Eu ropean Journal of Pharmacology, 209 (1991 ) 123-125 ¢'~ 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50
Improved posthypoxic recovery with a membrane-permeable calpain inhibitor L a u r e n A r l i n g h a u s , S h u j a a t h M e h d i l and Kevin S. L e e Department of Neurological Surgery, Health Sciences" Center, Unicersity of Virginia, Charlottesl,ille, VA 22908, U.S.A. and I Marion Merrell Dow Research Institute, Cincinnati, OH 45215, U.S.A. Received 22 August 1991, revised MS received 8 October 199l, accepted 22 October 1991
In vitro h i p p o c a m p a l slices from adult rats were subjected to t r a n s i e n t hypoxia in the p r e s e n c e of a cell-penetrating, calpain inhibitor ( C b z - V a l - P h e - H ; MDL-28170). T h e posthypoxic recovery of synaptic potentials was greatly improved in p r o t e a s e i n h i b i t o r - t r e a t e d slices relative to control slices. T h e s e findings s u p p o r t a role for calcium-activated proteolysis in the process of hypoxic pathophysiology.
Neuroprotection; Calpain inhibitor; Hypoxia; Hippocampal slice; Proteolysis; (Rat)
Elevated intracellular calcium and calcium-activated proteolysis have been implicated in the process of hypoxic/ischemic neuronal death (Siesj6 and Bengtsson, 1989; Seubert et al., 1989; Arai et al., 1990, in press; Lee et al., 1991). Critical cytoskeletal proteins such as spectrin and MAP2 are among the preferred substrates for calcium-activated proteases (calpains) and rapid proteolysis of these substrates has been demonstrated following neurodegenerative ischemic or hypoxic events (Kuwaki et al., 1989; Seubert et al., 1989; Arai et al., in press). This ischemic/hypoxic proteolytic response can be inhibited by treatment with the protease inhibitors, leupeptin and calpain inhibitor I. These inhibitors exert a neuroprotective effect, reducing post-hypoxic pathophysiology and post-ischemic cell death (Arai et al., 1990; Lee et al., 1991). To date, the potential for targeting calpain as a therapeutic treatment has been limited by the lack of specific calpain inhibitors that readily permeate cellular membranes. Recently, a new inhibitor of calpain, Cbz-ValPhe-H (MDL-28170), that exhibits substantial membrane permeability was introduced (Mehdi, 1991). CbzVal-Phe-H is an N-protected dipeptide aldehyde which exhibits potent and rather specific inhibition of calpain in intact cells (Mehdi, 1991). The studies presented here examined the effect of Cbz-Val-Phe-H on hypoxic
Correspondence to: K.S. Lee, Department of Neurological Surgery, Box 420, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, U.S.A. Tel. 1.804.924 0262, fax 1.804.982 3829.
cell death to ascertain whether this specific and permeable inhibitor of calpain is capable of modulating the outcome of transient hypoxia in vitro.
2. Materials and methods
Adult Sprague-Dawley rats (250-300 g) were sacrificed by decapitation, their brains removed rapidly and hippocampal slices prepared as described previously (Arai et al., 1990). Four to eight slices per preparation were maintained at interface in a recording chamber with artificial cerebrospinal fluid (ACSF) flowing continuously at a rate of approximately 0.5 ml/min. Electrophysiological recordings from the slices were initiated one hour after sacrifice by placing a stimulating electrode in the stratum radiatum near the junction of CA1 and CA3 and placing a recording electrode in the stratum radiatum of CAlb. These placements allow activation of the combined Schaffer collateral and commissural afferents to the apical dendrites of CA1 pyramidal ceils. Evoked synaptic responses (field excitatory postsynaptic potentials: fEPSPs) were then tested in a single slice for at least 10 min to establish the stability of the response. During this initial phase of testing, stimulation intensity was adjusted to elicit a submaximal fEPSP and stimuli were delivered once per 15 s. ACSF containing 100 /xM Cbz-Val-Phe-H and 0.2% DMSO was then perfused through the chamber for 1 h. Control slices were perfused with 0.2% DMSO for 1 h. Cbz-Val-Phe-H was supplied by Marion Merrell Dow, and the structure of this compound has been presented (Mehdi, 1991). During this treatment period,
124 fEPSPs were tested once per 15 s in a single slice. After 1 h, 4 - 8 slices per preparation were briefly tested to establish the m a x i m u m obtainable f E P S P amplitude in each slice. The stimulating and recording electrodes were then re-positioned in the original slice examined during the preceding hour and testing was resumed during the initial phases of hypoxia to determine the delay to disappearance of the f E P S P and disappearance of the fiber volley. Transient hypoxia was administered by replacing the flow of 95% O 2 into the c h a m b e r with 99% N 2. T h e flow of N 2 was continued until 3 min following disappearance of the fiber volley after which the flow of O 2 was resumed. T h e fiber volley is a short latency c o m p o n e n t of the evoked response which represents synchronous axonal discharge in response to stimulation. The fiber volley is more resistant to hypoxia than the f E P S P and, as described elsewhere (Arai et al., 1990), consistent levels of recovery from hypoxia can be achieved by standardizing the period between fiber volley disappearance and reoxygenation. A period of 3 rain between fiber volley disappearance and reoxygenation was chosen on the basis of previous studies in o r d e r to provide a m o d e r a t e to severe hypoxic challenge to the slices (Arai et al., 1990). All of the slices tested prior to hypoxia were retested 30 and 60 rain following the resumption of oxygen for the maximum obtainable f E P S P amplitude. T h e data for each slice were expressed as a percentage recovery of the maximum f E P S P amplitude following hypoxia.
3. Results In 11 experiments (6 control and 5 Cbz-Val-Phe-Htreated), a single slice was tested continuously (at 15 s intervals) beginning approximately 1 h after slice preparation and continuing for 70 min. T h e perfusion of A C S F containing Cbz-Val-Phe-H, or 0.2% D M S O in control slices, was begun after 10 min of baseline f E P S P testing; perfusion of the drug or vehicle was continued for 60 min. Synaptic responses (fEPSPs) were stable in C b z - V a l - P h e - H - t r e a t e d and control slices during this entire period. Just prior to and during the initial phases of the hypoxic episode, single slices were
50 >.. er" ~.~ ::>
O ~2 rv
Z kA or2
10 \\\\\ Control 30
Treated I h
Fig. 1. The effect of Cbz-VaI-Phe-H on the recovery of fEPSPs in hippocampal slices following hypoxia. The maximum fEPSP was tested (i) prior to hypoxia, (ii) 30 min after reoxygenation and (iii) 60 min after reoxygenation in 52 control and 45 Cbz-Val-Phe-H-treated slices. The percentage recovery relative to prehypoxic levels was calculated for each slice. The values shown are the means and S.E.M. for all slices in a group. Cbz-Val-Phe-H-treated slices exhibited significantly greater recovery at both the 30 and 60 min sampling periods (P < 0.001 on a two-tailed t-test).
again tested to ascertain the delays to fEPSP disapp e a r a n c e and fiber volley disappearance. Both groups of slices exhibited similar immediate changes in response to hypoxia. T h e delay to loss of fEPSPs and the delay to fiber volley disappearance during hypoxia did not differ significantly between Cbz-Val-Phe-H-treated and control slices (table 1). A total of 52 control slices from 9 animals and 44 C b z - V a l - P h e - H - t r e a t e d slices from 7 animals were tested for recovery from hypoxia. Control slices exhibited limited recovery following hypoxia. Sixty minutes following reoxygenation only 21% of control slices (11 of 52) exhibited maximum f E P S P responses of greater than 50% of their prehypoxic levels. In contrast, 73% of Cbz-Val-Phe-N-treated slices (33 of 45) displayed maximal responses of greater than 50% of their prehypoxic levels. The average values for post-hypoxic recovery in the two groups of slices are shown in fig. 1. C b z - V a l - P h e - H - t r e a t e d slices exhibited significantly greater recovery at both the 30 and 60 rain post-hypoxia sampling times.
TABLE 1 Delay to disappearance of responses during hypoxia (s). The delays to disappearance of the fEPSP and fiber volley during hypoxia are shown. Values are means -+S.E.M. Neither measure differed significantly between control and Cbz-VaI-Phe-H-treated groups (two-tailed t-test). fEPSPdisappearance Fiber volley disappearance
Control 134_+ 6 234 + 15
Treated 139+ 8 266 _4_13
4. Discussion Increased intracellular free calcium is t h o u g h t to play a central role in ischemic and hypoxic cell death (Choi, 1990; Siesj6 and Bengtsson, 1989). O n e m a n n e r in which calcium might exert a neurotoxic effect is via excessive activation of the calpains. Preferred substrates for these calcium-activated proteases include
125 m a j o r c y t o s k e l e t a l p r o t e i n s a n d key r e g u l a t o r y e n z y m e s ( M u r a c h i , 1989) which a r e p r e s u m a b l y essential to m a i n t a i n c e l l u l a r viability. R e c e n t studies i n d i c a t e t h a t t r e a t m e n t with p r o t e a s e i n h i b i t o r s not only a t t e n u a t e s the p r o t e o l y t i c r e s p o n s e to t r a n s i e n t i s c h e m i a or hypoxia b u t also a m e l i o r a t e s c e l l u l a r p a t h o l o g y . T h e inhibitors s t u d i e d to d a t e , i.e. c a l p a i n i n h i b i t o r I, leup e p t i n a n d C b z - V a l - P h e - H , are all inhibitors of calpain, however, effects on o t h e r p r o t e a s e s c a n n o t be r u l e d o u t entirely. I n h i b i t i o n o f p r o t e o l y s i s t h e r e f o r e a p p e a r s to be a p r o m i s i n g strategy for n e u r o p r o t e c t i o n in i s c h e m i c / h y p o x i c cell d e a t h a n d m a y also be useful for o t h e r t r a u m a t i c c e l l u l a r r e s p o n s e s that a r e aggrav a t e d by excessive p r o t e o l y t i c activity. T h e utility o f this t h e r a p e u t i c strategy will u l t i m a t e l y d e p e n d on the availability of specific a n d p e r m e a b l e p r o t e a s e inhibitors c a p a b l e of m o d u l a t i n g the isc h e m i c / h y p o x i c o u t c o m e . T h e studies p r e s e n t e d h e r e d e m o n s t r a t e that C b z - V a l - P h e - H , a r e c e n t l y d e v e l o p e d i n h i b i t o r of calpain, r e d u c e s hypoxic n e u r o n a l d a m a g e in h i p p o c a m p a l slices. D u e to its m o l e c u l a r c h a r a c t e r i s tics, this i n h i b i t o r may be of p a r t i c u l a r i n t e r e s t as a p o t e n t i a l n e u r o p r o t e c t i v e agent. C b z - V a l - P h e - H is a c e l l - p e n e t r a t i n g i n h i b i t o r of c a l p a i n ( M e h d i , 1991) which may be c a p a b l e o f passing the b l o o d b r a i n b a r rier ( M e h d i , u n p u b l i s h e d observations). In a d d i t i o n to its e n h a n c e d p e r m e a b i l i t y , C b z - V a l - P h e - H is an ext r e m e l y p o t e n t i n h i b i t o r of calpain. In in vitro assays, the r e p o r t e d IC50 values for c a l p a i n inhibition by CbzV a l - P h e - H , c a l p a i n inhibitor I a n d l e u p e p t i n are 0.1, 0 . 4 - 0 . 6 a n d 0 . 2 - 1 ~ M , respectively (as r e v i e w e d by W a n g , 1990). In an intact cell p r e p a r a t i o n (rat e r y t h r o cytes),the ICs0 for C b z - V a l - P h e - H is 1 /~M ( M e h d i et al., 1988). T h e p r e s e n t studies utilized a h i g h e r c o n c e n t r a t i o n b e c a u s e of the n a t u r e of the in vitro slice p r e p a r a t i o n w h e n m a i n t a i n e d at i n t e r f a c e with the a t m o s p h e r e of the c h a m b e r . In this m o d e l system, the A C S F is p e r f u s e d only at the base o f the slice. C o n s e quently, a d r u g m u s t diffuse t h r o u g h several h u n d r e d m i c r o m e t e r s of tissue to arrive at the cells from which
the r e c o r d i n g s a r e taken. T o e n s u r e a d e q u a t e availability t h r o u g h o u t t h e slice, a c o n c e n t r a t i o n of 100 ~ M C b z - V a l - P h e - H was utilized in t h e s e studies. T h e pot e n t i a l b e n e f i t of this c o m p o u n d as a n e u r o p r o t e c t i v e a g e n t will clearly r e q u i r e e v i d e n c e of its efficacy in vivo; n o n e t h e l e s s , the c u r r e n t studies suggest that it m a y b e a useful c o m p o u n d for limiting the d a m a g i n g effects of i s c h e m i a in the c e n t r a l nervous system.
References Arai, A., M. Kessler, K. Lee and G. Lynch, 1990, Calpain inhibitors improve the recovery of synaptic transmission from hypoxia in hippocampal slices, Brain Res. 532, 63. Arai, A., P. Vanderklish, M. Kessler, K. Lee and G. Lynch, A brief period of hypoxia causes proteolysis of cytoskeletal proteins in hippocampal slices, Brain Res. (in press). Choi, D., 1990, Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, 1988, Trends Neurol. Sci. 11,465. Kuwaki, T., H. Satoh, T. Ono, F. Shibayama, T. Yamashita and T. Nishimura, 1989, Nilvadipine attenuates ischemic degradation of gerbil brain cytoskeletal proteins, Stroke 20, 78. Lee, K., S. Frank, P. Vanderklish, A. Arai and G. Lynch, 1991, Inhibition of proteolysis protects hippocampal neurons from ischemia, Proc. Natl. Acad. Sci. (U.S.A.) 88, 7233. Mehdi, S., 1991, Cell-penetrating inhibitors of calpain, Trends Biochem. Sci. 16, 150. Mehdi, S., M. Angelastro, J. Wiseman and P. Bey, 1988, Inhibition of the proteolysis of rat erythrocyte membrane proteins by a synthetic inhibitor of calpain, Biochem. Biophys. Res. Commun. 157, 1117. Murachi, T., 1989, lntracellular regulators' system inw)lving calpain and calpastatin, Biochem. Int. 18. 263. Seubert, P., K. Lee and G. Lynch, 1989, Cerebral ischemia triggers NMDA receptor-linked cytoskeletal proteolysis in hippocampus, Brain Res. 492, 366. Siesj6, B. and F. Bengtsson, 1989, Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis, J. Cereb. Blood Flow Metab. 9, 127. Wang, K., 1990, Developing selective inhibitors of calpaim Trends Pharmacol. Sci. 11, 139.