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Possible involvement of dynorphinergic system in nociceptive transmission at spinal level
Neuropeptides
5: 425-428,
1985
POSSIBLE INVOLVEMENT OF DYNORPHINERGIC SYSTEM IN NOCICEPTIVE TRANSMISSION AT SPINAL LEVEL Santi Spampinato, Sanzio Candeletti,Patrizia Romualdi, Emanuela Cavicchini, Albert0 E. Panerai* and Sergio Ferri Institute of Pharmacology, University of Bologna and Milan*, Italy Irnerio 48, Bologna (reprint request to SF) ABSTR4CT The opioid peptide dynorphinl-32 (DYNl_32, 25 nmol) intrathecally administered causes, in the rat,an elevation of nociceptive threshold of longer duration than that of DYN A,as ascertained by vocalization test.Comparative findings obtained with tail flick test allow to differentiate antinociception from motor dysfunction. The breakdown of DYN A at spinal level is very rapid.The electrical stimulation of the tail associated to a restraint condition of the rat produces a significant increase of immunoreactive DYN in cervical,thoracic and lumbar segments of spinal cord,therefore indicating a correlative,if not causal,relationship between the s.pinaldynorphinergic system and aversive stimuli. INTRODUCTION DYN A and other related peptides,derived from the common precursor designated pre-pro-dynorphin,display antinociceptive properties after intrathecal (i.t.) administration (1). The aim of the present study was to compare the antinociceptive profiles of i.t. DYN A and DYNl_32,the 32-aminoacid peptide isolated from Fischli et al. (2) that comprises DYN A and DYN B linked by lysine-arginine.In order to ascertain the stability of DYN A,the labelled iodinated peptide was injected i.t. to the rat.Since stressful stimuli are capable to reduce the sensitivity to pain(3),the study was extended to examine the effects of an exposure to restraint and tail-shock upon levels of immunoreactive DYN (ir-dyn) at spinal level. MATERIALS AND METHODS Surgery.Male Sprague-Dawley rats(300-350 g)were implanted with chronic intrathecal catheters and seven days were elapsed before experiments. Evaluation of antinociceptive activity.The response to nociceptive stimuli was determined in the conscious rat by:(a) the vocalization test(maxima1 in425
;ensity of the current,2mA);(b) the tail flick test(cut-off time of 10 sec.). "In viva" breakdown of mono-iodinated DYN A. For assessment of "in vivo" stability of the peptide, mono-iodinated DYN A (1251-DYN A) was obtained(4) and injected to four rats i.t.(approx.10,000,000 cpm with 25 nmol of DYN A). The animals were killed after 8 min,their spinal cords rapidly removed and radioactivity extracted adopting the same procedure for extraction of ir-DYN (5). Stability of 1251-DYN A was evaluated by thin layer chromatography(TLC) as described by Leslie and Goldstein (4). Stress procedure.Male Sprague-Dawley rats(150-180 g) were subjected to stress by keeping them in restraining holders and applying a continuous electric shock to the tail for 70 min by two stainless steel 30 gauge electrodes inser ted into the tail(intensity of the current,0.4 mA).At the end of thestressful session the rats were killed,their spinal cords quickly removed and divided into cervical,thoracic and lumbar segments. Ir-DYN was extracted and measured by radioimmunoassay as reported previously (5).Non-stressed rats killed within lo-15 set after the removal from their cages were used as controls. RESULTS DYN A and DYNl_32 i.t. injected elevated the nociceptive threshold, measured by the tail flick and the vocalization test,but to different extent-figure 1 (top panel) reports the time-effect curves for the tail flick after injection of 25 nmol of DYN A or DYNl_32.Both peptides produced a maximal elevation of the tail flick latency in 5 min, lasting for the entire period of observation (2h). All rats exhibited flaccid hindlimb paralysis within 5 min, with flacci dity of the tail which gradually disappeared within 4 hours after injection. 1.t. administration of DYN A caused a rise in the vocalization test that had warn off within 60 min. DYNl_32 caused a rise of the same intensity, but its effect was longer in time than that of DYN A and still significantly different from control values 60 min after injection, 125I-DYN A appeared to be rapidly brokendown within a few minutes after i-t. administration.As shown in Table 1, while the radioactivity extracted from spinal cord of control rats was due to intact 1251-DYN A,the majority of radioactivity extracted from the spinal cord of rats killed 8 min after i.t. treatment, corresponded to 1251-tyrosine. The exposure to restraint and tail-shock procedure produced a marked increase of -ir-DYN occurring in all segments of the spinal cord (Table 2). DISCUSSION The two peptides DYN A and DYNl_32 caused maximal elevation of tail flick latency together with hindlimb paralysis lasting several hours,therefore confir ming previous reports(l).The vocalization test,however,showed that the rise of the nociceptive threshold had warn off within 60-90 min after i.t. injection.Thus it can be assumed that the prolonged depression of the tail flick reflex is related to the motor dysfunction and does not completely reflect the animal's response to painful stimuli.The rapid degradation of DYN A aster tained by TLC could somehow explain its short antinociceptive activity; thelonger activity of DYNl-32 could be ascribed to a less rapid breakdown. 426
TAIL FLICK
OJ 4’ VOCALIZATIONTEST
0’ -!200515 Ih r
s
r 30
1
4!5
’
00
I
120
TIME AFTER INTRATHECAL INJECTION(min)
Figure l.- Time-course of the effects of DYN A and DYNl_32 i.t. injected (25 nmol/rat) on tail flick latency and vocalization threshold. (0) DYN A, (0) DYNl_32, (0) saline. (n = 8-10). * p < 0.01, ** p < 0.05 vs pre-drug values.
Table l.- Rf values and relative recovery of radioactivity of extracted samples from rat spinal cord after i.t. injection of lL31-DYN A. Sample
Rf
Controls
0
Extracts from spinal cord of treated rats
0 0.57
% of total radioactivity 100 19 + 2 74 + - 4
Controls were performed by adding 1251-DYN A to 4 spinal cord samples from untreated animals prior to start extraction procedure. 1251-DYN A compound did not migrate (Rf = 0). 427
Table 2.- Effect of restraint and tail-shock on -ir-dyn content in spinal cord segments. Tissue
-ir-dyn (pmol/g) Non-stressed rats
Stressed rats
Cervical spinal cord
13.98 -t 0.79
22.24 t 0.95*
Thoracic spinal cord
22.22 -+ 0.70
29.14 t 1.78*
Lumbar spinal cord
12.55 -t 1.43
22.92 + 1.33*
Data are means + SEM from 2 experiments. Total n for each tissue is 14. * p < 0.01 vs non-stressed controls, The dynorphinergic system seems to be involved in restraint and tail shock, however,contrary to Millan et a1.(3) who showed that a brief foot-shock reduces ir-DYN at spinal level,we detected an increase.This may depend on the type oFstressor and on duration of the exposure(3). In any way these biochemical studies can provide evidence only for a correlative,not a causal, relationship between the dynorphinergic system at spinal level and aversive stimuli. The correlation,however,seems further strengthened by findings showing an increase of nociceptive threshold in rats exposed to restraint and tail shock (from 3.0520.3 to 5.220.4 p < 0.05, tail flick latency). ACKNOWLEDGEMENTS This work was supported by MPI and National Research Council (83.264056) grants. We thank Mrs.Elena Grazia for the expert secretarial assistance. REFERENCES 1.
Przewlocki, R., Shearman, G.T. and Herz, A. (1983). Mixed opioid/non opioid effects of dynorphin and dynorphin related peptides after their intrathecal injection in rats. Neuropeptides 3: 233-240.
2.
Fischli, W., Goldstein, A., Hunkapiller, M.W. and Hood, L.E. (1982). Two "big"dynorphins from porcine pituitary. Life Sci. 31: 1769-1772.
3.
Millan, M.J. (1981). Stress and endogenous opioid peptides: a review. In: Emrich, H.M. (ed.) The role of Endorphins in Psychopharmacology. Karger Press, Basel, p. 49-67.
4.
Leslie, F.M. and Goldstein, A. (1982). Degradation of dynorphin (1-13) by membrane-bound rat brain enzymes. Neuropeptides 2: 185-196.
5.
Spampinato, S. and Goldstein A. (1983). Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 3: 193-212.
428
5: 425-428,
1985
POSSIBLE INVOLVEMENT OF DYNORPHINERGIC SYSTEM IN NOCICEPTIVE TRANSMISSION AT SPINAL LEVEL Santi Spampinato, Sanzio Candeletti,Patrizia Romualdi, Emanuela Cavicchini, Albert0 E. Panerai* and Sergio Ferri Institute of Pharmacology, University of Bologna and Milan*, Italy Irnerio 48, Bologna (reprint request to SF) ABSTR4CT The opioid peptide dynorphinl-32 (DYNl_32, 25 nmol) intrathecally administered causes, in the rat,an elevation of nociceptive threshold of longer duration than that of DYN A,as ascertained by vocalization test.Comparative findings obtained with tail flick test allow to differentiate antinociception from motor dysfunction. The breakdown of DYN A at spinal level is very rapid.The electrical stimulation of the tail associated to a restraint condition of the rat produces a significant increase of immunoreactive DYN in cervical,thoracic and lumbar segments of spinal cord,therefore indicating a correlative,if not causal,relationship between the s.pinaldynorphinergic system and aversive stimuli. INTRODUCTION DYN A and other related peptides,derived from the common precursor designated pre-pro-dynorphin,display antinociceptive properties after intrathecal (i.t.) administration (1). The aim of the present study was to compare the antinociceptive profiles of i.t. DYN A and DYNl_32,the 32-aminoacid peptide isolated from Fischli et al. (2) that comprises DYN A and DYN B linked by lysine-arginine.In order to ascertain the stability of DYN A,the labelled iodinated peptide was injected i.t. to the rat.Since stressful stimuli are capable to reduce the sensitivity to pain(3),the study was extended to examine the effects of an exposure to restraint and tail-shock upon levels of immunoreactive DYN (ir-dyn) at spinal level. MATERIALS AND METHODS Surgery.Male Sprague-Dawley rats(300-350 g)were implanted with chronic intrathecal catheters and seven days were elapsed before experiments. Evaluation of antinociceptive activity.The response to nociceptive stimuli was determined in the conscious rat by:(a) the vocalization test(maxima1 in425
;ensity of the current,2mA);(b) the tail flick test(cut-off time of 10 sec.). "In viva" breakdown of mono-iodinated DYN A. For assessment of "in vivo" stability of the peptide, mono-iodinated DYN A (1251-DYN A) was obtained(4) and injected to four rats i.t.(approx.10,000,000 cpm with 25 nmol of DYN A). The animals were killed after 8 min,their spinal cords rapidly removed and radioactivity extracted adopting the same procedure for extraction of ir-DYN (5). Stability of 1251-DYN A was evaluated by thin layer chromatography(TLC) as described by Leslie and Goldstein (4). Stress procedure.Male Sprague-Dawley rats(150-180 g) were subjected to stress by keeping them in restraining holders and applying a continuous electric shock to the tail for 70 min by two stainless steel 30 gauge electrodes inser ted into the tail(intensity of the current,0.4 mA).At the end of thestressful session the rats were killed,their spinal cords quickly removed and divided into cervical,thoracic and lumbar segments. Ir-DYN was extracted and measured by radioimmunoassay as reported previously (5).Non-stressed rats killed within lo-15 set after the removal from their cages were used as controls. RESULTS DYN A and DYNl_32 i.t. injected elevated the nociceptive threshold, measured by the tail flick and the vocalization test,but to different extent-figure 1 (top panel) reports the time-effect curves for the tail flick after injection of 25 nmol of DYN A or DYNl_32.Both peptides produced a maximal elevation of the tail flick latency in 5 min, lasting for the entire period of observation (2h). All rats exhibited flaccid hindlimb paralysis within 5 min, with flacci dity of the tail which gradually disappeared within 4 hours after injection. 1.t. administration of DYN A caused a rise in the vocalization test that had warn off within 60 min. DYNl_32 caused a rise of the same intensity, but its effect was longer in time than that of DYN A and still significantly different from control values 60 min after injection, 125I-DYN A appeared to be rapidly brokendown within a few minutes after i-t. administration.As shown in Table 1, while the radioactivity extracted from spinal cord of control rats was due to intact 1251-DYN A,the majority of radioactivity extracted from the spinal cord of rats killed 8 min after i.t. treatment, corresponded to 1251-tyrosine. The exposure to restraint and tail-shock procedure produced a marked increase of -ir-DYN occurring in all segments of the spinal cord (Table 2). DISCUSSION The two peptides DYN A and DYNl_32 caused maximal elevation of tail flick latency together with hindlimb paralysis lasting several hours,therefore confir ming previous reports(l).The vocalization test,however,showed that the rise of the nociceptive threshold had warn off within 60-90 min after i.t. injection.Thus it can be assumed that the prolonged depression of the tail flick reflex is related to the motor dysfunction and does not completely reflect the animal's response to painful stimuli.The rapid degradation of DYN A aster tained by TLC could somehow explain its short antinociceptive activity; thelonger activity of DYNl-32 could be ascribed to a less rapid breakdown. 426
TAIL FLICK
OJ 4’ VOCALIZATIONTEST
0’ -!200515 Ih r
s
r 30
1
4!5
’
00
I
120
TIME AFTER INTRATHECAL INJECTION(min)
Figure l.- Time-course of the effects of DYN A and DYNl_32 i.t. injected (25 nmol/rat) on tail flick latency and vocalization threshold. (0) DYN A, (0) DYNl_32, (0) saline. (n = 8-10). * p < 0.01, ** p < 0.05 vs pre-drug values.
Table l.- Rf values and relative recovery of radioactivity of extracted samples from rat spinal cord after i.t. injection of lL31-DYN A. Sample
Rf
Controls
0
Extracts from spinal cord of treated rats
0 0.57
% of total radioactivity 100 19 + 2 74 + - 4
Controls were performed by adding 1251-DYN A to 4 spinal cord samples from untreated animals prior to start extraction procedure. 1251-DYN A compound did not migrate (Rf = 0). 427
Table 2.- Effect of restraint and tail-shock on -ir-dyn content in spinal cord segments. Tissue
-ir-dyn (pmol/g) Non-stressed rats
Stressed rats
Cervical spinal cord
13.98 -t 0.79
22.24 t 0.95*
Thoracic spinal cord
22.22 -+ 0.70
29.14 t 1.78*
Lumbar spinal cord
12.55 -t 1.43
22.92 + 1.33*
Data are means + SEM from 2 experiments. Total n for each tissue is 14. * p < 0.01 vs non-stressed controls, The dynorphinergic system seems to be involved in restraint and tail shock, however,contrary to Millan et a1.(3) who showed that a brief foot-shock reduces ir-DYN at spinal level,we detected an increase.This may depend on the type oFstressor and on duration of the exposure(3). In any way these biochemical studies can provide evidence only for a correlative,not a causal, relationship between the dynorphinergic system at spinal level and aversive stimuli. The correlation,however,seems further strengthened by findings showing an increase of nociceptive threshold in rats exposed to restraint and tail shock (from 3.0520.3 to 5.220.4 p < 0.05, tail flick latency). ACKNOWLEDGEMENTS This work was supported by MPI and National Research Council (83.264056) grants. We thank Mrs.Elena Grazia for the expert secretarial assistance. REFERENCES 1.
Przewlocki, R., Shearman, G.T. and Herz, A. (1983). Mixed opioid/non opioid effects of dynorphin and dynorphin related peptides after their intrathecal injection in rats. Neuropeptides 3: 233-240.
2.
Fischli, W., Goldstein, A., Hunkapiller, M.W. and Hood, L.E. (1982). Two "big"dynorphins from porcine pituitary. Life Sci. 31: 1769-1772.
3.
Millan, M.J. (1981). Stress and endogenous opioid peptides: a review. In: Emrich, H.M. (ed.) The role of Endorphins in Psychopharmacology. Karger Press, Basel, p. 49-67.
4.
Leslie, F.M. and Goldstein, A. (1982). Degradation of dynorphin (1-13) by membrane-bound rat brain enzymes. Neuropeptides 2: 185-196.
5.
Spampinato, S. and Goldstein A. (1983). Immunoreactive dynorphin in rat tissues and plasma. Neuropeptides 3: 193-212.
428