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Dihydropyridine ligands influence the evoked release of oxytocin and vasopressin dependent on stimulation conditions
ejp ELSEVIER
European Journal of Pharmacology 259 (1994) 157-163
Dihydropyridine ligands influence the evoked release of oxytocin and vasopressin dependent on stimulation conditions Annette J0rgensen a,,, Bjarne Fjalland a, Jens D. Christensen a, Marek Treiman b Department of Biological Sciences, The Royal Danish School of Pharmacy, UniL'ersitetsparken 2, DK-2100 Copenhagen O, Denmark, b Department of Medical Physiology, The Biotechnology Center for Signal Peptide Research, The Panum Institute, Blegdamsl,ej 3c, DK-2200 Copenhagen N, Denmark Received 22 March 1994; accepted 5 April 1994
Abstract
The effects of dihydropyridine ligands on the electrically evoked release of neurohypophysial hormones from isolated, rat neurointermediate lobes were investigated as a function of all combinations of two pulse widths (0.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz). The dihydropyridine agonist (S)-(+)-202-791 potentiated concentration dependently the release of both oxytocin and vasopressin at a pulse width of 2 ms and a frequency of 6.5 Hz. This effect of (S)-(+)-202-791 was abolished by the antagonist (-)-nitrendipine and stereospecifically by (R)-( )-202-791 (only vasopressin). The antagonist (R)-(-)-202-791 alone inhibited the release of oxytocin at 13 Hz and 2 ms. The results presented show that the effects of the dihydropyridine ligands are dependent on the stimulation conditions, and thus demonstrate that the entry of Ca 2~ through the dihydropyridine sensitive L-type Ca 2+ channel is associated with electrically evoked release of neurohypophysial hormones under certain conditions. Key words: Oxytocin; Vasopressin; Ca 2+ channel, L-type; Dihydropyridine; Neurointermediate lobe; (Electrical stimulation)
1. Introduction
The release of hormones from the neurohypophysis is initiated by action potentials propagated from the magnocellular cell bodies. Depolarization of the nerve terminals promotes entry of Ca 2+ from the extracellular environment (Brethes et al., 1987; Shibuki, 1990; Stuenkel, 1990; Fatatis et al., 1992), through voltageactivated Ca 2+ channels (Cazalis et al., 1987; Dayanithi et al., 1988; Obaid et al., 1989; Von Spreckelsen et al., 1990; Stuenkel, 1991; Kato et al., 1992). This rise in intraterminal free Ca 2+ triggers exocytose from neurosecretory nerve endings (Lim et al., 1990; Lindau et al., 1992). Evidence is available describing multiple types of voltage-activated Ca 2+ channels which differ in molecular, electrophysiological and pharmacological properties. The major classes of voltage-activated Ca 2+ channels are known as T, N, L and P (Tsien et al., 1988; Tsien, 1990; Scott et al., 1991).
* Corresponding author. Tel. + 45.35.37(/850, fax + 45.35.374457. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0[) 1 4 - 2 9 9 9 ( 9 4 ) 0 0 2 1 3 - Q
In isolated terminals from the neurohypophysis two types of high voltage-activated Ca 2+ channels have been characterized using patch-clamp techniques. One of these corresponds to the dihydropyridine-sensitive L-type channel, while the other is a dihydropyridineinsensitive channel of the N-type family (Lemos and Nowycky, 1989, 1991). The peptide toxin w-conotoxin G V I A has been shown to block both N- and L-type Ca 2+ currents in neurohypophysial nerve terminals (Wang et al., 1992) and to inhibit high K +- as well as electrically evoked release of vasopressin from isolated neurohypophysis (Dayanithi et al., 1988; Von Spreckelsen et al., 1990). The influence of the dihydropyridines seems to be more complex. During high K+-induced depolarizations the dihydropyridine agonist Bay K 8644 [(R,S)1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-trifluoromethylphenyl)-3-pyridinecarboxylic acid methyl ester] was shown to potentiate the release of vasopressin (Cazalis et al., 1987), while the dihydropyridine antagonists nicardipine, nitrendipine and nimodipine inhibited the peptide release (Cazalis et al., 1987; Dayanithi et al., 1988; Fatatis et al., 1992; Kato et al., 1992). On the
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other hand when isolated neurohypophysis were stimulated electrically using physiological patterns either no effects (Von Spreckelsen ct aI., 1990) or only very small effects (Dayanithi et al., 19881 of the dihydropyridine ligands were found. In the present work it has been studied whether the apparently lacking effects of the dihydropyridinc ligands on the electrically evoked release of neurohypophysial hormones depends on the choice of stimulation conditions. It is known that changes in intracellular Ca ~+ concentration and release of neurohypophysial hormones from isolated nerve terminals are a sensitive function of the applied frequency and pattern of electrical activity (Nordmann et al., 1987, Bicknell, 1988; Jackson et al., 1991). Increasing frequencies thus result in facilitation of the intracellular [Ca ~+] (Jackson et al., 19911 and the amount of peptide released per pulse (Nordmann ct a[., 1987). Therefore it was of interest to study whether different effects of dihydropyridine [igands could be found when changing the stimulation parameters. The effects of a pair of dihydropyridine stereoisomers, (S)-(+)-202-791 and (R)-( )-202-791 on the release of oxytocin and vasopressin from electrically stimulated, isolated, rat neurointermediate lobes have been investigated as function of combinations of different pulse widths and frequencies, iS)-( + )-202-791 and (R)-( - )-202-791 are known to exert opposite actions on L-type Ca 2~ channels, where (S)-(+)-202-791 acts as an agonist and (R)-( )-202-791 as an antagonist (Hosey and Lazdunski, 1988; Triggle and Rampe, 1989). Some of the data reported here have appeared in a preliminary form (J0rgensen et al., 1993).
2. Materials and methods
2.1. Sources of drugs" The enantiomers of 202-791 [1,4-dihydro-2,6-dimethyl-5-nitro-4-(2,1,3-benzoxadiazol-4-yl)-3-pyridinecarboxylic acid 2-propanester] and ( - ) - n i t r e n d i p i n e [( )- 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid ethyl methyl ester] were obtained as a generous gift from Sandoz (Copenhagen, Denmark) and Bayer (Leverkusen, Germany), respectively.
2.2. Preparation of isolated neurointermediate lobes Female Spraque-Dawley rats (18(I-250 g) in spontaneous oestrus were used and the neurointermediate lobe isolated following a procedure described earlier (Fjalland et al., 1987; Treiman et al., 1992). Briefly the pituitary stalk was tied to the cathode of a platinum wire electrode and immersed in 2 ml of a 37°C warm, carbogen bubbled medium, p H 7.4, of the following
composition (raM): NaCI 115, KCI 6.0, N a e H P O a (1.8, CaCI, 1.5, MgCI e 0.9, NaHCO~ 22, D-glucose 10. To allow equilibration of basal hormone release the preparation was preincubated for 1 h, during which time the medium was changed every 15 rain.
2.3. Stimulation protocol Electrical stimuli were applied between the clcctrode carrying the neurohypophysis and a platinum electrode dipped into the incubation medium. The gland was raised to the surface of the incubation medium and the supply of gas interrupted during stimulation. The neurointermediate lobes were stimulated three times (S I, S~ and S~) with an interval of 8(t rain by trains of rectangular pulses (0.8 V, for 10 s with an interval of 10 s for a total of 2.5 rain) generated by a HSE stimulator (type 215/I, Hugo Sachs Elcctronik, Freiburg, Germany). All combinations of two pulse widths ((/.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz) were studied. As the stimulation period was constant at 2.5 rain, the number of pulses were only dependent on the stimulation frequency. The frequencies 6.5, 13 and 30 Hz thus resulted in the delivery of 520, 1040 and 2400 pulses per stimulatkm, respectively. The interval between two stimulations was 80 rain. The incubation medium was changed 55, 40, 25, 10 and 0 rain before the stimulation. Drugs were added from 4(1 rain before and during the second stimulation. When testing two drugs in combination, the antagonist was added 15 rain before the agonist corresponding to 55 min before the stimulation. The incubation medium was collected 10 min after each stimulation and kept frozen at - 2 0 ° C untill assayed. The amount of oxytocin and vasopressin was analysed by radioimmunoassay. The basal hormone release was examined in 10 rain periods preceding the electrical stimulations S~ and S~. The dihydropyridine ligands were diluted in the incubation medium from stock solutions (1 m g / m l ) in ethanol. The final ethanol concentration did not exceed 1.8 m g / m l , and it was tested that there was no difference in the release of neurohypophysial hormones in the presence or absence of this ethanol concentration. All dihydropyridines as well as incubation chambers and other parts of equipment were protected from light during handling and experiments.
2.4. Determination of hormone release The amount of vasopressin in the incubation medium was measured by radioimmunoassay as previously described (Christensen and Fjalland, 19821. Briefly, to 0.2 ml of standard (synthetic arginine vasopressin, Ferring AB, Malm6, Sweden) or unknown, 0.1 ml of antibody
A. Jorgensen et al. / European Journal of Pharmacology 259 (19941 157-163 (final dilution 1 : 4 5 0 0 0 ) a n d 0.05 ml [1251]arginine vasopressin ( D u P o n t (UK), Stevenage, U K ) were added. T h e i n c u b a t i o n time was 48 h at 4°C. A m m o n i u m s u l p h a t e was used for s e p a r a t i o n of b o u n d a n d free [12SI]arginine vasopressin. T h e a n t i s e r u m did not disc r i m i n a t e b e t w e e n lysine a n d a r g i n i n e vasopressin a n d the m i n i m u m d e t e c t i o n limit was 10 p g / m l . Serial diluted test samples had d i l u t i o n curves parallel to the s t a n d a r d curve. Cross-reactivity of a n t i s e r u m to synthetic oxytocin was 0.01%. N o n e of the c o m p o u n d s used in this study i n t e r f e r e d with the assay. T h e within-assay a n d the b e t w e e n - a s s a y coefficients of variation were 4.2 a n d 7.8%, respectively. T h e oxytocin c o n t e n t was d e t e r m i n e d as described by F j a l l a n d et al. (19871. T o 0.1 ml of a n t i b o d y (final dilution 1 : 7 6 8 0 0 ) a n d 0.05 ml [125I]oxytocin ( D u P o n t (UK), Stevenage, U K ) were a d d e d 0.2 ml s t a n d a r d (synthetic oxytocin, F e r r i n g AB, Malta6, S w e d e n ) or u n k n o w n . T h e i n c u b a t i o n time was 72 h at 4°C. A m m o n i u m s u l p h a t e was used for s e p a r a t i o n of b o u n d a n d free [12Si]oxytocin. Cross-reactivity of a n t i s e r u m to lysine a n d a r g i n i n e vasopressin was 0.005%. T h e minimal d e t e c t a b l e level was 8 p g / m l . Serial diluted test samples were parallel to the s t a n d a r d curve. N o n e of the c o m p o u n d s used in the study i n t e r f e r e d with the assay. T h e within-assay a n d b e t w e e n - a s s a y coefficients of variation were 6.1 a n d 5.4%, respectively.
2.5. Bioassay T o o b t a i n a n i m m e d i a t e p r e l i m i n a r y m e a s u r e of the s t i m u l a t i o n a s e m i q u a n t i t a t i v e bioassay was used as described by F j a l l a n d et al. (1987) a n d T r e i m a n et al. (1992). Briefly the uteri from the rats were dissected out. A f t e r each s t i m u l a t i o n the i n c u b a t i o n m e d i u m c o n t a i n i n g the n e u r o h y p o p h y s i a l h o r m o n e s was led over the o r g a n before the collection for r a d i o i m m u n o a s s a y a n d c o n t r a c t i o n s of the u t e r i n e muscle were r e c o r d e d isotonically (Grass FT03 t r a n s d u c e r , Grass I n s t u m e n t s , Quincy, MA, USA).
Table 1 Absolute amounts of oxytocin and vasopressin released from isolated rat neurointermediate lobes after electrical stimulation Stimulation parameters width/frequency
3. Results 3.1 Basic parameters of hormone release T h e s p o n t a n e o u s release of oxytocin a n d vasopressin from the n e u r o i n t e r m e d i a t e lobes as m e a s u r e d
S I + S.E.M. (ng) Oxytocin
0.2 ms/6.5 t tz (1.2 ms/13 Hz 0.2 ms/30 Hz
(1.09+ 0.(12 I. 12+ 0.20 5.20+_0.71
2.0 ms/6.5 Hz 2.0 ms/13 Hz 2.0 ms/3(I Hz
0.44 + 0.02 2.57 + 0.26 13.9 + 1.4
Vasopressin (1.12+_0.02 2.32 + 0.37 4.05 +_0.47 0.94 + 0.05 4.49 ± 0.31 8.55_+0.72
All combinations of two pulse widths ((I.2 and 2.0 ms) and three stimulation frequencies (6.5, 13 and 30 Hz) were studied. The results are expressed as the amount of hormone released during the first of three stimulations (S K) in 2 ml of incubation medium when stimulated for 2.5 rain by trains of rectangular pulses ((I.8 V, for 10 s with and interval of 10 s). Each value is the mean+S.E.M, of 8 126 experiments. in a 10 min period prior to the electrical s t i m u l a t i o n s S~ a n d S 2 was always below 75 pg a n d not affected by the p r e s e n c e of (S)-( + )-202-791, (R)-( - )-202-791 or ( - ) - n i t r e n d i p i n e in c o n c e n t r a t i o n s up to 5 txM. Phasic, s u b m a x i m a l s t i m u l a t i o n of the pituitary stalk led to an evoked release of the n e u r o h y p o p h y s i a l horm o n e s that was strongly d e p e n d e n t on the choice of the p a t t e r n of electrical s t i m u l a t i o n (Table 1). T h e a m o u n t of h o r m o n e released per pulse was facilitated as a result of increasing frequencies. Vasopressin secretion was maximized a r o u n d 13 Hz, while oxytocin secretion was facilitated in the whole f r e q u e n c y range in q u e s t i o n ( 6 . 5 - 3 0 Hz) (Fig. 1). T h e choice of pulse
Hormone release (pg/pulse)
!
5i
2. 6. Quantitation of the results and statistical analysis T h e results were q u a n t i f i e d as the ratio (82/SI, $ 3 / S l) of h o r m o n e released d u r i n g the second and the third s t i m u l a t i o n to that of the first a n d p r e s e n t e d as m e a n s _+ S.E.M. V a l u e s were c o m p a r e d using the M a n n - W h i t n e y U-test.
159
/
I 1
0
4
0
T
5
•
'
10 15 20 25 Stimulation frequency (Hz)
"
30
35
Fig. 1. Frequency-dependent facilitation of electrically evoked release of oxytocin (o, e) and vasopressin (El, II) from isolated rat neurointermediate lobes. All combinations of two pulse widths (0.2 ms: O, D; 2.0 ms: *, II) and three stimulation frequencies (6.5, 13 and 30 Hz) were studied. The results are expressed as the amount of hormone released per pulse during the first of three stimulations (S I) when stimulated for 2.5 rain by trains of rectangular pulses (0.8 V, for 10 s with and interval of 10 s). Each value is the mean +S.E.M. of 8-126 experiments.
A. Jor~gensen et al. / European Journal of Pharmacolog.3, 259 11994) 157-1,63
160
Hormone release ($2/S1)
width was without significance in regard to these differences in the pattern of oxytocin and vasopressin release, but the absolute amount of hormone released per pulse was significantly higher when stimulating with 2 ms compared to 0.2 ms. The release of oxytocin and vasopressin (as measured by S2/S j and $3/S t) was constant during three subsequent control stimulations with an interval of 80 min and independent of stimulation parameters. Only at the combination 2 ms and 30 Hz a detectable change from ($2/S I) to ($3/S I) was found with a decrease in release of oxytocin of 20% and in release of vasopressin of 15%.
1.4
1.2 -
1.0
]
0,8
o
3.2. The influence of (S)-(+)-202-791 and ( R ) - ( - ) 202-791 on the hormone release at 6.5, 13 and 30 Hz The dihydropyridine agonist (S)-(+)-202-791 1 /.tM potentiated the electrically evoked release of both oxy-
O.Ol
Ol (s)-(+)-£o2-s9t
1
lO (~M)
Fig. 3. The effect of (S)-(+)-202-791 on the evoked releasc of oxytocin (e) and vasopressin ( • ) at a pulse width of 2 ms and a frequency of 6.5 Hz. iS)-( + )-202-791 was added from 40 min before and during the second stimulation. Results are expressed as the ratio ( $ 2 / S I) as explained in legends to Fig. 2 and in Materials and methods. Values are means+S.E.M, of h 41 experiments.* P < (I.05, ** P < 0.111.
Oxytocin release (S2/S1)
t.5
A
tocin and vasopressin compared to the relevant control by 22 and 28%, respectively, using the stimulation frequency 6.5 Hz and pulse width 2 ms. No significant effects of the agonist were seen at 13 or 30 Hz (Fig. 2A,B). The dihydropyridine antagonist ( R ) - ( - )-202-791 1 # M inhibited the release of oxytocin at 13 Hz and 2 ms. The antagonist did not influence the release of
i
0.5
•
o
Hormone release ($2/$1) 1.5
Vasopressin release ($2/$1) **
1.4
1.2
~
7 '
/2"
1
/ /,,'/
++ +++
++
, ./,
0.8 0.6 0.4
0.5
0.2
0
i 6.5 Hz
i 13 Hz
30 Hz
Fig. 2. The frequency dependence of the effects of the dihydropyridine agonist (S)-(+)-202-791 1 /xM (hatched bars) and the antagonist (R)-( - )-202-791 1 /xM (open bars) on the evoked release of oxytocin (A) and vasopressin (B) compared to the relevant control (closed bars). Each neurointermediate lobe was stimulated three times with an interval of 80 min for 2.5 min (0.8 V, for 10 s with an interval of 10 s) at a pulse width of 2 ms. The ligands were added from 40 min before and during the second stimulation. Results are expressed as the ratio (S 2 / S t) of the amount of hormone released by the second stimulation (S 2) in the presence or absence of test substance to that released by the first stimulation (S l) in control medium. Values are means_+S.E.M, of 4-41 experiments. Significance level (Mann-Whitney U-test) compared to control, ** P < I)./)l.
0 Oxytocin
Vasopressin
Fig. 4. Effects of the dihydropyridine antagonists (R)-( - )-202-791 1 /~M and (-)-nitrendipine 1 ,aM on the (S)-(+)-202-791 1 #M-induced facilitation of hormone release from electrically stimulated neurointermediate lobes at a pulse width of 2 ms and a frequency of 6.5 tlz. The antagonist was added from 55 mm and the agonist from 4/1 min before and during the second stimulation• The results are expressed as the ratio ( $ 2 / S I) (see legend to Fig. 2 and Materials and methods). Control (closed bars), (S)-(+)-202-791 alone (open bars), (S)-(+)-202-791+(R)-(-I-21)2 791 (hatched bars) and (S)( + ) - 2 0 2 - 7 9 1 + ( - ) - n i t r e n d i p i n e (cross-hatched bars). Values arc means+S.E.M, of 9-41 experiments. Significantly different from control (** P
A. Jorgensen et al. / European Journal o1"Pharmacology 259 (1994) 157-163 Table 2 Effects of dihydropyridine antagonists on the electrically evoked release of oxytocin and vasopressin from rat neurointermediate lobes Antagonist
Conc.
8 2 / S 1 ± S.E.M. (n)
p.M
Oxytocin
Vasopressin
0.96 + 0.05 (37)
0.97 _+0.04 (41)
Control (R)-(-)-2(12-791
1 5
0.92+_0.09 (7) 0.95_+0.11 (6)
0.99+0.08 (6) 1.06_+0.16 (6)
(
1 5
1.28_+0.20 (5) 0.97+_0.09 (6)
0.89_+0.08 (5) 1.02+0.10 (6)
)-nitrendipine
The results from (n) experiments are expressed as the ratio (S 2 / S t) ±S.E.M. of hormone release during the second stimulation in the presence of test substance to the release during the first stimulation in control medium at a frequency of 6.5 Hz and a pulse width of 2 ms.
vasopressin at these conditions or the release of oxytocin or vasopressin at any of the other frequencies tested (Fig. 2A,B). No effects of either the agonist or the antagonist were found stimulating with a pulse width of 0.2 ms in combination with any of the three frequencies. However, it must be emphasized that the absolute amount of hormone released was small, and that the combination 6.5 Hz and 0.2 ms did not induce hormone output significantly different from the spontaneous release.
3.3. The influence of (R)-(-)-202-791 and ( - ) nitrendipine on the (S)-(+)-202-791 induced hormone release ( S ) - ( + ) - 2 0 2 - 7 9 1 concentration dependently potentiated the release of neurohypophysial hormones at 2 ms and 6.5 Hz (Fig. 3). Maximal response was seen after a concentration of 1 p.M, producing a biphasic dose-response curve. The dihydropyridine antagonist ( - ) - n i t r e n d i p i n e 1 /xM abolished the agonist-induced facilitation of both oxytocin and vasopressin, while ( R ) - ( - )-202-791 1 /xM only abolished the facilitated effect on the release of vasopressin (Fig. 4). None of the antagonists influenced the hormone release when tested alone at the concentrations 1 and 5 / x M (Table 2).
4. Discussion
In the present study it has been shown that the effects of the dihydropyridine agonist, (S)-( + )-202-791 and the dihydropyridine antagonist, ( R ) - ( - ) - 2 0 2 - 7 9 1 on the electrically evoked release of oxytocin and vasopressin from the isolated, rat neurointermediate lobe are dependent on the stimulation conditions. Previous attempts to demonstrate pharmacologically a role for
161
L-type Ca 2+ channels in electrically stimulated hormone release from neurohypophysis did not provide a clear evidence. Dayanithi et al. (1988) found about 20% inhibition of electrically evoked vasopressin release using a high (5/xM) concentration of nicardipine, while no effects of dihydropyridines were found by Von Spreckelsen et al. (1990). To our knowledge, this is the first study where the influence of the dihydropyridine ligands on the electrically stimulated release of neurohypophysial hormones has been investigated as a function of the applied pulse pattern. The data obtained have allowed us to define stimulation conditions under which the release of vasopressin or oxytocin was dose dependently potentiated by ( S ) - ( + ) 202-791, and it is suggested that the influx of Ca 2+ through the L-type Ca 2+ channels at low stimulation frequency (6.5 Hz) is rate-limiting for hormone release. Small but potentiating effects of the dihydropyridine agonist were found on the release of both oxytocin and vasopressin at a stimulation frequency of 6.5 Hz. Although no significant effects of (S)-( + )-202-791 were achieved at higher stimulation frequencies, a tendency of the agonist to potentiate the release of oxytocin at 13 and 30 Hz was seen. According to Fig. 1 frequencydependent facilitation of the amount of oxytocin released per pulse was seen in the whole frequency range (6.5-30 Hz), while the secretion of vasopressin was maximal around 13 Hz. This could account for the observed tendency of the dihydropyridine agonist to potentiate the release of oxytocin at 13 and 30 Hz, as the secretion of oxytocin was not maximized at these stimulation frequencies. At a stimulation frequency of 6.5 Hz ( S ) - ( + ) - 2 0 2 791 enhanced the release of both oxytocin and vasopressin in a concentration-dependent manner, but apparently a maximal response was seen at a concentration of 1 /xM. A similar biphasic effect of the dihydropyridine agonist, ( S ) - ( - )-Bay K 8644 has been observed on the 45Ca2+ uptake into rat heart cells (Wei et al., 1989) and of the racemic Bay K 8644 on the release of amino acids from and uptake of 45Ca2+ into cerebrocortical synaptosomes (White and Bradford, 1986). In these studies this effect was explained as a tendency of the dihydropyridine agonist to behave as an antagonist in higher concentrations because of state-dependent interactions of the ligand with the Ca 2+ channel. The facilitating effect of (S)-( + )-202-791 at 6.5 Hz on the evoked release of oxytocin and vasopressin was abolished by ( - ) - n i t r e n d i p i n e and that of vasopressin stereospecifically by ( R ) - ( - ) - 2 0 2 - 7 9 1 , while the dihydropyridine antagonists failed to modify the hormone release when tested alone. An explanation for this apparent discrepancy could be a small contribution of the L-type Ca 2 + channel to the influx of Ca ~+, as also indicated by the size of the potentiating effects of the
162
A. Jorgensen el aL / European .hmrnal qf f~harmac¢)h)gv 259 (1994) 157 163
dihydropyridine agonist. Hence it was not possible to recognize suppressing effects of the antagonists alone. Sevcral explanations may account for the observed potentiation of vasopressin and oxytocin releasc by (S)-( + )-202-791. (1) A number of studies have suggested that electrical stimulation of isolated neurohypophysis or isolated neurohypophysial slices with increasing pulse frequencies results in action potential broadening (Gainer ct al., 1986; Bourque, 1990; Jackson et al., 1991). This broadening of action potentials has been proposcd to result from K+-current inactivation (Bondy et al., 1987). A consequence of inactivation of K + currents would be a prolonged period of depolarization, allowing an increased ( a influx through voltage-gatcd ( a - channels and hence enhanced secretion. In light of this mechanism, a dihydropyridine-mediated potentiation of hormone release would be most likely to occur at low stimulation frequencies, where thc K*-current inactivation is less prominent. This is in agreement with our results. (2) The potentiation of hormone release by (S)-( + )202-791 at 6.5, but not at higher stimulation frequencies could also reflect the presence of frequency-dependent facilitation due to a mechanism independent of the action potential broadening as demonstrated by Jackson et al. (1991). In this work, enhanced intracellular Ca -'~ with increasing frequencies was ew~ked by constant duration pulses in voltage-clamped neurohypophysial nerve terminals. This increase in Ca -'+ level could be accounted for by Ca ?+ channel facilitation due to a change in channel-gating behaviour. From single channel patch-clamp studies on neurons and cardiac cells, a three mode gating behaviour of the L-type Ca =+ channel has been proposed. Mode 1 is characterized by brief openings, mode 0 by no openings and mode 2 by long lasting openings and very brief closings. Mode 2 is according to this theory fawmred by the presence of dihydropyridine agonists, as well as for instance strong depolarizations and trains of rapidly delivered action potentials, while mode 0 is promoted by dihydropyridinc antagonists (Hess et al., 1984; Nowycky el al., 1985; Pietrobon and Hess, 1990). Thus, in the present study an increased influx of Ca = ' due to the action of (S)-(+)-202-791 at low stimulation frequency could reflect an agonist-promoted L-channel shift from mode I to mode 2. On the other hand, higher stimulation frequencies would be sufficient by itself to cause the mode 1 to mode 2 shift, thus masking the effect of the drug. (3) Additionally, the frequency dependent stimulation of (S)-( + )-202-791 could reflect a frequency-dependent change in the contribution of different types of Ca :+ channels to Ca =+ influx upon electrical stimulation. In addition to the dihydropyridine-sensitivc. Ltype Ca :~ channels, the neurohypophysial nerve termi-
nals have been suggested to contain N-type channels (Dayanithi et al., 1988; kemos and Nowycky, 1989; Von Spreckelsen et al., 1990; Nowycky, 1991; Wang et al., 1992). Therefore, it is possible that an enhanced recruitement of these channels at increased stimulation frequencies renders the contribution of L-channels to Ca :~ influx, and thus any pharmacoh)gical cffects upon these channels, less significant. In summary, these results dcmonstratc that the entry of Ca e- by the dihydropyridine-sensitivc L-type Ca -'~ channel under certain conditions plays a rolc in the release of oxytocin and vasopressin from isolated, rat neurointermediatc lobes when stimulated electrically.
Acknowledgements Ms. D. Christoflersen and Ms. G. Slevcn aic gratefully acknowledged for their excellent technical assistance. This work has bccn supported by the Danish Goverment Biotechnok)gy Program.
References Bicknell, R.J., 1988, Optimizing release from pepfide hormone sccr¢ tope nerve terminals, J. Exp. Biol. 139, 51. Bondy, C.A., H. Gainer and J.T. Russell. 1987, Effects of stimulus frequency and potassium channel blockade on tile secretion of vasopressin ;Jnd oxytocin from the ncurohypophysis. Neuroendocrinology 46, 258. Bourque, C.W., 199(/. lntratermina] recordings from the ral neurohy pophysis in vitro, J. Physiol. London 421,247. Brcthes, D., G. Dayanithi, 1., Letcllier and ,I.J. Nordmann. 1987. Dcpolarization-hldnced Ca 2" increase in isolated l)ctnosccl-elory nerve ternfinals measured with fura-2, Proc. Natl. Acad. Sci. USA 84. 1439. Cazalis, M., G. Dayanithi and .1.,1. Nordmann, 1987, 11ormonc re lease fronl isolated nm-vc endings of the rat neurohypophys[s, ,I. Physiol. l,ondon 390, 55. ('hristensen, ,I.D. and B. Fjalland, 1982, l,ack ol effect ol opiates on release of wlsopressin from isolated r'lt neurohypophysis, Acta Pharmacol. Toxicol. 50, 113. Dayanith[, G., N. Martin Moutot, S. Barlier, D.A. (olin. M. Krclz Zaeptel, F. Couraud and ,l.J. Nordmann, I¢)N8, Thc calcium channel antagonist w-conotoxin inhibits secretion from peptidcr gic nervc terminals, Biochcm. Biophys, Res. (kmmmn. 156. 255. Fatatis, A., L. Holtzclaw, K. Payza and J.T. Russel, 1~,~92, Secretion fronl fat neurohypophysial nerve terminals /neurosccrctosomcs) rapidly inactivates despite continued clevation ol intraccllular Ca -~, Brain Rcs. 574, 33. Fjalland, B., J.D. Christcnsen and 5. Grcll. 11}87, (;ABA receptor stimulation increases thc release of vasopressin and oxytocm in vitro, European. J. Pharmacol. 142, 155. Gainer, H., S.A. Wolfc, A.L, Obaid and B.M. Salzbcrg. 1986. Aclion potentials and frequency-dependent secretion in the mouse ncurohypophysis, Neuroendocrinology 43,557. Hess, P., J.B. kansman and R.W. Tsien, 1984, Different modes of Ca channel gating behaviour fawmred by dihydropyridine ('a agonists and antagonists, Nature 311, 538. Hosey. M.M. and M. Lazdunski, 1988, Calciunl channels: nlolectilaI pharmacology, struc'mre and regulation, ,1. Membrane Biol. 104, Sl.
A. J¢~rgensen et al. /European Journal of Pharmacologn/ 259 (1994) 157 1(i3 Jackson, M.B., A. Konnerth and G.J. Augustine, 1991, Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals, Proc. Natl. Acad. Sci. USA 88, 380. Jt~rgensen, A., B. Fjalland and J.D. Christensen, 1993, Influence of Ca 2+ channel agonists and antagonists on stimulated release of neurohypophysial hormones, Ann. NY Acad. Sci. 689, 593 Kato, M., C. C h a p m a n and R.J. Bicknell, 1992, Activation of K-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca 2~ in secretory nerve terminals of the neurohypophysis, Brain Res. 574, 138. Lemos, J.R. and M.C. Nowycky, 1989, Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals, Neuron 2, 1419. Lim, N.F., M.C. Nowycky and R.J. Bookman, 199(l, Direct measurement of exoeytosis and calcium currents in single vertebrate nerve terminals, Nature 344, 449. Lindau, M., E.L. Stuenkel and J.J. N o r d m a n n , 1992, Depolarization, intracellular calcium and exocytosis in single vertebrate nerve endings, Biophys. J. 61, 19. Nordmann, J.J., G. Dayanithi and J.R. Lemos, 1987, Isolated neurosecretory nerve endings as a tool for studying the mechanism of stimulus-secretion coupling, Biosci. Rep. 7, 411. No'~,ycky, M.C., 1991, Two high lreshold Ca 2~ channels contribute Ca 2 ~ for depolarization-secretion coupling in the m a m m a l i a n neurohypophysis, Ann. NY Acad. Sci. 635, 45. Nowyck-y', M.C., A.P. Fox and R.W. Tsien, 1985, Long opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644, Proc. Natl. Acad. Sci. U S A 82. 2178. Obaid, A.L., R. Flores and B.M. Salzberg, 1989, Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to w-eonotocin and relatively insensitive to dihydropyridines, J. Gen. Physiol. 93, 715. Pietrobon, D. and P. Hess, 1990, Novel mechanism of voltage-dependent gating in L-type calcium channels, Nature 346, 651. Scott, R.H., H.A. Pearson and A.C. Dolphin, 1991, Aspects of
163
vertebrate neuronal voltage-activated calcium currents and their regulation, Prog. Neurobiol. 36, 485. Shibuki, K., 1990, Activation of neurohypophysial wlsopressin release by Ca 2+ influx and intracellular Ca 2- accumulation in the rat, J. Physiol. London 422, 321. Stuenkel, E.L., 199(I, Effects of m e m b r a n e depolarization on intracellular calcium in single nerve terminals, Brain Rcs. 529, 96. Stuenkel, E.L., 1991, Relationship between m e m b r a n e depolarization and intracellular free calcium in individual nerve terminals from the nerurohypophysis, Ann. NY Acad. Sci. 635, 441. Treiman, M., K. Lollikc, J. Baldvinsdottir, A. J~rgensen, B. Fjalland and J.D. Christensen, 1992, Use of channel ligands to probe role of voltage-sensitive calcium ion channels in ncuropeptide release, in: Methods in Neurosciences, ed. P.M. Corm (Academic Press) Vol. 8, p. 86. Triggle, D.J. and D. Rampe, 1989, 1,4-Dihydropyridine activators and antagonists: structural and functional distinctions. Trends Pharmacol. Sci. 10, 507. Tsien, R.W., 1990, Calcium channels, stores and oscillations, Annu. Rev. Cell Biol. 6, 715. Tsien, R.W., D. Lipscombe, D.V. Madison, K.R. Bley and A.P. Fox, 1988, Multiple types of neuronal calcium channels and their selective modulation, Trends Neurosci. 11,431. Von Spreckelsen, S., K. Lollike and M. Treiman, 1990, Ca 2+ and vasopressin release in isolated rat neurohypophysis: differential effects of lk)ur classes of Ca 2+ channel ligands, Brain Res. 514, 68. Wang, X., S.N. Treistman and J.R. Lemos, 1992, Two types of high-threshold calcium currents inhibited by ¢o-conotoxin in nerve terminals of rat neurohypophysis, J. Physiol. London 445, 181. We(, X.Y., A. Rutlegde and D.J. Triggle, 1989, Voltage-dependent binding of 1,4-dihydropyridine Ca 2. channel antagonists and activators in cultured neonatal rat ventricular myocytes, Mol. Pharmacol. 35, 541. White, E.J., and H.F. Bradford, 1986, E n h a n c e m e n t of depolarization-induced synaptosomal calcium uptake and neurotransmitter release by Bay K8644, Biochem. Pharmacol. 35, 2193.
European Journal of Pharmacology 259 (1994) 157-163
Dihydropyridine ligands influence the evoked release of oxytocin and vasopressin dependent on stimulation conditions Annette J0rgensen a,,, Bjarne Fjalland a, Jens D. Christensen a, Marek Treiman b Department of Biological Sciences, The Royal Danish School of Pharmacy, UniL'ersitetsparken 2, DK-2100 Copenhagen O, Denmark, b Department of Medical Physiology, The Biotechnology Center for Signal Peptide Research, The Panum Institute, Blegdamsl,ej 3c, DK-2200 Copenhagen N, Denmark Received 22 March 1994; accepted 5 April 1994
Abstract
The effects of dihydropyridine ligands on the electrically evoked release of neurohypophysial hormones from isolated, rat neurointermediate lobes were investigated as a function of all combinations of two pulse widths (0.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz). The dihydropyridine agonist (S)-(+)-202-791 potentiated concentration dependently the release of both oxytocin and vasopressin at a pulse width of 2 ms and a frequency of 6.5 Hz. This effect of (S)-(+)-202-791 was abolished by the antagonist (-)-nitrendipine and stereospecifically by (R)-( )-202-791 (only vasopressin). The antagonist (R)-(-)-202-791 alone inhibited the release of oxytocin at 13 Hz and 2 ms. The results presented show that the effects of the dihydropyridine ligands are dependent on the stimulation conditions, and thus demonstrate that the entry of Ca 2~ through the dihydropyridine sensitive L-type Ca 2+ channel is associated with electrically evoked release of neurohypophysial hormones under certain conditions. Key words: Oxytocin; Vasopressin; Ca 2+ channel, L-type; Dihydropyridine; Neurointermediate lobe; (Electrical stimulation)
1. Introduction
The release of hormones from the neurohypophysis is initiated by action potentials propagated from the magnocellular cell bodies. Depolarization of the nerve terminals promotes entry of Ca 2+ from the extracellular environment (Brethes et al., 1987; Shibuki, 1990; Stuenkel, 1990; Fatatis et al., 1992), through voltageactivated Ca 2+ channels (Cazalis et al., 1987; Dayanithi et al., 1988; Obaid et al., 1989; Von Spreckelsen et al., 1990; Stuenkel, 1991; Kato et al., 1992). This rise in intraterminal free Ca 2+ triggers exocytose from neurosecretory nerve endings (Lim et al., 1990; Lindau et al., 1992). Evidence is available describing multiple types of voltage-activated Ca 2+ channels which differ in molecular, electrophysiological and pharmacological properties. The major classes of voltage-activated Ca 2+ channels are known as T, N, L and P (Tsien et al., 1988; Tsien, 1990; Scott et al., 1991).
* Corresponding author. Tel. + 45.35.37(/850, fax + 45.35.374457. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0[) 1 4 - 2 9 9 9 ( 9 4 ) 0 0 2 1 3 - Q
In isolated terminals from the neurohypophysis two types of high voltage-activated Ca 2+ channels have been characterized using patch-clamp techniques. One of these corresponds to the dihydropyridine-sensitive L-type channel, while the other is a dihydropyridineinsensitive channel of the N-type family (Lemos and Nowycky, 1989, 1991). The peptide toxin w-conotoxin G V I A has been shown to block both N- and L-type Ca 2+ currents in neurohypophysial nerve terminals (Wang et al., 1992) and to inhibit high K +- as well as electrically evoked release of vasopressin from isolated neurohypophysis (Dayanithi et al., 1988; Von Spreckelsen et al., 1990). The influence of the dihydropyridines seems to be more complex. During high K+-induced depolarizations the dihydropyridine agonist Bay K 8644 [(R,S)1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-trifluoromethylphenyl)-3-pyridinecarboxylic acid methyl ester] was shown to potentiate the release of vasopressin (Cazalis et al., 1987), while the dihydropyridine antagonists nicardipine, nitrendipine and nimodipine inhibited the peptide release (Cazalis et al., 1987; Dayanithi et al., 1988; Fatatis et al., 1992; Kato et al., 1992). On the
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el. Jor#ensen el al. / European Journal o/" IJharmacolo,w 25q (19941 157-103
other hand when isolated neurohypophysis were stimulated electrically using physiological patterns either no effects (Von Spreckelsen ct aI., 1990) or only very small effects (Dayanithi et al., 19881 of the dihydropyridine ligands were found. In the present work it has been studied whether the apparently lacking effects of the dihydropyridinc ligands on the electrically evoked release of neurohypophysial hormones depends on the choice of stimulation conditions. It is known that changes in intracellular Ca ~+ concentration and release of neurohypophysial hormones from isolated nerve terminals are a sensitive function of the applied frequency and pattern of electrical activity (Nordmann et al., 1987, Bicknell, 1988; Jackson et al., 1991). Increasing frequencies thus result in facilitation of the intracellular [Ca ~+] (Jackson et al., 19911 and the amount of peptide released per pulse (Nordmann ct a[., 1987). Therefore it was of interest to study whether different effects of dihydropyridine [igands could be found when changing the stimulation parameters. The effects of a pair of dihydropyridine stereoisomers, (S)-(+)-202-791 and (R)-( )-202-791 on the release of oxytocin and vasopressin from electrically stimulated, isolated, rat neurointermediate lobes have been investigated as function of combinations of different pulse widths and frequencies, iS)-( + )-202-791 and (R)-( - )-202-791 are known to exert opposite actions on L-type Ca 2~ channels, where (S)-(+)-202-791 acts as an agonist and (R)-( )-202-791 as an antagonist (Hosey and Lazdunski, 1988; Triggle and Rampe, 1989). Some of the data reported here have appeared in a preliminary form (J0rgensen et al., 1993).
2. Materials and methods
2.1. Sources of drugs" The enantiomers of 202-791 [1,4-dihydro-2,6-dimethyl-5-nitro-4-(2,1,3-benzoxadiazol-4-yl)-3-pyridinecarboxylic acid 2-propanester] and ( - ) - n i t r e n d i p i n e [( )- 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid ethyl methyl ester] were obtained as a generous gift from Sandoz (Copenhagen, Denmark) and Bayer (Leverkusen, Germany), respectively.
2.2. Preparation of isolated neurointermediate lobes Female Spraque-Dawley rats (18(I-250 g) in spontaneous oestrus were used and the neurointermediate lobe isolated following a procedure described earlier (Fjalland et al., 1987; Treiman et al., 1992). Briefly the pituitary stalk was tied to the cathode of a platinum wire electrode and immersed in 2 ml of a 37°C warm, carbogen bubbled medium, p H 7.4, of the following
composition (raM): NaCI 115, KCI 6.0, N a e H P O a (1.8, CaCI, 1.5, MgCI e 0.9, NaHCO~ 22, D-glucose 10. To allow equilibration of basal hormone release the preparation was preincubated for 1 h, during which time the medium was changed every 15 rain.
2.3. Stimulation protocol Electrical stimuli were applied between the clcctrode carrying the neurohypophysis and a platinum electrode dipped into the incubation medium. The gland was raised to the surface of the incubation medium and the supply of gas interrupted during stimulation. The neurointermediate lobes were stimulated three times (S I, S~ and S~) with an interval of 8(t rain by trains of rectangular pulses (0.8 V, for 10 s with an interval of 10 s for a total of 2.5 rain) generated by a HSE stimulator (type 215/I, Hugo Sachs Elcctronik, Freiburg, Germany). All combinations of two pulse widths ((/.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz) were studied. As the stimulation period was constant at 2.5 rain, the number of pulses were only dependent on the stimulation frequency. The frequencies 6.5, 13 and 30 Hz thus resulted in the delivery of 520, 1040 and 2400 pulses per stimulatkm, respectively. The interval between two stimulations was 80 rain. The incubation medium was changed 55, 40, 25, 10 and 0 rain before the stimulation. Drugs were added from 4(1 rain before and during the second stimulation. When testing two drugs in combination, the antagonist was added 15 rain before the agonist corresponding to 55 min before the stimulation. The incubation medium was collected 10 min after each stimulation and kept frozen at - 2 0 ° C untill assayed. The amount of oxytocin and vasopressin was analysed by radioimmunoassay. The basal hormone release was examined in 10 rain periods preceding the electrical stimulations S~ and S~. The dihydropyridine ligands were diluted in the incubation medium from stock solutions (1 m g / m l ) in ethanol. The final ethanol concentration did not exceed 1.8 m g / m l , and it was tested that there was no difference in the release of neurohypophysial hormones in the presence or absence of this ethanol concentration. All dihydropyridines as well as incubation chambers and other parts of equipment were protected from light during handling and experiments.
2.4. Determination of hormone release The amount of vasopressin in the incubation medium was measured by radioimmunoassay as previously described (Christensen and Fjalland, 19821. Briefly, to 0.2 ml of standard (synthetic arginine vasopressin, Ferring AB, Malm6, Sweden) or unknown, 0.1 ml of antibody
A. Jorgensen et al. / European Journal of Pharmacology 259 (19941 157-163 (final dilution 1 : 4 5 0 0 0 ) a n d 0.05 ml [1251]arginine vasopressin ( D u P o n t (UK), Stevenage, U K ) were added. T h e i n c u b a t i o n time was 48 h at 4°C. A m m o n i u m s u l p h a t e was used for s e p a r a t i o n of b o u n d a n d free [12SI]arginine vasopressin. T h e a n t i s e r u m did not disc r i m i n a t e b e t w e e n lysine a n d a r g i n i n e vasopressin a n d the m i n i m u m d e t e c t i o n limit was 10 p g / m l . Serial diluted test samples had d i l u t i o n curves parallel to the s t a n d a r d curve. Cross-reactivity of a n t i s e r u m to synthetic oxytocin was 0.01%. N o n e of the c o m p o u n d s used in this study i n t e r f e r e d with the assay. T h e within-assay a n d the b e t w e e n - a s s a y coefficients of variation were 4.2 a n d 7.8%, respectively. T h e oxytocin c o n t e n t was d e t e r m i n e d as described by F j a l l a n d et al. (19871. T o 0.1 ml of a n t i b o d y (final dilution 1 : 7 6 8 0 0 ) a n d 0.05 ml [125I]oxytocin ( D u P o n t (UK), Stevenage, U K ) were a d d e d 0.2 ml s t a n d a r d (synthetic oxytocin, F e r r i n g AB, Malta6, S w e d e n ) or u n k n o w n . T h e i n c u b a t i o n time was 72 h at 4°C. A m m o n i u m s u l p h a t e was used for s e p a r a t i o n of b o u n d a n d free [12Si]oxytocin. Cross-reactivity of a n t i s e r u m to lysine a n d a r g i n i n e vasopressin was 0.005%. T h e minimal d e t e c t a b l e level was 8 p g / m l . Serial diluted test samples were parallel to the s t a n d a r d curve. N o n e of the c o m p o u n d s used in the study i n t e r f e r e d with the assay. T h e within-assay a n d b e t w e e n - a s s a y coefficients of variation were 6.1 a n d 5.4%, respectively.
2.5. Bioassay T o o b t a i n a n i m m e d i a t e p r e l i m i n a r y m e a s u r e of the s t i m u l a t i o n a s e m i q u a n t i t a t i v e bioassay was used as described by F j a l l a n d et al. (1987) a n d T r e i m a n et al. (1992). Briefly the uteri from the rats were dissected out. A f t e r each s t i m u l a t i o n the i n c u b a t i o n m e d i u m c o n t a i n i n g the n e u r o h y p o p h y s i a l h o r m o n e s was led over the o r g a n before the collection for r a d i o i m m u n o a s s a y a n d c o n t r a c t i o n s of the u t e r i n e muscle were r e c o r d e d isotonically (Grass FT03 t r a n s d u c e r , Grass I n s t u m e n t s , Quincy, MA, USA).
Table 1 Absolute amounts of oxytocin and vasopressin released from isolated rat neurointermediate lobes after electrical stimulation Stimulation parameters width/frequency
3. Results 3.1 Basic parameters of hormone release T h e s p o n t a n e o u s release of oxytocin a n d vasopressin from the n e u r o i n t e r m e d i a t e lobes as m e a s u r e d
S I + S.E.M. (ng) Oxytocin
0.2 ms/6.5 t tz (1.2 ms/13 Hz 0.2 ms/30 Hz
(1.09+ 0.(12 I. 12+ 0.20 5.20+_0.71
2.0 ms/6.5 Hz 2.0 ms/13 Hz 2.0 ms/3(I Hz
0.44 + 0.02 2.57 + 0.26 13.9 + 1.4
Vasopressin (1.12+_0.02 2.32 + 0.37 4.05 +_0.47 0.94 + 0.05 4.49 ± 0.31 8.55_+0.72
All combinations of two pulse widths ((I.2 and 2.0 ms) and three stimulation frequencies (6.5, 13 and 30 Hz) were studied. The results are expressed as the amount of hormone released during the first of three stimulations (S K) in 2 ml of incubation medium when stimulated for 2.5 rain by trains of rectangular pulses ((I.8 V, for 10 s with and interval of 10 s). Each value is the mean+S.E.M, of 8 126 experiments. in a 10 min period prior to the electrical s t i m u l a t i o n s S~ a n d S 2 was always below 75 pg a n d not affected by the p r e s e n c e of (S)-( + )-202-791, (R)-( - )-202-791 or ( - ) - n i t r e n d i p i n e in c o n c e n t r a t i o n s up to 5 txM. Phasic, s u b m a x i m a l s t i m u l a t i o n of the pituitary stalk led to an evoked release of the n e u r o h y p o p h y s i a l horm o n e s that was strongly d e p e n d e n t on the choice of the p a t t e r n of electrical s t i m u l a t i o n (Table 1). T h e a m o u n t of h o r m o n e released per pulse was facilitated as a result of increasing frequencies. Vasopressin secretion was maximized a r o u n d 13 Hz, while oxytocin secretion was facilitated in the whole f r e q u e n c y range in q u e s t i o n ( 6 . 5 - 3 0 Hz) (Fig. 1). T h e choice of pulse
Hormone release (pg/pulse)
!
5i
2. 6. Quantitation of the results and statistical analysis T h e results were q u a n t i f i e d as the ratio (82/SI, $ 3 / S l) of h o r m o n e released d u r i n g the second and the third s t i m u l a t i o n to that of the first a n d p r e s e n t e d as m e a n s _+ S.E.M. V a l u e s were c o m p a r e d using the M a n n - W h i t n e y U-test.
159
/
I 1
0
4
0
T
5
•
'
10 15 20 25 Stimulation frequency (Hz)
"
30
35
Fig. 1. Frequency-dependent facilitation of electrically evoked release of oxytocin (o, e) and vasopressin (El, II) from isolated rat neurointermediate lobes. All combinations of two pulse widths (0.2 ms: O, D; 2.0 ms: *, II) and three stimulation frequencies (6.5, 13 and 30 Hz) were studied. The results are expressed as the amount of hormone released per pulse during the first of three stimulations (S I) when stimulated for 2.5 rain by trains of rectangular pulses (0.8 V, for 10 s with and interval of 10 s). Each value is the mean +S.E.M. of 8-126 experiments.
A. Jor~gensen et al. / European Journal of Pharmacolog.3, 259 11994) 157-1,63
160
Hormone release ($2/S1)
width was without significance in regard to these differences in the pattern of oxytocin and vasopressin release, but the absolute amount of hormone released per pulse was significantly higher when stimulating with 2 ms compared to 0.2 ms. The release of oxytocin and vasopressin (as measured by S2/S j and $3/S t) was constant during three subsequent control stimulations with an interval of 80 min and independent of stimulation parameters. Only at the combination 2 ms and 30 Hz a detectable change from ($2/S I) to ($3/S I) was found with a decrease in release of oxytocin of 20% and in release of vasopressin of 15%.
1.4
1.2 -
1.0
]
0,8
o
3.2. The influence of (S)-(+)-202-791 and ( R ) - ( - ) 202-791 on the hormone release at 6.5, 13 and 30 Hz The dihydropyridine agonist (S)-(+)-202-791 1 /.tM potentiated the electrically evoked release of both oxy-
O.Ol
Ol (s)-(+)-£o2-s9t
1
lO (~M)
Fig. 3. The effect of (S)-(+)-202-791 on the evoked releasc of oxytocin (e) and vasopressin ( • ) at a pulse width of 2 ms and a frequency of 6.5 Hz. iS)-( + )-202-791 was added from 40 min before and during the second stimulation. Results are expressed as the ratio ( $ 2 / S I) as explained in legends to Fig. 2 and in Materials and methods. Values are means+S.E.M, of h 41 experiments.* P < (I.05, ** P < 0.111.
Oxytocin release (S2/S1)
t.5
A
tocin and vasopressin compared to the relevant control by 22 and 28%, respectively, using the stimulation frequency 6.5 Hz and pulse width 2 ms. No significant effects of the agonist were seen at 13 or 30 Hz (Fig. 2A,B). The dihydropyridine antagonist ( R ) - ( - )-202-791 1 # M inhibited the release of oxytocin at 13 Hz and 2 ms. The antagonist did not influence the release of
i
0.5
•
o
Hormone release ($2/$1) 1.5
Vasopressin release ($2/$1) **
1.4
1.2
~
7 '
/2"
1
/ /,,'/
++ +++
++
, ./,
0.8 0.6 0.4
0.5
0.2
0
i 6.5 Hz
i 13 Hz
30 Hz
Fig. 2. The frequency dependence of the effects of the dihydropyridine agonist (S)-(+)-202-791 1 /xM (hatched bars) and the antagonist (R)-( - )-202-791 1 /xM (open bars) on the evoked release of oxytocin (A) and vasopressin (B) compared to the relevant control (closed bars). Each neurointermediate lobe was stimulated three times with an interval of 80 min for 2.5 min (0.8 V, for 10 s with an interval of 10 s) at a pulse width of 2 ms. The ligands were added from 40 min before and during the second stimulation. Results are expressed as the ratio (S 2 / S t) of the amount of hormone released by the second stimulation (S 2) in the presence or absence of test substance to that released by the first stimulation (S l) in control medium. Values are means_+S.E.M, of 4-41 experiments. Significance level (Mann-Whitney U-test) compared to control, ** P < I)./)l.
0 Oxytocin
Vasopressin
Fig. 4. Effects of the dihydropyridine antagonists (R)-( - )-202-791 1 /~M and (-)-nitrendipine 1 ,aM on the (S)-(+)-202-791 1 #M-induced facilitation of hormone release from electrically stimulated neurointermediate lobes at a pulse width of 2 ms and a frequency of 6.5 tlz. The antagonist was added from 55 mm and the agonist from 4/1 min before and during the second stimulation• The results are expressed as the ratio ( $ 2 / S I) (see legend to Fig. 2 and Materials and methods). Control (closed bars), (S)-(+)-202-791 alone (open bars), (S)-(+)-202-791+(R)-(-I-21)2 791 (hatched bars) and (S)( + ) - 2 0 2 - 7 9 1 + ( - ) - n i t r e n d i p i n e (cross-hatched bars). Values arc means+S.E.M, of 9-41 experiments. Significantly different from control (** P
A. Jorgensen et al. / European Journal o1"Pharmacology 259 (1994) 157-163 Table 2 Effects of dihydropyridine antagonists on the electrically evoked release of oxytocin and vasopressin from rat neurointermediate lobes Antagonist
Conc.
8 2 / S 1 ± S.E.M. (n)
p.M
Oxytocin
Vasopressin
0.96 + 0.05 (37)
0.97 _+0.04 (41)
Control (R)-(-)-2(12-791
1 5
0.92+_0.09 (7) 0.95_+0.11 (6)
0.99+0.08 (6) 1.06_+0.16 (6)
(
1 5
1.28_+0.20 (5) 0.97+_0.09 (6)
0.89_+0.08 (5) 1.02+0.10 (6)
)-nitrendipine
The results from (n) experiments are expressed as the ratio (S 2 / S t) ±S.E.M. of hormone release during the second stimulation in the presence of test substance to the release during the first stimulation in control medium at a frequency of 6.5 Hz and a pulse width of 2 ms.
vasopressin at these conditions or the release of oxytocin or vasopressin at any of the other frequencies tested (Fig. 2A,B). No effects of either the agonist or the antagonist were found stimulating with a pulse width of 0.2 ms in combination with any of the three frequencies. However, it must be emphasized that the absolute amount of hormone released was small, and that the combination 6.5 Hz and 0.2 ms did not induce hormone output significantly different from the spontaneous release.
3.3. The influence of (R)-(-)-202-791 and ( - ) nitrendipine on the (S)-(+)-202-791 induced hormone release ( S ) - ( + ) - 2 0 2 - 7 9 1 concentration dependently potentiated the release of neurohypophysial hormones at 2 ms and 6.5 Hz (Fig. 3). Maximal response was seen after a concentration of 1 p.M, producing a biphasic dose-response curve. The dihydropyridine antagonist ( - ) - n i t r e n d i p i n e 1 /xM abolished the agonist-induced facilitation of both oxytocin and vasopressin, while ( R ) - ( - )-202-791 1 /xM only abolished the facilitated effect on the release of vasopressin (Fig. 4). None of the antagonists influenced the hormone release when tested alone at the concentrations 1 and 5 / x M (Table 2).
4. Discussion
In the present study it has been shown that the effects of the dihydropyridine agonist, (S)-( + )-202-791 and the dihydropyridine antagonist, ( R ) - ( - ) - 2 0 2 - 7 9 1 on the electrically evoked release of oxytocin and vasopressin from the isolated, rat neurointermediate lobe are dependent on the stimulation conditions. Previous attempts to demonstrate pharmacologically a role for
161
L-type Ca 2+ channels in electrically stimulated hormone release from neurohypophysis did not provide a clear evidence. Dayanithi et al. (1988) found about 20% inhibition of electrically evoked vasopressin release using a high (5/xM) concentration of nicardipine, while no effects of dihydropyridines were found by Von Spreckelsen et al. (1990). To our knowledge, this is the first study where the influence of the dihydropyridine ligands on the electrically stimulated release of neurohypophysial hormones has been investigated as a function of the applied pulse pattern. The data obtained have allowed us to define stimulation conditions under which the release of vasopressin or oxytocin was dose dependently potentiated by ( S ) - ( + ) 202-791, and it is suggested that the influx of Ca 2+ through the L-type Ca 2+ channels at low stimulation frequency (6.5 Hz) is rate-limiting for hormone release. Small but potentiating effects of the dihydropyridine agonist were found on the release of both oxytocin and vasopressin at a stimulation frequency of 6.5 Hz. Although no significant effects of (S)-( + )-202-791 were achieved at higher stimulation frequencies, a tendency of the agonist to potentiate the release of oxytocin at 13 and 30 Hz was seen. According to Fig. 1 frequencydependent facilitation of the amount of oxytocin released per pulse was seen in the whole frequency range (6.5-30 Hz), while the secretion of vasopressin was maximal around 13 Hz. This could account for the observed tendency of the dihydropyridine agonist to potentiate the release of oxytocin at 13 and 30 Hz, as the secretion of oxytocin was not maximized at these stimulation frequencies. At a stimulation frequency of 6.5 Hz ( S ) - ( + ) - 2 0 2 791 enhanced the release of both oxytocin and vasopressin in a concentration-dependent manner, but apparently a maximal response was seen at a concentration of 1 /xM. A similar biphasic effect of the dihydropyridine agonist, ( S ) - ( - )-Bay K 8644 has been observed on the 45Ca2+ uptake into rat heart cells (Wei et al., 1989) and of the racemic Bay K 8644 on the release of amino acids from and uptake of 45Ca2+ into cerebrocortical synaptosomes (White and Bradford, 1986). In these studies this effect was explained as a tendency of the dihydropyridine agonist to behave as an antagonist in higher concentrations because of state-dependent interactions of the ligand with the Ca 2+ channel. The facilitating effect of (S)-( + )-202-791 at 6.5 Hz on the evoked release of oxytocin and vasopressin was abolished by ( - ) - n i t r e n d i p i n e and that of vasopressin stereospecifically by ( R ) - ( - ) - 2 0 2 - 7 9 1 , while the dihydropyridine antagonists failed to modify the hormone release when tested alone. An explanation for this apparent discrepancy could be a small contribution of the L-type Ca 2 + channel to the influx of Ca ~+, as also indicated by the size of the potentiating effects of the
162
A. Jorgensen el aL / European .hmrnal qf f~harmac¢)h)gv 259 (1994) 157 163
dihydropyridine agonist. Hence it was not possible to recognize suppressing effects of the antagonists alone. Sevcral explanations may account for the observed potentiation of vasopressin and oxytocin releasc by (S)-( + )-202-791. (1) A number of studies have suggested that electrical stimulation of isolated neurohypophysis or isolated neurohypophysial slices with increasing pulse frequencies results in action potential broadening (Gainer ct al., 1986; Bourque, 1990; Jackson et al., 1991). This broadening of action potentials has been proposcd to result from K+-current inactivation (Bondy et al., 1987). A consequence of inactivation of K + currents would be a prolonged period of depolarization, allowing an increased ( a influx through voltage-gatcd ( a - channels and hence enhanced secretion. In light of this mechanism, a dihydropyridine-mediated potentiation of hormone release would be most likely to occur at low stimulation frequencies, where thc K*-current inactivation is less prominent. This is in agreement with our results. (2) The potentiation of hormone release by (S)-( + )202-791 at 6.5, but not at higher stimulation frequencies could also reflect the presence of frequency-dependent facilitation due to a mechanism independent of the action potential broadening as demonstrated by Jackson et al. (1991). In this work, enhanced intracellular Ca -'~ with increasing frequencies was ew~ked by constant duration pulses in voltage-clamped neurohypophysial nerve terminals. This increase in Ca -'+ level could be accounted for by Ca ?+ channel facilitation due to a change in channel-gating behaviour. From single channel patch-clamp studies on neurons and cardiac cells, a three mode gating behaviour of the L-type Ca =+ channel has been proposed. Mode 1 is characterized by brief openings, mode 0 by no openings and mode 2 by long lasting openings and very brief closings. Mode 2 is according to this theory fawmred by the presence of dihydropyridine agonists, as well as for instance strong depolarizations and trains of rapidly delivered action potentials, while mode 0 is promoted by dihydropyridinc antagonists (Hess et al., 1984; Nowycky el al., 1985; Pietrobon and Hess, 1990). Thus, in the present study an increased influx of Ca = ' due to the action of (S)-(+)-202-791 at low stimulation frequency could reflect an agonist-promoted L-channel shift from mode I to mode 2. On the other hand, higher stimulation frequencies would be sufficient by itself to cause the mode 1 to mode 2 shift, thus masking the effect of the drug. (3) Additionally, the frequency dependent stimulation of (S)-( + )-202-791 could reflect a frequency-dependent change in the contribution of different types of Ca :+ channels to Ca =+ influx upon electrical stimulation. In addition to the dihydropyridine-sensitivc. Ltype Ca :~ channels, the neurohypophysial nerve termi-
nals have been suggested to contain N-type channels (Dayanithi et al., 1988; kemos and Nowycky, 1989; Von Spreckelsen et al., 1990; Nowycky, 1991; Wang et al., 1992). Therefore, it is possible that an enhanced recruitement of these channels at increased stimulation frequencies renders the contribution of L-channels to Ca :~ influx, and thus any pharmacoh)gical cffects upon these channels, less significant. In summary, these results dcmonstratc that the entry of Ca e- by the dihydropyridine-sensitivc L-type Ca -'~ channel under certain conditions plays a rolc in the release of oxytocin and vasopressin from isolated, rat neurointermediatc lobes when stimulated electrically.
Acknowledgements Ms. D. Christoflersen and Ms. G. Slevcn aic gratefully acknowledged for their excellent technical assistance. This work has bccn supported by the Danish Goverment Biotechnok)gy Program.
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