n o s i n e 3',
leased from cultures by freeze-thawing. D a t a are rep o r t e d as m e a n values + S . E . M . T h e significance of dif-
5'-cyclic m o n o p h o s p h a t e ( 8 - b r o m o - c l M P ) , which mimics some actions of c G M P 7. H y d r a l a z i n e , which has b e e n r e p o r t e d to activate soluble G C 26, increased L D H re-
ferences b e t w e e n m e a n s was assessed b y S t u d e n t ' s t-test for single c o m p a r i s o n s a n d by analysis of variance followed by post hoc S t u d e n t - N e w m a n - K e u l s tests for m u l t i p l e c o m p a r i s o n s , with P < 0.05 c o n s i d e r e d statisti-
lease. H o w e v e r , the m e c h a n i s m u n d e r l y i n g hydralazine toxicity is u n c l e a r since, in some systems, hydralazine has c G M P - i n d e p e n d e n t toxic effects m e d i a t e d t h r o u g h d a m a g e to D N A 7'29. D i p y r i d a m o l e , which inhibits the
cally significant. As shown in Table I, control cultures released a b o u t 20% of their total L D H c o n t e n t over 24 h. L D H release increased m o r e t h a n 2-fold following e x p o s u r e to 1 m M N M D A a n d almost 3-fold following exposure to 1 m M SNP ( P < 0.001); we f o u n d previously that the toxic effects of b o t h N M D A a n d SNP were m a x i m a l at this conc e n t r a t i o n (Lustig et al., s u b m i t t e d for p u b l i c a t i o n ) .
hydrolysis of c G M P by cGMP-specific (type V) P D E 1, did n o t affect L D H release. N e i t h e r the c G M P analogs n o r d i p y r i d a m o l e r e d u c e d the toxicity of N M D A or SNP, while hydralazine decreased SNP toxicity, p e r h a p s due to its ability to b i n d to SNP 2. Several G C inhibitors increased L D H release. These i n c l u d e d M e t h y l e n e blue, h y d r o x y l a m i n e , a n d N - m e t h -
L D H release has b e e n s h o w n to reflect the e x t e n t of m o r p h o l o g i c N M D A toxicity in cortical cultures in o t h e r studies 3, a n d we have f o u n d a similarly strong correla-
y l h y d r o x y l a m i n e , which are also toxic to g r a n u l e cells in cerebellar slices u , a n d LY 835836. H o w e v e r , the toxicity of these agents c a n n o t necessarily be a t t r i b u t e d to inhi-
TABLE I Effects of cGMP modulators on L D H release from cortical cultures
LDH release is expressed as a percent of total LDH released by freeze-thawing. Data shown are means + S.E.M. from the number of cultures indicated in brackets. Treatment
Treatment alone
Treatment + NMDA (1 mM)
Treatment + SNP (1 mM)
None
23 + 1 [141]
54 _+ 3 [39]
65 + 2 [36]
-
29 + 2 [16] 21 + 3 [11] 22 + 2 [11]
52 _+ 6 [7] 43 + 2 [7] 45 + 4 [8]
61 + 3 [12] 61 + 2 [11] 62 + 1 [11]
-
37 + 2 [25]*
43 + 4 [15]
44 + 4 [22]*
-
24 _+ 4 [15]
50 + 5 [8]
63 _+ 5 [12]
-
44 66 57 39 40
54 53 60 48 52
68 82 71 78 61
Activators cGMP analogs Dibutyryl-cGMP (1 mM) 8-Bromo-cGMP (1 mM) 8-Bromo-clMP (1 mM) Guanylate cyclase activator Hydralazine (1 mM) cGMP-specific (type V) PDE inhibitor Dipyridamole (10/tM) Inhibitors Guanylate cyclase inhibitors Methylene blue (10 #M) LY 83583 (1 mM) Hydroxylamine (1 mM) N-Methylhydroxylamine (1 mM) KC1 (50 mM) depolarization cGMP-dependent protein kinase inhibitors H-8 (100 ~M) H-9 (100 ~M) cOMP-inhibited (type III) PDE inhibitor Quazinone (10 ~M) *P < 0.05 relative to 'None' (Student-Newman-Keuls).
+ + + + +
3 4 4 5 2
[221" [161" [20]* [14]* [231"
+ + + + +
8 7 6 5 8
[lll [131 [6] [8] [81
-+ 5 -+ 5 _+ 4 ___6 + 3
Treatment + dibutyryl cGMP (1 mM)
I141 [131 16] [6] [81
49 70 70 44 34 -
26 + 4 [10] 41 _+ 7 [12]*
41 + 5 [10] 67 + 5 [91
59 + 9 [71 74 _+ 4 [12]
28 + 5 [131
56 + 2 [9]
61 ± 4 [121
-
+ + + + +
1 7 9 8 3
[4] [3] [10] [3] [4]
345 bition of GC, since hydroxylamine can be metabolized to N O 25 and LY 83583 generates superoxide radicals 8. Moreover, L D H release was not reduced by adding 1 mM dibutyryl c G M P to cultures treated with G C inhibitors. None of the G C inhibitors reduced the toxicity of N M D A or SNP. A small percentage of cultured cortical neurons express the enzyme N A D P H diaphorase, which has been identified as an N O synthase 17, and are comparatively resistant to N M D A toxicity 2°. Considering the apparent role of N O as a mediator of N M D A toxicity 5, this seems paradoxical. However, N M D A receptor activation elevates [Ca2+]i , which can inhibit soluble G C TM. Accordingly, it has been suggested that N M D A receptor-mediated increases in [Ca2+]i may protect NO-producing neurons from cGMP-dependent toxicity, while N O kills neighboring cells into which it diffuses, and in which [Ca2+]i is not elevated ~2. If this were the case, then increasing [Ca2+]i by an N M D A - i n d e p e n d e n t mechanism might protect against N M D A toxicity in the larger percentage of neurons that do not produce NO. To investigate this possibility, some cultures were depolarized by increasing the concentration of KC1 in the exposure buffer to 50 mM. Microspectrofluorometry on single cultured cortical neurons using the Ca 2+ indicator indo-1 showed that 50 mM KC1 increased [Ca2+]i from a resting value of 145 + 13 nM to 491 + 46 nM (n = 14, P < 0.001), which is within the range of [Ca2+]i reported to inhibit G C TM. However, KCI depolarization failed to protect against N M D A or SNP toxicity. Known intracellular targets for the effects of c G M P include cGMP-dependent protein kinase and cGMP-
stimulated (type II) and cGMP-inhibited (type III) cyclic nucleotide P D E s TM. If cGMP-dependent enzymes were involved in excitotoxicity, then inhibitors of these enzymes might reduce the toxic effects of N M D A and SNP. However, H-8 and H-9, protein kinase inhibitors with preferential high affinity for cGMP-dependent protein kinase 16, had no effect on N M D A or SNP toxicity, and H-9 was itself toxic. Selective inhibitors of cGMPstimulated (type II) P D E have not been described, but the selective type III (cGMP-inhibited) P D E inhibitor quazinone 1 was also ineffective as a neuroprotective agent. The major finding of this study is that the toxic effects of N M D A and SNP on cortical cultures are neither reproduced by cGMP analogs nor prevented by inhibitors of G C or of cGMP-dependent effector enzymes. These observations suggest that although both N M D A and SNP are capable of stimulating c G M P synthesis s, c G M P is unlikely to mediate their neurotoxic effects. The effects of N O on [Ca2+]i , mitogenesis, and proliferation in fibroblasts 9'1°, protein synthesis in hepatocytes 4 and osteoclast function 22 have also been shown to be independent of cGMP. O u r findings do not identify the specific mechanism underlying NO-mediated excitotoxic neural injury but, as proposed for immune complex-induced injury to lung and skin 23, directly toxic actions of N O or its oxidative metabolites may be involved.
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and -independent vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light, Blood Vessels, 28 (1991) 52-61. Garg, U.C. and Hassid, A., Nitric oxide-generating vasodilators inhibit mitogenesis and proliferation of BALB/C 3T3 fibroblasts by a cyclic GMP-independent mechanism, Biochern. Biophys. Res. Commun., 171 (1990) 474-479. Garg, U.C. and Hassid, A., Nitric oxide decreases cytosolic free calcium in Balb/c 3T3 fibroblasts by a cyclic GMP-independent mechanism, J. Biol. Chem., 266 (1991) 9-12. Garthwaite, G. and Garthwaite, J., Cyclic GMP and cell death in rat cerebellar slices, Neuroscience, 26 (1988) 321-326. Garthwaite, J., Glutamate, nitric oxide and cell-cell signalling in the nervous system, Trends Neurosci., 14 (1991) 60-68. Garthwaite, J., Charles, S.L. and Chess-Williams, R., Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain, Nature, 336 (1988) 385-388. Goy, M.F., cGMP: the wayward child of the cyclic nucleotide family, Trends Neurosci., 14 (1991) 293-299, Hartley, D.M. and Choi, D.W., Delayed rescue of N-methylD-aspartate receptor-mediated neuronal injury in cortical culture, J. Pharmacol. Exp. Ther., 250 (1989) 752-758. Hidaka, H., Inagaki, M., Kawamoto, S. and Sasaki, Y., Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nu-
Supported by grants from the USPHS (AA07032 and NS14543), the Dr. Louis Sklarow Memorial Fund, and the UCSF Academic Senate Committee on Research (D.A.G.), and by fellowships from the American Academy of Neurology and the American Heart Association, California Affiliate and Alameda County Chapter (J.C.).
9
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