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The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord
Brain Research, 413 (1987) 213-219 Elsevier
213
BRE 12620
The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord Ryszard Przewtocki 2, Wtadystaw Lasofi 2, Volker H611t1, Jerzy Silberring 2 and Albert Herz 1 1Department of Neuropharmacology, Max-Planck-lnstitut f~ir Psychiatrie, Martinsried (F. R. G.) and elnstitute of Pharmacology, Polish Academy of Sciences, Krakow (Poland) (Accepted 28 October 1986)
Key words: Opioid peptide; Spinal cord; Chronic foot shock; Pain
Recurrent exposure to intermittent electrical foot-shock (30 min, twice daily) for 7 days caused an increase in immunoreactive (ir) dynorphin and ir-a-neo-endorphin in lumbar and cervical (but not thoracic) spinal cord as measured 16 h following the final session. At this time the level of ir-Met-enkephalin-Arg6-Gly7-Leu s (MEAGL) was also increased at the lumbar level. An acute foot-shock depleted spinal cord dynorphin in chronically stressed but not in naive rats. No alterations in levels of ir-dynorphin or ir-MEAGL were seen in discrete brain tissues. In contrast to the brain, where no effects were seen, the levels offl-endorphin increased in both lobes of the pituitary. This change, however, was not accompanied by an alteration in levels of fl-endorphin in plasma. These data show that chronic foot-shock stress selectively influences particular pools of opioid peptides, predominantly those derived from proenkephalin B in the spinal cord and from proopiomelanocortin in the anterior pituitary. It is suggested that alterations observed in the spinal cord reflect enhanced activity of the proenkephalin B system in response to chronic nociceptive stimulation. INTRODUCTION Opioid systems seem to play an i m p o r t a n t role in the maintenance of homeostasis and there are many indications for an involvement of opioid peptides in stress p h e n o m e n a 6,1~,15,~9,e3. Stress-induced analgesia, which is generally at least partially naloxone reversible 1°'15'25, represents a well-studied example of the relationship between stress p h e n o m e n a and opioidergic mechanisms. The m a j o r i t y of these studies, however, have focussed upon acute stress and little is known concerning chronic or recurrent stress and the involvement of opioid peptides in the response to it24,26. Recently we have studied the p h e n o m e n o n of chronic pain and the involvement of opioid peptides by means of a m o d e l of chronic polyarthritis 14 or of chronic localized pain 2°. F r o m these studies a particular role for dynorphin-like peptides in the spinal
cord was concluded. In this context it seemed of interest to investigate a possible influence upon opioid peptides of chronic recurrent foot-shock, a m o d e l for stress. Such a comparison also seemed to be of interest in view of an evident, but ill-defined relationship between pain and stress. In the present study, levels of opioid peptides in the spinal cord, brain, pituitary and plasma following acute and chronic ( r e p e a t e d ) foot-shock were determined. Opioid peptides, representative of each of the 3 opioid peptide families known, were selected: Met-enkephalin and M e t - e n k e p h a l i n - A r g 6 - G l y7Leu 8 ( M E A G L ) for p r o e n k e p h a l i n A , dynorphin and a - n e o - e n d o r p h i n for p r o e n k e p h a l i n B and fl-endorphin for p r o o p i o m e l a n o c o r t i n ( P O M C ) . MATERIALS AND METHODS Male S p r a g u e - D a w l e y rats (190-210
g) were
Correspondence: A. Herz, Department of Neuropharmacology, Max-Planck-Institute fiir Psychiatrie, Am Klopferspitz 18a, D-8033 Martinsried, F.R.G. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
214 housed 8 per cage in a room with controlled humidity (60%) and temperature (22 °C) and with a fixed lighting schedule (lights on from 8.00 to 20.00 h). Food and water were given ad libitum. The rats were allowed to adapt to the environment for at least one week.
Foot-shock procedure The animals were placed in the test chamber which was surrounded by an isolating cubicle (Coulbourn Instruments, PA, U.S.A.), and provided with a grid floor, where they were subjected to foot-shock. Intermittent electric shocks (3 mA, 1 s duration every 5 s) were delivered in a scrambled fashion through the grid floor, according to Shiomi and Aki124. Each session lasted 30 min. The 'chronically' shocked rats were subjected to the foot-shock procedure twice daily (between 8.00 and 10.00 h, and 17.00 and 19.00 h) for a period of 1, 3 or 7 days and were decapitated immediately after or between 9.00 and 13.00 h on the day following the final foot-shock session. The acutely stressed rats were subjected to one foot-shock session and decapitated immediately afterwards. Naive, unstressed rats were used as the control group. After decapitation, the brain, pituitary and spinal cord were quickly removed. The brain, anterior and neurointermediate lobes of the pituitary were dissected; the spinal cord was divided into 3 parts corresponding to cervical, thoracic and lumbar sections. The trunk blood was collected in ice-cold tubes containing 20 mg EDTA, and centrifuged immediately for 10 min at 1500 g at 4 °C. Tissue processing and extraction Tissue parts were weighed and then incubated in 0.1 N HC1 (5 times the volume of the tissue) at 95 °C for 10 min. After homogenization, the homogenates were centrifuged at 10,000 g for 15 min and the supernatants adjusted to pH 7.5. After further centrifugation aliquots of the supernatants were appropriately diluted and assayed for the ir-dynorphin, ir-a-neoendorphin, ir-fl-endorphin, ir-MEAGL and ir-Metenkephalin, according to previously described procedures 9'12'18. The dynorphin radioimmunoassay (RIA) was performed with an antiserum directed against synthetic porcine dynorphin1_13. The antiserum recognized dynorphinl_17 with an equal avidity but exhibited a negligible avidity for a-neo-endor-
phin, Leu-enkephalin, Met-enkephalin, fl-endorphin and MEAGL. The antiserum to a-neo-endorphin did not recognize dynorphin or any of the peptides mentioned. The antiserum directed a g a i n s t h u m a n fl-endorphin recognized rat fl-endorphin, but did not cross-react with Leu-enkephalin, dynorphin, a-neoendorphin and MEAGL. T h e a n t i s e r u m directed against M E A G L did not cross-react with dynorphin, a-neoendorphin and fl-endorphin. The antiserum to Met-enkephalin did not recognize fl-endorphin, dynorphin, a-neo-endorphin and MEAGL, but showed about 10% cross-reactivity to Leu-enkephalin.
Statistics The statistical analysis of the results was assessed either by a one-way analysis of variance (ANOVA), and the inter-group differences were analyzed b~y applying Duncan's test.
20
10
CERVICAL
0
10
THORACIC
0
ai
20.
10
0 control
acute stress
LU~
(*acute| chronicstress
Fig. 1. Effect of acute and chronic foot-shock stress on the levels of ir-dynorphin in various segments of the spinal cord. Means __ S.E.M. are presented. Significance of difference between rats subjected to stress (n = 8) versus controls (n = 12) is indicated by asterisks and between subjects exposed to chronic stress and killed immediately or 16 h following the last session is indicated by triangles (*P ~ 0.01, Ap < 0.05, Duncan's test).
215 RESULTS
Spinal cord Fig. 1 shows the dynorphin content of various sections of the spinal cord following single or repeated foot-shock. There were no significant changes after acute foot-shock whereas significant increases in dynorphin were found in the cervical and lumbar level in repeatedly shocked rats (F3,31 - 9.80, P < 0.001; F3,32 = 7.83, P < 0.005, respectively). Interestingly, relative to this increase in levels, there was a significant decrease when the level of the peptide was estimated immediately after the last shock session (P < 0.05 and P < 0.01 on the cervical and lumbar level, respectively, Duncan's test). From Fig. 2 it can be seen that this increase in dynorphin content steadily develops in the cervical and lumbar enlargements (F3,46 = 5.79, P < 0.005; F3,44 - 5.54, P < 0.01, respectively) over one week as measured after 1 (not shown), 3 or 7 days. This also holds for a-neo-endorphin, another peptide derived from proenkephalin B. Also in this experiment, the changes are restricted to the cervical and lumbar level (F3,30 = 5.72, P < 0.01; F3,3s = 3.83, P < 0.05, respectively). Peptides derived from proenkephalin A, Met-enkephalin and
cervicat
o E
z: i:z L o
.c_
thoracic
lumbar
++2/
MEAGL, responded to a lesser degree than those derived from proenkephalin B; there was only a tendency for an increase in Met-enkephalin after chronic foot-shock and the increase in the case of MEAGLE measured in the lumbar cord, though significant (F3,48 = 3.82, P < 0.05), were less pronounced (Table I).
Brain Peptide levels were estimated in the hypothalamus and the thalamus, structures in which changes after acute stress or chronic pain had been observed previously 14A5'19'23. No significant changes in dynorphin or M E A G L E content were observed after acute or chronic stress. Acute stress tended to reduce the flendorphin content of the hypothalamus by about 20%, although this decrease was not significant and a tendency for an increase in the thalamus was seen in chronic stress (Table II).
Pituitary Acute stress tended to reduce the level offl-endorphin in the anterior pituitary, but not in the neurointermediate lobe (Fig. 3). The dynorphin content did not change under these conditions. Repeated footshock led to a significant increase in fl-endorphin above control levels in both anterior and neurointermediate lobes (F3.27 = 10.17, P < 0.001; F3,32 = 14.63, P < 0.001, respectively). In the case of dynorphin, an increase was seen only in the neurointermediate lobe (F3.25 = 3.07, P < 0.05).
10
/
TABLE I
Influence o f chronic foot-shock stress on levels o f ir-Met-enkephalin and ir-M EA G L in the lumbar section of the spinal cord of rats
0J o
i
6012
o
.-g
0
~
1
time (days) Fig. 2. Influence of chronic foot-shock stress on the levels of irdynorphin (O O), ir-a-neo-endorphin (A A) and i r - M E A G L ([3 D) in various segments of the spinal cord of rats. Rats were exposed to 30 rain intermittent footshock stress twice daily for 1 (not shown), 3 or 7 days. Means + S.E.M. are presented. Asterisks indicate significant differences as compared to controls (P ~<0.05, Duncan's test).
Means + S.E.M. presented. Significant differences between rats subjected to chronic stress and killed immediately or 16 h following the last session ( * P < 0.05 vs chronic stress group; ** P < 0.05 vs control and acute stress group are indicated).
Treatment
ir-Metenkephalin
n
irn M EA G L E
Control Acute stress Chronic stress Chronic + acute stress
445 411 490 377
12 8 8 9
55 57 68 60
+ + + +
27.7 20.4 22.9 32.1"
+ + + +
1.9 11 2.6 8 3.9** 8 3.6 9
216 TABLE II Levels of ir-fl-endorphin, ir-dynorphin and ir-MEA GL in the hypothalamus and thalamus of rats subjected to chronic foot-shock stress Means _+S.E.M. are presented. Treatment
ir-fl-Endorphin
Control Acute stress Chronic stress Chronic + acute stress
n
Hypothalamus
Thalamus
26.3 + 1.97 20.3 + 0.95 23.3 + 1.57 22.3 + 0.82
2.3 + 0.22 2.9 + 0.30 3.1 + 0.2 2.6 + 0.15
Plasma /3-Endorphin levels in plasma were increased after acute foot-shock in naive and chronically stressed rats (F3,s2 = 6.69, P < 0.01; Fig. 4). After chronic foot-shock, the levels were similar to control values; immediately after shock, similarly increased values as those seen after acute shock were observed. DISCUSSION The present study describes the effects of acute and chronic recurrent footshock u p o n opioid peptide systems in the spinal cord, brain and pituitary of rats. A striking finding concerns changes in spinal cord
11 7 7 7
ir-Dynorphin Hypothalamus
Thalamus
36.1 + 38.9 + 37.9 + 33.1 +
13,6 + 1.24 11.9 + 1.49 14.3 + 0.89 15.5 + 1.11
2.47 1.05 1.95 15.5
MEA GL
n
45.4 + 40.0 + 44.2 + 42.5 +
12 7 8 9
2.87 3.88 4.0 3.87
pools of proenkephalin B-derived peptides u n d e r chronic stress, although an acute stress of 30 rain did not cause significant changes in peptide levels in this study. Recurrent stress resulted in a marked elevation in the level of dynorphin and a - n e o - e n d o r p h i n in the l u m b a r and cervical (but not thoracic) segments; these segments correspond to the regions to which pathways conveying nociceptive information from the forelimbs and hindlimbs project. The alterations indicate an increase accumulation of these peptides in n e u r o n a l networks which may reflect either an increased biosynthesis and/or a reduced release. Furthermore, a significant depletion of this increased dynorphin level is seen when the chronically stressed
i iANTERO LOBE 1 llf 1
30"
20-
50
-~"
10.
•
I o
o
0i
.c
POSTERIOR -INTERMEDIATE LOBE i
6oo
,1000
D
400
iiii~
200
control ocutestre=s
(.ocute) chronic stress
control ocute s t r e l
(*ocule) ch~nic sb~m
-0
Fig. 3. Influence of chronic foot-shock stress on levels of ir-fl-endorphin and ir-dynorphin in anterior and neuro/intermediate pituitary of rats. Means + S.E.M. are presented. Significant difference between stressed (n = 8) and control rats (n = 12) is indicated by asterisks (*P ~<0.05).
217 Plasma level of fl- endorphin after s t r e s s
908070- - 6O
so "- 40
o
3o
,~
2o
•~
to
I
control
acute stress
(+acute) chronic stress
Fig. 4. The influence of foot-shock stress on levels of ir-fl-endorphin in plasma of intact rats, immediately after a single 30 min foot-shock (n = 8) or chronically stressed for 7 days (n = 9) 16 h and immediately after the last session. Significant differences between controls (n = 12) and stressed rats are indicated (P ~<0.01, Duncan's test).
rats were analyzed immediately after the last footshock session; this presumably reflects an enhancement of shock-induced peptide release as compared to naive rats. These changes are most probably related to noxious stimulation: thus, the peptides are present in maximal concentrations in the dorsal horn, and particularly in the substantia gelatinosa 1,2~ls, a key structure for modulation of nociception. Further, these peptides exert antinociceptive effects at the spinal level7'17,21,22. In addition, the observation that the changes are restricted to spinal cord sections which receive nociceptive input from the limbs favours this view. Similar changes (though less pronounced) can also be seen in the lumbar cord for the proenkephalin A system: a slight but significant elevation of M E A G L was seen following chronic stress, and a significant depletion of this peptide and of Met-enkephalin was seen upon analysis immediately after the last shock period. The most likely interpretation of these data is that spinal cord-localized opioid systems adapt to prolonged recurrent noxious stimulation by an enhancement in their activity in those areas receiving noxious input. The changes observed probably reflect a functional response to pain. In fact, similar alterations have
been reported in the spinal cord of rats suffering from polyarthritic pain 4,14 or chronic localized pain (monoarthritis) 5"2°. A combined biochemical and behavioral study in polyarthritic rats suggested that in this state, the activity of the spinal cord proenkephalin B system may be enhanced 13. A current in vivo and in vitro release study has shown, however, that the release of opioid peptides derived from proenkephalin A and B might not be enhanced but rather inhibited in this condition 3 (Przewtocki, in preparation). In contrast to the changes observed in the spinal cord, no significant alterations were seen in the opiold peptide content of the thalamus, a brain structure of importance for the receipt and processing of nociceptive information derived from ascending spinothalamic pathways. This finding contrasts with data obtained in polyarthritic rats, in which levels of proenkephalin B-derived peptides increased markedly 14. No such changes were observed, however, in the thalamus of rats subjected to chronic localized pain 2°. These differences might be due to the greater intensity and duration of polyarthritis. In line with another study 14, we found no evidence for an alteration of proenkephalin A and B peptides in the hypothalamus. There is also no evidence that the hypothalamic fl-endorphin system might be influenced either by chronic polyarthritis 14, recurrent foot-shock stress (this study) or chronic localized pain 2°. Nevertheless, this may not necessarily indicate that the hypothalamic fl-endorphin system is not involved in chronic pain or stress, since tissue levels may not necessarily reveal a change in activity. Further, one should bear in mind the fact that the system responds to acute stress by an enhanced release of the peptide 15'19'23. In agreement with certain previous studies, pituitary pools offl-endorphin were only slightly depleted upon acute stress, but increased following prolonged stress in both anterior and intermediate lobes. The elevated levels of peptide are accompanied by an elevation in levels of m R N A encoding POMC in the anterior but not in the intermediate lobe s , although acute foot-shock releases fl-endorphin not only from the anterior but also from the neurointermediate lobe 19. This may indicate an enhancement of the release and synthesis of fl-endorphin in the anterior lobe and in increase in peptide accumulation in the neurointermediate lobe. In line with the above data,
218 chronic arthritis is accompanied by an increase in anterior lobe pools of fl-endorphin and m R N A for P O M C , while, in contrast, no significant changes in fl-endorphin levels in the neurointermediate lobe occurred, and levels of m R N A coding for P O M C in this lobe tended to decrease 8. These shifts in pituitary opioid peptide levels may not seem to be directly related to the pain induced by foot-shock or chronic arthritis, since similar increases have been observed in rats merely placed in the foot-shock chamber but without being subjected to this stress (Millan, personal communication). Interestingly, however, no alterations of opioid peptides in spinal cord were visible in rats treated in this way. This might indicate that the changes observed in the spinal cord (in contrast to pituitary lobes) are directly related to the noxious pain. Plasma levels of fl-endorphin did not differ significantly between naive or chronically stressed rats exposed to an acute foot-shock. In other words, the releasable pool of the pituitary fl-endorphin responds in a similar way in naive and chronically stressed rats, even though there is an increase in pituitary lobe levels of peptide and prohormone synthesis in chronic stress. This observation differs from data obtained in polyarthritic rats, in which substantial increases in flendorphin levels in plasma were measured TM. An acute, short-term foot-shock stress was pre-
REFERENCES 1 Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 2 Botticelli, L.I., Cox, B.M. and Goldstein, A., Immunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7783-7786. 3 Cesselin, F., Bourgoin, S., Artaud, F. and Hamon, M., Basic and regulatory mechanisms of in vitro release of Met-enkephalin from dorsal horn of the rat spinal cord, J. Neurochem., 43 (1984) 763-773. 4 Cesselin, F., Montastruc, J.L., Gros, C., Bourgoin, S. and Hamon, M., Met-enkephalin levels and opiate receptors in the spinal cord of chronic suffering rats, Brain Research, 191 (1980) 289-293. 5 Faccini, E., Uzumaki, H., Govoni, S., Missale, C., Spano, P.F., Covelli, V. and Trabucchi, M., Afferent fibres mediate the increase of Met-enkephalin elicited in rat spinal cord by localized pain, Pain, 18 (1984) 25-31. 6 Guillemin, R., Vargo, T., Rossier, J., Minick, S., Ling, N., Rivier, C., Vale, W. and Bloom, R., fl-Endorphin and adrenocorticotropin are secreted concomitantly by the pitu-
viously reported to modify dynorphin levels in the anterior lobe but not to significantly affect the peptide levels in the neurointermediate lobe 16. As has been shown in the present study, neither acute nor chronic stress influenced the peptide levels in the anterior lobe. In chronically stressed rats, however, a slight increase in neurointermediate lobe level of the peptide was observed, which might indicate an increase in the activity of hypothalamo-pituitary dynorphin systems under prolonged stress. In summary, the present study demonstrates that recurrent chronic foot-shock stress is accompanied by an enhanced function of the proenkephalin A and/or B opioid systems in the spinal cord and of flendorphin in the anterior pituitary. It is noteworthy that to some extent similar changes seem to occur in rats with chronic localized pain, as well as in rats suffering from polyarthritic pain. These data might indicate the existence of a c o m m o n mechanism involved in the control of chronic pain and stress, in which the spinal cord proenkephalin A and B, on the one hand, and the anterior pituitary fl-endorphin system, on the other hand, may play an important regulatory role. ACKNOWLEDGEMENTS Supported by Deutsche Forschungsgemeinschaft, Bonn.
itary gland, Science, 197 (1977) 1367-1369. 7 Han, J.-S. and Xie, G.X., Dynorphin: potent analgetic effect in spinal cord of the rat, Life Sci., 31 (1982) 1781-1784. 8 H611t, V., Przewlocki, R. and Herz, A., Radioimmunoassay of/3-endorphin: basal and stimulated levels of extracted rat plasma, Naunyn-Schmiedeberg's Arch. Pharmacol., 303 (1978) 171-174. 9 HOllt, V., Przewlocki, R., Haarmann, I., Almeida, O., Kley, N., Millan, M. and Herz, A., Stress-induced alterations in the levels of messenger RNA encoding proopiomelanocortin in rat pituitary, Neuroendocrinology, 43 (1986) 277-282. 10 Lewis, J.W., Cannon, J.T. and Liebeskind, J.C., Opioid and non-opioid mechanisms of stress analgesia, Science, 208 (1980) 623-625. 11 Madden, J., Akil, H., Patrick, R.L. and Barchas, J.D., Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat, Nature (London), 265 (1977) 358-360. 12 Maysinger, D., H611t, V., Seizinger, B.R., Mehraein, P., Pasi, A. and Herz, A., Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissue, Brain Research, 280 (1983) 95-103. 13 Millan, M.J., Czlonkowski, A., Pilcher, C.W.T., Almeida,
219 O.F.X., Milran, M.H., Colpaert, F.C. and Herz, A., A model of chronic pain in the rat: functional correlates of alterations in the activity of opioid systems, J. Neurosci., in press. 14 Millan, M.J., Millan, M.H., Czlonkowski, A., H611t, V., Pilcher, C.W.T., Herz, A. and Colpaert, F.C., A model of chronic pain in the rat: response of multiple opioid systems to adjuvant-induced arthritis, J. Neurosci., 4 (1986) 889-906. 15 Millan, M.J., Przewlocki, R., Jerlicz, M., Gramsch, Ch.. H611t, V. and Herz, A., Stress-induced release of brain and pituitary fl-endorphin: major role of endorphins in generation of hyperthermia not analgesia, Brain Research, 208 (1981) 325-338. 16 Millan, M.J., Tsang, Y., Przewlocki, R., H611t, V. and Herz, A., The influence of foot-shock stress upon brain, pituitary and spinal cord pools of immunoreactive dynorphin in rats, Neurosci. Lett., 24 (1981) 75-79. 17 Piercey, M.F., Varner, K. and Schroeder, L.A., Analgesic activity of intraspinally-administered dynorphin and ethylketocyclazocine, Eur. J. Pharrnacol., 80 (1982) 283-284. 18 Przewlocki, R., Gramsch, Ch., Pasi, A. and Herz, A., Characterization and localization of immunoreactive dynorphin, a-neo-endorphin, Met-enkephalin and substance P in human spinal cord, Brain Research, 280 (1983) 95-103. 19 Przewlocki, R., Millan, M.J., Gramsch, Ch., Millan, M.H. and Herz, A., The influence of selective adeno- and neuro-
intermediate-hypophysectomy upon plasma and brain levels of fl-endorphin and their response to stress in rats, Brain Research, 242 (1982) 107-117. 20 Przewlocki, R., Przewlocka, B., Lasofi, W., Garz6n, J., Stala, L. and Herz, A., Opioid peptides, particularly dynorphin, and chronic pain, 1NSERM, 127 (1984) 159-170. 21 Przewlocki, R., Shearman, G.T. and Herz, A., Mixed opioid/non-opioid effects of dynorphin and dynorphin-related peptides after their intrathecal injection in rats, Neuropeptides, 3 (1983) 233-239. 22 Przewlocki, R., Stala, L., Greczek, M., Shearman, G.T., Przewlocka, B. and Herz, A., Analgesic effects of/~-, 6-and x-opiate agonists and, in particular, dynorphin at the spinal level, Life Sci. 33, Suppl. I (1983) 649-652. 23 Rossier, J., Guillemin, R. and Bloom, F.E., Foot-shock induced stress decreases LeuS-enkephalin immunoreactivity in rat hypothalamus, Eur. J. Pharrnacol., 48 (1978) 465-466. 24 Shiomi, H. and Akil, H., Pulse-chase studies of the POMC/fl-endorphin system in the pituitary of acutely and chronically stressed rats, Life Sci., 31 (1982) 2185-2188. 25 Watkins, L.R. and Mayer, D.J., Organization of endogenous opiate and non-opiate pain control systems, Science, 216 (1982) 1185-1192. 26 Young, E. and Akil, H., Changes in releasability of ACTH and beta-endorphin with chronic stress, Neuropeptides, 5 (1985) 545-548.
213
BRE 12620
The influence of chronic stress on multiple opioid peptide systems in the rat: pronounced effects upon dynorphin in spinal cord Ryszard Przewtocki 2, Wtadystaw Lasofi 2, Volker H611t1, Jerzy Silberring 2 and Albert Herz 1 1Department of Neuropharmacology, Max-Planck-lnstitut f~ir Psychiatrie, Martinsried (F. R. G.) and elnstitute of Pharmacology, Polish Academy of Sciences, Krakow (Poland) (Accepted 28 October 1986)
Key words: Opioid peptide; Spinal cord; Chronic foot shock; Pain
Recurrent exposure to intermittent electrical foot-shock (30 min, twice daily) for 7 days caused an increase in immunoreactive (ir) dynorphin and ir-a-neo-endorphin in lumbar and cervical (but not thoracic) spinal cord as measured 16 h following the final session. At this time the level of ir-Met-enkephalin-Arg6-Gly7-Leu s (MEAGL) was also increased at the lumbar level. An acute foot-shock depleted spinal cord dynorphin in chronically stressed but not in naive rats. No alterations in levels of ir-dynorphin or ir-MEAGL were seen in discrete brain tissues. In contrast to the brain, where no effects were seen, the levels offl-endorphin increased in both lobes of the pituitary. This change, however, was not accompanied by an alteration in levels of fl-endorphin in plasma. These data show that chronic foot-shock stress selectively influences particular pools of opioid peptides, predominantly those derived from proenkephalin B in the spinal cord and from proopiomelanocortin in the anterior pituitary. It is suggested that alterations observed in the spinal cord reflect enhanced activity of the proenkephalin B system in response to chronic nociceptive stimulation. INTRODUCTION Opioid systems seem to play an i m p o r t a n t role in the maintenance of homeostasis and there are many indications for an involvement of opioid peptides in stress p h e n o m e n a 6,1~,15,~9,e3. Stress-induced analgesia, which is generally at least partially naloxone reversible 1°'15'25, represents a well-studied example of the relationship between stress p h e n o m e n a and opioidergic mechanisms. The m a j o r i t y of these studies, however, have focussed upon acute stress and little is known concerning chronic or recurrent stress and the involvement of opioid peptides in the response to it24,26. Recently we have studied the p h e n o m e n o n of chronic pain and the involvement of opioid peptides by means of a m o d e l of chronic polyarthritis 14 or of chronic localized pain 2°. F r o m these studies a particular role for dynorphin-like peptides in the spinal
cord was concluded. In this context it seemed of interest to investigate a possible influence upon opioid peptides of chronic recurrent foot-shock, a m o d e l for stress. Such a comparison also seemed to be of interest in view of an evident, but ill-defined relationship between pain and stress. In the present study, levels of opioid peptides in the spinal cord, brain, pituitary and plasma following acute and chronic ( r e p e a t e d ) foot-shock were determined. Opioid peptides, representative of each of the 3 opioid peptide families known, were selected: Met-enkephalin and M e t - e n k e p h a l i n - A r g 6 - G l y7Leu 8 ( M E A G L ) for p r o e n k e p h a l i n A , dynorphin and a - n e o - e n d o r p h i n for p r o e n k e p h a l i n B and fl-endorphin for p r o o p i o m e l a n o c o r t i n ( P O M C ) . MATERIALS AND METHODS Male S p r a g u e - D a w l e y rats (190-210
g) were
Correspondence: A. Herz, Department of Neuropharmacology, Max-Planck-Institute fiir Psychiatrie, Am Klopferspitz 18a, D-8033 Martinsried, F.R.G. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
214 housed 8 per cage in a room with controlled humidity (60%) and temperature (22 °C) and with a fixed lighting schedule (lights on from 8.00 to 20.00 h). Food and water were given ad libitum. The rats were allowed to adapt to the environment for at least one week.
Foot-shock procedure The animals were placed in the test chamber which was surrounded by an isolating cubicle (Coulbourn Instruments, PA, U.S.A.), and provided with a grid floor, where they were subjected to foot-shock. Intermittent electric shocks (3 mA, 1 s duration every 5 s) were delivered in a scrambled fashion through the grid floor, according to Shiomi and Aki124. Each session lasted 30 min. The 'chronically' shocked rats were subjected to the foot-shock procedure twice daily (between 8.00 and 10.00 h, and 17.00 and 19.00 h) for a period of 1, 3 or 7 days and were decapitated immediately after or between 9.00 and 13.00 h on the day following the final foot-shock session. The acutely stressed rats were subjected to one foot-shock session and decapitated immediately afterwards. Naive, unstressed rats were used as the control group. After decapitation, the brain, pituitary and spinal cord were quickly removed. The brain, anterior and neurointermediate lobes of the pituitary were dissected; the spinal cord was divided into 3 parts corresponding to cervical, thoracic and lumbar sections. The trunk blood was collected in ice-cold tubes containing 20 mg EDTA, and centrifuged immediately for 10 min at 1500 g at 4 °C. Tissue processing and extraction Tissue parts were weighed and then incubated in 0.1 N HC1 (5 times the volume of the tissue) at 95 °C for 10 min. After homogenization, the homogenates were centrifuged at 10,000 g for 15 min and the supernatants adjusted to pH 7.5. After further centrifugation aliquots of the supernatants were appropriately diluted and assayed for the ir-dynorphin, ir-a-neoendorphin, ir-fl-endorphin, ir-MEAGL and ir-Metenkephalin, according to previously described procedures 9'12'18. The dynorphin radioimmunoassay (RIA) was performed with an antiserum directed against synthetic porcine dynorphin1_13. The antiserum recognized dynorphinl_17 with an equal avidity but exhibited a negligible avidity for a-neo-endor-
phin, Leu-enkephalin, Met-enkephalin, fl-endorphin and MEAGL. The antiserum to a-neo-endorphin did not recognize dynorphin or any of the peptides mentioned. The antiserum directed a g a i n s t h u m a n fl-endorphin recognized rat fl-endorphin, but did not cross-react with Leu-enkephalin, dynorphin, a-neoendorphin and MEAGL. T h e a n t i s e r u m directed against M E A G L did not cross-react with dynorphin, a-neoendorphin and fl-endorphin. The antiserum to Met-enkephalin did not recognize fl-endorphin, dynorphin, a-neo-endorphin and MEAGL, but showed about 10% cross-reactivity to Leu-enkephalin.
Statistics The statistical analysis of the results was assessed either by a one-way analysis of variance (ANOVA), and the inter-group differences were analyzed b~y applying Duncan's test.
20
10
CERVICAL
0
10
THORACIC
0
ai
20.
10
0 control
acute stress
LU~
(*acute| chronicstress
Fig. 1. Effect of acute and chronic foot-shock stress on the levels of ir-dynorphin in various segments of the spinal cord. Means __ S.E.M. are presented. Significance of difference between rats subjected to stress (n = 8) versus controls (n = 12) is indicated by asterisks and between subjects exposed to chronic stress and killed immediately or 16 h following the last session is indicated by triangles (*P ~ 0.01, Ap < 0.05, Duncan's test).
215 RESULTS
Spinal cord Fig. 1 shows the dynorphin content of various sections of the spinal cord following single or repeated foot-shock. There were no significant changes after acute foot-shock whereas significant increases in dynorphin were found in the cervical and lumbar level in repeatedly shocked rats (F3,31 - 9.80, P < 0.001; F3,32 = 7.83, P < 0.005, respectively). Interestingly, relative to this increase in levels, there was a significant decrease when the level of the peptide was estimated immediately after the last shock session (P < 0.05 and P < 0.01 on the cervical and lumbar level, respectively, Duncan's test). From Fig. 2 it can be seen that this increase in dynorphin content steadily develops in the cervical and lumbar enlargements (F3,46 = 5.79, P < 0.005; F3,44 - 5.54, P < 0.01, respectively) over one week as measured after 1 (not shown), 3 or 7 days. This also holds for a-neo-endorphin, another peptide derived from proenkephalin B. Also in this experiment, the changes are restricted to the cervical and lumbar level (F3,30 = 5.72, P < 0.01; F3,3s = 3.83, P < 0.05, respectively). Peptides derived from proenkephalin A, Met-enkephalin and
cervicat
o E
z: i:z L o
.c_
thoracic
lumbar
++2/
MEAGL, responded to a lesser degree than those derived from proenkephalin B; there was only a tendency for an increase in Met-enkephalin after chronic foot-shock and the increase in the case of MEAGLE measured in the lumbar cord, though significant (F3,48 = 3.82, P < 0.05), were less pronounced (Table I).
Brain Peptide levels were estimated in the hypothalamus and the thalamus, structures in which changes after acute stress or chronic pain had been observed previously 14A5'19'23. No significant changes in dynorphin or M E A G L E content were observed after acute or chronic stress. Acute stress tended to reduce the flendorphin content of the hypothalamus by about 20%, although this decrease was not significant and a tendency for an increase in the thalamus was seen in chronic stress (Table II).
Pituitary Acute stress tended to reduce the level offl-endorphin in the anterior pituitary, but not in the neurointermediate lobe (Fig. 3). The dynorphin content did not change under these conditions. Repeated footshock led to a significant increase in fl-endorphin above control levels in both anterior and neurointermediate lobes (F3.27 = 10.17, P < 0.001; F3,32 = 14.63, P < 0.001, respectively). In the case of dynorphin, an increase was seen only in the neurointermediate lobe (F3.25 = 3.07, P < 0.05).
10
/
TABLE I
Influence o f chronic foot-shock stress on levels o f ir-Met-enkephalin and ir-M EA G L in the lumbar section of the spinal cord of rats
0J o
i
6012
o
.-g
0
~
1
time (days) Fig. 2. Influence of chronic foot-shock stress on the levels of irdynorphin (O O), ir-a-neo-endorphin (A A) and i r - M E A G L ([3 D) in various segments of the spinal cord of rats. Rats were exposed to 30 rain intermittent footshock stress twice daily for 1 (not shown), 3 or 7 days. Means + S.E.M. are presented. Asterisks indicate significant differences as compared to controls (P ~<0.05, Duncan's test).
Means + S.E.M. presented. Significant differences between rats subjected to chronic stress and killed immediately or 16 h following the last session ( * P < 0.05 vs chronic stress group; ** P < 0.05 vs control and acute stress group are indicated).
Treatment
ir-Metenkephalin
n
irn M EA G L E
Control Acute stress Chronic stress Chronic + acute stress
445 411 490 377
12 8 8 9
55 57 68 60
+ + + +
27.7 20.4 22.9 32.1"
+ + + +
1.9 11 2.6 8 3.9** 8 3.6 9
216 TABLE II Levels of ir-fl-endorphin, ir-dynorphin and ir-MEA GL in the hypothalamus and thalamus of rats subjected to chronic foot-shock stress Means _+S.E.M. are presented. Treatment
ir-fl-Endorphin
Control Acute stress Chronic stress Chronic + acute stress
n
Hypothalamus
Thalamus
26.3 + 1.97 20.3 + 0.95 23.3 + 1.57 22.3 + 0.82
2.3 + 0.22 2.9 + 0.30 3.1 + 0.2 2.6 + 0.15
Plasma /3-Endorphin levels in plasma were increased after acute foot-shock in naive and chronically stressed rats (F3,s2 = 6.69, P < 0.01; Fig. 4). After chronic foot-shock, the levels were similar to control values; immediately after shock, similarly increased values as those seen after acute shock were observed. DISCUSSION The present study describes the effects of acute and chronic recurrent footshock u p o n opioid peptide systems in the spinal cord, brain and pituitary of rats. A striking finding concerns changes in spinal cord
11 7 7 7
ir-Dynorphin Hypothalamus
Thalamus
36.1 + 38.9 + 37.9 + 33.1 +
13,6 + 1.24 11.9 + 1.49 14.3 + 0.89 15.5 + 1.11
2.47 1.05 1.95 15.5
MEA GL
n
45.4 + 40.0 + 44.2 + 42.5 +
12 7 8 9
2.87 3.88 4.0 3.87
pools of proenkephalin B-derived peptides u n d e r chronic stress, although an acute stress of 30 rain did not cause significant changes in peptide levels in this study. Recurrent stress resulted in a marked elevation in the level of dynorphin and a - n e o - e n d o r p h i n in the l u m b a r and cervical (but not thoracic) segments; these segments correspond to the regions to which pathways conveying nociceptive information from the forelimbs and hindlimbs project. The alterations indicate an increase accumulation of these peptides in n e u r o n a l networks which may reflect either an increased biosynthesis and/or a reduced release. Furthermore, a significant depletion of this increased dynorphin level is seen when the chronically stressed
i iANTERO LOBE 1 llf 1
30"
20-
50
-~"
10.
•
I o
o
0i
.c
POSTERIOR -INTERMEDIATE LOBE i
6oo
,1000
D
400
iiii~
200
control ocutestre=s
(.ocute) chronic stress
control ocute s t r e l
(*ocule) ch~nic sb~m
-0
Fig. 3. Influence of chronic foot-shock stress on levels of ir-fl-endorphin and ir-dynorphin in anterior and neuro/intermediate pituitary of rats. Means + S.E.M. are presented. Significant difference between stressed (n = 8) and control rats (n = 12) is indicated by asterisks (*P ~<0.05).
217 Plasma level of fl- endorphin after s t r e s s
908070- - 6O
so "- 40
o
3o
,~
2o
•~
to
I
control
acute stress
(+acute) chronic stress
Fig. 4. The influence of foot-shock stress on levels of ir-fl-endorphin in plasma of intact rats, immediately after a single 30 min foot-shock (n = 8) or chronically stressed for 7 days (n = 9) 16 h and immediately after the last session. Significant differences between controls (n = 12) and stressed rats are indicated (P ~<0.01, Duncan's test).
rats were analyzed immediately after the last footshock session; this presumably reflects an enhancement of shock-induced peptide release as compared to naive rats. These changes are most probably related to noxious stimulation: thus, the peptides are present in maximal concentrations in the dorsal horn, and particularly in the substantia gelatinosa 1,2~ls, a key structure for modulation of nociception. Further, these peptides exert antinociceptive effects at the spinal level7'17,21,22. In addition, the observation that the changes are restricted to spinal cord sections which receive nociceptive input from the limbs favours this view. Similar changes (though less pronounced) can also be seen in the lumbar cord for the proenkephalin A system: a slight but significant elevation of M E A G L was seen following chronic stress, and a significant depletion of this peptide and of Met-enkephalin was seen upon analysis immediately after the last shock period. The most likely interpretation of these data is that spinal cord-localized opioid systems adapt to prolonged recurrent noxious stimulation by an enhancement in their activity in those areas receiving noxious input. The changes observed probably reflect a functional response to pain. In fact, similar alterations have
been reported in the spinal cord of rats suffering from polyarthritic pain 4,14 or chronic localized pain (monoarthritis) 5"2°. A combined biochemical and behavioral study in polyarthritic rats suggested that in this state, the activity of the spinal cord proenkephalin B system may be enhanced 13. A current in vivo and in vitro release study has shown, however, that the release of opioid peptides derived from proenkephalin A and B might not be enhanced but rather inhibited in this condition 3 (Przewtocki, in preparation). In contrast to the changes observed in the spinal cord, no significant alterations were seen in the opiold peptide content of the thalamus, a brain structure of importance for the receipt and processing of nociceptive information derived from ascending spinothalamic pathways. This finding contrasts with data obtained in polyarthritic rats, in which levels of proenkephalin B-derived peptides increased markedly 14. No such changes were observed, however, in the thalamus of rats subjected to chronic localized pain 2°. These differences might be due to the greater intensity and duration of polyarthritis. In line with another study 14, we found no evidence for an alteration of proenkephalin A and B peptides in the hypothalamus. There is also no evidence that the hypothalamic fl-endorphin system might be influenced either by chronic polyarthritis 14, recurrent foot-shock stress (this study) or chronic localized pain 2°. Nevertheless, this may not necessarily indicate that the hypothalamic fl-endorphin system is not involved in chronic pain or stress, since tissue levels may not necessarily reveal a change in activity. Further, one should bear in mind the fact that the system responds to acute stress by an enhanced release of the peptide 15'19'23. In agreement with certain previous studies, pituitary pools offl-endorphin were only slightly depleted upon acute stress, but increased following prolonged stress in both anterior and intermediate lobes. The elevated levels of peptide are accompanied by an elevation in levels of m R N A encoding POMC in the anterior but not in the intermediate lobe s , although acute foot-shock releases fl-endorphin not only from the anterior but also from the neurointermediate lobe 19. This may indicate an enhancement of the release and synthesis of fl-endorphin in the anterior lobe and in increase in peptide accumulation in the neurointermediate lobe. In line with the above data,
218 chronic arthritis is accompanied by an increase in anterior lobe pools of fl-endorphin and m R N A for P O M C , while, in contrast, no significant changes in fl-endorphin levels in the neurointermediate lobe occurred, and levels of m R N A coding for P O M C in this lobe tended to decrease 8. These shifts in pituitary opioid peptide levels may not seem to be directly related to the pain induced by foot-shock or chronic arthritis, since similar increases have been observed in rats merely placed in the foot-shock chamber but without being subjected to this stress (Millan, personal communication). Interestingly, however, no alterations of opioid peptides in spinal cord were visible in rats treated in this way. This might indicate that the changes observed in the spinal cord (in contrast to pituitary lobes) are directly related to the noxious pain. Plasma levels of fl-endorphin did not differ significantly between naive or chronically stressed rats exposed to an acute foot-shock. In other words, the releasable pool of the pituitary fl-endorphin responds in a similar way in naive and chronically stressed rats, even though there is an increase in pituitary lobe levels of peptide and prohormone synthesis in chronic stress. This observation differs from data obtained in polyarthritic rats, in which substantial increases in flendorphin levels in plasma were measured TM. An acute, short-term foot-shock stress was pre-
REFERENCES 1 Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 2 Botticelli, L.I., Cox, B.M. and Goldstein, A., Immunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 7783-7786. 3 Cesselin, F., Bourgoin, S., Artaud, F. and Hamon, M., Basic and regulatory mechanisms of in vitro release of Met-enkephalin from dorsal horn of the rat spinal cord, J. Neurochem., 43 (1984) 763-773. 4 Cesselin, F., Montastruc, J.L., Gros, C., Bourgoin, S. and Hamon, M., Met-enkephalin levels and opiate receptors in the spinal cord of chronic suffering rats, Brain Research, 191 (1980) 289-293. 5 Faccini, E., Uzumaki, H., Govoni, S., Missale, C., Spano, P.F., Covelli, V. and Trabucchi, M., Afferent fibres mediate the increase of Met-enkephalin elicited in rat spinal cord by localized pain, Pain, 18 (1984) 25-31. 6 Guillemin, R., Vargo, T., Rossier, J., Minick, S., Ling, N., Rivier, C., Vale, W. and Bloom, R., fl-Endorphin and adrenocorticotropin are secreted concomitantly by the pitu-
viously reported to modify dynorphin levels in the anterior lobe but not to significantly affect the peptide levels in the neurointermediate lobe 16. As has been shown in the present study, neither acute nor chronic stress influenced the peptide levels in the anterior lobe. In chronically stressed rats, however, a slight increase in neurointermediate lobe level of the peptide was observed, which might indicate an increase in the activity of hypothalamo-pituitary dynorphin systems under prolonged stress. In summary, the present study demonstrates that recurrent chronic foot-shock stress is accompanied by an enhanced function of the proenkephalin A and/or B opioid systems in the spinal cord and of flendorphin in the anterior pituitary. It is noteworthy that to some extent similar changes seem to occur in rats with chronic localized pain, as well as in rats suffering from polyarthritic pain. These data might indicate the existence of a c o m m o n mechanism involved in the control of chronic pain and stress, in which the spinal cord proenkephalin A and B, on the one hand, and the anterior pituitary fl-endorphin system, on the other hand, may play an important regulatory role. ACKNOWLEDGEMENTS Supported by Deutsche Forschungsgemeinschaft, Bonn.
itary gland, Science, 197 (1977) 1367-1369. 7 Han, J.-S. and Xie, G.X., Dynorphin: potent analgetic effect in spinal cord of the rat, Life Sci., 31 (1982) 1781-1784. 8 H611t, V., Przewlocki, R. and Herz, A., Radioimmunoassay of/3-endorphin: basal and stimulated levels of extracted rat plasma, Naunyn-Schmiedeberg's Arch. Pharmacol., 303 (1978) 171-174. 9 HOllt, V., Przewlocki, R., Haarmann, I., Almeida, O., Kley, N., Millan, M. and Herz, A., Stress-induced alterations in the levels of messenger RNA encoding proopiomelanocortin in rat pituitary, Neuroendocrinology, 43 (1986) 277-282. 10 Lewis, J.W., Cannon, J.T. and Liebeskind, J.C., Opioid and non-opioid mechanisms of stress analgesia, Science, 208 (1980) 623-625. 11 Madden, J., Akil, H., Patrick, R.L. and Barchas, J.D., Stress-induced parallel changes in central opioid levels and pain responsiveness in the rat, Nature (London), 265 (1977) 358-360. 12 Maysinger, D., H611t, V., Seizinger, B.R., Mehraein, P., Pasi, A. and Herz, A., Parallel distribution of immunoreactive a-neo-endorphin and dynorphin in rat and human tissue, Brain Research, 280 (1983) 95-103. 13 Millan, M.J., Czlonkowski, A., Pilcher, C.W.T., Almeida,
219 O.F.X., Milran, M.H., Colpaert, F.C. and Herz, A., A model of chronic pain in the rat: functional correlates of alterations in the activity of opioid systems, J. Neurosci., in press. 14 Millan, M.J., Millan, M.H., Czlonkowski, A., H611t, V., Pilcher, C.W.T., Herz, A. and Colpaert, F.C., A model of chronic pain in the rat: response of multiple opioid systems to adjuvant-induced arthritis, J. Neurosci., 4 (1986) 889-906. 15 Millan, M.J., Przewlocki, R., Jerlicz, M., Gramsch, Ch.. H611t, V. and Herz, A., Stress-induced release of brain and pituitary fl-endorphin: major role of endorphins in generation of hyperthermia not analgesia, Brain Research, 208 (1981) 325-338. 16 Millan, M.J., Tsang, Y., Przewlocki, R., H611t, V. and Herz, A., The influence of foot-shock stress upon brain, pituitary and spinal cord pools of immunoreactive dynorphin in rats, Neurosci. Lett., 24 (1981) 75-79. 17 Piercey, M.F., Varner, K. and Schroeder, L.A., Analgesic activity of intraspinally-administered dynorphin and ethylketocyclazocine, Eur. J. Pharrnacol., 80 (1982) 283-284. 18 Przewlocki, R., Gramsch, Ch., Pasi, A. and Herz, A., Characterization and localization of immunoreactive dynorphin, a-neo-endorphin, Met-enkephalin and substance P in human spinal cord, Brain Research, 280 (1983) 95-103. 19 Przewlocki, R., Millan, M.J., Gramsch, Ch., Millan, M.H. and Herz, A., The influence of selective adeno- and neuro-
intermediate-hypophysectomy upon plasma and brain levels of fl-endorphin and their response to stress in rats, Brain Research, 242 (1982) 107-117. 20 Przewlocki, R., Przewlocka, B., Lasofi, W., Garz6n, J., Stala, L. and Herz, A., Opioid peptides, particularly dynorphin, and chronic pain, 1NSERM, 127 (1984) 159-170. 21 Przewlocki, R., Shearman, G.T. and Herz, A., Mixed opioid/non-opioid effects of dynorphin and dynorphin-related peptides after their intrathecal injection in rats, Neuropeptides, 3 (1983) 233-239. 22 Przewlocki, R., Stala, L., Greczek, M., Shearman, G.T., Przewlocka, B. and Herz, A., Analgesic effects of/~-, 6-and x-opiate agonists and, in particular, dynorphin at the spinal level, Life Sci. 33, Suppl. I (1983) 649-652. 23 Rossier, J., Guillemin, R. and Bloom, F.E., Foot-shock induced stress decreases LeuS-enkephalin immunoreactivity in rat hypothalamus, Eur. J. Pharrnacol., 48 (1978) 465-466. 24 Shiomi, H. and Akil, H., Pulse-chase studies of the POMC/fl-endorphin system in the pituitary of acutely and chronically stressed rats, Life Sci., 31 (1982) 2185-2188. 25 Watkins, L.R. and Mayer, D.J., Organization of endogenous opiate and non-opiate pain control systems, Science, 216 (1982) 1185-1192. 26 Young, E. and Akil, H., Changes in releasability of ACTH and beta-endorphin with chronic stress, Neuropeptides, 5 (1985) 545-548.