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Regional brain concentrations of vasoactive intestinal polypeptide in normotensive Wistar-Kyoto and spontaneously hypertensive rats
Brain Research, 399 (1986) 173-177 Elsevier
173
BRE21911
Regional brain concentrations of vasoactive intestinal polypeptide in normotensive Wistar-Kyoto and spontaneously hypertensive rats S.J. Lewis 1, A.
Shulkes 2 and
B.
Jarrott 1
l University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Heidelberg, Vic. (Australia) and 2Department of Surgery, Austin Hospital, Heidelberg, Vic. (Australia) (Accepted 5 August 1986)
Key words: Vasoactive intestinal polypeptide (VIP); Wistar-Kyoto rat; Spontaneously hypertensive rat; Brain; Radioimmunoassay
The regional brain and spinal cord concentrations of vasoactive intestinal polypeptide immunoreactivity (VIP) were measured in age-matched normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats. The relative order of distribution of VIP in the WKY strain was cortex (44 pmol/g) > hippocampus = striatum > midbrain = hypothalamus > medulla oblongata/pons = lumbar spinal cord (SC) > cervical SC > thoracic SC (2.5 pmol/g) whereas in the SH strain this order was cortex (35 pmol/g) > striatum = midbrain > hippocampus = hypothalamus > medulla oblongata/pons = lumbar SC > cervical SC > thoracic SC (1 pmol/g). The VIP concentrations of the thalamus, cerebellum and pituitary were at the level of assay sensitivity (0.5 pmol/g) in both strains. In comparison to the WKY, the SH rats had significantly lower VIP levels in the hippocampus (-42%) and cervical (-46%) and thoracic (-56%) spinal cord but significantly higher levels in the midbrain (+64%). Vasoactive intestinal p o l y p e p t i d e (VIP) was originally isolated and purified from porcine small intestine 33 and this 28-amino acid residue p e p t i d e was found to be structurally and biologically r e l a t e d to secretin, and to a lesser extent, glucagon and gastric inhibitory p e p t i d e ( G I P ) 30'34. Studies employing radioi m m u n o a s s a y techniques have detected high concentrations of V I P t h r o u g h o u t the gastrointestinal tract 23 and subsequently in various regions of the central nervous system 2'11A5. In addition, immunohistochemical studies have r e v e a l e d the presence of VIP-containing nerve cell bodies and terminals t h r o u g h o u t the brain 14'22'23'26 and that these terminals densely innervate the cerebral arteries 22 and choroid plexus 25. M o r e o v e r , VIP-containing neurons have also been identified in the digestive tract and in the female and male genitourinary systems 12'23. The synaptic vesicular localization of V I P in the brain 11'15, the d e m o n stration of a c a l c i u m - d e p e n d e n t p o t a s s i u m - e v o k e d release of V I P from superfused rat h y p o t h a l a m u s 11 and from s y n a p t o s o m e s 15 and the finding that V I P causes excitation of central neurons after iontophoretic application 9 suggests that this p e p t i d e m a y be released from nerve terminals during physiological
stimulation (depolarization) and t h e r e f o r e may have a role in synaptic function either as a neurotransmitter or as a n e u r o m o d u l a t o r of synaptic events. Although the precise function(s) of V I P within the brain are not known 12, VIP-containing cell bodies and varicose terminals have b e e n found t h r o u g h o u t the cerebral cortex and in the n e o c o r t e x and these terminals are located mainly within layers I I - I V (ref 14). The innervation by VIP-containing terminals of various hypothalamic nuclei involved in h o r m o n e secretion 14'26 suggests that this p e p t i d e m a y be involved in n e u r o e n d o c r i n e function. The finding that VIP terminals occur in the perivascular plexuses of cerebral arteries 22 and that this p e p t i d e produces a dose-related dilatation of these vessels in vitro 1° suggests that V I P is involved in neural regulation of cerebral b l o o d flow. Both p e r i p h e r a l and central V I P containing neurones a p p e a r to be involved in cardiovascular control. In the p e r i p h e r y this is s u p p o r t e d by several observations including that, (1) postganglionic (noncholinergic, non-adrenergic) p a r a s y m p a t h e t i c neurons containing V I P widely innervate the vasculature of the gastrointestinal tract 13, (2) V I P is found in the
Correspondence: B. Jarrott, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Vic. 3084, Australia. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
174 carotid body 4° and adrenal glands 12, (3) electrical stimulation of the vagus releases VIP into the circulation 35 and (4) the systemic administration of VIP produces potent, dose-dependent vasodilator and hypotensive responses 4'18. A possible role of central VIP in cardiovascular control is suggested by the presence of VIP terminals in the amygdala, the parabrachial nucleus and several hypothalamic nuclei thought to be involved in the regulation of blood pressure 14'23'26. Moreover, the i.c.v, injections of VIP (1-25 ktg) produced dose-dependent hypertensive responses in conscious normotensive rats 4. To date, the possible involvement of central VIP-containing neurons in the pathogenesis of the arterial hypertension 31 and/or the behavioural abnormalities 7'3s of the spontaneously hypertensive (SH) strain of rat has received little attention. In view of this, the aim of the present study was to investigate the regional brain and spinal cord concentrations of VIP in the SH strain and to compare these concentrations to those of age-matched normotensive Wistar-Kyoto (WKY) rats. Female 16-17-week-old WKY (n = 7) and SH (n = 7) rats from our animal house were decapitated between 14.00-16.00 h and the brains, pituitaries and spinal cords were removed and cooled on ice. The brains were dissected into the cortex, cerebellum, striatum, hippocampus, thalamus, hypothalamus, midbrain and medulla oblongata/pons and the spinal cords were divided into cervical, thoracic and lumbar (including sacral) segments 16'19. The tissues were immediately frozen in liquid nitrogen and stored at - 7 0 °C for 7-10 days before extraction. A standard method of sequential boiling water and acid was used for the extraction of tissue VIP 32'37. Five vols. of distilled water were added to the frozen tissue and heated for 10 min in a boiling water bath. The mixture was then sonicated and 5 vols. of 6% acetic acid added and heated for a further 10 min in a boiling water bath. At this stage an aliquot of the pituitary homogenate was taken for subsequent protein estimation 27. The sample was cooled and centrifuged at 10,000 g for 10 min. An aliquot of the supernatant was taken for protein estimation and the remammg supernatant stored at -30 °C until VIP radioimmunoassay (3-4 weeks). The recoveries of VIP and other gut peptides with this extraction procedure have been reported previously and generally exceed 90% 3'5'41. In the radioimmunoassay procedure, all
extracts were prediluted at least 1:10 in 0.02 M veronal buffer, p H 8.2, containing 0.1% bovine serum albumin and 1% Trasylol (Bayer). Full details of the VIP antiserum and assay have been reported previously8'21'36. The VIP antiserum (No. 6) was directed towards the amino terminal of the molecule. High-specific-activity 125I-VIP was purified by sequential gel and ion exchange chromatography and the IC50 was 25 fmol/tube. For the sake of brevity, the concentrations of VIP immunoreactivity will be referred to in the text as VIP. All values represent the mean + S.E.M. of the VIP concentrations, which are expressed as pmol/g wet weight (corrected for protein recovery in the extracted supernatant) or, in the case of the pituitary, pmol/g of protein (corrected for protein recovery in the supernatant). The significances of differences between WKY and SH values were determined by analysis of variance (ANOVA) whereas differences between regions in each strain were determined by A N O V A and Student's modified t-tests with the Bonferroni adjustment (of the P value) for multiple comparisons 39. The A N O V A ' s were performed with the aid of the BMDP Statistical Package (Department of Mathematics, University of Los Angeles, CA, U.S.A.). The regional brain and spinal cord VIP concentrations in the WKY and SH rats are shown in Fig. 1. Generally, the relative distributions of VIP were similar in the two strains, with the cortex showing the highest concentrations and the lowest measurable levels were in the thoracic SC. The relative order of distribution of VIP in the W K Y strain was cortex > hippocampus = striatum > hypothalamus = midbrain > medulla oblongata/pons = lumbar SC > cervical SC > thoracic SC whereas in the SH this order was cortex > striatum = midbrain > hippocampus = hypothalamus > medulla oblongata/pons = lumbar SC > cervical SC > thoracic SC. The VIP concentrations of the thalamus, cerebellum and pituitary were at the level of assay sensitivity (0.5 pmol/g). The absolute concentrations in the spinal cord differed between the two strains with the SH having significantly lower concentrations in the cervical SC ( - 4 6 % ) and thoracic SC (-56%). Other significant differences between the WKY and SH strains were that the SH had lower VIP concentrations in the hippocampus (-42%) but higher levels in the midbrain (+64%).
175 5o,
•
WKY
..,, 40,
SHR
30, a. "~ 20,
G. >
lO,
CORT
HIPPO
CSC
TSC
STRIA
HYPO
M-B
PIO/P
6 5 4
23 v el
2
> 1
~
0
LSC
THAL
~
~ CER
PIT
Fig. 1. The regional brain and spinal cord concentrations of VIP immunoreactivity in 16-17-week-old WKY and SH rats. Asterisks denote significant differences between the two rat strains (P < 0.05, Student's unpaired t-test). Note the difference in scales of the two panels. CORT, cortex; HIPPO, hippocampus; STRIA, striatum; HYPO, hypothalamus; MB, midbrain; MOP, medulla oblongata/pons; CSC, cervical spinal cord; TSC, thoracicspinal cord; LSC, lumbar (including sacral) spinal cord; CER, cerebellum; THAL, thalamus; PIT, pituitary. In the present study the relative distribution of VIP within the brain of the WKY rat shows a similar trend to those previously described for normotensive strains 2'1]'12 with the cortex containing the highest levels of this peptide. VIP is generally present in high concentrations within the rat cortex 2'1m2 and this peptide is the putative neurotransmitter of a major intracortical neuronal system 14'26. Recent evidence suggests that VIP systems within the anterior cingulate cortex may be involved in the processing of afferent and efferent information in the conscious rat 29 and within the neocortex may have a synaptic role in memory or motor control 14. The absolute concentrations of VIP found in this study are also consistent with published values except for the cortex and hypothalamus which have levels 2-3 x lower than previously reported 2"v'12. In addition, the levels of VIP in the thalamus were at the level of assay sensitivity (0.5 pmol/g) whereas concentrations of 9-10 pmol/g have been reported for this rat brain region 2"12. Interestingly, our findings seem to be better correlated
with those in other species in that low levels (0.3-0.6 pmol/g) of thalamic VIP have been found in man, dog and pig 12. The discrepancies between our findings and those previously reported for the rat 2'12 may be due to several factors such as differences in rat strain; time of day the animals were killed; the extraction methods, and the properties of the antisera used for radioimmunoassay. Although there is some controversy regarding the suitability of the WKY strain as a control for SH rats 31, this comparison is generally considered to be the most appropriate for neurochemical studies 24. The concentrations of VIP in the hippocampus of the SH rats were considerably lower (-42%) that those of the W g Y animals. The lower levels of VIP may be due to a decreased number of VIP containing neurons in the SH or alternatively there may be genetic differences in the biosynthesis, processing or degradation of VIP in these two strains. Immunohistochemical studies of the hippocampus have demonstrated that VIP-immunoreactivity is present in a considerable number of interneurons in the area dentata, CA3 and CA1 regions, and in the subiculum 26. Electrophysiological studies indicate that these interneurones may be excitatory 9. The hippocampus is an important part of the limbic system, and although the precise functions of this brain region have not been elucidated, roles in the processing of auditory, sensory motor and locomotor information, stress, anxiety, memory, learning and the integration of complex behaviours have been proposed 17. Although the importance of the lower VIP content in the hippocampus of the SH strain must remain speculative, many behavioural differences between the WKY and SH strains have been identified. For example, the SH rat displays a higher level of exploratory behaviour, responds excessively, both physiologically and behaviourally, to stressful stimuli, has a lower anxiety level; and shows increased acquisition of avoidance tasks 7'38. Perhaps some of these behavioural differences may be causually related to the reduced levels (and perhaps function) of VIP within the hippocampus. Within the spinal cord of the WKY and SH strain, the relative order of the VIP concentrations was lumbar > cervical > thoracic with the levels of VIP in the lumbar SC being ca. two and 4 x higher, respectively, than in the thoracic segment. These findings are in
176 agreement with a recent immunohistochemical/rad i o i m m u n o a s s a y study I which has d e m o n s t r a t e d a distinctive distribution of VIP-containing fibres and terminals at the l u m b o s a c r a l segments of human spinal cord and that the levels of V I P in these segments are m a r k e d l y higher than those of the cervical and thoracic regions. The crucial role of the spinal cord in cardiovasular regulation, and in particular the thoracic segment which contains the bulk of the sympathetic preganglionic neurons, is well established 6. In comparison to the W K Y rats, the S H strain was found to have significantly lower levels of V I P within the cervical and thoracic spinal cord segments. Previous studies in this l a b o r a t o r y have shown that the concentrations of n o r e p i n e p h r i n e 2° and n e u r o p e p t i d e y28 in the thoracic spinal cord of SH rats were lower than those of agem a t c h e d W K Y animals. W h e t h e r or not the lower concentrations of these putative transmitters are causally involved in the higher b l o o d pressure of the SH strain remains to be established. The midbrain was the only region in which the concentration of V I P was found to be higher in the SH strain. This brain region contains discrete nuclear structures such as the a m y g d a l a and m o r e diffuse regions including the mesencephalic reticular formation and p e r i a q u e d u c t a l gray area, which have imp o r t a n t roles in b o t h cardiovascular and behavioural regulation 6'17'42. The a m y g d a l a receives a dense innervation of VIP-containing fibres 14'26, although
m a n y areas of the midbrain receive high to m o d e r a t e numbers of these fibres 26. A s such the increased levels of V I P within the m i d b r a i n of the SH rat m a y be of significance to this strain. In s u m m a r y , the concentrations of V I P i m m u n o r e activity within the h i p p o c a m p u s , cervical and thoracic spinal cord of the SH strain are lower whereas these concentrations within the m i d b r a i n are higher, than age-matched n o r m o t e n s i v e W i s t a r - K y o t o rats. These differences m a y reflect an alteration in the activities of VIP-containing neurons within the S H rat which m a y in turn contribute to the pathogenesis of the higher b l o o d pressure and behavioural differences in this strain. H o w e v e r , the m e a s u r e m e n t of VIP content cannot distinguish between changes in synthesis as c o m p a r e d to release nor can they determine w h e t h e r the altered V I P levels are causally involved in or result as a consequence of the h y p e r t e n sion. F u r t h e r studies including p e p t i d e t u r n o v e r rates and antihypertensive t r e a t m e n t will help d e t e r m i n e the significance of the strain differences in V I P contents.
1 Anand, P., Gibson, S.J., McGregor, G.P., Blank, M.A., Ghatei, M.A., Bacarese-Hamilton, A.J., Polak, J.M. and Bloom, S.R., A VIP-containing system concentrated in the lumbosacral region of the spinal cord, Nature (London), 305 (1983) 143-145. 2 Besson, J., Rotsztejn, W., Laburthe, M., Epelbaum, J., Beaudet, A., Kordon, C. and Rosselin, G., Vasoactive intestinal peptide (VIP): brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats, Brain Research, 165 (1979) 79-85. 3 Bryant, M.G., Bloom, S.R., Polak, J.M., Hobbs, S., Domscheke, W., Domscheke, S., Mitznegg, P., Ruppin, H:' and Demling, L., Measurement of gut hormonal peptides in biopsies from human stomach and proximal small intestine, Gut, 24 (1983) 11- 119. 4 Casio, R. and Phillips, M.I., The opposite effects of central and peripheral vasoactive intestinal polypeptide on blood pressure in rats, Neurosci. Lett., 28 (1983) 169-173. 5 Chayvialle, J.E., Miyata, M., Rayford, P.L. and Thompson, J.C., Immunoreactive somatostatin and vasoactive intestinal polypeptide in the digestive tract of cats, Gastroen.. terology, 79 (1980) 837-843.
6 Dampney, R.A.L., Functional organization of central cardiovascular pathways, Clin. Exp. Pharmacol. Physiol., 8 (1981) 241-259. 7 Danysz, W., Plaznik, A., Pucilowski, O., Plewako, M., Obersztyn, M. and Kostowski, W., Behavioural studies in spontaneously hypertensive rats, Behav. Neurol. Biol., 39 (1983) 22-29. 8 Dimaline, R., Vaillant, C. and Dockray, G.J., The use of region specific antibodies in the characterization and localization of vasoactive intestinal polypeptide like substances in the rat gastrointestinal tract, Regul. Peptides, 1 (1980) 1-16. 9 Dodd, J., Kelly, J.S. and Said, S.J., Excitation of CA1 neurons of the rat hippocampus by the octacosapeptide vasoactive intestinal polypeptide (VIP), Brit. J. Pharmacol., 66 (1979) 125P. 10 Duckies, S.P. and Said, S.I., Vasoactive intestinal peptide as a neurotransmitter in the cerebral circulation, Eur. J. Pharmacol., 78 (1982) 371-374. 11 Emson, P.C., Fahrenkrug, J., Schaffalitsky de Muckadell, O.B., Jessel, T.M. and Iversen, L.L., Vasoactive intestinal polypeptide (VIP): vesicular localization and potassium-
This w o r k was s u p p o r t e d by a p r o g r a m grant from the National H e a l t h and Medical Research Council of Australia. The authors would like to thank Brett Bodsworth and Michael C h r i s t o p h e r for expert technical assistance and Miss Allison Quinn for the efficient typing of the manuscript.
177 evoked release from rat hypothalamus, Brain Research, 143 (1978) 174-178. 12 Fahrenkrug, J., Vasoactive intestinal polypeptide: measurement, distribution and putative neurotransmitter function, Digestion, 19 (1979) 149-169. 13 Fahrenkrug, J., Galbo, H., Hoist, J.J. and Schaffalitzky de Muckadell, O.B., Influence of the autonomic nervous system on the release of vasoactive intestinal polypeptide from the porcine gastrointestinal tract, J. Physiol. (London), 280 (1978) 405-422. 14 Fuxe, K., H6kfelt, T., Said, S.I. and Mutt, V., Vasoactive intestinal polypeptide and the nervous system: immunohistochemical evidence for localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex, Neurosci. Lett., 5 (1977) 241-246. 15 Giachetti, A., Said, S.I., Reynolds, R. and Koniges, F.C., Vasoactive intestinal polypeptide in brain: localization in and release from isolated nerve terminals, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3424-3428. 16 Glowinski, J. and Iversen, L.L., Regional studies of catecholamines in rat brain. 1, J. Neurochem., 13 (1966) 655-669. 17 Guyton, A.C., Behavioural functions of the brain. The limbic system, and role of the hypothalamus. In A.C. Guyton (Ed.), Textbook of Medical Physiology, Saunders, Philadelphia, 1971, pp. 729-735. 18 Holliger, C,, Radzymer, M., Villiger, A., Anliker, M. and Knoblauch, M., Effects of glucagon, vasoactive intestinal polypeptide (VIP) and lysine-vasopressin on villous microcirculation and superior mesenteric blood flow in the rat, Bibl. Anat., 18 (1979) 129-133. 19 Holman, R.B., Angwin, P. and Barchas, J.D., Simultaneous determination of indoles and catecholamines in small brain regions in the rat using a weak cation exchange resin, Neuroscience, 1 (1976) 147-150. 20 Howes, L.G., Rowe, P.R., Summers, R.J. and Louis, W.J., Age related change s of noradrenaline content in brain regions of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats, Clin. Exp. Hypertens., A5 (1983) 857-874. 21 Krejs, G.J., Fortran, J.S., Bloom, S.R., Fahrenkrug, J.. Schaffalitzky de Muckadell, O.B., Fischer, J.E., Humphreys, D.S., O'Dorisio, T.M., Said, S.I., Walsh, J.A. and Shulkes, A., Effect of VIP infusion on water and ion transport in the human jejunum, Gastroenterology, 75 (1980) 722-727. 22 Larsson, L.I., Edvinsson, L., Fahrenkrug, J., Hakanson, R., Owman, Ch., Schaffalitzky de Muckadell, O.B. and Sundler, F., Immunohistochemical localization of a vasodilatory polypeptide (VIP) in cerebrovascular nerves, Brain Research, 113 (1976) 400-404. 23 Larsson, L.I., Fahrenkrug, J., Schaffalitzky de Muckadell, O.B., Sundler, F., Hakanson, R. and Rehfeld, J.F., Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons, Proc. Natl. Acad, Sci. U.S.A., 73 (1976) 3197-3200. 24 Lew, J.Y., Hata, F., Santer, A., Baba, Y., Engel, J. and Goldstein, M., Distribution of PNMT and epinephrine in the medulla oblongata of normotensive and spontaneously hypertensive rats, J. Neural. Transm., 44 (1979) 309-316. 25 Lindvall, M., Alumets, J., Edvinsson, L., Fahrenkrug, J., Hakanson, R., Hanko, J., Schaffalitzky de Muckadell, O.B. and Sundler, F., Peptidergic (VIP) nerves in the
mammalian choroid plexus, Neurosci. Lett., 9 (1978) 77-82. 26 Loren, I., Emson, P.C., Fahrenkrug, J., Bj6rklund, A., Alumets, J., Hakanson, R. and Sundler, F., Distribution of vasoactive intestinal polypeptide in the rat and mouse brain, Neuroscience, 4 (1979) 1953-1976. 27 Lowry, O., Rosebrough, N., Farr, A. and Randall, R., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 28 Maccarrone, C. and Jarrott, B., Differences in regional brain concentrations of neuropeptide Y in spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats, Brain Research, 345 (1985) 165-169. 29 McCulloch, J. and Kelly, P.A.T., A functional role for vasoactive intestine polypeptide in the anterior cingulate cortex, Nature (London), 304 (1983) 438-440. 30 Mutt, V. and Said, S.I., Structure of the porcine vasoactive intestinal octacosapeptide, the amino-acid sequence. Use of kallikrein in its determination, Europ. J. Biochem., 42 (1974) 581-589. 31 Rapp, J.P., A paradigm for identification of primary genetic causes of hypertension in rats, Hypertension, 5 (Suppl. 1) (1983) 198-203. 32 Rehfeld, J.F., Immunochemical studies on cholecystokinin, J. Biol Chem., 253 (1978) 4022-4030. 33 Said, S.I. and Mutt, V., Polypeptide with broad biological activity: isolation from small intestine, Science, 169 (1970) 1217-1218. 34 Said, S.I. and Mutt, V., Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and glucagon, Europ. J. Biochem., 28 (1972) 199-204. 35 Schaffalitzky de Muckadell, O.B., Fahrenkrug, J. and Hoist, J.J., Release of vasoactive intestinal polypeptide (VIP) by electrical stimulation of the vagal nerves, Gastroenterology, 72 (1977) 373-375. 36 Shulkes, A., Boden, R., Cook, I., Gallagher, N. and Furhess, J.B., Characterization of a pancreatic tumor containing vasoactive intestinal polypeptide, neurotensin and pancreatic polypeptide, J. Clin. Endocrinol. Metab., 58 (1984) 41-48. 37 Shulkes, A., Caussignas, Y., Lamers, C.B., Solomon, T.E., Yamada, T. and Walsh, J.H., Starvation in the rat: effect on peptides of the gut and brain, Aust. J. Exp. Biol. Med. Sci., 61 (1983) 581-587. 38 Tucker, D.C. and Johnson, A.K., Behavioural correlates of spontaneous hypertension, Neurosci. Biobehav. Rev., 5 (1981) 463-471. 39 Wallenstein, S., Zucker, C.L. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1-9. 40 Wharton, J., Polak, J.M., Pearce, A.G.E., McGregor, G.P., Bryant, M.G., Bloom, S.R., Emson, P.C., Bisgard, G.E. and Will, J.A., Enkephalin-, VIP- and substance Plike immunoreactivity in the carotid body, Nature (London), 284 (1980) 269-271. 41 Yamada, T., Marshak, D., Basinger, S., Walsh, J., Marley, J. and Stell, W., Somatostatin-like immunoreactivity in the retina, Proc. Nat. Acad. Sci. U.S.A., 77 (1980) 1691-1695. 42 Zanchetti, A., Subcortical and cortical mechanisms in arousal and emotional behaviour. In G.C. Quarton, T. Melnechuk and F.O. Schmitt (Eds.), The Neurosciences, The Rockefeller University Press, New York, 1967, pp. 6O2-624.
173
BRE21911
Regional brain concentrations of vasoactive intestinal polypeptide in normotensive Wistar-Kyoto and spontaneously hypertensive rats S.J. Lewis 1, A.
Shulkes 2 and
B.
Jarrott 1
l University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Heidelberg, Vic. (Australia) and 2Department of Surgery, Austin Hospital, Heidelberg, Vic. (Australia) (Accepted 5 August 1986)
Key words: Vasoactive intestinal polypeptide (VIP); Wistar-Kyoto rat; Spontaneously hypertensive rat; Brain; Radioimmunoassay
The regional brain and spinal cord concentrations of vasoactive intestinal polypeptide immunoreactivity (VIP) were measured in age-matched normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats. The relative order of distribution of VIP in the WKY strain was cortex (44 pmol/g) > hippocampus = striatum > midbrain = hypothalamus > medulla oblongata/pons = lumbar spinal cord (SC) > cervical SC > thoracic SC (2.5 pmol/g) whereas in the SH strain this order was cortex (35 pmol/g) > striatum = midbrain > hippocampus = hypothalamus > medulla oblongata/pons = lumbar SC > cervical SC > thoracic SC (1 pmol/g). The VIP concentrations of the thalamus, cerebellum and pituitary were at the level of assay sensitivity (0.5 pmol/g) in both strains. In comparison to the WKY, the SH rats had significantly lower VIP levels in the hippocampus (-42%) and cervical (-46%) and thoracic (-56%) spinal cord but significantly higher levels in the midbrain (+64%). Vasoactive intestinal p o l y p e p t i d e (VIP) was originally isolated and purified from porcine small intestine 33 and this 28-amino acid residue p e p t i d e was found to be structurally and biologically r e l a t e d to secretin, and to a lesser extent, glucagon and gastric inhibitory p e p t i d e ( G I P ) 30'34. Studies employing radioi m m u n o a s s a y techniques have detected high concentrations of V I P t h r o u g h o u t the gastrointestinal tract 23 and subsequently in various regions of the central nervous system 2'11A5. In addition, immunohistochemical studies have r e v e a l e d the presence of VIP-containing nerve cell bodies and terminals t h r o u g h o u t the brain 14'22'23'26 and that these terminals densely innervate the cerebral arteries 22 and choroid plexus 25. M o r e o v e r , VIP-containing neurons have also been identified in the digestive tract and in the female and male genitourinary systems 12'23. The synaptic vesicular localization of V I P in the brain 11'15, the d e m o n stration of a c a l c i u m - d e p e n d e n t p o t a s s i u m - e v o k e d release of V I P from superfused rat h y p o t h a l a m u s 11 and from s y n a p t o s o m e s 15 and the finding that V I P causes excitation of central neurons after iontophoretic application 9 suggests that this p e p t i d e m a y be released from nerve terminals during physiological
stimulation (depolarization) and t h e r e f o r e may have a role in synaptic function either as a neurotransmitter or as a n e u r o m o d u l a t o r of synaptic events. Although the precise function(s) of V I P within the brain are not known 12, VIP-containing cell bodies and varicose terminals have b e e n found t h r o u g h o u t the cerebral cortex and in the n e o c o r t e x and these terminals are located mainly within layers I I - I V (ref 14). The innervation by VIP-containing terminals of various hypothalamic nuclei involved in h o r m o n e secretion 14'26 suggests that this p e p t i d e m a y be involved in n e u r o e n d o c r i n e function. The finding that VIP terminals occur in the perivascular plexuses of cerebral arteries 22 and that this p e p t i d e produces a dose-related dilatation of these vessels in vitro 1° suggests that V I P is involved in neural regulation of cerebral b l o o d flow. Both p e r i p h e r a l and central V I P containing neurones a p p e a r to be involved in cardiovascular control. In the p e r i p h e r y this is s u p p o r t e d by several observations including that, (1) postganglionic (noncholinergic, non-adrenergic) p a r a s y m p a t h e t i c neurons containing V I P widely innervate the vasculature of the gastrointestinal tract 13, (2) V I P is found in the
Correspondence: B. Jarrott, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Vic. 3084, Australia. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
174 carotid body 4° and adrenal glands 12, (3) electrical stimulation of the vagus releases VIP into the circulation 35 and (4) the systemic administration of VIP produces potent, dose-dependent vasodilator and hypotensive responses 4'18. A possible role of central VIP in cardiovascular control is suggested by the presence of VIP terminals in the amygdala, the parabrachial nucleus and several hypothalamic nuclei thought to be involved in the regulation of blood pressure 14'23'26. Moreover, the i.c.v, injections of VIP (1-25 ktg) produced dose-dependent hypertensive responses in conscious normotensive rats 4. To date, the possible involvement of central VIP-containing neurons in the pathogenesis of the arterial hypertension 31 and/or the behavioural abnormalities 7'3s of the spontaneously hypertensive (SH) strain of rat has received little attention. In view of this, the aim of the present study was to investigate the regional brain and spinal cord concentrations of VIP in the SH strain and to compare these concentrations to those of age-matched normotensive Wistar-Kyoto (WKY) rats. Female 16-17-week-old WKY (n = 7) and SH (n = 7) rats from our animal house were decapitated between 14.00-16.00 h and the brains, pituitaries and spinal cords were removed and cooled on ice. The brains were dissected into the cortex, cerebellum, striatum, hippocampus, thalamus, hypothalamus, midbrain and medulla oblongata/pons and the spinal cords were divided into cervical, thoracic and lumbar (including sacral) segments 16'19. The tissues were immediately frozen in liquid nitrogen and stored at - 7 0 °C for 7-10 days before extraction. A standard method of sequential boiling water and acid was used for the extraction of tissue VIP 32'37. Five vols. of distilled water were added to the frozen tissue and heated for 10 min in a boiling water bath. The mixture was then sonicated and 5 vols. of 6% acetic acid added and heated for a further 10 min in a boiling water bath. At this stage an aliquot of the pituitary homogenate was taken for subsequent protein estimation 27. The sample was cooled and centrifuged at 10,000 g for 10 min. An aliquot of the supernatant was taken for protein estimation and the remammg supernatant stored at -30 °C until VIP radioimmunoassay (3-4 weeks). The recoveries of VIP and other gut peptides with this extraction procedure have been reported previously and generally exceed 90% 3'5'41. In the radioimmunoassay procedure, all
extracts were prediluted at least 1:10 in 0.02 M veronal buffer, p H 8.2, containing 0.1% bovine serum albumin and 1% Trasylol (Bayer). Full details of the VIP antiserum and assay have been reported previously8'21'36. The VIP antiserum (No. 6) was directed towards the amino terminal of the molecule. High-specific-activity 125I-VIP was purified by sequential gel and ion exchange chromatography and the IC50 was 25 fmol/tube. For the sake of brevity, the concentrations of VIP immunoreactivity will be referred to in the text as VIP. All values represent the mean + S.E.M. of the VIP concentrations, which are expressed as pmol/g wet weight (corrected for protein recovery in the extracted supernatant) or, in the case of the pituitary, pmol/g of protein (corrected for protein recovery in the supernatant). The significances of differences between WKY and SH values were determined by analysis of variance (ANOVA) whereas differences between regions in each strain were determined by A N O V A and Student's modified t-tests with the Bonferroni adjustment (of the P value) for multiple comparisons 39. The A N O V A ' s were performed with the aid of the BMDP Statistical Package (Department of Mathematics, University of Los Angeles, CA, U.S.A.). The regional brain and spinal cord VIP concentrations in the WKY and SH rats are shown in Fig. 1. Generally, the relative distributions of VIP were similar in the two strains, with the cortex showing the highest concentrations and the lowest measurable levels were in the thoracic SC. The relative order of distribution of VIP in the W K Y strain was cortex > hippocampus = striatum > hypothalamus = midbrain > medulla oblongata/pons = lumbar SC > cervical SC > thoracic SC whereas in the SH this order was cortex > striatum = midbrain > hippocampus = hypothalamus > medulla oblongata/pons = lumbar SC > cervical SC > thoracic SC. The VIP concentrations of the thalamus, cerebellum and pituitary were at the level of assay sensitivity (0.5 pmol/g). The absolute concentrations in the spinal cord differed between the two strains with the SH having significantly lower concentrations in the cervical SC ( - 4 6 % ) and thoracic SC (-56%). Other significant differences between the WKY and SH strains were that the SH had lower VIP concentrations in the hippocampus (-42%) but higher levels in the midbrain (+64%).
175 5o,
•
WKY
..,, 40,
SHR
30, a. "~ 20,
G. >
lO,
CORT
HIPPO
CSC
TSC
STRIA
HYPO
M-B
PIO/P
6 5 4
23 v el
2
> 1
~
0
LSC
THAL
~
~ CER
PIT
Fig. 1. The regional brain and spinal cord concentrations of VIP immunoreactivity in 16-17-week-old WKY and SH rats. Asterisks denote significant differences between the two rat strains (P < 0.05, Student's unpaired t-test). Note the difference in scales of the two panels. CORT, cortex; HIPPO, hippocampus; STRIA, striatum; HYPO, hypothalamus; MB, midbrain; MOP, medulla oblongata/pons; CSC, cervical spinal cord; TSC, thoracicspinal cord; LSC, lumbar (including sacral) spinal cord; CER, cerebellum; THAL, thalamus; PIT, pituitary. In the present study the relative distribution of VIP within the brain of the WKY rat shows a similar trend to those previously described for normotensive strains 2'1]'12 with the cortex containing the highest levels of this peptide. VIP is generally present in high concentrations within the rat cortex 2'1m2 and this peptide is the putative neurotransmitter of a major intracortical neuronal system 14'26. Recent evidence suggests that VIP systems within the anterior cingulate cortex may be involved in the processing of afferent and efferent information in the conscious rat 29 and within the neocortex may have a synaptic role in memory or motor control 14. The absolute concentrations of VIP found in this study are also consistent with published values except for the cortex and hypothalamus which have levels 2-3 x lower than previously reported 2"v'12. In addition, the levels of VIP in the thalamus were at the level of assay sensitivity (0.5 pmol/g) whereas concentrations of 9-10 pmol/g have been reported for this rat brain region 2"12. Interestingly, our findings seem to be better correlated
with those in other species in that low levels (0.3-0.6 pmol/g) of thalamic VIP have been found in man, dog and pig 12. The discrepancies between our findings and those previously reported for the rat 2'12 may be due to several factors such as differences in rat strain; time of day the animals were killed; the extraction methods, and the properties of the antisera used for radioimmunoassay. Although there is some controversy regarding the suitability of the WKY strain as a control for SH rats 31, this comparison is generally considered to be the most appropriate for neurochemical studies 24. The concentrations of VIP in the hippocampus of the SH rats were considerably lower (-42%) that those of the W g Y animals. The lower levels of VIP may be due to a decreased number of VIP containing neurons in the SH or alternatively there may be genetic differences in the biosynthesis, processing or degradation of VIP in these two strains. Immunohistochemical studies of the hippocampus have demonstrated that VIP-immunoreactivity is present in a considerable number of interneurons in the area dentata, CA3 and CA1 regions, and in the subiculum 26. Electrophysiological studies indicate that these interneurones may be excitatory 9. The hippocampus is an important part of the limbic system, and although the precise functions of this brain region have not been elucidated, roles in the processing of auditory, sensory motor and locomotor information, stress, anxiety, memory, learning and the integration of complex behaviours have been proposed 17. Although the importance of the lower VIP content in the hippocampus of the SH strain must remain speculative, many behavioural differences between the WKY and SH strains have been identified. For example, the SH rat displays a higher level of exploratory behaviour, responds excessively, both physiologically and behaviourally, to stressful stimuli, has a lower anxiety level; and shows increased acquisition of avoidance tasks 7'38. Perhaps some of these behavioural differences may be causually related to the reduced levels (and perhaps function) of VIP within the hippocampus. Within the spinal cord of the WKY and SH strain, the relative order of the VIP concentrations was lumbar > cervical > thoracic with the levels of VIP in the lumbar SC being ca. two and 4 x higher, respectively, than in the thoracic segment. These findings are in
176 agreement with a recent immunohistochemical/rad i o i m m u n o a s s a y study I which has d e m o n s t r a t e d a distinctive distribution of VIP-containing fibres and terminals at the l u m b o s a c r a l segments of human spinal cord and that the levels of V I P in these segments are m a r k e d l y higher than those of the cervical and thoracic regions. The crucial role of the spinal cord in cardiovasular regulation, and in particular the thoracic segment which contains the bulk of the sympathetic preganglionic neurons, is well established 6. In comparison to the W K Y rats, the S H strain was found to have significantly lower levels of V I P within the cervical and thoracic spinal cord segments. Previous studies in this l a b o r a t o r y have shown that the concentrations of n o r e p i n e p h r i n e 2° and n e u r o p e p t i d e y28 in the thoracic spinal cord of SH rats were lower than those of agem a t c h e d W K Y animals. W h e t h e r or not the lower concentrations of these putative transmitters are causally involved in the higher b l o o d pressure of the SH strain remains to be established. The midbrain was the only region in which the concentration of V I P was found to be higher in the SH strain. This brain region contains discrete nuclear structures such as the a m y g d a l a and m o r e diffuse regions including the mesencephalic reticular formation and p e r i a q u e d u c t a l gray area, which have imp o r t a n t roles in b o t h cardiovascular and behavioural regulation 6'17'42. The a m y g d a l a receives a dense innervation of VIP-containing fibres 14'26, although
m a n y areas of the midbrain receive high to m o d e r a t e numbers of these fibres 26. A s such the increased levels of V I P within the m i d b r a i n of the SH rat m a y be of significance to this strain. In s u m m a r y , the concentrations of V I P i m m u n o r e activity within the h i p p o c a m p u s , cervical and thoracic spinal cord of the SH strain are lower whereas these concentrations within the m i d b r a i n are higher, than age-matched n o r m o t e n s i v e W i s t a r - K y o t o rats. These differences m a y reflect an alteration in the activities of VIP-containing neurons within the S H rat which m a y in turn contribute to the pathogenesis of the higher b l o o d pressure and behavioural differences in this strain. H o w e v e r , the m e a s u r e m e n t of VIP content cannot distinguish between changes in synthesis as c o m p a r e d to release nor can they determine w h e t h e r the altered V I P levels are causally involved in or result as a consequence of the h y p e r t e n sion. F u r t h e r studies including p e p t i d e t u r n o v e r rates and antihypertensive t r e a t m e n t will help d e t e r m i n e the significance of the strain differences in V I P contents.
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6 Dampney, R.A.L., Functional organization of central cardiovascular pathways, Clin. Exp. Pharmacol. Physiol., 8 (1981) 241-259. 7 Danysz, W., Plaznik, A., Pucilowski, O., Plewako, M., Obersztyn, M. and Kostowski, W., Behavioural studies in spontaneously hypertensive rats, Behav. Neurol. Biol., 39 (1983) 22-29. 8 Dimaline, R., Vaillant, C. and Dockray, G.J., The use of region specific antibodies in the characterization and localization of vasoactive intestinal polypeptide like substances in the rat gastrointestinal tract, Regul. Peptides, 1 (1980) 1-16. 9 Dodd, J., Kelly, J.S. and Said, S.J., Excitation of CA1 neurons of the rat hippocampus by the octacosapeptide vasoactive intestinal polypeptide (VIP), Brit. J. Pharmacol., 66 (1979) 125P. 10 Duckies, S.P. and Said, S.I., Vasoactive intestinal peptide as a neurotransmitter in the cerebral circulation, Eur. J. Pharmacol., 78 (1982) 371-374. 11 Emson, P.C., Fahrenkrug, J., Schaffalitsky de Muckadell, O.B., Jessel, T.M. and Iversen, L.L., Vasoactive intestinal polypeptide (VIP): vesicular localization and potassium-
This w o r k was s u p p o r t e d by a p r o g r a m grant from the National H e a l t h and Medical Research Council of Australia. The authors would like to thank Brett Bodsworth and Michael C h r i s t o p h e r for expert technical assistance and Miss Allison Quinn for the efficient typing of the manuscript.
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