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Activation of κ-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca2+ in secretory nerve terminals of the neurohypophysis
138
Brain Research, 574 (1992) 138-14t~ © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17524
Activation of r-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca 2+ in secretory nerve terminals of the neurohypophysis M. Kato*, C. Chapman and R.J. Bicknell Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U. K.)
(Accepted 29 October 1991) Key words: Oxytocin; Vasopressin; Secretion; Calcium; Calcium channel; Opioid
Nerve endings of the magnocellular neurohypophysial neurones possess r-opioid receptors. Using a preparation of isolated terminals from the neurohypophysis we studied r- opioid effects on secretion of oxytocin and vasopressin and on intracellular Ca2+ concentration ([Ca2+]i) measured fluorimetrically or using digital video imaging with Fura-2. The dihydropyridine CaZ+-channel antagonist nicardipine reduced [Ca2+]i responses to K+-depolarisation (30-40 mM K +) by 55-75% and inhibited evoked secretion of oxytocin and vasopressin to a similar extent. The selective r-receptor agonist I)-Pro1° Dynorphin A 1-11 (DPDYN) substantially inhibited K + evoked secretion of oxytocin by 40-90% and secretion of arginine vasopressin (AVP) by 20-50%. DPDYN caused only a 10% reduction in the average total population [Ca2+]i response to K + depolarisation. No sub-population of inhibitory responses was observed when samples of individual terminal [Ca2+]i responses were examined with imaging. Although ~c-receptors are coupled to Ca2+-channels at neuronal somata our data suggest that alternative effector mechanisms operate in these secretory nerve endings. INTRODUCTION Opioid receptors in the rat neurohypophysis are predominantly or exclusively of the r-type 9'16'29. One population of r-receptors (rRs) is present on the nerve endings of the neurohypophysial magnocellular secretory neurones where their activation inhibits depolarisation evoked secretion of neurohormones, particularly oxytocin 7't3'33. Endogenous neurohypophysial opioids also act on opioid receptors within the tissue and antagonism of their action reveals an inhibitory influence again predominantly over secretion from terminals of oxytocin neurones 5'2°'34. The nerve terminal localisation of r R s at this site is analogous with that of presynaptic receptors elsewhere in the brain which modulate peptide and transmitter release occurring in response to invasion of the terminal by depolarising action potentials. Since the small size of mammalian nerve terminals has in general precluded their study by electrophysiological methods, the membrane and intracellular mechanisms of action of presynaptic receptors have generally been inferred from those seen at the cell body or from biochemical indices taken in highly heterogeneous synaptosome preparations.
Using the relatively homogeneous 'neurosecretosome' preparation derived from rat neurohypophysis we have now studied intracellular calcium concentrations [Ca2+]i with the Fura-2 fluorescent Ca 2÷ indicator in suspensions of nerve endings maintained in a fluorimeter or in individual terminals using digital video imaging technology. We have compared the effects of Ca2+-channel antagonists and r R activation using the highly selective r-agonist D-Pro 1° Dynorphin A 1-11 ( D P D Y N ) on the K ÷depolarisation evoked [Ca2+]i signal and on the secretion of oxytocin (OXT) and arginine vasopressin (AVP). Previous studies of r R effector mechanisms have suggested a coupling to 'N' type Ca2+-channels in the somatic cell membrane of cultured neurones to reduce Ca 2+ entry 14. A rise in [Ca2+]i caused by influx of Ca 2÷ during depolarisation is thought to be the primary trigger to exocytosis from nerve endings. MATERIALS AND METHODS Neurosecretosome preparation Neurosecretosomes were prepared from neurohypophyses of adult male rats (approx. 250 g) by a modification of the method of Cazalis et al 1°. Rats were stunned and killed by cervical dislocation. Neurohypophyses were dissected free of anterior and intermediate lobe tissue and gently disrupted in a teflon/glass homoge-
* Permanent address: Institute of Endocrinology, Gunma University, Maebashi, Gunma 371, Japan. Correspondence: R.J. Bicknell, Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
139
niser (clearance 50-100/zm) in 270 mM sucrose buffered with 10 mM HEPES neutralised to pH 7.2 with KOH and containing 10 /~M EGTA, maintained at 37°C. Following centrifugation at 600 g for 5 min to remove debris and larger elements, the supernatant was centrifuged at 3400 g for 15 rain. The resulting pellet (P2) was resuspended in a HEPES buffered salt solution (I-IBSS (in mM): NaCI 145, KC1 5, NaHCO 3 5, MgCI 2 2, CaC12 2, HEPES 10, o-glucose 5.6, Bovine serum albumin (Sigma Fraction V) 1 mg/ml; pH 7.35; filtered through a 0.22/~m membrane) and centrifuged at 3400 g for 15 min through an underlying layer of 7.5% Percoll (Pharmacia LKB) prepared in HBSS to produce a further pellet (P3). This Percoll step was found to produce a preparation giving lower basal levels of neurohormone release and more robust responses to depolarisation possibly because of the exclusion of larger nerve endings known to be less responsive 21. When examined in the electron microscope, P3 consisted predominantly of 1-2/~m diameter spherical membrane bound elements containing dense core secretory granules, clear microvesicles, mitochondria and vacuoles. P3 only differed from P2 in appearance in containing fewer relatively large elements (3-5/~m). Immediately prior to Fura-2 or secretion experiments the resuspended pellet was filtered through 30/~m nylon mesh.
Fura-2 loading For both cuvette and digital image measurements, prior to the Percoll step the P2 pellet was resuspended in HBSS containing 2 /~m Fura-2 acetoxymethylester and incubated for 15 mi'n at 37°C. Following Fura-2 loading the preparation was centrifuged through Percoll to yield P3 as described above.
Fluorimeter measurements P3 was resuspended in 5 ml of HBSS and centrifuged again for 15 min at 3400 g as an additional washing step. The resulting pellet was resuspended in HBSS to a concentration of neurosecretosomes equivalent to 3 neurohypophyses per ml. Three hundred and fifty /~l of suspension was placed in a stirred quartz microcuvette maintained at 37°C in a Perkin-Elmer LS-5B fluorimeter driven by a microcomputer programme supplied by the company to measure 340 nm: 380 nm emission ratio at 4-s intervals. Emission intensities were measured at 510 nm. Following lysis with Triton X-100 (0.14%) and calibration of the dye response with Ca :÷ and subsequently excess EGTA (20 mM), [Ca2+]i was derived in the programme according to the equation of Grynkiewicz et al, is using a Kd value for Fura 2 ~ a 2+ of 225 nM. Solutions were added to the cuvette by micropipette during a pause in the data collection. Elevated K + concentrations were achieved in this system by addition of isotonic HBSS in which KC1 was elevated at the expense of NaC1. No loss of response was seen upon storage of the Fura-2 loaded preparation at 22°C in the dark for up to 2 h. To reduce any problems of dye leakage from Fura-2 loaded neurosecretosomes into the extracellular medium all solutions used in the fluorimetric measurements contained 100/~M sulfinpyrazone, an inhibitor of organic anion transport 3~. Early experiments established that the apparent slow rise in [Ca2+]i seen during the course of each run was prevented by this agent. When the actions of the dynorphin peptide were examined, the HBSS for both vehicle and drug experiments contained a cocktail of peptidase inhibitors as previously described 33.
Digital imaging of [Ca2+]i The Fura-2 loaded P3 pellet was resuspended in HBSS to a concentration equivalent to 5 neurohypophyses per 100 /~1. Ten /~1 aliquots were pipetted on to the centre of 22 mm diameter glass coverslips previously washed in detergent and 1% dimethylsulfoxide and treated with 0.02% poly-o-lysine (Sigma). Following an attachment period of 10 min at 22-25°C the coverslip was fixed in a controlled perfusion chamber (150/~1 volume) mounted on the stage of a Nikon Diaphot microscope, maintained at 37°C and superfused continuously with HBSS at 500/d/min. Attached neurosecretosomes were visualised with a CF-Fluor 100 x oil
immersion quartz objective. The Joyce-Loebl (Gateshead, U.K.) 'Magical' system with TARDIS software was used for all dynamic video imaging and image processing. Excitation wavelengths of 340 nm and 380 nm were alternated by means of computer controlled rotating filter wheel between the xenon ultraviolet source and the microscope. Emission light at 510 nm (10 nm band width) was passed to an image intensifying charge coupled device camera (Photonic Science). The resulting image was averaged in real time (16 images), digitised, captured, stored and background subtracted as previously described 17. Time resolution between ratioed images was 3.6 s. The 340 nm: 380 nm ratios of emitted fluorescence were calculated for each frame on a pixel-by-pixel basis and converted to Ca 2÷ concentration according to the equation of Grynkiewicz et al. 15 using a Ka for Fura 2,Ca2+ of 225 nM. Calibration constants were not obtained in situ due to problems of loss of attachment during permeabilisation. Calibration constants were taken from experiments in the same set-up using Fura-2 loaded, permeabilised pituitary cells17 and thus accurately reflect the dye response in an intracellular milieu but do not report absolute levels of [Ca2+]i. Data for neurosecretosomes obtained by imaging are therefore expressed only as relative [Ca2÷]i values and quantitative analysis of drug effects performed with an internally controlled double stimulus protocol (see below). To produce a continuous trace of relative [Ca2+]i with time, each neurosecretosome was outlined on the video screen and the mean [Ca2+]i within the area defined was computed. Data for each terminal was exported to an ASCII file which was incorporated into a Lotus 123 spreadsheet for further calculations. For quantitative analysis of responses to two identical K ÷ depolarisations (S 1 and $2) the cumulative [Ca2+]i was calculated for each 120 s exposure to elevated K ÷ medium and a response ratio derived (S::S1; see Fig. 4).
Secretion experiments The P3 pellet was resuspended in HBSS at a concentration equivalent to 1 neurohypophysis per ml and 500/~1 aliquots inoculated through a 3-way tap on to 13 mm diameter, 0.45/~m fluoropolymer filter assemblies (Acro LC13; Gelman Sciences) containing a prefilter (Millipore AP20) fitted in the inlet barrel. Eight filters were prepared concurrently and perifused at 37°C at a flow rate of 200/~l/min with HBSS. Two minute fractions of perifusate were collected automatically and stored at -20°C until measurement of concentrations of OXT and AVP by specific radioimmuno,assays4. Following an equilibration period of 90 rain to allow basal secretion rates to stabilise, neurohormone release was evoked by depolarisation for 5 min with I-IBSS containing elevated levels of KCI (isotonically substituted for NaC1). In some experiments, two identical depolarisations, $1 and $2, were applied and ratios of total evoked release above basal calculated ($2:$1). Prior to calculations release rates were expressed as a percentage of tissue OXT and AVP content present on each filter at the beginning of the relevant collection period. Total OXT and AVP present on each filter at the end of the experiment were determined following lysis in 0.1% Triton X-100/0.1 M HCI for use in this calculation. Mean basal release rates per 2 min fraction immediately prior to depolarisation represented 0.054 + 0.004% and 0.066 + 0.005%, respectively, of tissue contents of OXT and AVP (n = 48; + S.E.M.).
Drugs Nicardipine-HC1 and sulfmpyrazone were purchased from Sigma (Poole, U.K.); ~o-conotoxin GVIA was from Peptide Institute Inc. (Osaka, Japan); Fura-2 acetoxymethylester was from Molecular Probes (Eugene, U.S.A.); o-Pro 1° Dynorphin A 1-11 was a gift from Dr. J.E. Gairin (Scripps Clinic, La Jolla, U.S.A.); norbinaltorphimine was a gift from Dr. C.EC. Smith (Reckitt and Colman, Hull, U.K.).
140 800
50 mM K ÷ qp-
600
~- 400 200.
400
] 10 m M EGTA
200,~
~
50 mM K÷
0 0
60
120
180
240
300
Time {s)
Fig. 1. Fluorimetric measurement of m e a n [Ca2+]i in the total neurosecretosome population. The rise in [Ca2+]i evoked by elevation of [K+]o (upper trace) is prevented by chelation of extracellular Ca2+ (lower trace). This experiment was repeated on 3 separate occasions. In subsequent analyses of fluorimetric measurements we analysed the peak [Ca2+]i response achieved, mean peak value achieved during the initial 20 s of depolarisation and an average value between 4-5 min after the onset of depolarisation (see Resuits). RESULTS
Fluorimeter measurement of [CaZ+] i Effects of depolarisation. In normal HBSS the average resting [Ca2+]i in the total neurosecretosome population was 113 + 4 nM (n = 37 from 13 preparations) and was stable during the 7 min duration of each experiment. Elevation of extracellular K ÷ ([K+]o) resulted in a prompt increase in [Ca2+]i (Fig. 1). Chelation of ex-
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tracellular Ca 2+ by addition of E G T A completely prevented the K+-induced increase in [Ca2+]i in 3 preparations tested (Fig. 1). The response to elevation of [K+]o consisted of an initial peak, very pronounced at higher levels of [K÷]o (compare Figs. 1 and 3), followed by a decline to a lower and relatively stable plateau value. For quantitative analysis we recorded the increment over basal (A) of the peak [Ca2+]i, a mean peak value taken from the first 5 readings after K ÷ addition, and a mean plateau value taken from readings 4-5 min after K ÷ addition. A dose-response relationship between these parameters and levels of [K÷]o between 20 and 70 m M was obtained (Fig. 2a). Effects of Ca2+-channel antagonists. The effects of the dihydropyridine Ca2÷-channel antagonist nicardipine (10 /~M) and of w-conotoxin G V I A (w-CTX; 1-10/~M, data combined) on the [Ca2÷]i response evoked by 30 mM [K+]o were assessed. Neurosecretosomes were pre-exposed to nicardipine for 1 min and to w-CTX for 10 min prior to depolarisation. For each antagonist, 3 separate preparations were used for multiple tests. On each occasion 1-3 neurosecretosome aliquots were used to obtain control data (30 mM [K+]o; vehicle only) and 1-3 aliquots used with antagonist. For each preparation, parameters of [Ca2+]i were averaged for the control and antagonist tests. Data for all preparations were then combined. Data were also analysed by expressing A[Ca2+]i as a percentage of the mean control value in each preparation prior to combination and statistical testing. Nicardipine did not affect resting [Ca2+]i but strongly suppressed all components of the K+-evoked increase (Fig. 3). Peak, mean peak and 4-5 min values were depressed by 74 + 2, 76 + 2 and 75 + 2%, respectively,
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30 50 70 [K+]o (mM) Fig. 2. K+-concentration dependence of (a) evoked increases above basal (A) of [Ca2+]i measured fluorimetrically and (b) evoked secretion of neuropeptides. Note log-scales for [K+]o. Data are mean + S.E.M. of 3-4 observations from 3 preparations ([Ca2+]i) and of 3-4 observations from 2 preparations for oxytocin (OXT) and arginine vasopressin (AVP). 20
141
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200
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Time (s)
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Fig. 3. A: effect of nicardipine (10/~M) on K ÷ evoked rise in [Ca2+]i measured fluorimetrically. Examples are sequential tests performed on aliquots of the same preparation. B: combined data from experiments exemplified above. Data are mean + S.E.M. for 6 observations from 3 preparations. For statistical analysis of control-normalised data, see text.
A. compared to control values in each preparation (Table I; n = 6 from 3 preparations; P < 0.01 in each case; Mann-Whitney U-test). ~0-CTX did not affect resting [Ca2+]i and only weakly inhibited the peak rise in [Ca2+]i evoked by 30 mM [K+]o (peak A[Ca2+]i control, 247 + 60 nM; to-CTX, 217 + 48 nm). Expression of the data relative to control values reveal significant inhibition of the peak A[Ca2+]i by 12 + 2% (P < 0.05), of the mean peak also by 12 + 2% (P < 0.01) but not of the 4-5 min A[Ca2+]i (Table I; n = 9 from 3 preparations; Mann Whitney U-test). Effects of rR activation. Pre-exposure of neurosecretosomes to the r-agonist DPDYN (1/zM) for 5 min prior to depolarisation did not affect resting [Ca2+]i b u t weakly inhibited the peak rise in [Ca2+]i evoked by 30 mM [K+]o (peak A[Ca2+]i control, 270 + 27 nM; DPDYN, 245 + 25 nM). Peak A[Ca2+]i, when expressed relative to control values, was depressed by 11 + 3% (P < 0.01), mean peak value was depressed by 7 + 3% (P < 0.05)
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AlCa;+]i (% control) Group
Peak
Mean peak 4-5 min
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Nicardipine (10/~M) w-CTX (1-10 ttM) DPDYN (1/~M)
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25 + 2** 92 :t: 5 99 + 3
* P < 0.05, **P < 0.01 vs relevant control; Mann Whitney U-test,
Fig. 4. Examples of relative [Ca2+]t responses (see Materials and Methods) of 4 individual Fura 2-loaded nerve endings measured using digital video imaging during superfusion on the microscope stage. In each experiment two 120 s periods of depolarisation with 40 mM K + (S1 and $2) were imposed, separated by a wash period in HBSS. A: control; B,C,D: l>Pro 1° Dynorphin A 1-11 (DPDYN) (1 /~M) present prior to and during S2. Derivation of $2:S 1 response ratios for each terminal is described in Materials and Methods.
142 0.8 A,
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[ ] 40 mM K +, DPDYN [ ] 40 mM K*; Nicardipine
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OXT
AVP 50 mm K + (n=6)
Fig. 7. Effects of to-conotoxin on release of OXT and AVP evoked by a single 40 or 50 mM K ÷ depolarisation. Data are mean _+ S.E.M. *P < 0.05 vs relative control level of evoked release; Mann Whitney U-test.
40
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~ 20 Z
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Fig. 5. Population distribution of $2:St response ratio derived from [Ca2+]i imaging experiments exemplified in Fig. 4. Data are the number of Sz:S~ values at each level indicated, plotted as a percentage of the total number of responses recorded for each treatment. A: 30 mM K + stimulus; data from 15 terminals under control conditions, 13 terminals in the presence of DPDYN during $2. B: 40 mM K + stimulus, data from 16 terminals under control conditions, 11 terminals in the presence of nicardipine and 9 terminals in the presence of DPDYN.
whilst the 4 - 5 min value was unaffected at 99 ___ 3% of the control (Table I; n = 15 from 9 preparations; M a n n Whitney U-test).
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Fig. 6. Effects of nicardipine on release of OXT and AVP evoked by a single 30 or 40 mM K + depolarisation during continual perifusion. Data are mean + S.E.M. **P < 0.01 vs relative control level of evoked release; Mann Whitney U-test.
In these experiments we e m p l o y e d a double stimulus protocol during superfusion of neurosecretosomes on the microscope stage. E x a m p l e s of profiles of relative [Ca2÷]i with time for 4 individual terminals are shown in Fig. 4. U n d e r control conditions (no drug), two identical depolarisations ($1 and $2) with 30 or 40 m M [K+]o for 120 s separated by a wash p e r i o d of 5 min p r o d u c e d very similar responses. Thus for 30 m M [K÷]o the m e a n control $2:S t response ratio of cumulative [Ca2+]i was 0.949 + 0.040 (n = 15 terminals for 4 experiments). In the presence of D P D Y N (1 ~ M ) during S 2, the m e a n [Ca2+]i rise caused by 30 m M [K÷]o was slightly enhanced ($2:$1 1.237 + 0.109, n = 13 in 3 experiments, examples in Fig.
4). With a 40 m M [K+]o stimulus m e a n control $2:$1 [Ca2+]i was 0.986 + 0.020 (n = 16 in 3 experiments). Inclusion of D P D Y N (1 /~M) did not alter the m e a n $2:$1 value (1.026 + 0.115, n = 9 in 3 experiments). U n d e r these conditions nicardipine (10/~M) depressed m e a n $2:$1 [Ca2+]i to 0.436 + 0.059 representing a 56% reduction (n = 11 in 3 experiments; P < 0.01, M a n n Whitney U-test). A t both levels of K ÷ depolarisation we looked to see if a subpopulation of terminals showed an inhibitory response to D P D Y N by plotting the distribution of S2:St ratios as a percentage of the total population analysed (Fig. 5). N o such subpopulation was revealed in either case, although at both levels of [K÷]o D P D Y N did app e a r to b r o a d e n the distribution of responses (Fig. 5). Since the control d a t a in both series a p p r o x i m a t e d to a normal distribution we c o m p a r e d the variances between control and D P D Y N groups. Significantly greater variance was found in the D P D Y N groups (at 30 m M [K÷]o FI2,14 --- 6.396; at 40 m M [K+]o F8,15 = 19.01; P < 0.01
143 0.3
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Fig. 8. Effects of DPDYN (1/~M) release of OXrI"and AVP evoked by a single 30 or 40 mM K+ depolarisation. Data are mean + S.E.M. *P < 0.05 and **P < 0.01 vs relative control level of evoked release; Mann Whitney U-test.
in both cases). In contrast to DPDYN, nicardipine clearly shifted the distribution of responses in the inhibited direction (Fig. 5). Broadening of the population distribution also occurred with nicardipine (Fl0,15 = 6.03; P < 0.01). Peptide secretion A single 5 min exposure to various levels of [K÷]o evoked dose-related increases in the secretion of O X T and AVP from neurosecretosomes during perifusion (Fig. 2b). Evoked secretory responses were dependent on the presence of extracellular Ca 2+ as previously described 11'33. Whilst the fluorimeter measurements readily revealed [Ca2+]i increases in the secretosome population in response to 20 mM [K+]o, the lowest level of [K+]o tested (Fig. 2a), this depolarisation led to barely detectable evoked release of peptides (approximately 0.02% of tissue content). The effects of antagonists on evoked release were examined following 5 min pre-exposure. None of the drugs affected basal output of OXT and AVP. Nicardipine (10 /~M) strongly inhibited 30 mM [K+]o evoked O X T and AVP secretion by 42 and 63%, respectively, and at 40 mM [K+]o inhibited evoked OXT and AVP secretion by 52 and 64%, respectively (Fig. 6). to-CTX (1/~M) had no major effect on peptide secretion evoked by 40 mM [K÷]o and inhibited OXT and AVP secretion evoked by 50 mM [K+]o by about 30% (P < 0.05 for AVP; Fig. 7). D P D Y N (1 btM) inhibited OXT secretion induced at 30 and 40 mM [K+]o by 52 and 36%, respectively. AVP secretion was also reduced but by a lesser extent in each case (38 and 20%, respectively; Fig. 8). In a further series the inhibitory effects of D P D Y N on 40 mM [K+]o evoked secretion were prevented by inclusion of the selective r R antagonist norbinaltorphinine (nor-BNI; 10
DPDYN
op2y O×T
AVP i
40 mM K+ (n=6)
Fig. 9. Effects of DPDYN (1 /~M) on OXT and AVP release evoked by 40 mM K÷ using a double stimulus protocol with DPDYN or no additions during the second stimulus ($2). Data are mean + S.E.M. $2:S1 response ratios of the amounts of evoked release. **P < 0.01 vs relative control; Mann Whitney U-test.
gM). Evoked release of OXT was 0.38 + 0.03% tissue content in the presence of nor-BNI alone and 0.36 + 0.03% tissue content in nor-BNI with DPDYN. Respective values for AVP release were 0.49 + 0.09 and 0.52 + 0.10% tissue content (n = 4 in 2 experiments). To more closely approximate the conditions achieved under the [Ca2+]i imaging experiments the actions of D P D Y N on peptide secretion were also examined using a double stimulus protocol. Two identical 4 min exposures to 40 mM [K+]o (S1 and $2) were separated by 12 rain and ratios of evoked peptide release calculated ( 5 2 : 5 1 ) . Inclusion of D P D Y N (1 gM) during S2 strongly reduced OXT and AVP release by 87% and 54%, respectively (Fig. 9; n = 5; P < 0.01 in each case, Mann Whitney U-test). DISCUSSION The neurosecretosome preparation The anatomically segregated and densely packed nerve terminals of the neurohypophysis have long served as a model in which to study mechanisms regulating release of neuropeptides. In recent years the development of a functional neurosecretosome preparation 1° has facilitated more direct study in particular of the roles of Ca 2÷. The preparation is analogous to synaptosomes prepared from brain but has the advantages of being uncontaminated by post-synaptic elements and of consisting predominantly of nerve endings of only two neuronal types - the magnocellular OXT and AVP secreting neurones zl. Our measurements reveal average resting [Ca2+]i in these terminals to be approximately 100 nM and peak levels achieved during K ÷ depolarisation were 500-1000 nM. It is probable that higher [Ca2+]i is achieved directly
144 between the nerve terminal membrane. In permeabilised neurosecretosomes, half maximal release of peptides occurs at about 1-2 /~M Ca 2+ (ref. 11). Our measured value of resting [Ca2+]i is somewhat lower than previously reported using fluorimeter measurements of Fura-2 in this preparation s and similar to a recent microspectrofluorometric study 2s. Ca2÷ channels in neurosecretosomes Our experiments indicate that a substantial part of the inward Ca 2+ movement during sustained depolarisation of secretosomes with K + is carded by dihydropyridinesensitive L-type Ca 2+ channels 32. Under these conditions to-CTX sensitive channels, which may define a separate population 26, appear to play a minor role. Secretion studies indicated that to-CTX sensitive channels may mediate more Ca 2÷ entry at higher levels of K ÷ depolarisation (>50 mM; Fig. 7). A recent fluorometric study of individual Fura-2 loaded neurosecretosomes came to a very similar conclusion regarding the predominance of L-channel mediated Ca2÷entry during K÷-depolarisa tion 2s.
The presence of both L-type and to-CTX sensitive putative N-type channels in secretosome membranes has been previously suggested from studies of neural lobe secretion, to-CTX sensitive channels appear to mediate the majority of Ca 2÷ entry and secretion when depolarisation is induced by Na + action potentials in the intact neurohypophysial tissue 12'23'27. Patch clamp recordings support the existence of populations of L- and N-type Ca 2÷ channels in the membrane of individual neurosecretosomes. Interestingly, although to-CTX sensitivity was not tested, the N-channels were defined as requiring a higher (more positive) threshold membrane potential for activation is. A range of pharmacological studies in heterogeneous synaptosome and other synaptic preparations from the brain also indicates that several channel types are involved in regulating Ca 2+ entry and transmitter release (e.g. Suszkiw et al.3°). r-Opioid receptor coupling Neurosecretosomes from rats possess rRs which when activated by selective opioid agonists or by dynorphins (the likely endogenous ligands) inhibit depolarisation evoked secretion of neuropeptides 13'32. Activation of KRs on the somatic membrane of cultured neurones has been reported to reduce a Ca 2+ conductance 14'19'25. Given the primary role of Ca 2÷ entry in mediating depolarisation evoked exocytosis in nerve terminals we decided to look directly for KR mediated inhibition of the depolarisation evoked [Ca2÷]i signal in neurosecretosomes. Surprisingly, we observed very little effect of r R activation on the average [Ca2+]i response of the total
secretosome population to moderate K*-depolarisation (7-11% inhibition of the peak response). Secretion of OXT and AVP evoked by the same level of K + depolarisation was inhibited by 52% and 38%, respectively, by DPDYN. Mediation of this effect via 7oRs was confirmed with a highly selective r R antagonist. The Ca 2+channel antagonist nicardipine resulted in similar levels of inhibition of OXT and AVP secretion induced by 30 mM [K+]o (42% and 63%, respectively). In this case nicardipine reduced all components of the total population [Ca2+]i response by 75% indicating blockade of C a 2+ entry as the mechanism of secretory inhibition. Depending to some extent on the choice of r-agonist, OXT secreting nerve endings are more sensitive than AVP secreting nerve endings to r R inhibition of exocytosis (Fig. 8; refs. 13, 33 and our further unpublished observations). This relative selectivity of opioid actions is particularly marked in the case of the actions of endogenous opioids in neurohypophysial tissue TM. We thus reasoned that in measuring the total s e c r e t o s o m e [Ca2+]i response to depolarisation we might be masking preferential r-opioid effects o n C a 2+ entry into OXT terminals or perhaps into some more actively secreting sub-population of terminals. This hypothesis was not supported by analysis of r-opioid effects on the [Ca2+]i response to depolarisation in individual nerve terminals using video imaging. Under the imaging conditions and using an internally controlled double-stimulus protocol, nicardipine again strongly suppressed t h e [Ca2+]i response (mean 56% reduction), shifting the distribution of response ratios in our sample in an inhibited direction. No sub-population of terminals exhibiting a similar shift was observed following r R activation and indeed, in the more thorough study using 30 mM K +, the trend was towards an elevated [Ca2+]i response in the presence of DPDYN. The small inhibition of the C a 2÷ signal by DPDYN seen in the fluorimeter study was not reproduced under the conditions of the imaging study. In these small terminals spatial resolution under imaging conditions limits study of subplasmalemmal C a 2+ concentrations that might still be modified by r R activation. Such an effect would, however, have to be in some way uncoupled from the total [Ca2+]i response which appears due entirely to C a 2+ entry. Whatever the explanation for this difference, using the double-stimulus protocol as for C a 2+ imaging in secretory studies, r R activation was highly effective in inhibiting exocytosis (88% inhibition of OXT and 59% inhibition of AVP secretion). We conclude that r R mediated inhibition of secretion from neurohypophysial nerve endings does not include a major suppression of C a 2+ entry. This implies a coupling mechanism able to inhibit the actions of C a 2+ o n the
145 secretory process and/or regulate some cellular compo-
intact tissue 6'24 (but see ref. 22). These second messen-
n e n t other than Ca 2÷ which is required together with
ger systems thus represent alternative targets for r R cou-
Ca 2+ for activation of exocytosis. Although r R s can be coupled to inhibition of Ca 2÷ channels at n e u r o n a l somata 3'14 and in cortical synaptosomes 1, negative coupling
pling to inhibition of exocytosis in the terminals of the
to adenylate cyclase has also been reported 2. Both inositol 1,4,5-trisphosphate and activators of protein kinase C potentiate Ca 2÷ stimulated exocytosis in permeabilised neurosecretosomes u and protein kinase C activation enhances electrically evoked neuropeptide release from the
REFERENCES 1 Adamson, P., Xiang, J-Z., Mantzourides, T., Brammer, M.J. and Campbell, I.C., Presynaptic a2-adrenoceptor and r-opiate receptor occupancy promotes closure of neuronal (N-type) calcium channels, Eur. J. Pharmacol., 174 (1989) 64-70. 2 Attali, B., Saya, D. and Vogel, Z., r-Opiate agonists inhibit adenylate cyclase and produce heterologous desensitisation in rat spinal cord, J. Neurochem., 52 (1989) 360-369. 3 Bean, B.P., Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence, Nature, 340 (1989) 153-156. 4 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. 5 Bicknell, R.J. and Leng, G., Endogenous opiates regulate oxytocin but not vasopressin secretion from the neurohypophysis, Nature, 298 (1982) 161-162. 6 Bondy, C.A. and Gainer, H., Activators of protein kinase C potentiate electrically stimulated hormone secretion from the rat's isolated neurohypophysis, Neurosci. Lett., 89 (1988) 97101. 7 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, Endocrinology, 122 (1988) 1321-1327. 8 Brethes, D., Dayanithi, G., Letellier, L. and Nordmann, J.J., Depolarisation-induced Ca2+ increase in isolated neurosecretory nerve terminals measured with fura-2, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1439-1443. 9 Bunn, S.J., Hanley, M.R. and Wilkin, G.P., Evidence for a kappa-opioid receptor on pituitary astrocytes: an autoradiographic study, Neurosci. Lea., 55 (1985) 317-323. 10 Cazalis, M., Dayanithi, G. and Nordmann, J.J., Hormone release from isolated nerve endings of the rat neurohypophysis, J. Physiol., 390 (1987a) 55-70. 11 Cazalis, M., Dayanithi, G. and Nordmann, J.J., Requirements for hormone release from permeabilised nerve endings isolated from the rat neurohypophysis, J. Physiol., 390 (1987b) 71-91. 12 Dayanithi, G., Martin-Moutot, N., Burlier, S., Colin, D.A., Kretz-Zaepfel, M., Couraud, E and Nordmann, J.J., The calcium channel antagonist w-conotoxin inhibits secretion from peptidergic nerve terminals, Biochem, Biophys. Res. Commun., 156 (1988) 255-262. 13 Falke, N., Dynorphin (1-8) inhibits stimulated release of oxytocin but not vasopressin from isolated neurosecretory endings of the rat neurohypophysis, Neuropeptides, 11 (1988) 163-167. 14 Gross, R.A. and MacDonald, R.L., Dynorphin A selectively reduces a large transient (N-type) calcium current of mouse dorsal root ganglion neurons in cell culture, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5469-5473. 15 Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. 16 Herkenham M., Rice, K.C., Jacobson, A.E. and Rothman,
magnocellular neurohypophysial neurones.
Acknowledgements. We are grateful to Dr. Simon Luckman for the electron microscopy, to Richard Bunting for data analysis and graphics and to Jennifer Cummings for preparing the manuscript. We thank Drs. Gareth Leng and Bill Mason for their helpful comments on the draft manuscript.
R.B., Opiate receptors in rat pituitary are confined to the neural lobe and are exclusively kappa, Brain Research, 382 (1986) 365-371. 17 Kato, M., Hoyland, J., Sikdar, S.K. and Mason, W.T., Dynamic video imaging of control pathways for intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor, J. Physiol., in press. 18 Lemos, J.R. and Nowycky, M.C., Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals, Neuron, 2 (1989) 1419-1426. 19 MacDonald, R.L. and Werz, M.A., Dynorphin A decreases voltage-dependent calcium conductance of mouse dorsal root ganglion neurones, J. Physiol., 377 (1986) 237-249. 20 Maysinger, D., Vermes, I., Tilders, F., Seizinger, B.R., Gramsch, C., Hollt, V. and Herz, A., Differential effects of various opioid peptides on vasopressin and oxytocin release from the rat pituitary in vitro, Naunyn-Schmiedeberg's Arch. Pharmacol., 328 (1984) 191-195. 21 Nordmann, J.J. and Dayanithi, G., Release of neuropeptides does not only occur at nerve terminals, Biosci. Rep., 8 (1988) 471-483. 22 Nordmann, J.J., Stuenkel, E.L. and Malviya, A.N., Exocytosis in neurohypophysial nerve terminals is not coupled to protein kinase C translocation, Biochem. J., 273 (1991) 493-496. 23 Obaid, A.L., Flores, R. and Salzberg, B.M., Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to to-conotoxin and relatively insensitive to dihydropyridines, J. Gen. Physiol., 93 (1989) 715-729. 24 Racke, K., Burns, F., Haas, B., Niebauer, J. and Pitzius, E., Frequency-dependent effects of activation and inhibition of protein kinase C on neurohypophysial release of oxytocin and vasopressin, Naunyn-Schmiedeberg's Arch. Pharmacol., 339 (1989) 617-624. 25 Schroeder, J.E., Fischbach, ES., Zheng, D. and McCleskey, E.W., Activation of/~ opioid receptors inhibits transient highand low-threshold Ca2+ currents, but spares a sustained current, Neuron, 6 (1991) 13-20. 26 Sher, E. and Clementi, E, to-Conotoxin-sensitive voltage-operated calcium channels in vertebrate cells, Neuroscience, 42 (1991) 301-307. 27 von Spreckelsen, S., Lollike, K. and Treiman, M., Ca 2+ and vasopressin release in isolated rat neurohypophysis: differential effects of four classes of Ca2+ channel ligands, Brain Research, 514 (1990) 68-76. 28 Stuenkel, E.L., Effect of membrane depolarisation on intracellular calcium in single nerve terminals, Brain Research, 529 (1990) 96-101. 29 Sumner, B.E.H., Coombes, J.E., Pumford, K.M. and Russell, J.A., Opioid receptor subtypes in the supraoptic nucleus and posterior pituitary gland of morphine-tolerant rats, Neuroscience, 37 (1990) 635-645. 30 Suszkiw, J.B., Murawsky, M.M. and Shi, M., Further characterisation of phasic calcium influx in rat cerebrocortical synaptosomes: inferences regarding calcium channel type(s) in nerve endings, J. Neurochem., 52 (1989) 1260-1269.
146 31 Tsien, R.Y., Fluorescence measurement and photochemical manipulation of cytosolic free calcium, Trends Neurosci.~ 11 (1988) 419-424. 32 Tsien, R.W., Lipscombe, D., Madison, D.V., Bley, K.R. and Fox, A.P., Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci., 11 (1988) 431-443. 33 Zhao, B.-G., Chapman, C. and Bicknell, R.J., Functional
~:-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis, Brain Research, 462 (1988) 62-66. 34 Zhao, B.-G., Chapman, C. and Bicknell, R.J., Opioid-noradrenergic interactions in the neurohypophysis, Neuroendocrinology, 48 (1988) 16-24.
Brain Research, 574 (1992) 138-14t~ © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17524
Activation of r-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca 2+ in secretory nerve terminals of the neurohypophysis M. Kato*, C. Chapman and R.J. Bicknell Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U. K.)
(Accepted 29 October 1991) Key words: Oxytocin; Vasopressin; Secretion; Calcium; Calcium channel; Opioid
Nerve endings of the magnocellular neurohypophysial neurones possess r-opioid receptors. Using a preparation of isolated terminals from the neurohypophysis we studied r- opioid effects on secretion of oxytocin and vasopressin and on intracellular Ca2+ concentration ([Ca2+]i) measured fluorimetrically or using digital video imaging with Fura-2. The dihydropyridine CaZ+-channel antagonist nicardipine reduced [Ca2+]i responses to K+-depolarisation (30-40 mM K +) by 55-75% and inhibited evoked secretion of oxytocin and vasopressin to a similar extent. The selective r-receptor agonist I)-Pro1° Dynorphin A 1-11 (DPDYN) substantially inhibited K + evoked secretion of oxytocin by 40-90% and secretion of arginine vasopressin (AVP) by 20-50%. DPDYN caused only a 10% reduction in the average total population [Ca2+]i response to K + depolarisation. No sub-population of inhibitory responses was observed when samples of individual terminal [Ca2+]i responses were examined with imaging. Although ~c-receptors are coupled to Ca2+-channels at neuronal somata our data suggest that alternative effector mechanisms operate in these secretory nerve endings. INTRODUCTION Opioid receptors in the rat neurohypophysis are predominantly or exclusively of the r-type 9'16'29. One population of r-receptors (rRs) is present on the nerve endings of the neurohypophysial magnocellular secretory neurones where their activation inhibits depolarisation evoked secretion of neurohormones, particularly oxytocin 7't3'33. Endogenous neurohypophysial opioids also act on opioid receptors within the tissue and antagonism of their action reveals an inhibitory influence again predominantly over secretion from terminals of oxytocin neurones 5'2°'34. The nerve terminal localisation of r R s at this site is analogous with that of presynaptic receptors elsewhere in the brain which modulate peptide and transmitter release occurring in response to invasion of the terminal by depolarising action potentials. Since the small size of mammalian nerve terminals has in general precluded their study by electrophysiological methods, the membrane and intracellular mechanisms of action of presynaptic receptors have generally been inferred from those seen at the cell body or from biochemical indices taken in highly heterogeneous synaptosome preparations.
Using the relatively homogeneous 'neurosecretosome' preparation derived from rat neurohypophysis we have now studied intracellular calcium concentrations [Ca2+]i with the Fura-2 fluorescent Ca 2÷ indicator in suspensions of nerve endings maintained in a fluorimeter or in individual terminals using digital video imaging technology. We have compared the effects of Ca2+-channel antagonists and r R activation using the highly selective r-agonist D-Pro 1° Dynorphin A 1-11 ( D P D Y N ) on the K ÷depolarisation evoked [Ca2+]i signal and on the secretion of oxytocin (OXT) and arginine vasopressin (AVP). Previous studies of r R effector mechanisms have suggested a coupling to 'N' type Ca2+-channels in the somatic cell membrane of cultured neurones to reduce Ca 2+ entry 14. A rise in [Ca2+]i caused by influx of Ca 2÷ during depolarisation is thought to be the primary trigger to exocytosis from nerve endings. MATERIALS AND METHODS Neurosecretosome preparation Neurosecretosomes were prepared from neurohypophyses of adult male rats (approx. 250 g) by a modification of the method of Cazalis et al 1°. Rats were stunned and killed by cervical dislocation. Neurohypophyses were dissected free of anterior and intermediate lobe tissue and gently disrupted in a teflon/glass homoge-
* Permanent address: Institute of Endocrinology, Gunma University, Maebashi, Gunma 371, Japan. Correspondence: R.J. Bicknell, Department of Neuroendocrinology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
139
niser (clearance 50-100/zm) in 270 mM sucrose buffered with 10 mM HEPES neutralised to pH 7.2 with KOH and containing 10 /~M EGTA, maintained at 37°C. Following centrifugation at 600 g for 5 min to remove debris and larger elements, the supernatant was centrifuged at 3400 g for 15 rain. The resulting pellet (P2) was resuspended in a HEPES buffered salt solution (I-IBSS (in mM): NaCI 145, KC1 5, NaHCO 3 5, MgCI 2 2, CaC12 2, HEPES 10, o-glucose 5.6, Bovine serum albumin (Sigma Fraction V) 1 mg/ml; pH 7.35; filtered through a 0.22/~m membrane) and centrifuged at 3400 g for 15 min through an underlying layer of 7.5% Percoll (Pharmacia LKB) prepared in HBSS to produce a further pellet (P3). This Percoll step was found to produce a preparation giving lower basal levels of neurohormone release and more robust responses to depolarisation possibly because of the exclusion of larger nerve endings known to be less responsive 21. When examined in the electron microscope, P3 consisted predominantly of 1-2/~m diameter spherical membrane bound elements containing dense core secretory granules, clear microvesicles, mitochondria and vacuoles. P3 only differed from P2 in appearance in containing fewer relatively large elements (3-5/~m). Immediately prior to Fura-2 or secretion experiments the resuspended pellet was filtered through 30/~m nylon mesh.
Fura-2 loading For both cuvette and digital image measurements, prior to the Percoll step the P2 pellet was resuspended in HBSS containing 2 /~m Fura-2 acetoxymethylester and incubated for 15 mi'n at 37°C. Following Fura-2 loading the preparation was centrifuged through Percoll to yield P3 as described above.
Fluorimeter measurements P3 was resuspended in 5 ml of HBSS and centrifuged again for 15 min at 3400 g as an additional washing step. The resulting pellet was resuspended in HBSS to a concentration of neurosecretosomes equivalent to 3 neurohypophyses per ml. Three hundred and fifty /~l of suspension was placed in a stirred quartz microcuvette maintained at 37°C in a Perkin-Elmer LS-5B fluorimeter driven by a microcomputer programme supplied by the company to measure 340 nm: 380 nm emission ratio at 4-s intervals. Emission intensities were measured at 510 nm. Following lysis with Triton X-100 (0.14%) and calibration of the dye response with Ca :÷ and subsequently excess EGTA (20 mM), [Ca2+]i was derived in the programme according to the equation of Grynkiewicz et al, is using a Kd value for Fura 2 ~ a 2+ of 225 nM. Solutions were added to the cuvette by micropipette during a pause in the data collection. Elevated K + concentrations were achieved in this system by addition of isotonic HBSS in which KC1 was elevated at the expense of NaC1. No loss of response was seen upon storage of the Fura-2 loaded preparation at 22°C in the dark for up to 2 h. To reduce any problems of dye leakage from Fura-2 loaded neurosecretosomes into the extracellular medium all solutions used in the fluorimetric measurements contained 100/~M sulfinpyrazone, an inhibitor of organic anion transport 3~. Early experiments established that the apparent slow rise in [Ca2+]i seen during the course of each run was prevented by this agent. When the actions of the dynorphin peptide were examined, the HBSS for both vehicle and drug experiments contained a cocktail of peptidase inhibitors as previously described 33.
Digital imaging of [Ca2+]i The Fura-2 loaded P3 pellet was resuspended in HBSS to a concentration equivalent to 5 neurohypophyses per 100 /~1. Ten /~1 aliquots were pipetted on to the centre of 22 mm diameter glass coverslips previously washed in detergent and 1% dimethylsulfoxide and treated with 0.02% poly-o-lysine (Sigma). Following an attachment period of 10 min at 22-25°C the coverslip was fixed in a controlled perfusion chamber (150/~1 volume) mounted on the stage of a Nikon Diaphot microscope, maintained at 37°C and superfused continuously with HBSS at 500/d/min. Attached neurosecretosomes were visualised with a CF-Fluor 100 x oil
immersion quartz objective. The Joyce-Loebl (Gateshead, U.K.) 'Magical' system with TARDIS software was used for all dynamic video imaging and image processing. Excitation wavelengths of 340 nm and 380 nm were alternated by means of computer controlled rotating filter wheel between the xenon ultraviolet source and the microscope. Emission light at 510 nm (10 nm band width) was passed to an image intensifying charge coupled device camera (Photonic Science). The resulting image was averaged in real time (16 images), digitised, captured, stored and background subtracted as previously described 17. Time resolution between ratioed images was 3.6 s. The 340 nm: 380 nm ratios of emitted fluorescence were calculated for each frame on a pixel-by-pixel basis and converted to Ca 2÷ concentration according to the equation of Grynkiewicz et al. 15 using a Ka for Fura 2,Ca2+ of 225 nM. Calibration constants were not obtained in situ due to problems of loss of attachment during permeabilisation. Calibration constants were taken from experiments in the same set-up using Fura-2 loaded, permeabilised pituitary cells17 and thus accurately reflect the dye response in an intracellular milieu but do not report absolute levels of [Ca2+]i. Data for neurosecretosomes obtained by imaging are therefore expressed only as relative [Ca2÷]i values and quantitative analysis of drug effects performed with an internally controlled double stimulus protocol (see below). To produce a continuous trace of relative [Ca2+]i with time, each neurosecretosome was outlined on the video screen and the mean [Ca2+]i within the area defined was computed. Data for each terminal was exported to an ASCII file which was incorporated into a Lotus 123 spreadsheet for further calculations. For quantitative analysis of responses to two identical K ÷ depolarisations (S 1 and $2) the cumulative [Ca2+]i was calculated for each 120 s exposure to elevated K ÷ medium and a response ratio derived (S::S1; see Fig. 4).
Secretion experiments The P3 pellet was resuspended in HBSS at a concentration equivalent to 1 neurohypophysis per ml and 500/~1 aliquots inoculated through a 3-way tap on to 13 mm diameter, 0.45/~m fluoropolymer filter assemblies (Acro LC13; Gelman Sciences) containing a prefilter (Millipore AP20) fitted in the inlet barrel. Eight filters were prepared concurrently and perifused at 37°C at a flow rate of 200/~l/min with HBSS. Two minute fractions of perifusate were collected automatically and stored at -20°C until measurement of concentrations of OXT and AVP by specific radioimmuno,assays4. Following an equilibration period of 90 rain to allow basal secretion rates to stabilise, neurohormone release was evoked by depolarisation for 5 min with I-IBSS containing elevated levels of KCI (isotonically substituted for NaC1). In some experiments, two identical depolarisations, $1 and $2, were applied and ratios of total evoked release above basal calculated ($2:$1). Prior to calculations release rates were expressed as a percentage of tissue OXT and AVP content present on each filter at the beginning of the relevant collection period. Total OXT and AVP present on each filter at the end of the experiment were determined following lysis in 0.1% Triton X-100/0.1 M HCI for use in this calculation. Mean basal release rates per 2 min fraction immediately prior to depolarisation represented 0.054 + 0.004% and 0.066 + 0.005%, respectively, of tissue contents of OXT and AVP (n = 48; + S.E.M.).
Drugs Nicardipine-HC1 and sulfmpyrazone were purchased from Sigma (Poole, U.K.); ~o-conotoxin GVIA was from Peptide Institute Inc. (Osaka, Japan); Fura-2 acetoxymethylester was from Molecular Probes (Eugene, U.S.A.); o-Pro 1° Dynorphin A 1-11 was a gift from Dr. J.E. Gairin (Scripps Clinic, La Jolla, U.S.A.); norbinaltorphimine was a gift from Dr. C.EC. Smith (Reckitt and Colman, Hull, U.K.).
140 800
50 mM K ÷ qp-
600
~- 400 200.
400
] 10 m M EGTA
200,~
~
50 mM K÷
0 0
60
120
180
240
300
Time {s)
Fig. 1. Fluorimetric measurement of m e a n [Ca2+]i in the total neurosecretosome population. The rise in [Ca2+]i evoked by elevation of [K+]o (upper trace) is prevented by chelation of extracellular Ca2+ (lower trace). This experiment was repeated on 3 separate occasions. In subsequent analyses of fluorimetric measurements we analysed the peak [Ca2+]i response achieved, mean peak value achieved during the initial 20 s of depolarisation and an average value between 4-5 min after the onset of depolarisation (see Resuits). RESULTS
Fluorimeter measurement of [CaZ+] i Effects of depolarisation. In normal HBSS the average resting [Ca2+]i in the total neurosecretosome population was 113 + 4 nM (n = 37 from 13 preparations) and was stable during the 7 min duration of each experiment. Elevation of extracellular K ÷ ([K+]o) resulted in a prompt increase in [Ca2+]i (Fig. 1). Chelation of ex-
(a)
~
(b)
peak 400
.
t-
r-" ¢, tO O ¢D :D
OXT
1.00
AVP
mean peak ~
in
o <
tracellular Ca 2+ by addition of E G T A completely prevented the K+-induced increase in [Ca2+]i in 3 preparations tested (Fig. 1). The response to elevation of [K+]o consisted of an initial peak, very pronounced at higher levels of [K÷]o (compare Figs. 1 and 3), followed by a decline to a lower and relatively stable plateau value. For quantitative analysis we recorded the increment over basal (A) of the peak [Ca2+]i, a mean peak value taken from the first 5 readings after K ÷ addition, and a mean plateau value taken from readings 4-5 min after K ÷ addition. A dose-response relationship between these parameters and levels of [K÷]o between 20 and 70 m M was obtained (Fig. 2a). Effects of Ca2+-channel antagonists. The effects of the dihydropyridine Ca2÷-channel antagonist nicardipine (10 /~M) and of w-conotoxin G V I A (w-CTX; 1-10/~M, data combined) on the [Ca2÷]i response evoked by 30 mM [K+]o were assessed. Neurosecretosomes were pre-exposed to nicardipine for 1 min and to w-CTX for 10 min prior to depolarisation. For each antagonist, 3 separate preparations were used for multiple tests. On each occasion 1-3 neurosecretosome aliquots were used to obtain control data (30 mM [K+]o; vehicle only) and 1-3 aliquots used with antagonist. For each preparation, parameters of [Ca2+]i were averaged for the control and antagonist tests. Data for all preparations were then combined. Data were also analysed by expressing A[Ca2+]i as a percentage of the mean control value in each preparation prior to combination and statistical testing. Nicardipine did not affect resting [Ca2+]i but strongly suppressed all components of the K+-evoked increase (Fig. 3). Peak, mean peak and 4-5 min values were depressed by 74 + 2, 76 + 2 and 75 + 2%, respectively,
""i..........' ".c,..... ........ .o 4 - 5 m i n
200 ••
.... o..'"
'
0.50
/
== ~
"0
j
/
s
/S
tU S
I
l
I
0
i
30 50 [K+]o (mM)
70
20
i
!
!
30 50 70 [K+]o (mM) Fig. 2. K+-concentration dependence of (a) evoked increases above basal (A) of [Ca2+]i measured fluorimetrically and (b) evoked secretion of neuropeptides. Note log-scales for [K+]o. Data are mean + S.E.M. of 3-4 observations from 3 preparations ([Ca2+]i) and of 3-4 observations from 2 preparations for oxytocin (OXT) and arginine vasopressin (AVP). 20
141
BI
A. 500
400 30 mM K+
300 ~¢= 250 (9
,_¢ ~'~ 200 Z
.< i
30 mM K + 10 ~ Nicardipine
200
100
0
0
21'0
Time (s)
peak
420
mean )eak
4-5 rain
Fig. 3. A: effect of nicardipine (10/~M) on K ÷ evoked rise in [Ca2+]i measured fluorimetrically. Examples are sequential tests performed on aliquots of the same preparation. B: combined data from experiments exemplified above. Data are mean + S.E.M. for 6 observations from 3 preparations. For statistical analysis of control-normalised data, see text.
A. compared to control values in each preparation (Table I; n = 6 from 3 preparations; P < 0.01 in each case; Mann-Whitney U-test). ~0-CTX did not affect resting [Ca2+]i and only weakly inhibited the peak rise in [Ca2+]i evoked by 30 mM [K+]o (peak A[Ca2+]i control, 247 + 60 nM; to-CTX, 217 + 48 nm). Expression of the data relative to control values reveal significant inhibition of the peak A[Ca2+]i by 12 + 2% (P < 0.05), of the mean peak also by 12 + 2% (P < 0.01) but not of the 4-5 min A[Ca2+]i (Table I; n = 9 from 3 preparations; Mann Whitney U-test). Effects of rR activation. Pre-exposure of neurosecretosomes to the r-agonist DPDYN (1/zM) for 5 min prior to depolarisation did not affect resting [Ca2+]i b u t weakly inhibited the peak rise in [Ca2+]i evoked by 30 mM [K+]o (peak A[Ca2+]i control, 270 + 27 nM; DPDYN, 245 + 25 nM). Peak A[Ca2+]i, when expressed relative to control values, was depressed by 11 + 3% (P < 0.01), mean peak value was depressed by 7 + 3% (P < 0.05)
40raM.K~(SO
~(S2)
150 S2:N , 1.~1
100
0
200
400
600
800
1p.U DPDYN
B.
1 / 1 1 1 1 1 1 1 1 1 / / / / / / 1 1 / / / / / / i / 1 1 1
2OO 150 .~
t~
100 5O
0
>
C.N ff.
400
600
~
t / / / / / / / / / / / i / / / / / / / / / / / / / / / / l
150
0
D.
200
200
200
400
600
800
i / / / / i / / / / / / / / / / / / / / / / / / / / ( / i l
150 TABLE I
100
cts o f Ca2+-channel antagonists and D P D Y N on the rise in +]~ evoked by 30 m M K +
5O
AlCa;+]i (% control) Group
Peak
Mean peak 4-5 min
n
Nicardipine (10/~M) w-CTX (1-10 ttM) DPDYN (1/~M)
26 ___ 2** 88 + 2* 89 + 3**
24 + 2** 88 + 2* 93 + 3*
6 9 15
25 + 2** 92 :t: 5 99 + 3
* P < 0.05, **P < 0.01 vs relevant control; Mann Whitney U-test,
Fig. 4. Examples of relative [Ca2+]t responses (see Materials and Methods) of 4 individual Fura 2-loaded nerve endings measured using digital video imaging during superfusion on the microscope stage. In each experiment two 120 s periods of depolarisation with 40 mM K + (S1 and $2) were imposed, separated by a wash period in HBSS. A: control; B,C,D: l>Pro 1° Dynorphin A 1-11 (DPDYN) (1 /~M) present prior to and during S2. Derivation of $2:S 1 response ratios for each terminal is described in Materials and Methods.
142 0.8 A,
50
•
i ....... i I
E [ ] 30 mM K ÷, control [ ] 30 mM K +, DPDYN
40
o
0.6-
o~
0.4 4
~
0.2
l
-~--
l
i
i~il
o
~ 30 "6 20
E= lo
[•,
Z
0
B.
0.1
o.'3 0.s
0.7
0.'9
1.1
1.3
1.5
m, 1 .'7 1.9
2.'1
OXT
(s~s~)
40 mM K + (n=4)
[ ] 40 mM K +, DPDYN [ ] 40 mM K*; Nicardipine
60
_ _ J AVP
OXT
AVP 50 mm K + (n=6)
Fig. 7. Effects of to-conotoxin on release of OXT and AVP evoked by a single 40 or 50 mM K ÷ depolarisation. Data are mean _+ S.E.M. *P < 0.05 vs relative control level of evoked release; Mann Whitney U-test.
40
"6
[Ca2+]i imaging
~ 20 Z
o o11
0.3
0.5
0.7
0.9
1.1
1:3 G
1:7 G 211
(S2/S 1)
Fig. 5. Population distribution of $2:St response ratio derived from [Ca2+]i imaging experiments exemplified in Fig. 4. Data are the number of Sz:S~ values at each level indicated, plotted as a percentage of the total number of responses recorded for each treatment. A: 30 mM K + stimulus; data from 15 terminals under control conditions, 13 terminals in the presence of DPDYN during $2. B: 40 mM K + stimulus, data from 16 terminals under control conditions, 11 terminals in the presence of nicardipine and 9 terminals in the presence of DPDYN.
whilst the 4 - 5 min value was unaffected at 99 ___ 3% of the control (Table I; n = 15 from 9 preparations; M a n n Whitney U-test).
0.5
0.4
8 ~=
t_
8O
._o
8o
X
o1',;
[ ] 40 mM K*, control v
•
0.3 i
0.2 d 0.1 tJJ 0
OXT
AVP 30 mM K + (n=7)
OXT
AVP 40 mM K + (n=4)
Fig. 6. Effects of nicardipine on release of OXT and AVP evoked by a single 30 or 40 mM K + depolarisation during continual perifusion. Data are mean + S.E.M. **P < 0.01 vs relative control level of evoked release; Mann Whitney U-test.
In these experiments we e m p l o y e d a double stimulus protocol during superfusion of neurosecretosomes on the microscope stage. E x a m p l e s of profiles of relative [Ca2÷]i with time for 4 individual terminals are shown in Fig. 4. U n d e r control conditions (no drug), two identical depolarisations ($1 and $2) with 30 or 40 m M [K+]o for 120 s separated by a wash p e r i o d of 5 min p r o d u c e d very similar responses. Thus for 30 m M [K÷]o the m e a n control $2:S t response ratio of cumulative [Ca2+]i was 0.949 + 0.040 (n = 15 terminals for 4 experiments). In the presence of D P D Y N (1 ~ M ) during S 2, the m e a n [Ca2+]i rise caused by 30 m M [K÷]o was slightly enhanced ($2:$1 1.237 + 0.109, n = 13 in 3 experiments, examples in Fig.
4). With a 40 m M [K+]o stimulus m e a n control $2:$1 [Ca2+]i was 0.986 + 0.020 (n = 16 in 3 experiments). Inclusion of D P D Y N (1 /~M) did not alter the m e a n $2:$1 value (1.026 + 0.115, n = 9 in 3 experiments). U n d e r these conditions nicardipine (10/~M) depressed m e a n $2:$1 [Ca2+]i to 0.436 + 0.059 representing a 56% reduction (n = 11 in 3 experiments; P < 0.01, M a n n Whitney U-test). A t both levels of K ÷ depolarisation we looked to see if a subpopulation of terminals showed an inhibitory response to D P D Y N by plotting the distribution of S2:St ratios as a percentage of the total population analysed (Fig. 5). N o such subpopulation was revealed in either case, although at both levels of [K÷]o D P D Y N did app e a r to b r o a d e n the distribution of responses (Fig. 5). Since the control d a t a in both series a p p r o x i m a t e d to a normal distribution we c o m p a r e d the variances between control and D P D Y N groups. Significantly greater variance was found in the D P D Y N groups (at 30 m M [K÷]o FI2,14 --- 6.396; at 40 m M [K+]o F8,15 = 19.01; P < 0.01
143 0.3
c
8
.m.o.8
0.2
8 ~ 0.6
w
OXT
AVP
i
OXT i
30 mM K+ (n=7)
i
~
0.4
"6 .9 0.2 rr AVP
0
40 mM K+ (n=6)
Fig. 8. Effects of DPDYN (1/~M) release of OXrI"and AVP evoked by a single 30 or 40 mM K+ depolarisation. Data are mean + S.E.M. *P < 0.05 and **P < 0.01 vs relative control level of evoked release; Mann Whitney U-test.
in both cases). In contrast to DPDYN, nicardipine clearly shifted the distribution of responses in the inhibited direction (Fig. 5). Broadening of the population distribution also occurred with nicardipine (Fl0,15 = 6.03; P < 0.01). Peptide secretion A single 5 min exposure to various levels of [K÷]o evoked dose-related increases in the secretion of O X T and AVP from neurosecretosomes during perifusion (Fig. 2b). Evoked secretory responses were dependent on the presence of extracellular Ca 2+ as previously described 11'33. Whilst the fluorimeter measurements readily revealed [Ca2+]i increases in the secretosome population in response to 20 mM [K+]o, the lowest level of [K+]o tested (Fig. 2a), this depolarisation led to barely detectable evoked release of peptides (approximately 0.02% of tissue content). The effects of antagonists on evoked release were examined following 5 min pre-exposure. None of the drugs affected basal output of OXT and AVP. Nicardipine (10 /~M) strongly inhibited 30 mM [K+]o evoked O X T and AVP secretion by 42 and 63%, respectively, and at 40 mM [K+]o inhibited evoked OXT and AVP secretion by 52 and 64%, respectively (Fig. 6). to-CTX (1/~M) had no major effect on peptide secretion evoked by 40 mM [K÷]o and inhibited OXT and AVP secretion evoked by 50 mM [K+]o by about 30% (P < 0.05 for AVP; Fig. 7). D P D Y N (1 btM) inhibited OXT secretion induced at 30 and 40 mM [K+]o by 52 and 36%, respectively. AVP secretion was also reduced but by a lesser extent in each case (38 and 20%, respectively; Fig. 8). In a further series the inhibitory effects of D P D Y N on 40 mM [K+]o evoked secretion were prevented by inclusion of the selective r R antagonist norbinaltorphinine (nor-BNI; 10
DPDYN
op2y O×T
AVP i
40 mM K+ (n=6)
Fig. 9. Effects of DPDYN (1 /~M) on OXT and AVP release evoked by 40 mM K÷ using a double stimulus protocol with DPDYN or no additions during the second stimulus ($2). Data are mean + S.E.M. $2:S1 response ratios of the amounts of evoked release. **P < 0.01 vs relative control; Mann Whitney U-test.
gM). Evoked release of OXT was 0.38 + 0.03% tissue content in the presence of nor-BNI alone and 0.36 + 0.03% tissue content in nor-BNI with DPDYN. Respective values for AVP release were 0.49 + 0.09 and 0.52 + 0.10% tissue content (n = 4 in 2 experiments). To more closely approximate the conditions achieved under the [Ca2+]i imaging experiments the actions of D P D Y N on peptide secretion were also examined using a double stimulus protocol. Two identical 4 min exposures to 40 mM [K+]o (S1 and $2) were separated by 12 rain and ratios of evoked peptide release calculated ( 5 2 : 5 1 ) . Inclusion of D P D Y N (1 gM) during S2 strongly reduced OXT and AVP release by 87% and 54%, respectively (Fig. 9; n = 5; P < 0.01 in each case, Mann Whitney U-test). DISCUSSION The neurosecretosome preparation The anatomically segregated and densely packed nerve terminals of the neurohypophysis have long served as a model in which to study mechanisms regulating release of neuropeptides. In recent years the development of a functional neurosecretosome preparation 1° has facilitated more direct study in particular of the roles of Ca 2÷. The preparation is analogous to synaptosomes prepared from brain but has the advantages of being uncontaminated by post-synaptic elements and of consisting predominantly of nerve endings of only two neuronal types - the magnocellular OXT and AVP secreting neurones zl. Our measurements reveal average resting [Ca2+]i in these terminals to be approximately 100 nM and peak levels achieved during K ÷ depolarisation were 500-1000 nM. It is probable that higher [Ca2+]i is achieved directly
144 between the nerve terminal membrane. In permeabilised neurosecretosomes, half maximal release of peptides occurs at about 1-2 /~M Ca 2+ (ref. 11). Our measured value of resting [Ca2+]i is somewhat lower than previously reported using fluorimeter measurements of Fura-2 in this preparation s and similar to a recent microspectrofluorometric study 2s. Ca2÷ channels in neurosecretosomes Our experiments indicate that a substantial part of the inward Ca 2+ movement during sustained depolarisation of secretosomes with K + is carded by dihydropyridinesensitive L-type Ca 2+ channels 32. Under these conditions to-CTX sensitive channels, which may define a separate population 26, appear to play a minor role. Secretion studies indicated that to-CTX sensitive channels may mediate more Ca 2÷ entry at higher levels of K ÷ depolarisation (>50 mM; Fig. 7). A recent fluorometric study of individual Fura-2 loaded neurosecretosomes came to a very similar conclusion regarding the predominance of L-channel mediated Ca2÷entry during K÷-depolarisa tion 2s.
The presence of both L-type and to-CTX sensitive putative N-type channels in secretosome membranes has been previously suggested from studies of neural lobe secretion, to-CTX sensitive channels appear to mediate the majority of Ca 2÷ entry and secretion when depolarisation is induced by Na + action potentials in the intact neurohypophysial tissue 12'23'27. Patch clamp recordings support the existence of populations of L- and N-type Ca 2÷ channels in the membrane of individual neurosecretosomes. Interestingly, although to-CTX sensitivity was not tested, the N-channels were defined as requiring a higher (more positive) threshold membrane potential for activation is. A range of pharmacological studies in heterogeneous synaptosome and other synaptic preparations from the brain also indicates that several channel types are involved in regulating Ca 2+ entry and transmitter release (e.g. Suszkiw et al.3°). r-Opioid receptor coupling Neurosecretosomes from rats possess rRs which when activated by selective opioid agonists or by dynorphins (the likely endogenous ligands) inhibit depolarisation evoked secretion of neuropeptides 13'32. Activation of KRs on the somatic membrane of cultured neurones has been reported to reduce a Ca 2+ conductance 14'19'25. Given the primary role of Ca 2÷ entry in mediating depolarisation evoked exocytosis in nerve terminals we decided to look directly for KR mediated inhibition of the depolarisation evoked [Ca2÷]i signal in neurosecretosomes. Surprisingly, we observed very little effect of r R activation on the average [Ca2+]i response of the total
secretosome population to moderate K*-depolarisation (7-11% inhibition of the peak response). Secretion of OXT and AVP evoked by the same level of K + depolarisation was inhibited by 52% and 38%, respectively, by DPDYN. Mediation of this effect via 7oRs was confirmed with a highly selective r R antagonist. The Ca 2+channel antagonist nicardipine resulted in similar levels of inhibition of OXT and AVP secretion induced by 30 mM [K+]o (42% and 63%, respectively). In this case nicardipine reduced all components of the total population [Ca2+]i response by 75% indicating blockade of C a 2+ entry as the mechanism of secretory inhibition. Depending to some extent on the choice of r-agonist, OXT secreting nerve endings are more sensitive than AVP secreting nerve endings to r R inhibition of exocytosis (Fig. 8; refs. 13, 33 and our further unpublished observations). This relative selectivity of opioid actions is particularly marked in the case of the actions of endogenous opioids in neurohypophysial tissue TM. We thus reasoned that in measuring the total s e c r e t o s o m e [Ca2+]i response to depolarisation we might be masking preferential r-opioid effects o n C a 2+ entry into OXT terminals or perhaps into some more actively secreting sub-population of terminals. This hypothesis was not supported by analysis of r-opioid effects on the [Ca2+]i response to depolarisation in individual nerve terminals using video imaging. Under the imaging conditions and using an internally controlled double-stimulus protocol, nicardipine again strongly suppressed t h e [Ca2+]i response (mean 56% reduction), shifting the distribution of response ratios in our sample in an inhibited direction. No sub-population of terminals exhibiting a similar shift was observed following r R activation and indeed, in the more thorough study using 30 mM K +, the trend was towards an elevated [Ca2+]i response in the presence of DPDYN. The small inhibition of the C a 2÷ signal by DPDYN seen in the fluorimeter study was not reproduced under the conditions of the imaging study. In these small terminals spatial resolution under imaging conditions limits study of subplasmalemmal C a 2+ concentrations that might still be modified by r R activation. Such an effect would, however, have to be in some way uncoupled from the total [Ca2+]i response which appears due entirely to C a 2+ entry. Whatever the explanation for this difference, using the double-stimulus protocol as for C a 2+ imaging in secretory studies, r R activation was highly effective in inhibiting exocytosis (88% inhibition of OXT and 59% inhibition of AVP secretion). We conclude that r R mediated inhibition of secretion from neurohypophysial nerve endings does not include a major suppression of C a 2+ entry. This implies a coupling mechanism able to inhibit the actions of C a 2+ o n the
145 secretory process and/or regulate some cellular compo-
intact tissue 6'24 (but see ref. 22). These second messen-
n e n t other than Ca 2÷ which is required together with
ger systems thus represent alternative targets for r R cou-
Ca 2+ for activation of exocytosis. Although r R s can be coupled to inhibition of Ca 2÷ channels at n e u r o n a l somata 3'14 and in cortical synaptosomes 1, negative coupling
pling to inhibition of exocytosis in the terminals of the
to adenylate cyclase has also been reported 2. Both inositol 1,4,5-trisphosphate and activators of protein kinase C potentiate Ca 2÷ stimulated exocytosis in permeabilised neurosecretosomes u and protein kinase C activation enhances electrically evoked neuropeptide release from the
REFERENCES 1 Adamson, P., Xiang, J-Z., Mantzourides, T., Brammer, M.J. and Campbell, I.C., Presynaptic a2-adrenoceptor and r-opiate receptor occupancy promotes closure of neuronal (N-type) calcium channels, Eur. J. Pharmacol., 174 (1989) 64-70. 2 Attali, B., Saya, D. and Vogel, Z., r-Opiate agonists inhibit adenylate cyclase and produce heterologous desensitisation in rat spinal cord, J. Neurochem., 52 (1989) 360-369. 3 Bean, B.P., Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence, Nature, 340 (1989) 153-156. 4 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. 5 Bicknell, R.J. and Leng, G., Endogenous opiates regulate oxytocin but not vasopressin secretion from the neurohypophysis, Nature, 298 (1982) 161-162. 6 Bondy, C.A. and Gainer, H., Activators of protein kinase C potentiate electrically stimulated hormone secretion from the rat's isolated neurohypophysis, Neurosci. Lett., 89 (1988) 97101. 7 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, Endocrinology, 122 (1988) 1321-1327. 8 Brethes, D., Dayanithi, G., Letellier, L. and Nordmann, J.J., Depolarisation-induced Ca2+ increase in isolated neurosecretory nerve terminals measured with fura-2, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 1439-1443. 9 Bunn, S.J., Hanley, M.R. and Wilkin, G.P., Evidence for a kappa-opioid receptor on pituitary astrocytes: an autoradiographic study, Neurosci. Lea., 55 (1985) 317-323. 10 Cazalis, M., Dayanithi, G. and Nordmann, J.J., Hormone release from isolated nerve endings of the rat neurohypophysis, J. Physiol., 390 (1987a) 55-70. 11 Cazalis, M., Dayanithi, G. and Nordmann, J.J., Requirements for hormone release from permeabilised nerve endings isolated from the rat neurohypophysis, J. Physiol., 390 (1987b) 71-91. 12 Dayanithi, G., Martin-Moutot, N., Burlier, S., Colin, D.A., Kretz-Zaepfel, M., Couraud, E and Nordmann, J.J., The calcium channel antagonist w-conotoxin inhibits secretion from peptidergic nerve terminals, Biochem, Biophys. Res. Commun., 156 (1988) 255-262. 13 Falke, N., Dynorphin (1-8) inhibits stimulated release of oxytocin but not vasopressin from isolated neurosecretory endings of the rat neurohypophysis, Neuropeptides, 11 (1988) 163-167. 14 Gross, R.A. and MacDonald, R.L., Dynorphin A selectively reduces a large transient (N-type) calcium current of mouse dorsal root ganglion neurons in cell culture, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5469-5473. 15 Grynkiewicz, G., Poenie, M. and Tsien, R.Y., A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. 16 Herkenham M., Rice, K.C., Jacobson, A.E. and Rothman,
magnocellular neurohypophysial neurones.
Acknowledgements. We are grateful to Dr. Simon Luckman for the electron microscopy, to Richard Bunting for data analysis and graphics and to Jennifer Cummings for preparing the manuscript. We thank Drs. Gareth Leng and Bill Mason for their helpful comments on the draft manuscript.
R.B., Opiate receptors in rat pituitary are confined to the neural lobe and are exclusively kappa, Brain Research, 382 (1986) 365-371. 17 Kato, M., Hoyland, J., Sikdar, S.K. and Mason, W.T., Dynamic video imaging of control pathways for intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor, J. Physiol., in press. 18 Lemos, J.R. and Nowycky, M.C., Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals, Neuron, 2 (1989) 1419-1426. 19 MacDonald, R.L. and Werz, M.A., Dynorphin A decreases voltage-dependent calcium conductance of mouse dorsal root ganglion neurones, J. Physiol., 377 (1986) 237-249. 20 Maysinger, D., Vermes, I., Tilders, F., Seizinger, B.R., Gramsch, C., Hollt, V. and Herz, A., Differential effects of various opioid peptides on vasopressin and oxytocin release from the rat pituitary in vitro, Naunyn-Schmiedeberg's Arch. Pharmacol., 328 (1984) 191-195. 21 Nordmann, J.J. and Dayanithi, G., Release of neuropeptides does not only occur at nerve terminals, Biosci. Rep., 8 (1988) 471-483. 22 Nordmann, J.J., Stuenkel, E.L. and Malviya, A.N., Exocytosis in neurohypophysial nerve terminals is not coupled to protein kinase C translocation, Biochem. J., 273 (1991) 493-496. 23 Obaid, A.L., Flores, R. and Salzberg, B.M., Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to to-conotoxin and relatively insensitive to dihydropyridines, J. Gen. Physiol., 93 (1989) 715-729. 24 Racke, K., Burns, F., Haas, B., Niebauer, J. and Pitzius, E., Frequency-dependent effects of activation and inhibition of protein kinase C on neurohypophysial release of oxytocin and vasopressin, Naunyn-Schmiedeberg's Arch. Pharmacol., 339 (1989) 617-624. 25 Schroeder, J.E., Fischbach, ES., Zheng, D. and McCleskey, E.W., Activation of/~ opioid receptors inhibits transient highand low-threshold Ca2+ currents, but spares a sustained current, Neuron, 6 (1991) 13-20. 26 Sher, E. and Clementi, E, to-Conotoxin-sensitive voltage-operated calcium channels in vertebrate cells, Neuroscience, 42 (1991) 301-307. 27 von Spreckelsen, S., Lollike, K. and Treiman, M., Ca 2+ and vasopressin release in isolated rat neurohypophysis: differential effects of four classes of Ca2+ channel ligands, Brain Research, 514 (1990) 68-76. 28 Stuenkel, E.L., Effect of membrane depolarisation on intracellular calcium in single nerve terminals, Brain Research, 529 (1990) 96-101. 29 Sumner, B.E.H., Coombes, J.E., Pumford, K.M. and Russell, J.A., Opioid receptor subtypes in the supraoptic nucleus and posterior pituitary gland of morphine-tolerant rats, Neuroscience, 37 (1990) 635-645. 30 Suszkiw, J.B., Murawsky, M.M. and Shi, M., Further characterisation of phasic calcium influx in rat cerebrocortical synaptosomes: inferences regarding calcium channel type(s) in nerve endings, J. Neurochem., 52 (1989) 1260-1269.
146 31 Tsien, R.Y., Fluorescence measurement and photochemical manipulation of cytosolic free calcium, Trends Neurosci.~ 11 (1988) 419-424. 32 Tsien, R.W., Lipscombe, D., Madison, D.V., Bley, K.R. and Fox, A.P., Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci., 11 (1988) 431-443. 33 Zhao, B.-G., Chapman, C. and Bicknell, R.J., Functional
~:-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis, Brain Research, 462 (1988) 62-66. 34 Zhao, B.-G., Chapman, C. and Bicknell, R.J., Opioid-noradrenergic interactions in the neurohypophysis, Neuroendocrinology, 48 (1988) 16-24.