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Vasoactive intestinal peptide inhibits release of somatostatin from
493
European Journal o f Pharmacology, 58 (1979) 493--495 © Elsevier/North-Holland Biomedical Press
Short communication VASOACTIVE INTESTINAL PEPTIDE INHIBITS RELEASE OF SOMATOSTATIN FROM HYPOTHALAMUS IN VITRO * JACQUES EPELBAUM, LUCIA TAPIA-ARANCIBIA, JACQUELINE BESSON 1, WILLIAM H. ROTSZTEJN and CLAUDE KORDON
Unitd 159 de Neuroendocrinologie, Centre Paul Broca de I'INSERM, 2ter rue d'Aldsia, ~5014 Paris, France and 1Unitd 55 INSERM, H6pital Saint-Antoine, 75012 Paris, France Received 15 August 1979, accepted 17 August 1979
J. EPELBAUM, L. TAPIA-ARANCIBIA, J. BESSON, W.H. ROTSZTEJN and C. KORDON, Vasoactive intestinal peptide inhibits release of somatostatin from hypothalamus in vitro, European J. Pharmacol. 58 (1979) 493--495. The effect of vasoactive intestinal peptide (VIP) was studied on the release of somatostatin (SRIF) from slices of several regions of the rat brain in vitro. VIP induced a dose-dependent inhibition of SRIF release from mediobasal hypothalamic slices but did not interfere with SRIF release from preoptic area, amygdala or cortex. VIP inhibition had an apparent affinity: K d -- 6.8 - 3.9 × 10 -11 M. Secretin had a similar effect but at 600-fold higher concentrations (K d secretin = 4.2 + 0.6 × 10 -s M). Glucagon was ineffective in concentrations ranging from 10 -1° M to 10 -7 M. The data are consistent with a role of VIP in the hypothalamic control of growth hormone secretion. Mediobasal hypothalamus
Neuropeptide release
1. Introduction
Somatostatin (SRIF) and vasoactive intestinal peptide (VIP) are found in relatively high concentrations in both the gut and the central nervous system. As most other neuropeptides tested so far, VIP and SRIF are preferentially distributed to subcellular fractions corresponding to nerve terminals (Epelbaum et al., 1977; Besson et al., 1979), from which they can be released in vitro b y depolarization in a calcium-dependent manner (Emson et al., 1978; Iversen et al., 1978). Furthermore, they have been shown by iontophoresis to modulate neuronal activity (Renaud et al., 1975; Phillis et al., 1978). These data suggest that these neuropeptides may act as neurotransmitter-like substances. Since VIP seems to be involved in the control of growth hormone secretion (Vijayan et al., 1979), it was of * Supported by INSERM (contract CRL to W.H.R. no. 79-5-236-4).
Somatostatin
Vasoactive intestinal peptide
interest to test whether it could influence SRIF release from neuroendocrine cells in the mediobasal hypothalamus or from other parts of the brain.
2. Materials and methods Adult male Wistar rats (280--300 g b o d y weight) were killed b y decapitation. The mediobasal hypothalamus (MBH) and anterior hypothalamus (POA), as well as the amygdala (AMY) and the cortex (Cx) were rapidly dissected from chilled brains as already described (Epelbaum et al., 1977). 250-pM thick slices were cross-cut from each structure with a McIlwain tissue chopper and incubated under constant gassing with 95% 02-5% CO2 in a shaking water bath at 37°C in a modified Locke medium (NaC1 154 (mM), KC1 5.6, CaC12 2.2, MgC12 1, NaHCO3 6, glucose 10, buffered to pH 7.2 with 2 mM Hepes (Calbiochem). Bacitracin (2 × 10 -s M
494
J. E P E L B A U M ET AL.
Sigma) and trasylol (500 IU/ml, gift of Bayer) were added to prevent peptidasic degradation. The experimental pr oc e dur e includes a washing step in cold medium and a 40-min preincubation before a 10-min incubation period. After the incubation, slices were separated from the medium by 2-min centrifugation in an E p p e n d o r f microfuge. Supernatants were collected in cold 2 N CHaCOOH (1 vol acid/ 1 0 v o l supernatant); slices were extracted with 0.2 N acetic acid and sonicated for 15 sec. SRIF concentrations were determined by radioimmunoassay. The SRIF release was calculated as a percentage o f S R I F tissue c o n t e n t and was plotted against the logarithm of the doses of VIP or secretin. Statistical significance was assessed by analysis of covariance. The SRIF c o n t e n t o f the tissues was n o t significantly affected b y th e incubation (values. f ound before and after preincubation + incubation were respectively 22.2 -+ 2.2 and 18.1 + 1.6 ng/structure equivalent for MBH, 4.4 + 0.4 and 3.9 -+ 0.2 for APO, 20.4 -+ 2.0 and 16.1 + 1.3 for AMY and 3.7 + 0.4 and 4.5 + 0.3 for Cx; mean + S.E.M. of 7 determinations in each structure). A fair correlation was obtained in all cases between the a m o u n t of SRIF released into the medium and the tissue c o n t e n t at the end of the e xpe r i m e nt ( r = 0.81 for 22 paired determinations in MBH, 0.66 for 20 determinations in APO, 0.72 for 22 determinations in AMY and 0.58 for 14 determinations in Cx), so that the
4.Ill
== E 3.01
== v
~=
2.01
10g[d0se1 Fig. 1. Effects o f V I P and secretin on in v i t r o s o m a t o statin release f r o m mediobasal h y p o t h a l a m u s . Values r e p r e s e n t the m e a n -+ S.E.M. N u m b e r s o f e x p e r i m e n tal p o i n t s in each group are given in p a r e n t h e s e s . Ordinate: S R I F release e x p r e s s e d as pg m e d i u m over pg tissue c o n t e n t x l 0 0 . Abscissa: Log dose o f VIP (~ e ) or secretin (o ©). (for 3 x 10 -1° M VIP F = 1 2 . 0 5 8 2 , highly significantly d i f f e r e n t f r o m c o n t r o l : for 10 -6 M secretin F = 15.9436, highly significantly d i f f e r e n t f r o m control).
SRIF release was subsequently expressed as percent o f the tissue c o n t e n t in corresponding incubates.
3. Results As shown in fig. 1, addition of VIP to the incubation medium induced a dose-dependent inhibition of SRIF release from MBH slices; effective doses ranged from 3 × 10 -I' to 10 -9.
TABLE 1 S R I F release (pg released/pg c o n t e n t x 100). Lack o f effect o f various VIP c o n c e n t r a t i o n s (M) o n in vitro S R I F release f r o m slices o f a n t e r i o r h y p o t h a l a m u s - - p r e o p t i c area (POA), amygdala (AMY) and c o r t e x (Cx). Same i n c u b a t i o n c o n d i t i o n s as for MBH slices. N u m b e r o f samples in p a r e n t h e s e s . Data e x p r e s s e d as in fig. 1. Structure
Control
10 -12
3 x 10 -12
10 -11
3 x 10 -11
10 -1°
POA
3.14 -+ 0.43 (21)
2.45 + 0.43
1.5
+ 0.18
3.26 -+ 0.37
3.26 + 0.40
2.76 -+ 0.32
AMY
1.30 -+ 0.21 (21)
1.55 -+ 0.41
1.28 + 0.28
1.38 -+ 0.22
1.31 -+ 0.15
1.25 + 0.32
Cx
3.43 + 0.45 (14)
4.44 -+ 0.74
4.16 -+ 0.45
4.31 -+ 0.91
3.42 -+ 0.48
3 X 10 -10
10 -9
10 -8
3.07 + 0.60
3.65 --4"0.44
1.32 -+ 0.08
1.89 -+ 0.29
1.42 -+ 0.23
4.28 -+ 0.63
3.76 + 0.56
495
VIP AND SOMATOSTATIN RELEASE
Apparent affinity (Kd) and maximal response (Emax) calculated according to Parker and Wand (1971) were, respectively, K d = 6 . 8 + 3 . 9 × 1 0 -11M and Ema x = 1 . 8 + 0 . 3 p g medium/tissue inhibition. Secretin had a similar effect, but at about 600-fold higher concentrations (K a = 4.2 + 0.6 × 10 -s, Em,x = 2.2 + 0.8, not significantly different from VIP inhibition). In contrast, glucagon was ineffective, in molar concentrations ranging from 10 -1° M to 10 -7 M (data not shown). In addition, the inhibitory effect of VIP on SRIF release was restricted to the MBH, and was not obtained from slices prepared from the other structures we have tested (table 1).
4. Discussion
The present data show that the inhibitory effect of VIP on SRIF release was restricted to the MBH and thus suggest that VIP receptors are present on SRIF neurons of the MBH, but not of the other structures rich in SRIFand VIP-containing neurons. This conclusion is further substantiated by the finding that higher concentrations of secretin, but not glucagon, can inhibit SRIF release from the MBH. VIP, secretin and glucagon are closely related structurally and secretin, but not glucagon, has been shown to bind to VIPbinding sites in hypothalamic membrane preparations (Robberecht et al., 1979), with affinity ratios between VIP and secretin comparable to our data. The apparent affinity ratio for VIP and secretin calculated from our doseresponse curve is also very similar to that computed on the basis of binding experiments in isolated intestinal epithelial cells from rats (Prieto et al., 1979). The effect of VIP on the release of somatostatin from infundibular nerve terminals, which are mostly located in the median eminence, is in good agreement with recent in vivo data showing stimulation of plasma growth hormone after -intraventricular infusion of VIP; this last observation could be well explained by an inhibition of SRIF release into the hypothalamo-hypophyseal portal system resulting in a subsequent dis-
inhibition of pituitary growth hormone secretion. Acknowledgements We thank Dr. S. Said for gi~ing VIP and Pr. G. Rosselin for providing us with secretin and glucacon.
References Besson, J., W.H. Rotsztejn, M. Laburthe, J. Epelbaum, A. Beaudet, C. Kordon and G. Rosselin, 1979, Vasoactive intestinal peptide (VIP): brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats, Brain Res. 165, 79. Emson, P.C., J. Fahrenkrug, O.B.S. De Muckadel, T.M. Jessel and L.L. Iversen, 1978, Vasoactive intestinal peptide (VII)): Vesicular localization and potassium evoked relea'se from rat hypothalamus, Brain Res. 143,174. Epelbaum, J., P. Brazeau, D. Tsang, J. Brawer and J.B. Martin, 1977, Subcellular distribution of radioimmunoassayable somatostatin in rat brain, Brain Res. 123,309. Iversen, L.L., S.D. Iversen, F. Bloom, C. Douglas, M. Brown and W. Vale, 1978, Calcium dependent release of somatostatin and neurotensin from rat brain in vitro, Nature 273,161. Parker, R.B. and D.R.J. Wand, 1971, Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. I: Agonists, J. Pharmacol. Exp. Therap. 177, 1. Phillis, J.W., J.R. Kirkpatrick and S.I. Said, 1978, Vasoactive intestinal polypeptide excitation of central neurons, Can. J. Physiol. Pharmacol. 56, 337. Prieto, J.C., M. Laburthe and G. Rosselin, 1979, Interaction of vasoactive intestinal peptide with isolated epithelial cells from rat. I: Characterization, quantitation aspects and structural requirements of binding sites, Eur. J. Biochem. (in press). Renaud, L.P., J.B. Martin and P. Brazeau, 1975, Depressant action of TRH, LHRH and somatostatin on activity of central neurones, Nature 255, 233. Robberecht, P., P. de Neef, M. Lammens, M. Deschodt Lanckman and J.P. Christophe, 1979, Specific binding of vasoactive intestinal peptide in brain membranes from the guinea pig, Eur. J. Biochem. 9 0 , 1 4 7 . Vijayan, E., W.K. Samson, S.I. Said and S.M. McCann, 1979, Vasoactive intestinal peptide: evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone and prolactin in conscious ovariectomized rats, Endocrinology 104, 53.
European Journal o f Pharmacology, 58 (1979) 493--495 © Elsevier/North-Holland Biomedical Press
Short communication VASOACTIVE INTESTINAL PEPTIDE INHIBITS RELEASE OF SOMATOSTATIN FROM HYPOTHALAMUS IN VITRO * JACQUES EPELBAUM, LUCIA TAPIA-ARANCIBIA, JACQUELINE BESSON 1, WILLIAM H. ROTSZTEJN and CLAUDE KORDON
Unitd 159 de Neuroendocrinologie, Centre Paul Broca de I'INSERM, 2ter rue d'Aldsia, ~5014 Paris, France and 1Unitd 55 INSERM, H6pital Saint-Antoine, 75012 Paris, France Received 15 August 1979, accepted 17 August 1979
J. EPELBAUM, L. TAPIA-ARANCIBIA, J. BESSON, W.H. ROTSZTEJN and C. KORDON, Vasoactive intestinal peptide inhibits release of somatostatin from hypothalamus in vitro, European J. Pharmacol. 58 (1979) 493--495. The effect of vasoactive intestinal peptide (VIP) was studied on the release of somatostatin (SRIF) from slices of several regions of the rat brain in vitro. VIP induced a dose-dependent inhibition of SRIF release from mediobasal hypothalamic slices but did not interfere with SRIF release from preoptic area, amygdala or cortex. VIP inhibition had an apparent affinity: K d -- 6.8 - 3.9 × 10 -11 M. Secretin had a similar effect but at 600-fold higher concentrations (K d secretin = 4.2 + 0.6 × 10 -s M). Glucagon was ineffective in concentrations ranging from 10 -1° M to 10 -7 M. The data are consistent with a role of VIP in the hypothalamic control of growth hormone secretion. Mediobasal hypothalamus
Neuropeptide release
1. Introduction
Somatostatin (SRIF) and vasoactive intestinal peptide (VIP) are found in relatively high concentrations in both the gut and the central nervous system. As most other neuropeptides tested so far, VIP and SRIF are preferentially distributed to subcellular fractions corresponding to nerve terminals (Epelbaum et al., 1977; Besson et al., 1979), from which they can be released in vitro b y depolarization in a calcium-dependent manner (Emson et al., 1978; Iversen et al., 1978). Furthermore, they have been shown by iontophoresis to modulate neuronal activity (Renaud et al., 1975; Phillis et al., 1978). These data suggest that these neuropeptides may act as neurotransmitter-like substances. Since VIP seems to be involved in the control of growth hormone secretion (Vijayan et al., 1979), it was of * Supported by INSERM (contract CRL to W.H.R. no. 79-5-236-4).
Somatostatin
Vasoactive intestinal peptide
interest to test whether it could influence SRIF release from neuroendocrine cells in the mediobasal hypothalamus or from other parts of the brain.
2. Materials and methods Adult male Wistar rats (280--300 g b o d y weight) were killed b y decapitation. The mediobasal hypothalamus (MBH) and anterior hypothalamus (POA), as well as the amygdala (AMY) and the cortex (Cx) were rapidly dissected from chilled brains as already described (Epelbaum et al., 1977). 250-pM thick slices were cross-cut from each structure with a McIlwain tissue chopper and incubated under constant gassing with 95% 02-5% CO2 in a shaking water bath at 37°C in a modified Locke medium (NaC1 154 (mM), KC1 5.6, CaC12 2.2, MgC12 1, NaHCO3 6, glucose 10, buffered to pH 7.2 with 2 mM Hepes (Calbiochem). Bacitracin (2 × 10 -s M
494
J. E P E L B A U M ET AL.
Sigma) and trasylol (500 IU/ml, gift of Bayer) were added to prevent peptidasic degradation. The experimental pr oc e dur e includes a washing step in cold medium and a 40-min preincubation before a 10-min incubation period. After the incubation, slices were separated from the medium by 2-min centrifugation in an E p p e n d o r f microfuge. Supernatants were collected in cold 2 N CHaCOOH (1 vol acid/ 1 0 v o l supernatant); slices were extracted with 0.2 N acetic acid and sonicated for 15 sec. SRIF concentrations were determined by radioimmunoassay. The SRIF release was calculated as a percentage o f S R I F tissue c o n t e n t and was plotted against the logarithm of the doses of VIP or secretin. Statistical significance was assessed by analysis of covariance. The SRIF c o n t e n t o f the tissues was n o t significantly affected b y th e incubation (values. f ound before and after preincubation + incubation were respectively 22.2 -+ 2.2 and 18.1 + 1.6 ng/structure equivalent for MBH, 4.4 + 0.4 and 3.9 -+ 0.2 for APO, 20.4 -+ 2.0 and 16.1 + 1.3 for AMY and 3.7 + 0.4 and 4.5 + 0.3 for Cx; mean + S.E.M. of 7 determinations in each structure). A fair correlation was obtained in all cases between the a m o u n t of SRIF released into the medium and the tissue c o n t e n t at the end of the e xpe r i m e nt ( r = 0.81 for 22 paired determinations in MBH, 0.66 for 20 determinations in APO, 0.72 for 22 determinations in AMY and 0.58 for 14 determinations in Cx), so that the
4.Ill
== E 3.01
== v
~=
2.01
10g[d0se1 Fig. 1. Effects o f V I P and secretin on in v i t r o s o m a t o statin release f r o m mediobasal h y p o t h a l a m u s . Values r e p r e s e n t the m e a n -+ S.E.M. N u m b e r s o f e x p e r i m e n tal p o i n t s in each group are given in p a r e n t h e s e s . Ordinate: S R I F release e x p r e s s e d as pg m e d i u m over pg tissue c o n t e n t x l 0 0 . Abscissa: Log dose o f VIP (~ e ) or secretin (o ©). (for 3 x 10 -1° M VIP F = 1 2 . 0 5 8 2 , highly significantly d i f f e r e n t f r o m c o n t r o l : for 10 -6 M secretin F = 15.9436, highly significantly d i f f e r e n t f r o m control).
SRIF release was subsequently expressed as percent o f the tissue c o n t e n t in corresponding incubates.
3. Results As shown in fig. 1, addition of VIP to the incubation medium induced a dose-dependent inhibition of SRIF release from MBH slices; effective doses ranged from 3 × 10 -I' to 10 -9.
TABLE 1 S R I F release (pg released/pg c o n t e n t x 100). Lack o f effect o f various VIP c o n c e n t r a t i o n s (M) o n in vitro S R I F release f r o m slices o f a n t e r i o r h y p o t h a l a m u s - - p r e o p t i c area (POA), amygdala (AMY) and c o r t e x (Cx). Same i n c u b a t i o n c o n d i t i o n s as for MBH slices. N u m b e r o f samples in p a r e n t h e s e s . Data e x p r e s s e d as in fig. 1. Structure
Control
10 -12
3 x 10 -12
10 -11
3 x 10 -11
10 -1°
POA
3.14 -+ 0.43 (21)
2.45 + 0.43
1.5
+ 0.18
3.26 -+ 0.37
3.26 + 0.40
2.76 -+ 0.32
AMY
1.30 -+ 0.21 (21)
1.55 -+ 0.41
1.28 + 0.28
1.38 -+ 0.22
1.31 -+ 0.15
1.25 + 0.32
Cx
3.43 + 0.45 (14)
4.44 -+ 0.74
4.16 -+ 0.45
4.31 -+ 0.91
3.42 -+ 0.48
3 X 10 -10
10 -9
10 -8
3.07 + 0.60
3.65 --4"0.44
1.32 -+ 0.08
1.89 -+ 0.29
1.42 -+ 0.23
4.28 -+ 0.63
3.76 + 0.56
495
VIP AND SOMATOSTATIN RELEASE
Apparent affinity (Kd) and maximal response (Emax) calculated according to Parker and Wand (1971) were, respectively, K d = 6 . 8 + 3 . 9 × 1 0 -11M and Ema x = 1 . 8 + 0 . 3 p g medium/tissue inhibition. Secretin had a similar effect, but at about 600-fold higher concentrations (K a = 4.2 + 0.6 × 10 -s, Em,x = 2.2 + 0.8, not significantly different from VIP inhibition). In contrast, glucagon was ineffective, in molar concentrations ranging from 10 -1° M to 10 -7 M (data not shown). In addition, the inhibitory effect of VIP on SRIF release was restricted to the MBH, and was not obtained from slices prepared from the other structures we have tested (table 1).
4. Discussion
The present data show that the inhibitory effect of VIP on SRIF release was restricted to the MBH and thus suggest that VIP receptors are present on SRIF neurons of the MBH, but not of the other structures rich in SRIFand VIP-containing neurons. This conclusion is further substantiated by the finding that higher concentrations of secretin, but not glucagon, can inhibit SRIF release from the MBH. VIP, secretin and glucagon are closely related structurally and secretin, but not glucagon, has been shown to bind to VIPbinding sites in hypothalamic membrane preparations (Robberecht et al., 1979), with affinity ratios between VIP and secretin comparable to our data. The apparent affinity ratio for VIP and secretin calculated from our doseresponse curve is also very similar to that computed on the basis of binding experiments in isolated intestinal epithelial cells from rats (Prieto et al., 1979). The effect of VIP on the release of somatostatin from infundibular nerve terminals, which are mostly located in the median eminence, is in good agreement with recent in vivo data showing stimulation of plasma growth hormone after -intraventricular infusion of VIP; this last observation could be well explained by an inhibition of SRIF release into the hypothalamo-hypophyseal portal system resulting in a subsequent dis-
inhibition of pituitary growth hormone secretion. Acknowledgements We thank Dr. S. Said for gi~ing VIP and Pr. G. Rosselin for providing us with secretin and glucacon.
References Besson, J., W.H. Rotsztejn, M. Laburthe, J. Epelbaum, A. Beaudet, C. Kordon and G. Rosselin, 1979, Vasoactive intestinal peptide (VIP): brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats, Brain Res. 165, 79. Emson, P.C., J. Fahrenkrug, O.B.S. De Muckadel, T.M. Jessel and L.L. Iversen, 1978, Vasoactive intestinal peptide (VII)): Vesicular localization and potassium evoked relea'se from rat hypothalamus, Brain Res. 143,174. Epelbaum, J., P. Brazeau, D. Tsang, J. Brawer and J.B. Martin, 1977, Subcellular distribution of radioimmunoassayable somatostatin in rat brain, Brain Res. 123,309. Iversen, L.L., S.D. Iversen, F. Bloom, C. Douglas, M. Brown and W. Vale, 1978, Calcium dependent release of somatostatin and neurotensin from rat brain in vitro, Nature 273,161. Parker, R.B. and D.R.J. Wand, 1971, Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. I: Agonists, J. Pharmacol. Exp. Therap. 177, 1. Phillis, J.W., J.R. Kirkpatrick and S.I. Said, 1978, Vasoactive intestinal polypeptide excitation of central neurons, Can. J. Physiol. Pharmacol. 56, 337. Prieto, J.C., M. Laburthe and G. Rosselin, 1979, Interaction of vasoactive intestinal peptide with isolated epithelial cells from rat. I: Characterization, quantitation aspects and structural requirements of binding sites, Eur. J. Biochem. (in press). Renaud, L.P., J.B. Martin and P. Brazeau, 1975, Depressant action of TRH, LHRH and somatostatin on activity of central neurones, Nature 255, 233. Robberecht, P., P. de Neef, M. Lammens, M. Deschodt Lanckman and J.P. Christophe, 1979, Specific binding of vasoactive intestinal peptide in brain membranes from the guinea pig, Eur. J. Biochem. 9 0 , 1 4 7 . Vijayan, E., W.K. Samson, S.I. Said and S.M. McCann, 1979, Vasoactive intestinal peptide: evidence for a hypothalamic site of action to release growth hormone, luteinizing hormone and prolactin in conscious ovariectomized rats, Endocrinology 104, 53.