- Email: [email protected]
Potentiation by naloxone of reflex activity in the isolated spinal cord of Xenopus
European Journal of Pharmacology, 55 (1979) 431--433 © Elsevier/North-Holland Biomedical Press
431
Rapid communication POTENTIATION BY NALOXONE OF REFLEX ACTIVITY IN THE ISOLATED SPINAL CORD OF XENOPUS WOUT WOUTERS, MARIA C.E. ERMES-BUSIO and JOEP VAN DEN BERCKEN
Institute of Veterinary Pharmacology and Toxicology, University of Utrecht, Biltstraat 172, 3572 BP Utrecht, The Netherlands Received 5 April 1979, accepted 10 April 1979
Opiate receptors, and enkephalins which are endogenous ligands for these receptors, are found in many parts of the vertebrate nervous system (Snyder and Simantov, 1977). A high receptor density together with a high concentration of enkephalins occurs in the dorsal horn of the spinal cord and is probably associated with primary afferent nerve terminals and interneurons (Atweh and Kuhar, 1977; H5kfelt et al., 1977). The widespread distribution of the enkephalins makes it highly unlikely that their only physiological function is in the control of pain perception. Recently it has been suggested that the enkephalins act as inhibitory neurotransmitters (Frederickson, 1977). If the enkephalins have a more general physiological function, the application of pure narcotic antagonists such as naloxone should procedure significant effects. So far, however, evidence for such direct actions by narcotic antagonists is rather scarce (Frederickson, 1977). We report here that naloxone in concentrations starting as low as 5 × 10 -s M potentiated the reflex activity in the isolated spinal cord of Xenopus. This effect was concentrationdependent and could be reversed by D-alamet-enkephalinamide. The experiments were carried out on the isolated spinal cord of the clawed frog, Xenopus laevis, at a temperature of 20 + 1 ° C. The caudal part of the spinal cord with spinal roots IX and X was dissected by dorsal laminectomy from a spinal animal anaesthetized with
tricaine methanesulfonate (MS 222, Sandoz). The spinal cord was placed on its side in a central groove of an experimental chamber, which was continuously perfused with standard frog Ringer solution. The spinal roots were pulled through silicone-sealed slits on either side of the groove and mounted on a pair of silver wire electrodes. Either the IXth or Xth dorsal root was stimulated at 1 min -1 with supramaximum pulses of 0.1 msec duration. Ventral root responses were recorded from the ipsilateral ventral root by a differential, ac-coupled amplifier (time constant 0.1 sec), displayed on an oscilloscope and digitized. Five successive responses were averaged by computer and plotted. Naloxone hydrochloride (ACF) and D-ala-met-enkephalinamide (Beckman) were applied to the spinal cord by switching from normal superfusion to Ringer solution which contained the desired concentration of the drug. A single stimulus to the dorsal root evoked a synchronized monosynaptic response in the ventral root, followed by polysynaptic activity. Shortly after the application of naloxone, the amplitude of the ventral root response started to increase. The effect of different concentrations of naloxone on the peak amplitude of the monosynaptic response is illustrated in fig. 1. A concentration of 5 X 1 0 -s M produced a slight increase in the monosynaptic response, which did not differ significantly from the control experiments. Naloxone concentrations between 8 × 10 -s M and 5 X
432
300 200~ o
/
/
100 V////////////~A/fsZTJ
0 40 d
I'0 2'0 30 4'0
Fig. 1. P o t e n t i a t i o n o f t h e m o n o s y n a p t i c v e n t r a l r o o t r e s p o n s e o f t h e isolated spinal c o r d o f Xenopus b y various c o n c e n t r a t i o n s o f n a l o x o n e . N a l o x o n e was applied at t i m e 0 for a p e r i o d o f 35 rain as i n d i c a t e d b y t h e h a t c h e d bar. A m p l i t u d e s o f m o n o s y n a p t i c r e s p o n s e s are e x p r e s s e d as a p e r c e n t a g e of t h e a m p l i t u d e a t t i m e 0. (A) 5 x 10 -$ M, n = 2; (©) 8 × 10 -a M--5 × 10 -7 M, n = 5 a n d (~) 10 -6 M n a l o x o n e , n = 3. Ordinate: a m p l i t u d e (%);abscissa: time (min).
10 -7 M caused a marked potentiation of the monosynaptic response and after 1 0 - 6 M naloxone, its amplitude increased to 300% of the value prior to the application of naloxone. The effect of naloxone could n o t be reversed by washing with normal Ringer solution. Although the increase in amplitude leveled off, the monosynaptic response did n o t start to decrease, indicating that naloxone was n o t easily washed out. Therefore, an a t t e m p t was made to reverse the effect o f naloxone b y treating the preparation with D-ala-met-enkephalinamide, which on its own produces a potent suppression of spinal reflex activity that is rapidly reversed b y washing with normal Ringer (W. Wouters et al., in preparation). After application of D-ala-met-enkephalinamide at 10 -s M for 10 min, followed b y a 50 min washing period with normal Ringer, the amplitude of the monosynaptic response decreased to the control level from a value of 274 + 15% (mean + s.d.; n = 3) just before the application o f D-ala-met-enkephalinamide. The complete reversal of the naloxoneinduced potentiation b y a short treatment with D-ala-met-enkephalinamide strongly suggests that the effect of naloxone is mediated via specific opiate receptors. Naloxone m a y
potentiate the spinal reflex activity by antagonizing the inhibitory action of endogenous enkephalins or other endorphins which may be present in high levels because of the stress induced by the dissection. A dose-independent increase in spinal reflex activity has been observed in the acute spinal cat after i.v. injection of naloxone and may be due to a similar mechanism (Goldfarb and Hu, 1976). The present results clearly demonstrate that naloxone alone produces a significant effect in an isolated preparation and that this effect can be reversed b y D-ala-met-enkephalinamide.
Acknowledgement This w o r k was s u p p o r t e d in p a r t b y t h e F o u n d a t i o n f o r Medical R e s e a r c h F U N G 0 / Z W O .
References A t w e h , S.F. a n d M.J. K u h a r , 1 9 7 7 , A u t o r a d i o g r a p h i c l o c a l i z a t i o n of o p i a t e r e c e p t o r s in rat brain. I. Spinal c o r d a n d l o w e r m e d u l l a , B r a i n Res. 124, 53. F r e d e r i c k s o n , R.C.A., 1 9 7 7 , E n k e p h a l i n p e n t a p e p tides. A review o f c u r r e n t e v i d e n c e for a physiological role in v e r t e b r a t e n e u r o t r a n s m i s s i o n , Life Sci. 21, 23.
433 Goldfarb, J. and J.W. Hu, 1976, Enhancement of reflexes by naloxone in spinal cats, Neuropharmacology 15,785. HSkfelt, T., •. Ljungdahl, L. Terenius, R. Elde and G. Nilsson, 1977, Immunohistochemical analysis of peptide pathways possibly .related to pain and
analgesia: Enkephalin and substance P, Proc. Nat. Acad. Sci. U.S.A. 74, 3081. Snyder, S.H. and R. Simantov, 1977, The opiate receptor and opioid peptides, J. Neurochemistry 28, 13.
431
Rapid communication POTENTIATION BY NALOXONE OF REFLEX ACTIVITY IN THE ISOLATED SPINAL CORD OF XENOPUS WOUT WOUTERS, MARIA C.E. ERMES-BUSIO and JOEP VAN DEN BERCKEN
Institute of Veterinary Pharmacology and Toxicology, University of Utrecht, Biltstraat 172, 3572 BP Utrecht, The Netherlands Received 5 April 1979, accepted 10 April 1979
Opiate receptors, and enkephalins which are endogenous ligands for these receptors, are found in many parts of the vertebrate nervous system (Snyder and Simantov, 1977). A high receptor density together with a high concentration of enkephalins occurs in the dorsal horn of the spinal cord and is probably associated with primary afferent nerve terminals and interneurons (Atweh and Kuhar, 1977; H5kfelt et al., 1977). The widespread distribution of the enkephalins makes it highly unlikely that their only physiological function is in the control of pain perception. Recently it has been suggested that the enkephalins act as inhibitory neurotransmitters (Frederickson, 1977). If the enkephalins have a more general physiological function, the application of pure narcotic antagonists such as naloxone should procedure significant effects. So far, however, evidence for such direct actions by narcotic antagonists is rather scarce (Frederickson, 1977). We report here that naloxone in concentrations starting as low as 5 × 10 -s M potentiated the reflex activity in the isolated spinal cord of Xenopus. This effect was concentrationdependent and could be reversed by D-alamet-enkephalinamide. The experiments were carried out on the isolated spinal cord of the clawed frog, Xenopus laevis, at a temperature of 20 + 1 ° C. The caudal part of the spinal cord with spinal roots IX and X was dissected by dorsal laminectomy from a spinal animal anaesthetized with
tricaine methanesulfonate (MS 222, Sandoz). The spinal cord was placed on its side in a central groove of an experimental chamber, which was continuously perfused with standard frog Ringer solution. The spinal roots were pulled through silicone-sealed slits on either side of the groove and mounted on a pair of silver wire electrodes. Either the IXth or Xth dorsal root was stimulated at 1 min -1 with supramaximum pulses of 0.1 msec duration. Ventral root responses were recorded from the ipsilateral ventral root by a differential, ac-coupled amplifier (time constant 0.1 sec), displayed on an oscilloscope and digitized. Five successive responses were averaged by computer and plotted. Naloxone hydrochloride (ACF) and D-ala-met-enkephalinamide (Beckman) were applied to the spinal cord by switching from normal superfusion to Ringer solution which contained the desired concentration of the drug. A single stimulus to the dorsal root evoked a synchronized monosynaptic response in the ventral root, followed by polysynaptic activity. Shortly after the application of naloxone, the amplitude of the ventral root response started to increase. The effect of different concentrations of naloxone on the peak amplitude of the monosynaptic response is illustrated in fig. 1. A concentration of 5 X 1 0 -s M produced a slight increase in the monosynaptic response, which did not differ significantly from the control experiments. Naloxone concentrations between 8 × 10 -s M and 5 X
432
300 200~ o
/
/
100 V////////////~A/fsZTJ
0 40 d
I'0 2'0 30 4'0
Fig. 1. P o t e n t i a t i o n o f t h e m o n o s y n a p t i c v e n t r a l r o o t r e s p o n s e o f t h e isolated spinal c o r d o f Xenopus b y various c o n c e n t r a t i o n s o f n a l o x o n e . N a l o x o n e was applied at t i m e 0 for a p e r i o d o f 35 rain as i n d i c a t e d b y t h e h a t c h e d bar. A m p l i t u d e s o f m o n o s y n a p t i c r e s p o n s e s are e x p r e s s e d as a p e r c e n t a g e of t h e a m p l i t u d e a t t i m e 0. (A) 5 x 10 -$ M, n = 2; (©) 8 × 10 -a M--5 × 10 -7 M, n = 5 a n d (~) 10 -6 M n a l o x o n e , n = 3. Ordinate: a m p l i t u d e (%);abscissa: time (min).
10 -7 M caused a marked potentiation of the monosynaptic response and after 1 0 - 6 M naloxone, its amplitude increased to 300% of the value prior to the application of naloxone. The effect of naloxone could n o t be reversed by washing with normal Ringer solution. Although the increase in amplitude leveled off, the monosynaptic response did n o t start to decrease, indicating that naloxone was n o t easily washed out. Therefore, an a t t e m p t was made to reverse the effect o f naloxone b y treating the preparation with D-ala-met-enkephalinamide, which on its own produces a potent suppression of spinal reflex activity that is rapidly reversed b y washing with normal Ringer (W. Wouters et al., in preparation). After application of D-ala-met-enkephalinamide at 10 -s M for 10 min, followed b y a 50 min washing period with normal Ringer, the amplitude of the monosynaptic response decreased to the control level from a value of 274 + 15% (mean + s.d.; n = 3) just before the application o f D-ala-met-enkephalinamide. The complete reversal of the naloxoneinduced potentiation b y a short treatment with D-ala-met-enkephalinamide strongly suggests that the effect of naloxone is mediated via specific opiate receptors. Naloxone m a y
potentiate the spinal reflex activity by antagonizing the inhibitory action of endogenous enkephalins or other endorphins which may be present in high levels because of the stress induced by the dissection. A dose-independent increase in spinal reflex activity has been observed in the acute spinal cat after i.v. injection of naloxone and may be due to a similar mechanism (Goldfarb and Hu, 1976). The present results clearly demonstrate that naloxone alone produces a significant effect in an isolated preparation and that this effect can be reversed b y D-ala-met-enkephalinamide.
Acknowledgement This w o r k was s u p p o r t e d in p a r t b y t h e F o u n d a t i o n f o r Medical R e s e a r c h F U N G 0 / Z W O .
References A t w e h , S.F. a n d M.J. K u h a r , 1 9 7 7 , A u t o r a d i o g r a p h i c l o c a l i z a t i o n of o p i a t e r e c e p t o r s in rat brain. I. Spinal c o r d a n d l o w e r m e d u l l a , B r a i n Res. 124, 53. F r e d e r i c k s o n , R.C.A., 1 9 7 7 , E n k e p h a l i n p e n t a p e p tides. A review o f c u r r e n t e v i d e n c e for a physiological role in v e r t e b r a t e n e u r o t r a n s m i s s i o n , Life Sci. 21, 23.
433 Goldfarb, J. and J.W. Hu, 1976, Enhancement of reflexes by naloxone in spinal cats, Neuropharmacology 15,785. HSkfelt, T., •. Ljungdahl, L. Terenius, R. Elde and G. Nilsson, 1977, Immunohistochemical analysis of peptide pathways possibly .related to pain and
analgesia: Enkephalin and substance P, Proc. Nat. Acad. Sci. U.S.A. 74, 3081. Snyder, S.H. and R. Simantov, 1977, The opiate receptor and opioid peptides, J. Neurochemistry 28, 13.