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Release of vasoactive intestinal peptide from rat brain slices by various depolarizing agents
Neuroscience Letters, 28 (! 982) 281-285 Elsevier/North-Holland Scientific Publishers Ltd.
281
RELEASE OF VASOAC'/IVE INTESTINAL PEPTIDE FROM RAT BRAIN SLICES BY VARIOUS DEPOLARIZING AGENTS
J. BESSON, W. ROTSZTEJN, B. POUSSIN, A.M. LttlAUBET and G. ROSSELIN
INSERM U. 5.L Service de Diab~tologie et d'Etudes Radioimmunoiogiques des Hormones Protdiques, H~pital Saint-Antoine, 184, rue du Faubourg Salnt-Am'oine, 75012 Paris (France) (Received October 22nd, 1981; Revised version received December 8th, 1981; Accepted December 9th,
1981)
The relea~e ,)f vasoactive intestinal peptide (VIP) from rat brain cortical a ed amygdala slices was studied by using various depolarizing agents such as potassium (K *), veratridine (VER) and batrachotoxin (BTX). Tl:e basal release of VIP observed is of the same order of magnitude for both structures and represents less than 0.1% of the tissue content per miaute inca.tared by a specific radioimmunoassay. Maximal stimulation o b t a i n ~ with 56 mM K +, 50 ~tM VER and 1 #M BTX corre.,;ponds to a mean 3-fold increase above the basa! release of VIP in both cotter, and amygdala. When the incubation medium did not contain any calcium, the action of potassium on the release of VIP was mppressed. When tetr,xlotoxin (1 t~M) was added to the incubation medium, the veratridine- and ba~,acS-~c.~oxin-induced release of VIP was inhibited whereas K ~-indnced relea.v- was unaffected. These r, ~ults support the hypothesis that VIP can be a neurotransmitter iu the cemra! nervous system.
Vasoactive intestinal peptide (VIP) has been found in several structures of the brain by both imnmnocytochemical and radioimmunoiogical techniques [2, 7, 15, 16]. Its distribution is uneven, with the highes.: cc,ncentrafions i:a ~:hecerebral cortex. It is also localized in several limbic structures such as the amygdala. In bo~h cortex and amygdala VIP was found in perikarya and in nerve terrainals [8, 11, 17]. Although the role of VIP in brain has not been elucidated as yet, its localization suggests that the peptide may act as a neurotransmitter. To test this hypothesis, various depolarizing agents such as potassium, veratridine, and '~atrachotoxin were used to induct: VIP rele~.se from cortical and amygdala slices and to evaluate the possible involvement of ionic channels in this process. Wistar rats were obtained from our breeding colony. Male rat; weighing 250-300 g were housed under diurmtl lighting conditions and were given food and water ad libitum. They were killed by decapitation and then parietal cortex and amygdala were dissected out. Both structures were cross-cu, into slic~'g (250 ttm) with a Mcllwain tissue choplx,r. The slices were washed 5 or 6 times with a Krebs-RingerBicarbonate buffer (KRB), pH 7.4~ containing CraM): NaC1 118, KC1 5, CaCI2.2H20 2.5, KH2PO4 1.2, MgSO.,.7H20 1.2, NaHCO, 25, glucose 10, 0304-.3940/82/00(O-0000/$ 0235 ~) Elsevier/North.Holland ,.~ientific Publishers Ltd.
28:~
bacitracin 2 x 10 -s M and Trasylol 500 KIU/ml. Trasyloi and bacitracin were added to the incubation medium because we observed that the degradation of the ~ptide was lower with than without the~;e anti*proteasic factors. The slic~.,s were preincubated in KRB under 95% 02-5% CO2 during 30 min at 37"C. Incubation was started by addition of 200 ~1 of the suspension of cortex (63 rag, i.e, 1/2 equivalent cortex) or I20 ~1 of the s u s i ~ i o n of amygdala (lO rng, i.e. !/2 equivalent amygdaia) to the polyethylene tubes containing the various substances to test. Incubation was carried out for 10 rain a~ 37 °C and was ended by centrifuiation in an Eppendorf-type microfuge (10,000 >: g for 2 min). VIP content was assayed both in the supernatant and the tissue extracts by using a specific radioimmunoassay [tl. The dose-response curves of VIP to I:~tassium (K+), veratridine (VER), and batrachotoxin (BTX) are shown in Fig. I. The basal release in the medium represents less than 0.1% of the VIP content per minute of the tissue in both prepa. rations, in the cortex, the maximum stimulation, corresponding to a 4-fold increase of basal level, is obtained with a concerttr~tion of 56 mM K +. The maximum stimulation obtained with 50 I~M VER and 1 ~zM BTX corresponds to a 3- and 2.5fold increase of the control respectively, la the amygdala a 3.3 and 3.5-fold increase of the basal level represents the maximal response obtained with 56 mM K +, I ttM BTX and 50 I~M VER, respectively. Figure 2 shows the effect of calcium on the release of VIP induced by K +. The plateau of potassium effect is reached at 2~5 and 5 ntM calcium. A decrease or absence of calcium in the medium results in inhibition of the effect of K +, and in both corlex and amygdala the release of VIP is calcium dependent. On the other hand, the spontaneous release of VIP is unaffected by either removal or addition of calcium imo the incubation medium. When sodium CORI[X
AMYGOALA
,!
4
3 2
2
I
10 ,
j
.
.
10
50 .
.
.
.
.
.
.
100 K+ m~ (.)
"
5b . . . . .
A ....
50 1
100 VER IjM(A} 2 BT× IJM(')
1
2
I'ig. !. Dose---response curves of potassium (K +), veratridine (VER) and batrachotoxiv (BTX), The release: of VIP is expressed as the percentage of VlP secreted in the medium (M) versus the amount of VIP present in the tissue (T). Results are the mean + S.E,M. of 6 determinations, i.e. 6 s,:parate tissue !;amples in the same experiment assayed singly.
283 CORTEX
%
2
. •A M Y ~ A
I
2
0 06 12 25 ~; CALCIUMmM 0 06 12 25 5 Fig, 2. Effect of calcium oa potassium-induced release of VIP. Controls (I ]) ~.nd potassiunt-stimulatcd (iS) values are expressed as in Fig. I. Results are the mean :t S.E.M. of 6 determinations, i.e. 6 ~cf~aratc tissue samples in the same experiment assayed si,~gly,
channels and early calcium channels were blocked by 1 #M tcttodotoxin ('FrX) the stimulatory effect o f K ~ 56 m M in both st:'uctures was unaffected, wherea~ Ihc release o f VIP induced by VER (50 ,aM) and BTX (1 pM) was significantly decreased
(Fig. 3). Our results confirm that high concentrations o f K ' induce release o f V l P in a calcium d e p e n d e n t manner as previously described by Ciacheui et al. [9] and Emson et al. [6] using either cortical synaptosomes or superfused hypothalamie slices. Furthermore, our results show that other depolarizing agents :,uch as VER and BTX are able to induce the release o f the pepfide at concentrations much lower than dmt o f K +. In contra.~t to K +, VER and BT~,: are k n o w n ~o act through ~odium channels [4]. We d e m o n s t r a t e that tetrodotoxin, a blocker of ~.hese ionic cha,mels, inhibits the release o f V I P induced by these drugs. This indicates that TTX-:~ensitive sodium chaanels as well as calcium channels are present on VIP nerve termiuals. 'l'he t~ct
CORTEX
% 4
% ~ catrd VER BIX
50#
I#
K+ S6mM
AMYGOALA
4
3 2 1 4.
4.
4"
4.
'fin 4.
4'
4,
4.
Fig. 3. Effect of tetrodotoxin (TTX) on potassium (K ~)-, veratridine (VER)- and batrachotoxiu ~BTX)induced release of VIP. The addition of TTX (1 f,M) oct'urs at the beginning ot" the incubation a~d i~ indicated by (+). The results represent the mean ± S..E.M. of 6 dete~'mi,ations, i+e. 6 separate tisale samples in the same experiment assayed singly. They are expressed as in Fig. I.
284
that the stimulating effect of K + wa~ not abolished by TTX shows that sodium and e~riy calcium channels are not essent:ial for the K +-induced release of VtP in agr~'emeat with data concerning other neuropeptideg such as luteinizing hormone-rclez.sing hormone and somatostatin relea~', from hypothalamic sl;,ces [5]. On the contrary, the effect of K + is calcium dependent and the amplitude of the re.sporsse depends upon :he calcium concentration of the medium. It has been reported that depolarization induces a discharge of neuropcptMes and neurotransmitters from neurosecretory terminals [3, 10, 12-14, 18]. The observation that VIP ~ n b¢ relea3ed by various membrane del~31arizin~ agents in the present work furth;:r suggests that VIP can be a neurotransmitter in the central nervous system. We thank Dr. Daly who gave us batrachotoxin and C. Brunet for the good presentation of the manuscript. This work was supported by iNSERM CRI+ 79.5.236.4. ' Bcsst~t:, J., Laburthe, M., BataiUe, D., Dul~ont, C. and Ro~lin, G. Va~active intestinal peptide (VIP): tissue distribution in the rat as measmed by radioimmunoassay ~ad radioreeeptorassay, Acta ¢nd.3cr. 87 (!978) 799-810. 2 8e~'++on,J., Rotszteja, W., Laburthe, M., Epelbaum, J., Beaudet, A., Kordon, C. and Rosselin, G., Vaso.~:tive intestinal peptide (VIP): brain distribution, subcellolar localization and effect of deafferentation of the hypothalamus i~ male rats, Brain Res., 165 (1979) 79-85. 3 Biaustein, M.P., Jobt-~son, E.M. and l'!~01cn~an, P., Calcium+dependent nor,:Finephrine release ~rom presynl, ptic her ca endings ia vitro, Proc. nat. ~:a:.l. Sci. U.S.A., 69 (1972) 2237 -2240. 4 Caueral, W.A., Neu:otoxin+ 'hat act on voltage-sensitive se,;lium chau~els in excitable membranes, Ann. Rev. Pharmacol. Toxi:ol., 20 (1980), 15-43. 5 Drouva, S.V., Ep¢lbaum, J , Hery, M., Tapia-Arancibia, I.., Laplante E. and Kordon, C., Io~i¢ channels involved in the I,HRH and SRIF reie~..,e from rat mediobasal bypothalamus, NeuroetJdocrinology, 32 (1981) 155-162. 6 Emson, P.C., Fahrenkrug, J., ~haff~,litzky de Muckadell, O.B., Jessel, T.M. and lvcrsen, L.L., Vasoact~ve intestinal peptide (VIP~: vesicular localigation and potassium evoked release from rat hypothalamus, Brain Res., 143 (1978) 174-178. 7 Fahrenkrug, J, and Schaffalitzky de Muckade[I, O.B., Distribution of Vosoactive intesginai polypeptide (VtP) in the porcine central nervous system, J. Neurochem., 31 (197g) 1445-1451. 8 Fuxe, K., HOkfelt, T., Sa~'d, S.I. and Mutt, V., Vasoactive int~tina! polypeptide and the nervous system: immunohistochemical evidence for localizalion in central and p~riphcral r~eutons, particularly intracortical neuron~ of the ~erebral cortex, Nem'~ci. Le~t., 5 0977) 241-246. 9 Giachctti, A., Safd, S.I, Rcynoids, R.C. and Koniges, F.C., Vasoactive intestinal pclypeptide in broin" localization in and rctease from isolated nerve term:,nals, Proc. nat. Acad. Sci, U,S.A., 74 (1977) 4324-4328. l0 [versen, L.L.. Iversen, S.D., Bloom, F., r..~ugia~, C., ~ro~a, M, and Vain, W., Calcium-deper~dent r~lca~e of somatostatin and ncurolensin from rat brain in vitro, Nature (Load.), 273 (197~ 161-163. 11 Loren, l.. Emson, P.C., Fab.~enkrug, J., B.iOrPSuctd,A., Alumets, J., Hakanson, R. and Sandier, F., Distribution of v~.oa,::iv~ int~tina| poiypeplide in the rat and mouse brain, Neuro~cience, 4 (1919~ 1953-1976. 12 Mt~lder, A.~,'.., Van D¢.~ Berg, W.B. and Stoof, J.C., Catc~.umglepcndent r~lease of radioiabelted cat~holamit;es and serotonin from t'at brain synaptoson~ in a sup:rfusion system, Brain Res., 99 (19"~5) 419--424
285 13 Osborne, H., Holtt, V. and Herz, A., Potassium-induced relea~e of enkephalin from rat striatal slices, Europ. J. Pharmacol., 48 (1978) 219-221. 14 Rotsztejn, W.H., Charli, J.L., Pat~ou, E., Epelbaum, I. and Kotdon. C., In vitro reieasc of iuteinizi~g hormone.releasing hormone (LHRH) from [at mediobasal hy[bothalamu.~. Effect:, of go~assium calcium a~ad dopamine, Endocrinology, 99 (1976~ 1663-- 1¢~66. 15 Said, S.I. and Rosea~erg, R.N., Vasoactive intL.stinal polypepfide: abudant immunorc~,clivi:y in neural cell lines and no~nal nervous tissue, Science. 192 (1976) 907--908. 16 Samson, W.K., Said, S.I. and McCann, S.M., Radioimmurto!ogic localization (,f vasoactivc intestinal polvpcptide in hypothalamie and extrahypothalamic sites in the rat brain. Ncuro~ci. [elt., 12 (1979) 265 -269. 17 Sims, K.B., ~-toffman, D.L., Said, S.I. and Zimmerman, E.A., Vasoactive intes~izaal potypcp~id¢ (VIP) in rr~ouse and rat brain: an immunocytochemical ~tudy, Brain Res., 186 C|9~;0) 165-183 18 Subramanian, N, and Muldet, A.M., Potassium-induced release of tritiated his~a.tl~.i~.cfrom rat [:t'3~.q tissue slice~ Euron. J. Pharmacol., 35 (1976) 377-380.
281
RELEASE OF VASOAC'/IVE INTESTINAL PEPTIDE FROM RAT BRAIN SLICES BY VARIOUS DEPOLARIZING AGENTS
J. BESSON, W. ROTSZTEJN, B. POUSSIN, A.M. LttlAUBET and G. ROSSELIN
INSERM U. 5.L Service de Diab~tologie et d'Etudes Radioimmunoiogiques des Hormones Protdiques, H~pital Saint-Antoine, 184, rue du Faubourg Salnt-Am'oine, 75012 Paris (France) (Received October 22nd, 1981; Revised version received December 8th, 1981; Accepted December 9th,
1981)
The relea~e ,)f vasoactive intestinal peptide (VIP) from rat brain cortical a ed amygdala slices was studied by using various depolarizing agents such as potassium (K *), veratridine (VER) and batrachotoxin (BTX). Tl:e basal release of VIP observed is of the same order of magnitude for both structures and represents less than 0.1% of the tissue content per miaute inca.tared by a specific radioimmunoassay. Maximal stimulation o b t a i n ~ with 56 mM K +, 50 ~tM VER and 1 #M BTX corre.,;ponds to a mean 3-fold increase above the basa! release of VIP in both cotter, and amygdala. When the incubation medium did not contain any calcium, the action of potassium on the release of VIP was mppressed. When tetr,xlotoxin (1 t~M) was added to the incubation medium, the veratridine- and ba~,acS-~c.~oxin-induced release of VIP was inhibited whereas K ~-indnced relea.v- was unaffected. These r, ~ults support the hypothesis that VIP can be a neurotransmitter iu the cemra! nervous system.
Vasoactive intestinal peptide (VIP) has been found in several structures of the brain by both imnmnocytochemical and radioimmunoiogical techniques [2, 7, 15, 16]. Its distribution is uneven, with the highes.: cc,ncentrafions i:a ~:hecerebral cortex. It is also localized in several limbic structures such as the amygdala. In bo~h cortex and amygdala VIP was found in perikarya and in nerve terrainals [8, 11, 17]. Although the role of VIP in brain has not been elucidated as yet, its localization suggests that the peptide may act as a neurotransmitter. To test this hypothesis, various depolarizing agents such as potassium, veratridine, and '~atrachotoxin were used to induct: VIP rele~.se from cortical and amygdala slices and to evaluate the possible involvement of ionic channels in this process. Wistar rats were obtained from our breeding colony. Male rat; weighing 250-300 g were housed under diurmtl lighting conditions and were given food and water ad libitum. They were killed by decapitation and then parietal cortex and amygdala were dissected out. Both structures were cross-cu, into slic~'g (250 ttm) with a Mcllwain tissue choplx,r. The slices were washed 5 or 6 times with a Krebs-RingerBicarbonate buffer (KRB), pH 7.4~ containing CraM): NaC1 118, KC1 5, CaCI2.2H20 2.5, KH2PO4 1.2, MgSO.,.7H20 1.2, NaHCO, 25, glucose 10, 0304-.3940/82/00(O-0000/$ 0235 ~) Elsevier/North.Holland ,.~ientific Publishers Ltd.
28:~
bacitracin 2 x 10 -s M and Trasylol 500 KIU/ml. Trasyloi and bacitracin were added to the incubation medium because we observed that the degradation of the ~ptide was lower with than without the~;e anti*proteasic factors. The slic~.,s were preincubated in KRB under 95% 02-5% CO2 during 30 min at 37"C. Incubation was started by addition of 200 ~1 of the suspension of cortex (63 rag, i.e, 1/2 equivalent cortex) or I20 ~1 of the s u s i ~ i o n of amygdala (lO rng, i.e. !/2 equivalent amygdaia) to the polyethylene tubes containing the various substances to test. Incubation was carried out for 10 rain a~ 37 °C and was ended by centrifuiation in an Eppendorf-type microfuge (10,000 >: g for 2 min). VIP content was assayed both in the supernatant and the tissue extracts by using a specific radioimmunoassay [tl. The dose-response curves of VIP to I:~tassium (K+), veratridine (VER), and batrachotoxin (BTX) are shown in Fig. I. The basal release in the medium represents less than 0.1% of the VIP content per minute of the tissue in both prepa. rations, in the cortex, the maximum stimulation, corresponding to a 4-fold increase of basal level, is obtained with a concerttr~tion of 56 mM K +. The maximum stimulation obtained with 50 I~M VER and 1 ~zM BTX corresponds to a 3- and 2.5fold increase of the control respectively, la the amygdala a 3.3 and 3.5-fold increase of the basal level represents the maximal response obtained with 56 mM K +, I ttM BTX and 50 I~M VER, respectively. Figure 2 shows the effect of calcium on the release of VIP induced by K +. The plateau of potassium effect is reached at 2~5 and 5 ntM calcium. A decrease or absence of calcium in the medium results in inhibition of the effect of K +, and in both corlex and amygdala the release of VIP is calcium dependent. On the other hand, the spontaneous release of VIP is unaffected by either removal or addition of calcium imo the incubation medium. When sodium CORI[X
AMYGOALA
,!
4
3 2
2
I
10 ,
j
.
.
10
50 .
.
.
.
.
.
.
100 K+ m~ (.)
"
5b . . . . .
A ....
50 1
100 VER IjM(A} 2 BT× IJM(')
1
2
I'ig. !. Dose---response curves of potassium (K +), veratridine (VER) and batrachotoxiv (BTX), The release: of VIP is expressed as the percentage of VlP secreted in the medium (M) versus the amount of VIP present in the tissue (T). Results are the mean + S.E,M. of 6 determinations, i.e. 6 s,:parate tissue !;amples in the same experiment assayed singly.
283 CORTEX
%
2
. •A M Y ~ A
I
2
0 06 12 25 ~; CALCIUMmM 0 06 12 25 5 Fig, 2. Effect of calcium oa potassium-induced release of VIP. Controls (I ]) ~.nd potassiunt-stimulatcd (iS) values are expressed as in Fig. I. Results are the mean :t S.E.M. of 6 determinations, i.e. 6 ~cf~aratc tissue samples in the same experiment assayed si,~gly,
channels and early calcium channels were blocked by 1 #M tcttodotoxin ('FrX) the stimulatory effect o f K ~ 56 m M in both st:'uctures was unaffected, wherea~ Ihc release o f VIP induced by VER (50 ,aM) and BTX (1 pM) was significantly decreased
(Fig. 3). Our results confirm that high concentrations o f K ' induce release o f V l P in a calcium d e p e n d e n t manner as previously described by Ciacheui et al. [9] and Emson et al. [6] using either cortical synaptosomes or superfused hypothalamie slices. Furthermore, our results show that other depolarizing agents :,uch as VER and BTX are able to induce the release o f the pepfide at concentrations much lower than dmt o f K +. In contra.~t to K +, VER and BT~,: are k n o w n ~o act through ~odium channels [4]. We d e m o n s t r a t e that tetrodotoxin, a blocker of ~.hese ionic cha,mels, inhibits the release o f V I P induced by these drugs. This indicates that TTX-:~ensitive sodium chaanels as well as calcium channels are present on VIP nerve termiuals. 'l'he t~ct
CORTEX
% 4
% ~ catrd VER BIX
50#
I#
K+ S6mM
AMYGOALA
4
3 2 1 4.
4.
4"
4.
'fin 4.
4'
4,
4.
Fig. 3. Effect of tetrodotoxin (TTX) on potassium (K ~)-, veratridine (VER)- and batrachotoxiu ~BTX)induced release of VIP. The addition of TTX (1 f,M) oct'urs at the beginning ot" the incubation a~d i~ indicated by (+). The results represent the mean ± S..E.M. of 6 dete~'mi,ations, i+e. 6 separate tisale samples in the same experiment assayed singly. They are expressed as in Fig. I.
284
that the stimulating effect of K + wa~ not abolished by TTX shows that sodium and e~riy calcium channels are not essent:ial for the K +-induced release of VtP in agr~'emeat with data concerning other neuropeptideg such as luteinizing hormone-rclez.sing hormone and somatostatin relea~', from hypothalamic sl;,ces [5]. On the contrary, the effect of K + is calcium dependent and the amplitude of the re.sporsse depends upon :he calcium concentration of the medium. It has been reported that depolarization induces a discharge of neuropcptMes and neurotransmitters from neurosecretory terminals [3, 10, 12-14, 18]. The observation that VIP ~ n b¢ relea3ed by various membrane del~31arizin~ agents in the present work furth;:r suggests that VIP can be a neurotransmitter in the central nervous system. We thank Dr. Daly who gave us batrachotoxin and C. Brunet for the good presentation of the manuscript. This work was supported by iNSERM CRI+ 79.5.236.4. ' Bcsst~t:, J., Laburthe, M., BataiUe, D., Dul~ont, C. and Ro~lin, G. Va~active intestinal peptide (VIP): tissue distribution in the rat as measmed by radioimmunoassay ~ad radioreeeptorassay, Acta ¢nd.3cr. 87 (!978) 799-810. 2 8e~'++on,J., Rotszteja, W., Laburthe, M., Epelbaum, J., Beaudet, A., Kordon, C. and Rosselin, G., Vaso.~:tive intestinal peptide (VIP): brain distribution, subcellolar localization and effect of deafferentation of the hypothalamus i~ male rats, Brain Res., 165 (1979) 79-85. 3 Biaustein, M.P., Jobt-~son, E.M. and l'!~01cn~an, P., Calcium+dependent nor,:Finephrine release ~rom presynl, ptic her ca endings ia vitro, Proc. nat. ~:a:.l. Sci. U.S.A., 69 (1972) 2237 -2240. 4 Caueral, W.A., Neu:otoxin+ 'hat act on voltage-sensitive se,;lium chau~els in excitable membranes, Ann. Rev. Pharmacol. Toxi:ol., 20 (1980), 15-43. 5 Drouva, S.V., Ep¢lbaum, J , Hery, M., Tapia-Arancibia, I.., Laplante E. and Kordon, C., Io~i¢ channels involved in the I,HRH and SRIF reie~..,e from rat mediobasal bypothalamus, NeuroetJdocrinology, 32 (1981) 155-162. 6 Emson, P.C., Fahrenkrug, J., ~haff~,litzky de Muckadell, O.B., Jessel, T.M. and lvcrsen, L.L., Vasoact~ve intestinal peptide (VIP~: vesicular localigation and potassium evoked release from rat hypothalamus, Brain Res., 143 (1978) 174-178. 7 Fahrenkrug, J, and Schaffalitzky de Muckade[I, O.B., Distribution of Vosoactive intesginai polypeptide (VtP) in the porcine central nervous system, J. Neurochem., 31 (197g) 1445-1451. 8 Fuxe, K., HOkfelt, T., Sa~'d, S.I. and Mutt, V., Vasoactive int~tina! polypeptide and the nervous system: immunohistochemical evidence for localizalion in central and p~riphcral r~eutons, particularly intracortical neuron~ of the ~erebral cortex, Nem'~ci. Le~t., 5 0977) 241-246. 9 Giachctti, A., Safd, S.I, Rcynoids, R.C. and Koniges, F.C., Vasoactive intestinal pclypeptide in broin" localization in and rctease from isolated nerve term:,nals, Proc. nat. Acad. Sci, U,S.A., 74 (1977) 4324-4328. l0 [versen, L.L.. Iversen, S.D., Bloom, F., r..~ugia~, C., ~ro~a, M, and Vain, W., Calcium-deper~dent r~lca~e of somatostatin and ncurolensin from rat brain in vitro, Nature (Load.), 273 (197~ 161-163. 11 Loren, l.. Emson, P.C., Fab.~enkrug, J., B.iOrPSuctd,A., Alumets, J., Hakanson, R. and Sandier, F., Distribution of v~.oa,::iv~ int~tina| poiypeplide in the rat and mouse brain, Neuro~cience, 4 (1919~ 1953-1976. 12 Mt~lder, A.~,'.., Van D¢.~ Berg, W.B. and Stoof, J.C., Catc~.umglepcndent r~lease of radioiabelted cat~holamit;es and serotonin from t'at brain synaptoson~ in a sup:rfusion system, Brain Res., 99 (19"~5) 419--424
285 13 Osborne, H., Holtt, V. and Herz, A., Potassium-induced relea~e of enkephalin from rat striatal slices, Europ. J. Pharmacol., 48 (1978) 219-221. 14 Rotsztejn, W.H., Charli, J.L., Pat~ou, E., Epelbaum, I. and Kotdon. C., In vitro reieasc of iuteinizi~g hormone.releasing hormone (LHRH) from [at mediobasal hy[bothalamu.~. Effect:, of go~assium calcium a~ad dopamine, Endocrinology, 99 (1976~ 1663-- 1¢~66. 15 Said, S.I. and Rosea~erg, R.N., Vasoactive intL.stinal polypepfide: abudant immunorc~,clivi:y in neural cell lines and no~nal nervous tissue, Science. 192 (1976) 907--908. 16 Samson, W.K., Said, S.I. and McCann, S.M., Radioimmurto!ogic localization (,f vasoactivc intestinal polvpcptide in hypothalamie and extrahypothalamic sites in the rat brain. Ncuro~ci. [elt., 12 (1979) 265 -269. 17 Sims, K.B., ~-toffman, D.L., Said, S.I. and Zimmerman, E.A., Vasoactive intes~izaal potypcp~id¢ (VIP) in rr~ouse and rat brain: an immunocytochemical ~tudy, Brain Res., 186 C|9~;0) 165-183 18 Subramanian, N, and Muldet, A.M., Potassium-induced release of tritiated his~a.tl~.i~.cfrom rat [:t'3~.q tissue slice~ Euron. J. Pharmacol., 35 (1976) 377-380.