- Email: [email protected]
Dynorphin (1–8) inhibits stimulated release of oxytoxin but not vasopressin from isolated neurosecretory endings of the rat neurohypophysis
Nercropeprides (198X) II. 163-167 @I I.ongman Group UK Ltd 1988
Dynorphin (1-8) Inhibits Stimulated Release of Oxytocin but not Vasopressin from Isolated Neurosecretory Endings of the Rat Neurohypophysis N. FALKE Sektion
Eiektronenmikroskopie,
lJniversit8t
Urn, Oberer Eselsberg, 0-7goo U/m, F.R.G.
Abstract-The effects of the opioid peptide dynorphin (l-8) on oxytocin and vasopressin release at the level of isolated neurosecretory endings were investigated. Neurosecretory endings prepared by homogenization and centrifugation were placed on a filter and constantly superfused. Stimulated hormone release was evoked by potassium depolarization (30mM) and simultaneous increase of the osmolarity (20mosmoVl). Stimulation resulted in two peaks of hormone release - a short first peak and a longer second one. Addition of dynorphin (l-8) (10W7M) to the superfusion buffer significantly diminished the first peak of oxytocin release and totally abolished the second. There was no effect of dynorphin (l-8) on vasopressin release.
Introduction In autoradiographic studies opioid binding sites in the rat neurohypophysis were found to be exclusively of the kappa subtype (1, 2). The occurrence of only kappa opioid binding sites in the neurohypophysis seems to be a common feature of various species. Kappa opioid binding sites were localized in isolated neurosecretory terminals from the rat (3), and in purified preparations of terminals from bovine (4) and pig (5) neurohypophyses. In preparations from pig neurowhere oxytocin and vasopressin hypophyses, terminals were enriched in different fractions, Date received Date accepted
27 January 8 February
3988 1988
opioid binding was found to be associated preferentially with oxytocin endings (5). Evidence has been reported for coexistence of dynorphin (1-8) with vasopressin in the same neurosecretory terminals and granules of the rat neurohypophysis in about 300 times lower concentration (6). Presumably dynorphin (l-8) is released when vasopressin is secreted (7). Dynorphin (l-8) is a preferential kappa-ligand (8). With the typical delta-ligand D-Ala-D-Leu-enkephalin no effects on hormone release and calcium-uptake were found at the level of the neurosecretory endings (9). Therefore, it appears likely that under conditions favouring vasopressin release, neighbouring oxytocin endings are affected by co-released dynorphin (l-8). We studied the action of dynorphin (l-8) on the stimulated release of oxytocin and
163
164
vasopressin from isolated neurosecretory terminals and found inhibition of oxytocin, but not vasopressin release.
NELJROPEPTIDES
0.1% . The oxytocin antiserum was obtained from Ferring (Kiel, FRG). It was diluted 1.67000,its crossreactivity with Arg-vasopressin was 0.01%. Results
Methods For each experiment two neurohypophyses from male rats (Chbb, a Wistar-derived stock) of approximately 200g were carefully cleaned from intermediate lobe tissue. In order to minimize hormone release which is enhanced by cooling the tissue, the entire preparation was carried out at room temperature. The neurohypophyses were homogenized in a buffer composed of 0.27M sucrose, 10mM MOPS (morpholinopropanesulfonic acid), 0.1mM EGTA (ethyleneglycol-tetraacetic acid), pH 7.3. After homogenization (5 times at 500 rpm and one time at 1000 rpm) the tissue suspension was centrifuged for 5 min at 40xg. The pellet was discarded, and the supernatant from this centrifugation was collected and centrifuged for 10 min at 5000xg. The pellet from the second centrifugation, the secretsosome fraction, was resuspended in basal super-fusion buffer (140mM NaCl, 2.2mM CaClz, 1mM MgC12, 1OmM MOPS, 0.01% BSA; pH 7.3,285mosmoyl). The secretosome suspension was placed on a Millipore filter (0.22 pm pore size) and superfused for 30 min at a flow rate of 50 pl/min. Then the flow rate was accelerated to 100 @min and after 10 min superfusion at this flow rate, 500 ~1 fractions were collected. After 15 min (3 fractions) the basal buffer was exchanged for the stimulatory buffer (120mM NaCl, 30mM KCl, 2.2mM CaC12,lmM MgCL, 1OmM MOPS, 0.01% BSA; pH 7.3, 305 mosmol/l) for 10 min. Then the neurosecretosomes were superfused again with basal buffer until the end of the experiment. At t = 55 min collection of fractions was stopped and filters were extracted in 1M acetic acid for oxytocin ,and vasopressin radioimmunoassays. Dynorphin (l-8) was obtained from Serva (Heidelberg, FRG) and was added during the entire super-fusion at 100 @min, both to basal and stimulatory buffer. Oxytocin was obtained from Sigma (Munich, FRG) and Argvasopressin from Bachem (Switzerland). For the radiommunoassays the peptides were labeled with 1251 by addition of chloramine T. The labeled peptides were purified by chromatography on Sephadex GlO. The vasopressin antiserum was kindly provided by R.E. Lang, Heidelberg. It was diluted 1:42000, its crossreactivity with oxytocin was
At the superfusion rate of lOOl~J/min the basal release of untreated neurosecretosomes was 0.44pmol/min (SEM = 0.10, n = 6) for oxytocin and 0.87pmoYmin (SEM = 0.10, n = 6) for vasopressin. At the end of the experiment a considerable reservoir of hormones remained within the neurosecretosomes on the filter. The mean value for oxytocin was 384pmol (SEM = 135, n = 6) and for vasopressin 506pmol (SEM = 80, n = 6). There was no significant effect of dynorphin (l-8) on basal release of oxytocin or vasopressin. In the dynorphin-treated neurosecretosomes the basal release of oxytocin was 0.72pmol/min (SEM = 0.24, n = 6) and the basal release of vasopressin was l.O8pmol/min (SEM = 0.37, n = 6). As compared to the controls higher values were also found in the filter extracts of the dynorphin-treated samples for both hormones, the values were 461pmol (SEM = 145, n = 6) for oxytocin and Sllpmol (SEM = 136, n = 6) for vasopressin. The slightly higher basal release of both hormones, oxytocin and vasopressin, in the dynorphin-treated neurosecretosomes, which was not statistically significant, could be explained by a higher quantity of secretosomes on the filter in some of these experiments since there were also higher amounts of hormones in the extracts. In order to standardize and compare the results from different experiments with different quantities of secretosomes on the filters the basal release (=mean value of the first 4 fractions) in each experiment was set = 1. By superfusion of the filter with a coloured solution it was found that the stimulatory buffer arrived at the secretosomes 7.5 min after the buffer change. The colour in the eluate was 66% of the original colour in fraction 5 (25 min). In fraction 6 (30 min) it was maximal (93%) and in fraction 7 (35 min) it diminished again to 29%. In untreated neurosecretosomes the K+-depolarization and the increase in osmolarity induced a peak of oxytocin release in fraction 5 (25 min). Oxytocin release in this fraction was increased 3.23 fold relative to basal release (see Table and Figure). After a decrease in the next two fractions a second increase of oxytocin release occurred without external stimulus which was maximal in fraction 9 (45 min).
DYNORPHIN
(t-8) INHIBITS STIMULATED
165
RELEASE OF OXYTOCIN
Table Hormone-Release is Expressed Relative to Basal Release (=Mean Value of Fraction l-4) which was set = 1. The Number of Experiments was 6. SEM = Standard Error of the Mean oxytocin release control
fract. no.lmin
x
SEM
11 5 2/10 311.5 4120 5125 6130 7135 s/40 9145 lo/so 11155
1.09 1.05 0.98 0.89 3.23 1.77 1.50 2.97 3.51 3.24 2.16
.I2 .07 .lO .13 .44 .27 .21 .87 .84 .85 .57
dyn. (IO-‘M) x SEM .02 .08 .08 .04 .28 .04 .06 .08 .I2 .05 .I7
0.95 0.98 1.06 1.00 1.57 0.71 0.54 0.94 1.08 1.05 1.07
In the dynorphin-treated neurosecretosomes the first peak of oxytocin release in fraction 5 was significantly diminished (p = 0.01). The decrease of oxytocin release in fractions 6 and 7 was more pronounced as compared to the control (p = 0.02 and 0.01 respectively). The second peak of oxytotin release in fraction 9 was totally abolished by addition of dynorphin (l-8) (p = 0.05; p was established by the student t-test for groups with different variances). In untreated neurosecretosomes the stimulated vasopressin release in fraction 5 was lower than oxytocin release: a 1.8 fold increase over the basal level was seen (Table). Similar to the pattern of oxytocin release, after a decrease in the next two fractions a second more extended peak of vaso-
vasopressin release dw. (111-‘M) SEM .I? SEM
control .u 0.84 0.98 0.96 1.22 1.80 0.99 1.02 1.68 1.79 1.79 1.73
.05
.04 .04 .11 .20 .09 .09 .16 .21 .22 .22
1.00 0.94 1.01 1.05 1.75 1.32 1.30 1.52 1.66 1.48 1.36
.02
.03 .01 .02 .I’ .35 30
18 .21 .?9 .2u
pressin release occurred. There was no effect of dynorphin (1-8) on vasopressin release. The stimulatory effect on hormone release of other buffer systems was investigated using untreated neurosecretosomes. If the stimulatory superfusion buffer contained 30mM Kf and other components described previously, but had the same tonicity as the basal superfusion buffer (285mosmol/l) the evoked hormone release in fraction 5 was much lower. In two experiments oxytocin release (relative to the basal level) was 1.27 and 1.18 respectively; vasopressin release in this fraction was increased 1.43 fold and 1.47 fold. Furthermore, the second peak was attenuated. In fraction 9 oxytocin release was 1.14 and 1.60 times the basal release value. So the combination of
3~-----a
contrd dynorphin
(l-6)
lo-‘M
1 SEM n=6
L
I
5 Figure
I
15
1
25
1
36
I
45
55
[min]
Effect of dynorphin on stimulated release of oxytocin. Values relative to basal release (O-20 min) which was set = 1.
166 K+-depolarization and increase in osmolarity seemed optimal for investigating the effects of dynorphin (1-8) on evoked oxytocin and vasopressin release.
Discussion In the present study hormone release from isolated neurosecretory endings was stimulated by potassium depolarisation and a simultaneous increase in osmolarity. Interestingly, the maximal response was obtained (in fraction 5) when the stimulatory superfusion buffer had not reached its maximal concentration (only 66%). In the next fraction, the stimulatory superfusion buffer was much more concentrated (93%) but the hormone release decreased; this means that the hyperpolarizing current in the nerve endings may already have started. The osmosensitivity of the magnocellular neurosecretory cells originating in the region of the supraoptic nucleus of the hypothalamus has been shown electrophysiologically (10). The neurosecretory cells responded to an increase in osmolarity with depolarization and - if synaptic input from neighbouring cells were allowed - with an increase in spike frequency. With small changes in osmolarity (
NfIIIKOPfzf’TIDES
current (12). Dynorphin (l-8) which has been shown to bind to kappa-receptors associated with Ca’+-channels (13) is a very effective inhibitor of this later event as well as the earlier events in the oxytocin neurons which are dependent on K+-conductance, namely the first action potential and the subsequent hyperpolarization. Most probably the Ca’+-dependent K+-conductance which is rapidly activated during a spike and contributes to spike repolarization and to the hyperpolarizing afterpotential (14) is influenced by the effect of dynorphin (l-8) on Ca’+-channels. The selectivity and specificity of the effects of dynorphin (l-8) are evident in the fact that only the stimulated release of oxytocin but not vasopressin is inhibited. So far our results are in accordance with earlier physiological studies using whole neurohypophyses (15). Acknowledgement I am indebted to Eberhard Schmid for excellent technical assistance and I wish to thank Prof. R. Martin for critical discussion. This work was supported by the Deutsche Forschungsgemeinschaft Projekt MA 259111.
References 1. Bunn. S. J., Hanley, M. R. and Wilkin, G. P. (1985). Evidence for a kappa-opioid receptor on pituitary astrocytes: An autoradiographic study. Neuroscience Letters 5.5: 317-323. 2. Herkenham, M., Rice, R. C., Jacobsen, R. E. and Rothman. R. B. (1986). Opiate receptors in the rat pituitary are confined to the neural lobe and are exclusively kappa. Brain Res. 382: 365371. 3. Falke. N. and Martin, R. (1986). Characterization and localization of opioid binding sites in a rat neurohypophysial fraction enriched in secretory nerve endings. Neuroendocrine Perspectives 5: 291-295. 4. Pesce, G., Lang, M. A., Russel, J. T., Rodbard, D. and Gainer. H. (1987). Characterization of kappa opioid receptors in neurosecretosomes from bovine posterior pituitary. J. Neurochem. 49: 421-427. 5. Falke. N. and Martin, R. (in press). Opiate binding differentially associated with oxytocin and vasopressin nerve endings from porcine neurohypophyses. Exp. Brain Res. 6. Whitnall, M. H., Gainer, H., Cox. B. M. and Molineaux, C. J. (1983). Dynorphin Ai_s is contained within vasopressin neurosecretory vesicles in rat pituituary. Science 222: 1137-1139. I. Zamir, N., Zamir, D., Eiden, L. E., et al. (1985). Methionine and leucine enkephalin in rat neurohypophysis: Different responses to osmotic stimuli and Tz Toxin. Science 228: 606-608. 8. Corbett, A., Paterson, S. J., McKnight, A. T., Magnan, J. and Kosterlitz H. W. (1982). Dynorphin l-8 and dynorphin 1-9 are ligands of the kappa subtype of opiate receptor. Nature (London) 299: 79-81.
DYNORPHIN
9.
(l-8) INHIBITS STIMULATED
RELEASE
OF OXYTOCIN
Nordmann, J. J., Dayanithi, G. and Cazalis, M. (1986). Do opioid peptides modulate, at the level of the nerve endings, the release of neurohypophysial hormons? Exp. Brain Res. 61: 560-566. 10. Mason. W. T. (1980). Supraoptic neurones of rat hypothalamus are osmosensitive. Nature 287: 154-157. I I. Andrew. R. D. and Dudek F. E. (1983). Burst discharge in mammalian neuroendocrine cells involves an intrinsic regenerative mechanism. Science 221: 1050-1052. 12. Bourque. C. W. (1986). Calcium-dependent spike aftercurrent induces burst firing in magnocellular neurosecretory cells. Neuroscience Letters 70: 204-209.
167
13. North, R. A. (1986). Opioid receptor types and membrane ion channels. TINS 9: 114-117. 14. Bourque. C. W., Randle, J. C. R. and Renaud, L. P. (1985). Calcium-dependent potassium conductance in rat supraoptic nucleus neurosecretory neurons. J. Neurophysiol. 54: 13751382. 15. Bicknell. R. J. and Leng. G. (1982). Endogenous opiates regulate oxytocin but not vasopressin secretion from the neuropophysis. Nature 298: 161-162.
Dynorphin (1-8) Inhibits Stimulated Release of Oxytocin but not Vasopressin from Isolated Neurosecretory Endings of the Rat Neurohypophysis N. FALKE Sektion
Eiektronenmikroskopie,
lJniversit8t
Urn, Oberer Eselsberg, 0-7goo U/m, F.R.G.
Abstract-The effects of the opioid peptide dynorphin (l-8) on oxytocin and vasopressin release at the level of isolated neurosecretory endings were investigated. Neurosecretory endings prepared by homogenization and centrifugation were placed on a filter and constantly superfused. Stimulated hormone release was evoked by potassium depolarization (30mM) and simultaneous increase of the osmolarity (20mosmoVl). Stimulation resulted in two peaks of hormone release - a short first peak and a longer second one. Addition of dynorphin (l-8) (10W7M) to the superfusion buffer significantly diminished the first peak of oxytocin release and totally abolished the second. There was no effect of dynorphin (l-8) on vasopressin release.
Introduction In autoradiographic studies opioid binding sites in the rat neurohypophysis were found to be exclusively of the kappa subtype (1, 2). The occurrence of only kappa opioid binding sites in the neurohypophysis seems to be a common feature of various species. Kappa opioid binding sites were localized in isolated neurosecretory terminals from the rat (3), and in purified preparations of terminals from bovine (4) and pig (5) neurohypophyses. In preparations from pig neurowhere oxytocin and vasopressin hypophyses, terminals were enriched in different fractions, Date received Date accepted
27 January 8 February
3988 1988
opioid binding was found to be associated preferentially with oxytocin endings (5). Evidence has been reported for coexistence of dynorphin (1-8) with vasopressin in the same neurosecretory terminals and granules of the rat neurohypophysis in about 300 times lower concentration (6). Presumably dynorphin (l-8) is released when vasopressin is secreted (7). Dynorphin (l-8) is a preferential kappa-ligand (8). With the typical delta-ligand D-Ala-D-Leu-enkephalin no effects on hormone release and calcium-uptake were found at the level of the neurosecretory endings (9). Therefore, it appears likely that under conditions favouring vasopressin release, neighbouring oxytocin endings are affected by co-released dynorphin (l-8). We studied the action of dynorphin (l-8) on the stimulated release of oxytocin and
163
164
vasopressin from isolated neurosecretory terminals and found inhibition of oxytocin, but not vasopressin release.
NELJROPEPTIDES
0.1% . The oxytocin antiserum was obtained from Ferring (Kiel, FRG). It was diluted 1.67000,its crossreactivity with Arg-vasopressin was 0.01%. Results
Methods For each experiment two neurohypophyses from male rats (Chbb, a Wistar-derived stock) of approximately 200g were carefully cleaned from intermediate lobe tissue. In order to minimize hormone release which is enhanced by cooling the tissue, the entire preparation was carried out at room temperature. The neurohypophyses were homogenized in a buffer composed of 0.27M sucrose, 10mM MOPS (morpholinopropanesulfonic acid), 0.1mM EGTA (ethyleneglycol-tetraacetic acid), pH 7.3. After homogenization (5 times at 500 rpm and one time at 1000 rpm) the tissue suspension was centrifuged for 5 min at 40xg. The pellet was discarded, and the supernatant from this centrifugation was collected and centrifuged for 10 min at 5000xg. The pellet from the second centrifugation, the secretsosome fraction, was resuspended in basal super-fusion buffer (140mM NaCl, 2.2mM CaClz, 1mM MgC12, 1OmM MOPS, 0.01% BSA; pH 7.3,285mosmoyl). The secretosome suspension was placed on a Millipore filter (0.22 pm pore size) and superfused for 30 min at a flow rate of 50 pl/min. Then the flow rate was accelerated to 100 @min and after 10 min superfusion at this flow rate, 500 ~1 fractions were collected. After 15 min (3 fractions) the basal buffer was exchanged for the stimulatory buffer (120mM NaCl, 30mM KCl, 2.2mM CaC12,lmM MgCL, 1OmM MOPS, 0.01% BSA; pH 7.3, 305 mosmol/l) for 10 min. Then the neurosecretosomes were superfused again with basal buffer until the end of the experiment. At t = 55 min collection of fractions was stopped and filters were extracted in 1M acetic acid for oxytocin ,and vasopressin radioimmunoassays. Dynorphin (l-8) was obtained from Serva (Heidelberg, FRG) and was added during the entire super-fusion at 100 @min, both to basal and stimulatory buffer. Oxytocin was obtained from Sigma (Munich, FRG) and Argvasopressin from Bachem (Switzerland). For the radiommunoassays the peptides were labeled with 1251 by addition of chloramine T. The labeled peptides were purified by chromatography on Sephadex GlO. The vasopressin antiserum was kindly provided by R.E. Lang, Heidelberg. It was diluted 1:42000, its crossreactivity with oxytocin was
At the superfusion rate of lOOl~J/min the basal release of untreated neurosecretosomes was 0.44pmol/min (SEM = 0.10, n = 6) for oxytocin and 0.87pmoYmin (SEM = 0.10, n = 6) for vasopressin. At the end of the experiment a considerable reservoir of hormones remained within the neurosecretosomes on the filter. The mean value for oxytocin was 384pmol (SEM = 135, n = 6) and for vasopressin 506pmol (SEM = 80, n = 6). There was no significant effect of dynorphin (l-8) on basal release of oxytocin or vasopressin. In the dynorphin-treated neurosecretosomes the basal release of oxytocin was 0.72pmol/min (SEM = 0.24, n = 6) and the basal release of vasopressin was l.O8pmol/min (SEM = 0.37, n = 6). As compared to the controls higher values were also found in the filter extracts of the dynorphin-treated samples for both hormones, the values were 461pmol (SEM = 145, n = 6) for oxytocin and Sllpmol (SEM = 136, n = 6) for vasopressin. The slightly higher basal release of both hormones, oxytocin and vasopressin, in the dynorphin-treated neurosecretosomes, which was not statistically significant, could be explained by a higher quantity of secretosomes on the filter in some of these experiments since there were also higher amounts of hormones in the extracts. In order to standardize and compare the results from different experiments with different quantities of secretosomes on the filters the basal release (=mean value of the first 4 fractions) in each experiment was set = 1. By superfusion of the filter with a coloured solution it was found that the stimulatory buffer arrived at the secretosomes 7.5 min after the buffer change. The colour in the eluate was 66% of the original colour in fraction 5 (25 min). In fraction 6 (30 min) it was maximal (93%) and in fraction 7 (35 min) it diminished again to 29%. In untreated neurosecretosomes the K+-depolarization and the increase in osmolarity induced a peak of oxytocin release in fraction 5 (25 min). Oxytocin release in this fraction was increased 3.23 fold relative to basal release (see Table and Figure). After a decrease in the next two fractions a second increase of oxytocin release occurred without external stimulus which was maximal in fraction 9 (45 min).
DYNORPHIN
(t-8) INHIBITS STIMULATED
165
RELEASE OF OXYTOCIN
Table Hormone-Release is Expressed Relative to Basal Release (=Mean Value of Fraction l-4) which was set = 1. The Number of Experiments was 6. SEM = Standard Error of the Mean oxytocin release control
fract. no.lmin
x
SEM
11 5 2/10 311.5 4120 5125 6130 7135 s/40 9145 lo/so 11155
1.09 1.05 0.98 0.89 3.23 1.77 1.50 2.97 3.51 3.24 2.16
.I2 .07 .lO .13 .44 .27 .21 .87 .84 .85 .57
dyn. (IO-‘M) x SEM .02 .08 .08 .04 .28 .04 .06 .08 .I2 .05 .I7
0.95 0.98 1.06 1.00 1.57 0.71 0.54 0.94 1.08 1.05 1.07
In the dynorphin-treated neurosecretosomes the first peak of oxytocin release in fraction 5 was significantly diminished (p = 0.01). The decrease of oxytocin release in fractions 6 and 7 was more pronounced as compared to the control (p = 0.02 and 0.01 respectively). The second peak of oxytotin release in fraction 9 was totally abolished by addition of dynorphin (l-8) (p = 0.05; p was established by the student t-test for groups with different variances). In untreated neurosecretosomes the stimulated vasopressin release in fraction 5 was lower than oxytocin release: a 1.8 fold increase over the basal level was seen (Table). Similar to the pattern of oxytocin release, after a decrease in the next two fractions a second more extended peak of vaso-
vasopressin release dw. (111-‘M) SEM .I? SEM
control .u 0.84 0.98 0.96 1.22 1.80 0.99 1.02 1.68 1.79 1.79 1.73
.05
.04 .04 .11 .20 .09 .09 .16 .21 .22 .22
1.00 0.94 1.01 1.05 1.75 1.32 1.30 1.52 1.66 1.48 1.36
.02
.03 .01 .02 .I’ .35 30
18 .21 .?9 .2u
pressin release occurred. There was no effect of dynorphin (1-8) on vasopressin release. The stimulatory effect on hormone release of other buffer systems was investigated using untreated neurosecretosomes. If the stimulatory superfusion buffer contained 30mM Kf and other components described previously, but had the same tonicity as the basal superfusion buffer (285mosmol/l) the evoked hormone release in fraction 5 was much lower. In two experiments oxytocin release (relative to the basal level) was 1.27 and 1.18 respectively; vasopressin release in this fraction was increased 1.43 fold and 1.47 fold. Furthermore, the second peak was attenuated. In fraction 9 oxytocin release was 1.14 and 1.60 times the basal release value. So the combination of
3~-----a
contrd dynorphin
(l-6)
lo-‘M
1 SEM n=6
L
I
5 Figure
I
15
1
25
1
36
I
45
55
[min]
Effect of dynorphin on stimulated release of oxytocin. Values relative to basal release (O-20 min) which was set = 1.
166 K+-depolarization and increase in osmolarity seemed optimal for investigating the effects of dynorphin (1-8) on evoked oxytocin and vasopressin release.
Discussion In the present study hormone release from isolated neurosecretory endings was stimulated by potassium depolarisation and a simultaneous increase in osmolarity. Interestingly, the maximal response was obtained (in fraction 5) when the stimulatory superfusion buffer had not reached its maximal concentration (only 66%). In the next fraction, the stimulatory superfusion buffer was much more concentrated (93%) but the hormone release decreased; this means that the hyperpolarizing current in the nerve endings may already have started. The osmosensitivity of the magnocellular neurosecretory cells originating in the region of the supraoptic nucleus of the hypothalamus has been shown electrophysiologically (10). The neurosecretory cells responded to an increase in osmolarity with depolarization and - if synaptic input from neighbouring cells were allowed - with an increase in spike frequency. With small changes in osmolarity (
NfIIIKOPfzf’TIDES
current (12). Dynorphin (l-8) which has been shown to bind to kappa-receptors associated with Ca’+-channels (13) is a very effective inhibitor of this later event as well as the earlier events in the oxytocin neurons which are dependent on K+-conductance, namely the first action potential and the subsequent hyperpolarization. Most probably the Ca’+-dependent K+-conductance which is rapidly activated during a spike and contributes to spike repolarization and to the hyperpolarizing afterpotential (14) is influenced by the effect of dynorphin (l-8) on Ca’+-channels. The selectivity and specificity of the effects of dynorphin (l-8) are evident in the fact that only the stimulated release of oxytocin but not vasopressin is inhibited. So far our results are in accordance with earlier physiological studies using whole neurohypophyses (15). Acknowledgement I am indebted to Eberhard Schmid for excellent technical assistance and I wish to thank Prof. R. Martin for critical discussion. This work was supported by the Deutsche Forschungsgemeinschaft Projekt MA 259111.
References 1. Bunn. S. J., Hanley, M. R. and Wilkin, G. P. (1985). Evidence for a kappa-opioid receptor on pituitary astrocytes: An autoradiographic study. Neuroscience Letters 5.5: 317-323. 2. Herkenham, M., Rice, R. C., Jacobsen, R. E. and Rothman. R. B. (1986). Opiate receptors in the rat pituitary are confined to the neural lobe and are exclusively kappa. Brain Res. 382: 365371. 3. Falke. N. and Martin, R. (1986). Characterization and localization of opioid binding sites in a rat neurohypophysial fraction enriched in secretory nerve endings. Neuroendocrine Perspectives 5: 291-295. 4. Pesce, G., Lang, M. A., Russel, J. T., Rodbard, D. and Gainer. H. (1987). Characterization of kappa opioid receptors in neurosecretosomes from bovine posterior pituitary. J. Neurochem. 49: 421-427. 5. Falke. N. and Martin, R. (in press). Opiate binding differentially associated with oxytocin and vasopressin nerve endings from porcine neurohypophyses. Exp. Brain Res. 6. Whitnall, M. H., Gainer, H., Cox. B. M. and Molineaux, C. J. (1983). Dynorphin Ai_s is contained within vasopressin neurosecretory vesicles in rat pituituary. Science 222: 1137-1139. I. Zamir, N., Zamir, D., Eiden, L. E., et al. (1985). Methionine and leucine enkephalin in rat neurohypophysis: Different responses to osmotic stimuli and Tz Toxin. Science 228: 606-608. 8. Corbett, A., Paterson, S. J., McKnight, A. T., Magnan, J. and Kosterlitz H. W. (1982). Dynorphin l-8 and dynorphin 1-9 are ligands of the kappa subtype of opiate receptor. Nature (London) 299: 79-81.
DYNORPHIN
9.
(l-8) INHIBITS STIMULATED
RELEASE
OF OXYTOCIN
Nordmann, J. J., Dayanithi, G. and Cazalis, M. (1986). Do opioid peptides modulate, at the level of the nerve endings, the release of neurohypophysial hormons? Exp. Brain Res. 61: 560-566. 10. Mason. W. T. (1980). Supraoptic neurones of rat hypothalamus are osmosensitive. Nature 287: 154-157. I I. Andrew. R. D. and Dudek F. E. (1983). Burst discharge in mammalian neuroendocrine cells involves an intrinsic regenerative mechanism. Science 221: 1050-1052. 12. Bourque. C. W. (1986). Calcium-dependent spike aftercurrent induces burst firing in magnocellular neurosecretory cells. Neuroscience Letters 70: 204-209.
167
13. North, R. A. (1986). Opioid receptor types and membrane ion channels. TINS 9: 114-117. 14. Bourque. C. W., Randle, J. C. R. and Renaud, L. P. (1985). Calcium-dependent potassium conductance in rat supraoptic nucleus neurosecretory neurons. J. Neurophysiol. 54: 13751382. 15. Bicknell. R. J. and Leng. G. (1982). Endogenous opiates regulate oxytocin but not vasopressin secretion from the neuropophysis. Nature 298: 161-162.