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Functional κ-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis
62
Brairt Research, 462 (1988) 62 -6~ Elscvicr
BRE 13968
Functional c-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis B.-G. Zhao, C. Chapman and R.J. Bicknell Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Cambridge (U. K. ) (Accepted 26 April 1988) Key words: Oxytocin; Vasopressin; Opioid receptor; K-Opioid; Dynorphin; Neurohypophysis; Presynaptic localization
Opioids intrinsic to the rat neurohypophysial system act to inhibit secretion from the terminals of magnocellular neurones. Opioid receptors in the neurohypophysis are predominantly of the x-subtype and selective K-agonists suppress electrically evoked release of oxytocin (OXT) and vasopressin (AVP). We have looked for the presence of functional K-receptors on neurohypophysial nerve terminals by examining effects of K-agonists on secretion from suspensions of isolated neurohypophysial nerve terminals (neurosecretosomes) retained on filters in a perifusion system. Release of both OXT and AVP evoked by K+-depolarisation was inhibited by the K-agonists U-50,488H (34% and 45% respectively) and dynorphin Al_13 (68% and 51% respectively). Inhibition by dynorphin A was only observed in the presence of peptidase inhibitors. The actions of both ~-agonists were prevented by the opioid receptor antagonist naloxone. The experiments indicate the presence of x-receptors on terminals of OXT and AVP neurones. This receptor population is in addition to those previously described on pituicytes and those influencing release of neurohypophysial noradrenaline.
INTRODUCTION Opioids contained within n e u r o h y p o p h y s i a l nerve terminals act to suppress secretion from terminals of the magnocellular n e u r o s e c r e t o r y neurones 3. The suppression is very m a r k e d in the case of oxytocin t "~(OXT) where antagonism of the actions of intrinsic opioids with naloxone increases e v o k e d release by 100-200%. Secretion from vasopressin (AVP) terminals in these experiments is either unaffected by n a loxone or shows a m o d e s t e n h a n c e m e n t ~'3'6'~s'23. Naloxone t r e a t m e n t in vivo will also selectively elevate plasma O X T rather than AVP levels under a n u m b e r of physiological and e x p e r i m e n t a l conditions t2'2° although the site of opioid actions may not be restricted to the neurohypophysis 4'21. We have p r o p o s e d a m o d e l of opioid actions in the neurohypophysis which involves both 'direct' and n o r a d r e n a l i n e - m e d i a t e d influences on secretion
from magnocellular nerve terminals 23,24. Direct effects a p p e a r to p r e d o m i n a t e s and to be m e d i a t e d by ~-opioid receptors 23 which are the m a j o r or exclusive subtype in the rat neurohypophysis 8,13. The cellular localisation of these receptors is not yet clear. O n e population of opioid receptors appears to be present on neurohypophysial astrocytes, pituicytes 8~4. In the present study we r e p o r t the presence of functional ~receptors on the magnocellular nerve terminals themselves by demonstrating inhibitory effects of ~copioid agonists on secretion of O X T and AVP from suspensions of isolated n e u r o h y p o p h y s i a l terminals (neurosecretosomes, NSS). Some of these data have been presented in a preliminary report 2. MATERIALS AND METHODS Pituitaries were r e m o v e d from male Wistar rats (220-240 g) and the n e u r o h y p o p h y s e s dissected free
Correspondence: R.J. Bicknell, Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
63 of intermediate lobe tissue. Suspensions of NSS were prepared following gentle homogenisation according to the method of Nordmann et al. 9. We have confirmed the findings of these authors that upon examination with the electron microscope the preparation consists primarily of spherical membrane-bound elements, 1-2 ~m in diameter, containing neurosecretory granules and microvesicles. NSS were resuspended in a bicarbonate-buffered isotonic solution 23 and aliquots, each equivalent to one neurohypophysis, gently inoculated onto 13 mm diameter, 0.45/~m fluoropolymer filter assemblies (Acro LC13, Gelman Sciences) containing a prefilter (Millipore AP20) fitted in the inlet barrel. Up to 8 chambers were prepared concurrently, each connected to the outflow of a peristaltic pump and perifused at 37 °C at a flow rate of 100 ~tl/min with medium as described. Fiveminute fractions of perifusate were collected automatically and stored at -20 °C until measurement of contents of OXT and AVP by specific radioimmunoassay 1. Following an equilibration period of 120 min neurohormone release was evoked by depolarisation for 5 min with isotonic medium containing 60 mM K ÷ (substituted for Na+). The amounts of OXT and AVP release evoked above the immediately preceding basal release were calculated. These absolute amounts were found to vary between preparations and a double stimulus protocol was therefore used in which a first depolarising stimulation (Sl) was always performed in the absence of test compounds and a second identical stimulation ($2) performed 45 min later in the presence of test compounds introduced 20 min previously. The ratios $2:S l of the absolute amounts of evoked OXT and AVP release were compared with control experiments in which no test drugs were included. Significant differences between groups were assessed by non-parametric statistics (Wilcoxon test). Dynorphin A1_13 and [o-Ala2,MePheS-Gly-ol]en kephalin ( D A G O ) were obtained from Cambridge Research Biochemicals. D,L-Thiorphan was from Bachem (Switzerland) and bestatin from Sigma (U.K.). We are grateful to ICI Pharmaceuticals (U.K.) for the gift of U-50,488H, to Squibb laboratories for captopril and to DuPont (U.K.) for naloxone hydrochloride.
RESULTS Mean basal rates of release (B 0 of OXT and AVP immediately preceding the first stimulation were 93 + 6 pg/min and 78 + 6 pg/min respectively (n = 66). S 1 evoked release of an additional 124 + 7 pg/min OXT and 150 + 11 pg/min AVP. Evoked release was dependent on the presence of extracellular Ca 2÷ ($2:S l, when Ca 2÷ omitted during S2 was 0.007 for OXT and 0.09 for AVP, n = 4, compare Table I), and prevented by the Ca2+-channel blocker Co 2÷ as previously reported 9 (data not shown). The selective ~c-receptor agonist U-50,488H (10 -6 M) strongly inhibited evoked release of both OXT and AVP (Table I, 34% and 45% inhibitions respectively). These effects were prevented by inclusion of the non-selective opioid receptor antagonist naloxone, which alone was without significant influence (Table I). Neither drug affected basal release as judged by comparison of ratios of basal release immediately prior to each stimulation (B2:B1, Table I). Dynorphin AI_I3 (10 -6 M) was also tested in this series but did not alter evoked release of either OXT or AVP ($2:S 1 0 X T 1.00 + 0.05, AVP 0.96 + 0.07, n = 10, compare Table I). This study with dynorphin A was repeated in the presence, during $2, of a cocktail of amino-, carboxyand endopeptidase inhibitors 1° to try and prevent any enzymatic breakdown of dynorphin. The peptidase inhibitors did not alter the control $2:S 1 ratio but in their presence dynorphin A strongly inhibited evoked release of both OXT and AVP (68% and 51% inhibitions respectively, Table II). Under identical conditions the highly selective /~-opioid receptor agonist D A G O was without effect. The inhibitory actions of dynorphin A were completely prevented by naloxone (Table II). Selective d-opioid receptor agonists were not tested since this class of receptor is absent in the rat neurohypophysis s,13 and d-antagonists are ineffective in promoting neurohormone secretion ~. DISCUSSION The selective ~c-agonist U-50,488H and dynorphin Al_13 but not the p-agonist D A G O inhibited secretion of both OXT and AVP evoked by direct depolarisation of isolated neurosecretory terminals. Both
64 TABLE 1 Effects (~f U-50.488H on secretion ~ff O X T and AVPJ?om NSS
Calculation of basal release ratios (B2:B 0 and stimulus-evoked release ratios ($2:S~) is described in Materials and Methods. Dala arc mean +_ S.E.M. Treatment
n
OXT
AVP
B2.'B l
$2:S l
B2.'B l
$2:5 l
Control U-50,488H (10 -0 M) Natoxone (10 -5 M) U-50,488H (10 6 M) + naloxone (10 -5 M)
10 11 7 8
0.72 _+ 0.08 [I.63 + 0.07 (/.72 ± 0.06 0.62 + 0.06
0.98 ± 0.09 0.65 _+0.09 ~' 1.25 _+(I.20 1.38 _+ 0.15 ~"
(t.61 0.54 0.70 0.68
0.96 ± 0.10 0.53 _±(I.(18h 0.98 _+ 0.14 1.(/5 ± 0.13 ~
_+ 0.07 _+ 0.07 +_ (I.(t3 _+ 0.06
~P < 0.05 vs control. bp < 0.01 vs control. ~P < 0.01 vs U-50,488H (Wilcoxon text).
inhibitory actions w e r e n a l o x o n e - r e v e r s i b l e . W e in-
tors to suppress Ca2+-currents 17. T h u s , it is likely that
terpret these findings as indicating the p r e s e n c e of ~:-
the m e c h a n i s m by which ;c-receptors inhibit O X T and
receptors on the t e r m i n a l s (axonal swellings are gen-
A V P release f r o m NSS is via inhibition of depolarisat i o n - e v o k e d Ca2+-entry.
erally e x c l u d e d in this N S S p r e p a r a t i o n 9) of both Opioid
In the intact n e u r o h y p o p h y s i s U - 5 0 , 4 8 8 H will also
binding sites with ;c-characteristics are k n o w n to be
inhibit electrically e v o k e d release of b o t h O X T and
present on b o t h p o r c i n e and b o v i n e N S S lt'ls. This lo-
A V P x3, so this action p r o b a b l y i n v o l v e s the K-recep-
calisation is thus a n a l o g o u s to a p r e s y n a p t i c t e r m i n a l
tors we h a v e n o w described. O u r p r e v i o u s studies in
localisation e l s e w h e r e in the brain. D i r e c t effects of
the intact tissue with d y n o r p h i n A did not r e v e a l any
OXT
and
AVP
magnocellular
neurones.
o p i o i d agonists on o u t p u t of n o n - p e p t i d e t r a n s m i t t e r s
inhibitory actions I. A l t h o u g h
from
previously
m e n t s suggest that inactivation of d y n o r p h i n by p e p -
b e e n t a k e n as d e m o n s t r a t i n g a p r e s y n a p t i c r e c e p t o r
tidases m i g h t a c c o u n t for this failure, a r e c e n t study
localisation 1~.
has d e s c r i b e d inhibitory effects of u n p r o t e c t e d dyn-
synaptosomal
preparations
have
the c u r r e n t
experi-
o r p h i n Al_ 8 (ref. 6). Interestingly, this inhibition is
D e p o l a r i s a t i o n - e v o k e d release f r o m NSS i n v o l v e s entry of e x t r a c e l l u l a r Ca -~+ t h r o u g h v o l t a g e - s e n s i t i v e
selective for O X T 6, and intrinsic n e u r o h y p o p h y s i a l
Ca2+-channels
neuronal
opioids also relatively selectively inhibit o u t p u t f r o m O X T t e r m i n a l s in the intact t i s s u e 1'3"~'L5"23. T h u s ,
p e r i k a r y a have indicated a direct action of K-recep-
whilst A V P n e r v e t e r m i n a l s possess K-receptors these
Previous
in t e r m i n a l
plasma
electrophysiological
m e m b r a n e s 7'9.
studies
at
TABLE I1 Effects of x- and .u-selective opioid peptides on secretion o f O X T and A VP from NSS
All treatment and control groups included peptidase inhibitors as follows: bestatin 10 5 M, captopri110 -~ M, D.L-thiorphan 3 × l[) ~M. Calculation of basal release ratios (Bz:B 0 and stimulus-evoked release ratios ($2:S 0 is described in Materials and Methods. Data are mean + S.E.M. Treatment
-
Control Dynorphin A t i:~( 1()-¢'M) Dynorphin Al_t3 ( 10 " M) + naloxone (10-5 M) DAGO(10 ~'M) "P < 0.01 vs control. hp < 0.05 vs dynorphin alone (Wilcoxon test).
n
5 5 4 6
--O X T
....
AVP
B2.'B I
$2.'S I
Be.'B l
.S':.'Sl
0.79 -+ 0.11 0.66±0.04 (I.68+0.02 0.82+-0.05
1.20 + 0.15 0.31+0.04 u 1.38 + 0.15 h 1.01±0.11
0.70 + 0.06 0.58±(t.06 0,70+0.04 0.72+-0.03
1.02 ± (I.06 0.47 + (L06'' 1.27+0.10 I' 1.01 ±0.13
65 appear hardly activated by intrinsic opioids. Whether
in the neurohypophysis 24. For O X T neurones, r-opi-
peptidase inactivation of intrinsic opioids is involved in this selectivity is under investigation. Prodynorphin-derived peptides in the neurohypophysis are coproduced in the AVP neurones 19'22 and may thus exert a cross-inhibitory paracrine influence over O X T terminals as has been previously speculated6,20, 23.
oid receptors are localised on their terminals and also on pituicytes. Intrinsic opioids, possibly prodynorphin peptides released from adjacent AVP terminals, act on either or both of these populations to reduce O X T release in response to depolarising action potentials. A further indirect modulatory action of intrinsic opioids, acting via receptors probably not of the ~c-subtype which regulate release of facilitatory noradrenaline, also exists 24. Catecholamine-dependent regulatory mechanisms appear to play a minor role under conditions so far examined 5. Terminals of AVP neurones also possess r-receptors but, for reasons as yet unknown, these are not strongly activated by intrinsic opioids although the potential exists for powerful actions of opioids to inhibit AVP secretion at this site.
Unlike the intact neurohypophysis where opioid antagonists alone promote neurohormone secretion, there appears to be no significant influence of intrinsic opioids in the perifused NSS preparation, although there may be a small residual effect of naloxone on O X T secretion (Table I). This finding is thus consistent with the breakdown of paracrine opioid influences upon disruption of the tissue, presumably since released opioids will now be rapidly removed from the vicinity of the nerve terminals. However, the NSS preparation also destroys the intimate relationships existing in the intact tissue between neurosecretory elements and pituicytes. These glial cells also possess opioid receptors of the r-subtype and the function of this receptor population will require further study 8A4. In conclusion we wish to extend our model of opioid mechanisms regulating neurohormone secretion REFERENCES 1 Bicknell, R.J., Chapman, C. and Leng, G., Effects of opioid agonists and antagonists on oxytocin and vasopressin release in vitro, Neuroendocrinology, 41 (1985) 142-148. 2 Bicknell, R.J., Chapman, C. and Zhao, B.-G., Functional kappa-opioid receptors on oxytocin and vasopressin secretory terminals isolated from rat neurohypophysis, J. Physiol. (Lond.), 394 (1987) 123P. 3 Bicknell, R.J. and Leng, G., Endogenous opiates regulate oxytocin but not vasopressin secretion from the neurohypophysis, Nature (Lond.), 298 (1982) 161-162. 4 Bicknell, R.J., Leng, G., Lincoln, D.W. and Russell, J.A., Naloxone excites oxytocin neurones in the supraoptic nucleus of lactating rats after chronic morphine treatment, J. Physiol. (Lond.), 396 (1988) 297-317. 5 Bicknell, R.J., Zhao, B.-G., Chapman, C., Heavens, R.P. and Sirinathsinghji, D.J.S., Opioid inhibition of secretion, from oxytocin and vasopressin nerve terminals following selective depletion of neurohypophysial catecholamines, Endocrinology 122 (1988) 1321-1327. 6 Bondy, C.A., Gainer, H. and Russell, J.T., Dynorphin A inhibits and naloxone increases the electrically stimulated release of oxytocin but not vasopressin from the terminals of the neural lobe, in press. 7 Brethes, D., Dayanithi, G., Letellier, L. and Nordmann, J.J., Depolarisation-induced Ca2+ increase in isolated
ACKNOWLEDGEMENTS Bai-ge Zhao is supported by the World Health Organisation. Permanent address: Shanghai Institute of Planned Parenthood Research, Shanghai, People's Republic of China. We thank Dr. F.B.P. Wooding for producing electron micrographs, and Jennifer Cummings for typing the manuscript. neurosecretory nerve terminals measured with Fura-2, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1439-1443. 8 Bunn, S.J., Hanley, M.R. and Wilkin, G.P., Evidence for a kappa-opioid receptor on pituitary astrocytes: an autoradiographic study, Neurosci. Lett., 55 (1985) 317-323. 9 Cazalis, M., Dayanithi, G. and Nordmann, J.J., Hormone release from isolated nerve endings of the rat neurohypophysis, J. Physiol. (Lond.), 390 (1987) 55-70. 10 Corbett, A.D., Paterson, S.J., McKnight, A.T., Magnan, J. and Kosterlitz, H.W., Dynorphinl_ 8 and dynorphinl_ 9 are ligands for the n-subtype of opiate receptor, Nature (Lond.), 299 (1982) 79-81. 11 Falke, N. and Martin, R., Opioid binding in a rat neurohypophysial fraction enriched in oxytocin and vasopressin nerve endings, Neurosci. Lett., 61 (1985) 37-41. 12 Hartman, R.D., Rosella-Dampman, L.M., Emmert, S.E. and Summy-Long, J.Y., Inhibition of release of neurohypophysial hormones by endogenous opioid peptides in pregnant and parturient rats, Brain Research, 382 (1986) 352-359. 13 Herkenham, M., Rice, K.C., Jacobson, A.E. and Rothman, R.B., Opiate receptors in rat pituitary are confined to the neural lobe and are exclusively kappa, Brain Research, 382 (1986) 365-371. 14 Lightman, S.L., Ninkovic, M., Hunt, S.P. and Iversen, L.L., Evidence for opiate receptors on pituicytes, Nature (Lond.), 305 (1983) 235-237.
66 15 Maysinger, D., Vermes, I., Tilders, F., Seizinger, B.R., Gramsch, C., H611t, H. and Herz. A., Differential effects of various opioid peptides on vasopressin and oxytocin release from the rat pituitary in vitro, Arch. Pharmacol., 328 (1984) 191-195. 16 Mulder, A.H., Hogenboom, F.. Wardeh, G. and Schoffelmeer, A.N.M., Morphine and enkephalins potently inhibit [3H]noradrenaline release from rat brain cortex synaptosomes: further evidence for a presynaptic localisation of g~opioid receptors, J. Neurochem., 48 (1987) 1043-1047. 17 North, R.A., Opioid receptor types and membrane ion channels, Trends Neurosci., 9 (1986) 114-117. 18 Pesce, G., Lang, M.A., Russell, J.T.. Rodbard, D. and Gainer, H., Characterisation of ~-opioid receptors in neurosecretosomes from bovine posterior pituitary, J. Neurochem., 49 (1987) 421-427. 19 Sherman, T.G., Civelli, O., Douglass, J., Herbert, E. and Watson, S.J., Co-ordinate expression of hypothalamic prodynorphin and pro-vasopressin mRNAs with osmotic stimulation, Neuroendocrinology, 44 (1986) 222-228. 20 Summy-Long, J.Y., Miller, R.S.. Rosella-Dampman,
21
22
23
24
L.M., Hartman, R.D. and Emmert, S.E,, A functional role for opioid peptides in the differential secretion of vasopressin and oxytocin, Brain Research, 309 (1984) 362-366. Wakerley, J.B., Noble, R. and Clarke, G., Effects of morphine and D-AIa. D-Leu enkephalin on the electrical activity of supraoptie neurosecretory cells in vitro. Neuroscience, 10 (1983) 73-81. Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Greidanus, T.B., Dynorphin and vasopressin: common localisation in magnocellular neurons, Science, 216 (1982) 85-87. Zhao, B.-G.. Chapman, C. and Bicknell, R,J., Opioid-noradrenergic interactions in the neurohypophysis. I. Differential opioid receptor regulation of oxytocin, vasopressin and noradrenaline release, Neuroendocrinology. in press. Zhao, B.-G., Chapman, C., Brown, D, and Bicknell, R.J., Opioid-noradrenergic interactions in the neurohypophysis. II. Does noradrenaline mediate the action of endogenous opioids on oxytoein and vasopressin release'?, Neuroendocrinology, in press.
Brairt Research, 462 (1988) 62 -6~ Elscvicr
BRE 13968
Functional c-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis B.-G. Zhao, C. Chapman and R.J. Bicknell Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Cambridge (U. K. ) (Accepted 26 April 1988) Key words: Oxytocin; Vasopressin; Opioid receptor; K-Opioid; Dynorphin; Neurohypophysis; Presynaptic localization
Opioids intrinsic to the rat neurohypophysial system act to inhibit secretion from the terminals of magnocellular neurones. Opioid receptors in the neurohypophysis are predominantly of the x-subtype and selective K-agonists suppress electrically evoked release of oxytocin (OXT) and vasopressin (AVP). We have looked for the presence of functional K-receptors on neurohypophysial nerve terminals by examining effects of K-agonists on secretion from suspensions of isolated neurohypophysial nerve terminals (neurosecretosomes) retained on filters in a perifusion system. Release of both OXT and AVP evoked by K+-depolarisation was inhibited by the K-agonists U-50,488H (34% and 45% respectively) and dynorphin Al_13 (68% and 51% respectively). Inhibition by dynorphin A was only observed in the presence of peptidase inhibitors. The actions of both ~-agonists were prevented by the opioid receptor antagonist naloxone. The experiments indicate the presence of x-receptors on terminals of OXT and AVP neurones. This receptor population is in addition to those previously described on pituicytes and those influencing release of neurohypophysial noradrenaline.
INTRODUCTION Opioids contained within n e u r o h y p o p h y s i a l nerve terminals act to suppress secretion from terminals of the magnocellular n e u r o s e c r e t o r y neurones 3. The suppression is very m a r k e d in the case of oxytocin t "~(OXT) where antagonism of the actions of intrinsic opioids with naloxone increases e v o k e d release by 100-200%. Secretion from vasopressin (AVP) terminals in these experiments is either unaffected by n a loxone or shows a m o d e s t e n h a n c e m e n t ~'3'6'~s'23. Naloxone t r e a t m e n t in vivo will also selectively elevate plasma O X T rather than AVP levels under a n u m b e r of physiological and e x p e r i m e n t a l conditions t2'2° although the site of opioid actions may not be restricted to the neurohypophysis 4'21. We have p r o p o s e d a m o d e l of opioid actions in the neurohypophysis which involves both 'direct' and n o r a d r e n a l i n e - m e d i a t e d influences on secretion
from magnocellular nerve terminals 23,24. Direct effects a p p e a r to p r e d o m i n a t e s and to be m e d i a t e d by ~-opioid receptors 23 which are the m a j o r or exclusive subtype in the rat neurohypophysis 8,13. The cellular localisation of these receptors is not yet clear. O n e population of opioid receptors appears to be present on neurohypophysial astrocytes, pituicytes 8~4. In the present study we r e p o r t the presence of functional ~receptors on the magnocellular nerve terminals themselves by demonstrating inhibitory effects of ~copioid agonists on secretion of O X T and AVP from suspensions of isolated n e u r o h y p o p h y s i a l terminals (neurosecretosomes, NSS). Some of these data have been presented in a preliminary report 2. MATERIALS AND METHODS Pituitaries were r e m o v e d from male Wistar rats (220-240 g) and the n e u r o h y p o p h y s e s dissected free
Correspondence: R.J. Bicknell, Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
63 of intermediate lobe tissue. Suspensions of NSS were prepared following gentle homogenisation according to the method of Nordmann et al. 9. We have confirmed the findings of these authors that upon examination with the electron microscope the preparation consists primarily of spherical membrane-bound elements, 1-2 ~m in diameter, containing neurosecretory granules and microvesicles. NSS were resuspended in a bicarbonate-buffered isotonic solution 23 and aliquots, each equivalent to one neurohypophysis, gently inoculated onto 13 mm diameter, 0.45/~m fluoropolymer filter assemblies (Acro LC13, Gelman Sciences) containing a prefilter (Millipore AP20) fitted in the inlet barrel. Up to 8 chambers were prepared concurrently, each connected to the outflow of a peristaltic pump and perifused at 37 °C at a flow rate of 100 ~tl/min with medium as described. Fiveminute fractions of perifusate were collected automatically and stored at -20 °C until measurement of contents of OXT and AVP by specific radioimmunoassay 1. Following an equilibration period of 120 min neurohormone release was evoked by depolarisation for 5 min with isotonic medium containing 60 mM K ÷ (substituted for Na+). The amounts of OXT and AVP release evoked above the immediately preceding basal release were calculated. These absolute amounts were found to vary between preparations and a double stimulus protocol was therefore used in which a first depolarising stimulation (Sl) was always performed in the absence of test compounds and a second identical stimulation ($2) performed 45 min later in the presence of test compounds introduced 20 min previously. The ratios $2:S l of the absolute amounts of evoked OXT and AVP release were compared with control experiments in which no test drugs were included. Significant differences between groups were assessed by non-parametric statistics (Wilcoxon test). Dynorphin A1_13 and [o-Ala2,MePheS-Gly-ol]en kephalin ( D A G O ) were obtained from Cambridge Research Biochemicals. D,L-Thiorphan was from Bachem (Switzerland) and bestatin from Sigma (U.K.). We are grateful to ICI Pharmaceuticals (U.K.) for the gift of U-50,488H, to Squibb laboratories for captopril and to DuPont (U.K.) for naloxone hydrochloride.
RESULTS Mean basal rates of release (B 0 of OXT and AVP immediately preceding the first stimulation were 93 + 6 pg/min and 78 + 6 pg/min respectively (n = 66). S 1 evoked release of an additional 124 + 7 pg/min OXT and 150 + 11 pg/min AVP. Evoked release was dependent on the presence of extracellular Ca 2÷ ($2:S l, when Ca 2÷ omitted during S2 was 0.007 for OXT and 0.09 for AVP, n = 4, compare Table I), and prevented by the Ca2+-channel blocker Co 2÷ as previously reported 9 (data not shown). The selective ~c-receptor agonist U-50,488H (10 -6 M) strongly inhibited evoked release of both OXT and AVP (Table I, 34% and 45% inhibitions respectively). These effects were prevented by inclusion of the non-selective opioid receptor antagonist naloxone, which alone was without significant influence (Table I). Neither drug affected basal release as judged by comparison of ratios of basal release immediately prior to each stimulation (B2:B1, Table I). Dynorphin AI_I3 (10 -6 M) was also tested in this series but did not alter evoked release of either OXT or AVP ($2:S 1 0 X T 1.00 + 0.05, AVP 0.96 + 0.07, n = 10, compare Table I). This study with dynorphin A was repeated in the presence, during $2, of a cocktail of amino-, carboxyand endopeptidase inhibitors 1° to try and prevent any enzymatic breakdown of dynorphin. The peptidase inhibitors did not alter the control $2:S 1 ratio but in their presence dynorphin A strongly inhibited evoked release of both OXT and AVP (68% and 51% inhibitions respectively, Table II). Under identical conditions the highly selective /~-opioid receptor agonist D A G O was without effect. The inhibitory actions of dynorphin A were completely prevented by naloxone (Table II). Selective d-opioid receptor agonists were not tested since this class of receptor is absent in the rat neurohypophysis s,13 and d-antagonists are ineffective in promoting neurohormone secretion ~. DISCUSSION The selective ~c-agonist U-50,488H and dynorphin Al_13 but not the p-agonist D A G O inhibited secretion of both OXT and AVP evoked by direct depolarisation of isolated neurosecretory terminals. Both
64 TABLE 1 Effects (~f U-50.488H on secretion ~ff O X T and AVPJ?om NSS
Calculation of basal release ratios (B2:B 0 and stimulus-evoked release ratios ($2:S~) is described in Materials and Methods. Dala arc mean +_ S.E.M. Treatment
n
OXT
AVP
B2.'B l
$2:S l
B2.'B l
$2:5 l
Control U-50,488H (10 -0 M) Natoxone (10 -5 M) U-50,488H (10 6 M) + naloxone (10 -5 M)
10 11 7 8
0.72 _+ 0.08 [I.63 + 0.07 (/.72 ± 0.06 0.62 + 0.06
0.98 ± 0.09 0.65 _+0.09 ~' 1.25 _+(I.20 1.38 _+ 0.15 ~"
(t.61 0.54 0.70 0.68
0.96 ± 0.10 0.53 _±(I.(18h 0.98 _+ 0.14 1.(/5 ± 0.13 ~
_+ 0.07 _+ 0.07 +_ (I.(t3 _+ 0.06
~P < 0.05 vs control. bp < 0.01 vs control. ~P < 0.01 vs U-50,488H (Wilcoxon text).
inhibitory actions w e r e n a l o x o n e - r e v e r s i b l e . W e in-
tors to suppress Ca2+-currents 17. T h u s , it is likely that
terpret these findings as indicating the p r e s e n c e of ~:-
the m e c h a n i s m by which ;c-receptors inhibit O X T and
receptors on the t e r m i n a l s (axonal swellings are gen-
A V P release f r o m NSS is via inhibition of depolarisat i o n - e v o k e d Ca2+-entry.
erally e x c l u d e d in this N S S p r e p a r a t i o n 9) of both Opioid
In the intact n e u r o h y p o p h y s i s U - 5 0 , 4 8 8 H will also
binding sites with ;c-characteristics are k n o w n to be
inhibit electrically e v o k e d release of b o t h O X T and
present on b o t h p o r c i n e and b o v i n e N S S lt'ls. This lo-
A V P x3, so this action p r o b a b l y i n v o l v e s the K-recep-
calisation is thus a n a l o g o u s to a p r e s y n a p t i c t e r m i n a l
tors we h a v e n o w described. O u r p r e v i o u s studies in
localisation e l s e w h e r e in the brain. D i r e c t effects of
the intact tissue with d y n o r p h i n A did not r e v e a l any
OXT
and
AVP
magnocellular
neurones.
o p i o i d agonists on o u t p u t of n o n - p e p t i d e t r a n s m i t t e r s
inhibitory actions I. A l t h o u g h
from
previously
m e n t s suggest that inactivation of d y n o r p h i n by p e p -
b e e n t a k e n as d e m o n s t r a t i n g a p r e s y n a p t i c r e c e p t o r
tidases m i g h t a c c o u n t for this failure, a r e c e n t study
localisation 1~.
has d e s c r i b e d inhibitory effects of u n p r o t e c t e d dyn-
synaptosomal
preparations
have
the c u r r e n t
experi-
o r p h i n Al_ 8 (ref. 6). Interestingly, this inhibition is
D e p o l a r i s a t i o n - e v o k e d release f r o m NSS i n v o l v e s entry of e x t r a c e l l u l a r Ca -~+ t h r o u g h v o l t a g e - s e n s i t i v e
selective for O X T 6, and intrinsic n e u r o h y p o p h y s i a l
Ca2+-channels
neuronal
opioids also relatively selectively inhibit o u t p u t f r o m O X T t e r m i n a l s in the intact t i s s u e 1'3"~'L5"23. T h u s ,
p e r i k a r y a have indicated a direct action of K-recep-
whilst A V P n e r v e t e r m i n a l s possess K-receptors these
Previous
in t e r m i n a l
plasma
electrophysiological
m e m b r a n e s 7'9.
studies
at
TABLE I1 Effects of x- and .u-selective opioid peptides on secretion o f O X T and A VP from NSS
All treatment and control groups included peptidase inhibitors as follows: bestatin 10 5 M, captopri110 -~ M, D.L-thiorphan 3 × l[) ~M. Calculation of basal release ratios (Bz:B 0 and stimulus-evoked release ratios ($2:S 0 is described in Materials and Methods. Data are mean + S.E.M. Treatment
-
Control Dynorphin A t i:~( 1()-¢'M) Dynorphin Al_t3 ( 10 " M) + naloxone (10-5 M) DAGO(10 ~'M) "P < 0.01 vs control. hp < 0.05 vs dynorphin alone (Wilcoxon test).
n
5 5 4 6
--O X T
....
AVP
B2.'B I
$2.'S I
Be.'B l
.S':.'Sl
0.79 -+ 0.11 0.66±0.04 (I.68+0.02 0.82+-0.05
1.20 + 0.15 0.31+0.04 u 1.38 + 0.15 h 1.01±0.11
0.70 + 0.06 0.58±(t.06 0,70+0.04 0.72+-0.03
1.02 ± (I.06 0.47 + (L06'' 1.27+0.10 I' 1.01 ±0.13
65 appear hardly activated by intrinsic opioids. Whether
in the neurohypophysis 24. For O X T neurones, r-opi-
peptidase inactivation of intrinsic opioids is involved in this selectivity is under investigation. Prodynorphin-derived peptides in the neurohypophysis are coproduced in the AVP neurones 19'22 and may thus exert a cross-inhibitory paracrine influence over O X T terminals as has been previously speculated6,20, 23.
oid receptors are localised on their terminals and also on pituicytes. Intrinsic opioids, possibly prodynorphin peptides released from adjacent AVP terminals, act on either or both of these populations to reduce O X T release in response to depolarising action potentials. A further indirect modulatory action of intrinsic opioids, acting via receptors probably not of the ~c-subtype which regulate release of facilitatory noradrenaline, also exists 24. Catecholamine-dependent regulatory mechanisms appear to play a minor role under conditions so far examined 5. Terminals of AVP neurones also possess r-receptors but, for reasons as yet unknown, these are not strongly activated by intrinsic opioids although the potential exists for powerful actions of opioids to inhibit AVP secretion at this site.
Unlike the intact neurohypophysis where opioid antagonists alone promote neurohormone secretion, there appears to be no significant influence of intrinsic opioids in the perifused NSS preparation, although there may be a small residual effect of naloxone on O X T secretion (Table I). This finding is thus consistent with the breakdown of paracrine opioid influences upon disruption of the tissue, presumably since released opioids will now be rapidly removed from the vicinity of the nerve terminals. However, the NSS preparation also destroys the intimate relationships existing in the intact tissue between neurosecretory elements and pituicytes. These glial cells also possess opioid receptors of the r-subtype and the function of this receptor population will require further study 8A4. In conclusion we wish to extend our model of opioid mechanisms regulating neurohormone secretion REFERENCES 1 Bicknell, R.J., Chapman, C. and Leng, G., Effects of opioid agonists and antagonists on oxytocin and vasopressin release in vitro, Neuroendocrinology, 41 (1985) 142-148. 2 Bicknell, R.J., Chapman, C. and Zhao, B.-G., Functional kappa-opioid receptors on oxytocin and vasopressin secretory terminals isolated from rat neurohypophysis, J. Physiol. (Lond.), 394 (1987) 123P. 3 Bicknell, R.J. and Leng, G., Endogenous opiates regulate oxytocin but not vasopressin secretion from the neurohypophysis, Nature (Lond.), 298 (1982) 161-162. 4 Bicknell, R.J., Leng, G., Lincoln, D.W. and Russell, J.A., Naloxone excites oxytocin neurones in the supraoptic nucleus of lactating rats after chronic morphine treatment, J. Physiol. (Lond.), 396 (1988) 297-317. 5 Bicknell, R.J., Zhao, B.-G., Chapman, C., Heavens, R.P. and Sirinathsinghji, D.J.S., Opioid inhibition of secretion, from oxytocin and vasopressin nerve terminals following selective depletion of neurohypophysial catecholamines, Endocrinology 122 (1988) 1321-1327. 6 Bondy, C.A., Gainer, H. and Russell, J.T., Dynorphin A inhibits and naloxone increases the electrically stimulated release of oxytocin but not vasopressin from the terminals of the neural lobe, in press. 7 Brethes, D., Dayanithi, G., Letellier, L. and Nordmann, J.J., Depolarisation-induced Ca2+ increase in isolated
ACKNOWLEDGEMENTS Bai-ge Zhao is supported by the World Health Organisation. Permanent address: Shanghai Institute of Planned Parenthood Research, Shanghai, People's Republic of China. We thank Dr. F.B.P. Wooding for producing electron micrographs, and Jennifer Cummings for typing the manuscript. neurosecretory nerve terminals measured with Fura-2, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1439-1443. 8 Bunn, S.J., Hanley, M.R. and Wilkin, G.P., Evidence for a kappa-opioid receptor on pituitary astrocytes: an autoradiographic study, Neurosci. Lett., 55 (1985) 317-323. 9 Cazalis, M., Dayanithi, G. and Nordmann, J.J., Hormone release from isolated nerve endings of the rat neurohypophysis, J. Physiol. (Lond.), 390 (1987) 55-70. 10 Corbett, A.D., Paterson, S.J., McKnight, A.T., Magnan, J. and Kosterlitz, H.W., Dynorphinl_ 8 and dynorphinl_ 9 are ligands for the n-subtype of opiate receptor, Nature (Lond.), 299 (1982) 79-81. 11 Falke, N. and Martin, R., Opioid binding in a rat neurohypophysial fraction enriched in oxytocin and vasopressin nerve endings, Neurosci. Lett., 61 (1985) 37-41. 12 Hartman, R.D., Rosella-Dampman, L.M., Emmert, S.E. and Summy-Long, J.Y., Inhibition of release of neurohypophysial hormones by endogenous opioid peptides in pregnant and parturient rats, Brain Research, 382 (1986) 352-359. 13 Herkenham, M., Rice, K.C., Jacobson, A.E. and Rothman, R.B., Opiate receptors in rat pituitary are confined to the neural lobe and are exclusively kappa, Brain Research, 382 (1986) 365-371. 14 Lightman, S.L., Ninkovic, M., Hunt, S.P. and Iversen, L.L., Evidence for opiate receptors on pituicytes, Nature (Lond.), 305 (1983) 235-237.
66 15 Maysinger, D., Vermes, I., Tilders, F., Seizinger, B.R., Gramsch, C., H611t, H. and Herz. A., Differential effects of various opioid peptides on vasopressin and oxytocin release from the rat pituitary in vitro, Arch. Pharmacol., 328 (1984) 191-195. 16 Mulder, A.H., Hogenboom, F.. Wardeh, G. and Schoffelmeer, A.N.M., Morphine and enkephalins potently inhibit [3H]noradrenaline release from rat brain cortex synaptosomes: further evidence for a presynaptic localisation of g~opioid receptors, J. Neurochem., 48 (1987) 1043-1047. 17 North, R.A., Opioid receptor types and membrane ion channels, Trends Neurosci., 9 (1986) 114-117. 18 Pesce, G., Lang, M.A., Russell, J.T.. Rodbard, D. and Gainer, H., Characterisation of ~-opioid receptors in neurosecretosomes from bovine posterior pituitary, J. Neurochem., 49 (1987) 421-427. 19 Sherman, T.G., Civelli, O., Douglass, J., Herbert, E. and Watson, S.J., Co-ordinate expression of hypothalamic prodynorphin and pro-vasopressin mRNAs with osmotic stimulation, Neuroendocrinology, 44 (1986) 222-228. 20 Summy-Long, J.Y., Miller, R.S.. Rosella-Dampman,
21
22
23
24
L.M., Hartman, R.D. and Emmert, S.E,, A functional role for opioid peptides in the differential secretion of vasopressin and oxytocin, Brain Research, 309 (1984) 362-366. Wakerley, J.B., Noble, R. and Clarke, G., Effects of morphine and D-AIa. D-Leu enkephalin on the electrical activity of supraoptie neurosecretory cells in vitro. Neuroscience, 10 (1983) 73-81. Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Greidanus, T.B., Dynorphin and vasopressin: common localisation in magnocellular neurons, Science, 216 (1982) 85-87. Zhao, B.-G.. Chapman, C. and Bicknell, R,J., Opioid-noradrenergic interactions in the neurohypophysis. I. Differential opioid receptor regulation of oxytocin, vasopressin and noradrenaline release, Neuroendocrinology. in press. Zhao, B.-G., Chapman, C., Brown, D, and Bicknell, R.J., Opioid-noradrenergic interactions in the neurohypophysis. II. Does noradrenaline mediate the action of endogenous opioids on oxytoein and vasopressin release'?, Neuroendocrinology, in press.