The polyamine synthesis inhibitor α-difluoromethylornithine blocks NMDA-induced neurotoxicity

The polyamine synthesis inhibitor α-difluoromethylornithine blocks NMDA-induced neurotoxicity

European Journal of Pharmacology, 182 (1990) 607-609 607 Elsevier EJP 0259R Rapid communication The polyamine synthesis inhibitor a-difluoromethyl...

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European Journal of Pharmacology,

182 (1990) 607-609

607

Elsevier EJP 0259R Rapid communication

The polyamine synthesis inhibitor a-difluoromethylornithine blocks NMDA-induced neurotoxicity M a r y A n n K. Markwell, S. Paul Berger and Steven M. Paul Section on Molecular Pharmacology, Clinical Neuroscience Branch, National Institute of Mental Health, 9000 Rockoille Pike, Bethesda, MD 20892, U.S.A.

Received11 June 1990, accepted12 June 1990

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor has been implicated in the neuronal damage produced by a wide variety of environmental and metabolic insults to the CNS (Hartley and Choi, 1989). Although the neurotoxic properties of NMDA receptor agonists as well as the neuroprotective efficacy of NMDA receptor antagonists have been well-documented both in vitro and in vivo, the exact cellular mechanism(s) underlying NMDA receptor-mediated neurotoxicity is still unknown. Recently, distinct binding sites for glycine and polyamines have been shown to be associated with the NMDA receptor channel complex (Ransom and Stec, 1988). These serve to allosterically enhance the binding of radiolabeled antagonists and to augment channel activation induced by agonists. Drugs that antagonize either the glycine or polyamine modulatory sites have been shown to block NMDA receptor-mediated neurotoxicity (Carter et al., 1988). These data, coupled with the finding of Siddiqui et al. (1988) that the NMDA-induced release of neurotransmitters from synaptosomes is dependent on polyamine synthesis, prompted a study of whether the neurotoxic (presumably post-synaptic) effects of NMDA may also involve a polyamine-dependent mechanism. We now report that the selective,

Correspondence to: S.M. Paul, Section on Molecular Phar-

macology, Clinical Neuroscience Branch, Building 10, Room 4N224, Bethesda,MD 20892, U.S.A.

enzyme-activated, irreversible inhibitor of ornithine decarboxylase, a-difluoromethylornithine (DFMO) blocks NMDA-induced neurotoxicity in cultured cortical neurons. Primary cultures of cerebral cortical neurons were prepared from the embryos of C57B1/6 mice (14-18 days gestation) according to the method described by Hartley and Choi (1989) with minor modifications. The cultures, which contained both neurons and astrocytes, were maintained in minimal essential medium containing 21 mM glucose, 2 mM glutamine, 10% fetal bovine serum, and 10% heat-inactivated horse serum for 8 days, then without fetal bovine serum until used on day 20. Neuronal viability was assessed by phase contrast microscopy and by measuring the high affinity binding of [3H]ouabain to neuronal Na+K ÷ ATPase. The latter has been recently documented to be a rapid and simple method for quantifying neurons in mixed cultures (Markwell et al., submitted). Exposure of cultured mouse cortical neurons to NMDA (100/~M) for 5 days resulted in an almost complete loss of neurons (as visually assessed using phase contrast microscopy) and a marked (>~ 95%) reduction in specific [3H]ouabain binding (fig. 1). Preincubation of cultures with DFMO (5 mM) for 1 h prior to the addition of NMDA blocked neuronal cell loss to the same degree as the noncompetitive N M D A receptor antagonist phencyclidine (PCP) (100 gM). Specific [3H]ouabain binding for both conditions was the same as in the

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Fig. 1. Effect of DFMO on NMDA-induced toxicity in cultured mouse cortical neurons. Neuronal survival was assessed by the specific binding of [3H]ouabain (mean+S.E.) 5 days after exposure of the cultures to the various drugs. Treatment with NMDA (100 #M) resulted in significant neuronal loss (Dunnett's comparison of means, n = 3 , ** P<0.01)). (A) One hour preincubation (followed by coincubation) with DFMO (5 mM) or PCP (100 #M) completely protected neurons against NMDA-induced neurotoxicity (P = N.S. vs. control). DFMO (5 raM) alone had no effect on neuronal viability or [3H]ouabain binding. The inclusion of 2% dialyzed horse serum had no effect on the blockade of NMDA-induced neurotoxicity by DFMO (B).

c o n t r o l cultures (fig. 1). D F M O a l o n e (fig. 1) o r P C P alone ( d a t a n o t shown) h a d n o effect on neuronal viability or [3H]ouabain binding. DFMO

at c o n c e n t r a t i o n s u p to 5 m M h a d no effect on [ 3 H ] M K - 8 0 1 b i n d i n g to N M D A r e c e p t o r s m e a s u r e d in r a t b r a i n m e m b r a n e s ( d a t a n o t shown). T h e s e d a t a d e m o n s t r a t e that p r e i n c u b a t i o n of m o u s e c o r t i c a l n e u r o n s with D F M O , a selective, e n z y m e - a c t i v a t e d , irreversible i n h i b i t o r of ornit h i n e d e c a r b o x y l a s e , the r a t e - l i m i t i n g e n z y m e in p o l y a m i n e b i o s y n t h e s i s , b l o c k s the n e u r o t o x i c i t y i n d u c e d b y e x p o s u r e to N M D A . A l t h o u g h unlikely, it is c o n c e i v a b l e that the b l o c k a d e of NMDA-induced neurotoxicity by DFMO may be u n r e l a t e d to i n h i b i t i o n of o r n i t h i n e d e c a r b o x y l a s e . H o w e v e r , D F M O , at the c o n c e n t r a t i o n used in o u r e x p e r i m e n t s , has b e e n p r e v i o u s l y s h o w n to m a r k e d l y r e d u c e the c o n c e n t r a t i o n o f p u t r e s c i n e a n d s p e r m i d i n e in p r i m a r y cultures of chick cortical neurons, w i t h o u t altering cellular m o r p h o l o g y o r the cellular c o n c e n t r a t i o n of p r o t e i n or R N A (Seiler et al., 1984). O u r findings suggest that: (1) a c t i v a t i o n of o r n i t h i n e d e c a r b o x y l a s e m a y represent a critical p o s t - r e c e p t o r event m e d i a t i n g the n e u r o t o x i c i t y of N M D A a n d t h a t (2) p o l y a m i n e s , either as a result of their r e c e n t l y d e s c r i b e d actions at N M D A r e c e p t o r s o r via their r e p o r t e d ability to release i n t r a c e l l u l a r C a 2+ ( S i d d i q u i et al., 1988), o r b o t h , m a y b e i n v o l v e d in N M D A i n d u c e d n e u r o t o x i c i t y . I n h i b i t o r s of p o l y a m i n e synthesis t h a t cross the b l o o d - b r a i n b a r r i e r may, therefore, r e p r e s e n t a novel class of n e u r o p r o t e c tive agents.

Acknowledgement The authors thank Dr. P. McCann of the MerreU Dow Research Institute for supplying the a difluoromethylornithine.

References Carter, D., J. Benavides, P. Legendre, J.D. Vincent, F. Noel, F. Thuret, K.G. Lloyd, S. Arbilla, B. Zikovic, E.T. MacKenzie, B. Scatton and S.Z. Langer, 1988, Ifenprodil and SI 82.0715 as cerebral anti-ischemic agents. II. Evidence for N-methyl-D-aspartate receptor antagonist properties, J. Pharmacol. Exp. Ther. 247, 1222. Hartley, D.M. and D.W. Choi, 1989, Delayed rescue of Nmethyl-D-aspartate receptor-mediated neuronal injury in cortical culture, J. Pharmacol. Exp. Ther. 250, 752.

609 Ransom, R.W. and N.L. Stec, 1988, Cooperative modulation at [3H]MK-801 binding to the NMDA receptor-ion channel complex by L-glutamate, glycine and polyamines, J. Neurochem. 51, 830. Seiler, N., S. Sarhan and B.F. Roth-Schechter, 1984, Polyamines and the development of isolated neurons in cell culture, Neurochem. Res. 9, 871.

Siddiqui, F., Z. Iqbqal and H. Koenig, 1988, Polyamine dependence on NMDA receptor-mediated Ca 2+ fluxes and transmitter release from rat hippocampus, Soc. Neurosci. Abstr. 14, 1048.