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Subcellular distribution of neuropeptide Y-like immunoreactivity in guinea pig neocortex
354
Brain Researcfi, 3~5 ~'?,?,~5)354 ~59
BRE 20829
Subcellular distribution of neuropeptide Y-like immunoreactivity in guinea pig neocortex MARION E. DE QUIDT 1, PETER J. RICHARDSON 2 and PIERS C. EMSON 1 1MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School and 2Department of Clinical Biochemistry, University of Cambridge, A ddenbrooke's Hospital, Cambridge CB2 2QR (U, K, ) (Accepted January 3rd, 1985) Key words: neuropeptide Y - - somatostatin - - noradrenaline - - subcellular fractionation - - neocortex - - chromatography
Neuropeptide Y-like immunoreactivity (NPY-LI) was enriched in synaptosomal fractions of neocortex, which on lysis yielded vesicle-rich fractions. The distribution of NPY-LI on a sucrose density gradient was similar to that of somatostatin, with a concentration in heavy vesicles. The peptides were not found in light vesicles in contrast to the distribution of noradrenaline. Both homogenate and vesicular NPY-LI coeluted with synthetic NPY on reverse-phase HPLC.
N e u r o p e p t i d e Y (NPY) was isolated from porcine brain extracts 30 and its amino acid sequence determined in 198229. Subsequently, a h u m a n N P Y sequence has been d e t e r m i n e d from p h a e o c h r o m o c y toma 4. M o r e recently, the h u m a n m R N A encoding the N P Y precursor has been cloned and its nucleotide sequence d e t e r m i n e d 2t, confirming the N P Y sequence of C o r d e r et al. 4. NPY-like immunoreactivity ( N P Y - L I ) has been m e a s u r e d by r a d i o i m m u n o a s s a y in extracts from m a n y regions of m a m m a l i a n brain~,56,12 and found to have a non-uniform distribution. In addition, immunohistochemical studies have revealed a neuronal localization of N P Y - L I in the CNS of ratl,12A 4, h u m a n 6, m o n k e y 17 and cat ( D e Quidt, unpublished observations). The present study investigated the subcellular distribution of N P Y - L I in neocortex, using biochemical fractionation techniques and by comparing the NPY distribution with that of a variety of m a r k e r enzymes and two putative n e u r o t r a n s m i t t e r s - - somatostatin (SRIF) and n o r a d r e n a l i n e ( N A ) . A d u l t female guinea pigs (250-400 g) were stunned and decapitated. T h e brain was rapidly removed and the n e o c o r t e x p e e l e d off and placed on ice. Subcellular fractionation was carried out in two
stages. The first involved the p r e p a r a t i o n of a crude mitochondrial fraction (P2), largely by the m e t h o d of Gray and W h i t t a k e r 16 as described by Richardson et al. 25. The second stage of fractionation involved hypo-osmotic lysis of the s y n a p t o s o m e fraction P2 by suspension in 10 n M H e p e s p H 7.4 for 10 min at 4 °C. followed by fractionation of the lysate on a discontinuous sucrose density gradient 31, Samples of all fractions were assayed for total protein content 3 and a series of enzyme activities. 5'-Nucleotidase (EC 3.1.3.5) was used as a m a r k e r for plasma m e m b r a n e s and assayed by the m e t h o d of Stanley et al. 27. Lactate d e h y d r o g e n a s e ( L D H : EC 1.1.1.27), a cytoplasmic m a r k e r , and fumarase (EC 4.2.1.2), a mitochondrial m a r k e r , were assayed by the methods of Keiding et al.18 and Racker23. respectively. A d d i t i o n a l samples of all fractions were m a d e to 50 mM HCI, in o r d e r both to inhibit enzyme activity (proteases, catecholamine catabolising enzymes) and to extract both peptides and N A . This was carried out at 4 °C to minimise the oxidation of N A . After centrifugation (10,000 g, 4 min) to r e m o v e cell debris, the supernatants were assayed directly for NPYand S R I F - L I by radioimmunoassay. Both assays
Correspondence: M. E. de Quidt, MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Hills Road, Cambridge CB2 2QH, U.K. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
355 were able to tolerate up to one third total assay volume of concentrated (1 M) sucrose with 50 mM HC1 or 10 mM Hepes per 50 mM HC1 without non-specific interference in antiserum-tracer binding. The antisera crossreactivities and assay characteristics have been described previously (SRIF: C-terminal assayS; NPY: ref. 11, with modifications using Bolton-Hunter [125I]NPY as tracer (Amersham, S.A. - 2 0 0 0 Ci/mmol) and synthetic porcine NPY as standard (Bachem, U.S.A.), which did not alter antiserum crossreactivity characteristics (De Quidt, in preparation)). The assay buffer (50 mM phosphate/0.1% bovine serum albumin (BSA; R I A grade, Sigma)/50 mM E D T A , pH 7.4) contained trasylol (200 kIU/ml, Sigma) as an additional peptidase inhibitor, which did not alter the assay characteristics. Serial dilution of the subcellular fractions in both NPY and SRIF radioimmunoassays demonstrated that the extracted immunoreactivities diluted in parallel with the synthetic peptide standards. NA was measured by reverse-phase HPLC with electrochemical detection essentially as described by Reynolds 24. Fractions were diluted 1:3 to 1:5 with 1.5 Tris/0.2 mM E D T A and mixed with 20 mg activated alumina for 20 min. The HPLC buffer was 0.1 M phosphate pH 3.6, not phosphate-citrate as previously described 24. Recovery of internal standard (3,4-dihydroxybenzylamine) was 81.2 + 2.5% (n = 24) from homogenate and fractions P 1 - S 2 , and 71.5 _+ 1.6% (n = 40) from fractions W (supernatant)-I (mean _+ S.E.M.). Further characterization of the subcellular NPYLI was carried out by means of reverse-phase HPLC using a Waters Associates/~Bondapak C18 column (30 x 0.39 cm) with an acetonitrile gradient (2
ml/min) as shown in Fig. 2D. 0.1% trifluoroacetic acid (TFA) was present throughout. 2-ml fractions were collected, dried at 50 °C (12-18 h) and the remaining aqueous solvents then freeze-dried. Fractions were reconstituted in assay buffer for radioimmunoassay. The following NPY-like synthetic peptides were used to characterise the gradient: porcine NPY, porcine peptide YY (PYY, Bachem, U.S.A.) and avian pancreatic polypeptide (APP, Bachem, U.S.A.). Differential centrifugation. The separation of enzyme, peptide and NA activities are summarised in Table I. The absolute amounts of substances measured in the homogenate were as follows (mean _+ S.E.M. from 3 or 4 experiments unless otherwise stated): total protein 124.7 _+ 33.4 mg; 5'-nucleotidase 1.93 + 0.23 ymol/h/mg protein; L D H 1.98 + 0.60 k~mol/min/mg protein; fumarase 0.46 + 0.20 ¢tmol/min/mg protein; SRIF-LI 3.65 + 2.22 pmol/mg protein; NPY-LI 4.65 + 1.05 pmol/mg protein; NA 3.85, 3.42 pmol/mg protein (n = 2). The recovery of all activities in P1 with S 1 from the homogenate was > 95% and in P2 with $2, from Sl, was > 90% for protein, enzymes and NA, 82.0 + 7.3% for SRIF-LI and 65.5 _ 10.2% for NPY-LI. An enrichment (relative specific activity (RSA) > 1) of 5'-nucleotidase, fumarase, SRIF-LI, NPY-LI and NA was detected in P1 which has been shown to contain cell debris including cell bodies and nuclei 16. This represented between one third to one half of total homogenate enzyme and transmitter activity. Fraction P2 contained enriched levels of these enzymes and both peptide immunoreactivities. Isopycnic centrifugation. The fractionation of subcellular markers and putative neurotransmitters is
TABLE I
Fractionation of guinea pig neocortex homogenate by differential centrifugation Fractions were prepared by two centrifugation steps, the first at 1000 g, 10 min at 4 °C to produce P1 and S 1 from the homogenate, the second at 2,000 g for 20 rain at 4 °C to form P2 and S2 from S v RSA values of enzyme of neurochemical markers are given as means _+ S.E.M. for 3 - 4 separate experiments (NA two experiments). The RSA values are defined as (% of total recovered activity in fraction)/(% of total recovered protein in fraction).
Fraction
P1 S1 P2 S2
Relative specific activity 5'-Nucleotidase
Lactate dehydrogenase
Fumarase
SRIF-L1
NPY-LI
NA
1.16 0.95 1.23 0.59
0.94 1.05 0.62 1.89
1.66 0.78 1.38 0.31
1.47 0.82 1.45 0.26
1.54 0.78 1.37 0.44
1.95, 1.87 0.65, 0.62 0.6, 1.14 1.97, 1.09
_+ 0.09 + 0.05 + 0.09 + 0.09
+ 0.06 + 0.04 + 0.12 +_ 0.32
_+ 0.52 _+ 0.22 _+ 0.10 _+ 0.07
+ + + +
0.20 0.10 0.19 0.08
+ 0.18 + 0.07 + 0.19 _+ 0.13
356 Noradrenaline
5'- Nucleotidase
!!
o! 11
i
SRIF-like immunoreactivity
Lactate dehydrogenase _
-t-! ~m
(D ¢J
2-
2-
1
-t
(J
== >= 3
O
I
i
0
i
NPY-like irnmunoreactivity
Fumarase 3-
3-
2-
2--
1
S
+
--
O
O
O
5O
1
5'0
O
1OO
1OO
% recovered protein W
ODE
F
G
H
I
W
ODE
F
G
H
I
Fraction Fig. 1. Fractionation of guinea pig neocortex synaptosomes by isopycnic centrifugation. Fraction P2 was layered onto a discontinuous sucrose density gradient (0, 0.2 M Sucrose to I, 1.2 M sucrose) after hypo,osmotic shock. RSAs of enzymes and putative neurotransmitters were calculated as described in the legend to Table I. Values given are means _+S.E.M. for 3 or 4 separate experiments.
presented in Fig. t according to the scheme proposed by De Duve et al.7. The enrichment of L D H in W and the mitochondrial marker (fumarase) in fraction I indicated that the hypo-osmotic shock had successfully lysed the majority of the synaptosomes present in P2. The small peak of L D H in fractions F and G indicated that a small proportion of the synaptosomes was resistant to complete lysis. Membrane fragments and synaptic vesicles of various densities had been separated mainly into fractions E - H as shown by the distribution of 5'-nucleotidase. Further characterization of the density gradient
was provided by the distribution of NA. which was found significantly enriched (P < 0.05. paired Student's t-test) in fraction G but was also evenly distributed in W - F . In sympathetic nerves heterogeneity of adrenergic vesicles has been demonstrated by both morphological and biochemical techniques (see Zimmerman 33 for references). Our results are consistent with the localization of cortical N A stores within dense or 'heavy' vesicles (fraction G) but also within 'light' vesicles equilibrating at 0.4 M sucrose (fraction D). The recovery of 15% of the N A within W suggests either that this soluble NA was originally
357 free in the cytosol within synaptosomes, or that it was released from vesicles damaged by hypo-osmotic shock 26. In contrast to the NA distribution both peptide immunoreactivities concentrated at the inferface between 0.8 and 1.0 M sucrose, in the 'heavy' or densecored vesicle fraction, (SRIF, P < 0.005, NPY, P < 0.05; paired sample t-test). Only low levels of immunoreactivity were detected in the light vesicle fraction D. The enrichment of both peptides in fractions F and G indicated a location within dense vesicles and synaptic vesicles closely attached to presynaptic membranes respectively 31. The low levels of peptide recovered with mitochondria (fraction I) indicate that the majorities of both SRIF- and NPY-LI in the original P2 fraction were contained in synaptosomes and not in the mitochondrial contaminants of that fraction. The recoveries of all enzymes, protein, NA and NPY-LI on the sucrose density gradient were between 72 and 95%, but the recovery of SRIF-L! was lower, 45.3 +_ 6.3% (n = 3), perhaps indicating that a proportion (35%) of synaptosomal SRIF-LI was contained in the cytoplasm or released from vesicles damaged by hypo-osmotic shock and degraded during the preparation of the density gradient. NPY-LI, however, was recovered by 79.0 _+ 18.2% (n = 4), of which 75% was found in the dense vesicle fractions F-H. HPLC analysis of the NPY-LI extracted from the original homogenate and from the heavy vesicle fractions of the density gradient (G and H) indicated that the majority of the material was similar to the synthetic porcine NPY standard (Fig. 2), with a small proportion of material (9%), possibly proteinbound, failing to adhere to the column, eluting in the breakthrough peak (Fig. 2A). These results demonstrate that guinea-pig cortical NPY- and SRIF-LI are found in synaptosomes, particularly concentrated in a dense vesicular compartment and not in a light vesicular compartment. In addition, approximately 45% of the peptides may be found inside cell bodies as indicated by their concentration in P r Previous studies have shown the fractionation of SRIF-LI in synaptosomes prepared from rat brain 2,]3,28. However, these studies 2,28 did not demonstrate a convincing enrichment in vesicular fractions because peptide recovery was matched by protein recovery or no controls were included. To
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Fig. 2. Reverse phase HPLC purification of NPY-LI in subcellular fractions (0.1 ml). A, homogenate; B, fraction G; C, fraction H; D, acetonitrile gradient used; 0.1% TFA was included throughout. our knowledge this is the first biochemical demonstration of a heavy vesicular localization of peptide immunoreactivity in the CNS. In the periphery, a similar localization of neuronal vasoactive intestinal polypeptide-like immunoreactivity has been demonstrated biochemically 19. The storage of a range of CNS peptides within nerve endings or synaptic vesicles has been demonstrated in several studies (see Emsonl0 for references). Morphological studies have indicated that CNS peptide storage granules are large (80-120 nm) and dense-cored in most cases 22. However, peptide storage in peripheral nerves such as bovine splenic nerve may involve small vesicles 15. The role of differently sized vesicles is unclear but may be related to usage or vesicle recycling mechanisms. An interesting possibility would be the costorage of NPY with NA, or NPY with SRIF within large vesicles in the cortex, in a similar way to the costorage of
358 e n k e p h a l i n with N A in b o v i n e splenic n e r v e ~:. N P Y -
this p e p t i d e . F u r t h e r c o n f i r m a t i o n of such a role will
LI has b e e n s h o w n to coexist with N A in locus c o e r u -
r e q u i r e the d e v e l o p m e n t of a c o m p e t i t i v e p h a r m a c o -
leus cells t4 which m a y give rise to p r o j e c t i o n s to n e o -
logical a n t a g o n i s t to facilitate studies of its biological
cortex, and also to coexist with S R 1 F in cortical inter-
actions.
n e u r o n e s 17. F u r t h e r m o r e , N P Y is f o u n d in high conc e n t r a t i o n s in s y m p a t h e t i c ganglia > and a p p e a r s to
T h e w o r k was, in part, s u p p o r t e d
by grants to
be c o n t a i n e d within the s u p e r i o r cervical g a n g l i o n in-
P . J . R . f r o m t h e M u s c u l a r D y s t r o p h y G r o u p of G r e a t
n e r v a t i o n to c e r e b r a l a r t e r i e s s. A n i n t e r a c t i o n with
Britain and N o r t h e r n I r e l a n d and the S m i t h K l i n e
N A at the p o s t s y n a p t i c level in the c o n t r o l of b l o o d
F o u n d a t i o n . W e t h a n k G a v i n R e y n o l d s for the use of
flow s e e m s likely f r o m b o t h central and p e r i p h e r a l
c a t e c h o l a m i n e H P L C and d e t e c t o r , P e t e r H o r s f i e l d
studies (see E d v i n s s o n et al. 9 for r e f e r e n c e s ) .
for technical assistance and M a r y W y n n for typing
T h e p r e s e n t d e m o n s t r a t i o n of N P Y - L I in synaptic
the m a n u s c r i p t . M . E . de Q. is an M R C Scholar.
vesicles is consistent with a n e u r o t r a n s m i t t e r role for 1 Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T. J., Bloom, S. R. and Polak, J. M., Neuropeptide Y distribution in the rat brain, Science, 221 (1983) 877-879. 2 Berelowitz, M., Hudson, A., Pimstone, B., Kronheim, S. and Bennett, G. W., Subcellular localization of growth hormone release inhibiting hormone in rat hypothalamus, cerebral cortex, striatum and thalamus, J. Neurochem., 31 (1978) 751-753. 3 Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem.. 72 (1976) 248-254. 4 Corder, R., Emson, P. C. and Lowry, P. J., Purification and characterization of human neuropeptide Y from adrenal medullary phaeochromocytoma tissue, Biochem. J., 219 (1984) 699-706. 5 Dawbarn, D., De Quidt, M. E. and Emson, P. C.. Survival of basal ganglia neuropeptide Y/somatostatin neurones in Huntington's disease, Brain Research, (in press). 6 Dawbarn, D., Hunt, S. P. and Emson, P. C., Neuropeptide Y: regional distribution, chromatographic characterization and immunohistochemical demonstration in post-mortem human brain, Brain Research, 296 (1984) 168-173. 7 De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R. and Appelmans, F., Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue, Biochern. J., 60 (1955) 604-617. 8 Edvinsson, L., Emson, P. C., McCulloch, J., Tatemoto, K. and Uddman, R., Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat, Neurosci. Lett., 43 (1983) 79-84. 9 Edvinsson, L., Ekblad, E., Hfikanson, R. and Wahlestedt, C., Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels, Brit. J. Pharmacol., 83 (1984) 519-525. 10 Emson, P. C., Peptides as neurotransmitter candidates in the mammalian CNS, Progr. Neurobiol., 13 (1979) 61-116. 11 Emson, P. C., Corder, R. and Lowry, P. J., Demonstration of a neuropeptide Y-like immunoreactivity in human phaeochromocytoma extracts, Regul. Pept., 8 (1984) 89-94. 12 Emson, P. C. and de Quidt, M. E., NPY - a new member of the pancreatic polypeptide family, Trends Neurosci., 7 (1984) 31-35. 13 Epelbaum, J., Brazeau, P., Tsang, D., Brawer, J. and Mar-
tin, J. B., Subcellular distribution of radimmmunoassayable somatostatin in rat brain. Brain Research, 126 (1977) 309-323. 14 Everitt, B. J., H6kfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M., Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat, Neuroscience, 11 (1984) 443-462. 15 Fried, G., Lundberg, J. M., H6kfelt, T., Lagercrantz, tt., Fahrenkrug, J., Lundgren, G., Holmstedt, B., Brodin, E., Efendic, S. and Terenius, L., Do peptides coexist with classical transmitters in the same neuronal storage vesicles? In L. L. Stjarne (Ed.), Chemical Transmission: 75 years, Academic Press, New York, 1981, pp. 105-Ill. 16 Gray, E. G. and Whittaker, V. P., The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79-87. 17 Hendry, S. H. C., Jones, E. G. and Emson, P. C., Morphology, distribution and synaptic relations of somatostatin and neuropeptide Y immunoreactive neurons in rat and monkey neocortex, J. Neurosci., 4 (1984) 2497-2517. 18 Keiding, R., Horder. M.. Gerhardt. W.. Pitkanen. E.. Tenhunen, R., Stromme. J. H.. Theordersen. L. Waldenstrom, J., Tryding, N. and Westlund. L., Recommended methods for the determination of four enzymes in blood. Scand. J. clin. Lab. Invest.. 33 (1974) 291-306 19 Lundberg, J. M., Fried, G.. Fahrenkrug, J.. Holmstedt, B.. HOkfelt, T., Lagercrantz. H.. Lundgren. G. and/~,ngghrd. A., Subcellular fractionation of cat submandibular gland: comparative studies on the distribution of acetylcholine and vasoactive intestinal polypeptide (VIPL Neuroscience (1981) 1001-1010. 20 Lundberg, J. M.. Terenius. L.. Htikfelt, T. and Goldstein. M., High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man, Neurosci. Lett., 42 (1983) 167-172 21 Minth, C. D., Bloom, S. R., Polak. J. M. and Dixon. J. E.. Cloning, characterization, and DNA sequence of a human cDNA encoding neuropeptide tyrosine. Proc. nat. Acad. Sci. U.S.A.. 81 (1984) 4577-4581. 22 Priestley, J. V. and Cuelto. A. C.. Electron nricroscoDc lmmunocytochemistry for CNS transmitters and transmitter markers. In A C. Cuello (Ed.), Methods in the Neurosctences, Vol. 3, IBRO (1983) pp. 273-322.
359 23 Racker, E., Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids, Biochim. biophys. Acta, 4 (1950) 211-214. 24 Reynolds, G. P., Increased concentrations and lateral asymmetry of amygdala dopamine in schizophrenia, Nature (Lond.), 305 (1983) 527-529. 25 Richardson, P. J., Siddle, K. and Luzio, J. P., Immunoaffinity purification of intact, metabolically active, cholinergic nerve terminals from mammalian brain, Biochem. J., 219 (1984) 647-654. 26 Smith, A. D., Subcellular localisation of noradrenaline in sympathetic neurons, Pharm Rev., 24 (1972) 435-457. 27 Stanley, K. K., Edwards, M. R, and Luzio, J. P., Subcellular distribution and movement of 5'-nucleotidase in rat cells, Biochem. J., 186 (1980) 59-69. 28 Styne, D. M., Goldsmith, P. C., Burstein, S. R., Kaplan, S. L. and Grumbach, M. M., Immunoreactive somatostatin and luteinizing hormone releasing hormone in median eminence synaptosomes of the rat: detection by immunohisto-
29
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chemistry and quantification by radioimmunoassay, Endocrinology, 101 (1977) 1099-1103. Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 5485-5489. Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (Lond.). 296 (1982) 659-660. Von Schwarzenfeld, I., Origin of transmitters released by electrical stimulation from a small metabolically very active vesicular pool of cholinergic synapses in guinea-pig cerebral cortex, Neuroscience, 4 (1979) 477-493. Wilson, S. P., Klein, R. L., Chang, K.-J., Gasparis, M. S., Viveros, O. H. and Yang, W.-H., Are opioid peptides cotransmitters in noradrenergic vesicles of sympathetic nerves? Nature (Lond.), 288 (1980) 707-709. Zimmerman, H., Vesicle recycling and transmitter release, Neuroscience, 4 (1979) 1773-1804. -
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32
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Brain Researcfi, 3~5 ~'?,?,~5)354 ~59
BRE 20829
Subcellular distribution of neuropeptide Y-like immunoreactivity in guinea pig neocortex MARION E. DE QUIDT 1, PETER J. RICHARDSON 2 and PIERS C. EMSON 1 1MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School and 2Department of Clinical Biochemistry, University of Cambridge, A ddenbrooke's Hospital, Cambridge CB2 2QR (U, K, ) (Accepted January 3rd, 1985) Key words: neuropeptide Y - - somatostatin - - noradrenaline - - subcellular fractionation - - neocortex - - chromatography
Neuropeptide Y-like immunoreactivity (NPY-LI) was enriched in synaptosomal fractions of neocortex, which on lysis yielded vesicle-rich fractions. The distribution of NPY-LI on a sucrose density gradient was similar to that of somatostatin, with a concentration in heavy vesicles. The peptides were not found in light vesicles in contrast to the distribution of noradrenaline. Both homogenate and vesicular NPY-LI coeluted with synthetic NPY on reverse-phase HPLC.
N e u r o p e p t i d e Y (NPY) was isolated from porcine brain extracts 30 and its amino acid sequence determined in 198229. Subsequently, a h u m a n N P Y sequence has been d e t e r m i n e d from p h a e o c h r o m o c y toma 4. M o r e recently, the h u m a n m R N A encoding the N P Y precursor has been cloned and its nucleotide sequence d e t e r m i n e d 2t, confirming the N P Y sequence of C o r d e r et al. 4. NPY-like immunoreactivity ( N P Y - L I ) has been m e a s u r e d by r a d i o i m m u n o a s s a y in extracts from m a n y regions of m a m m a l i a n brain~,56,12 and found to have a non-uniform distribution. In addition, immunohistochemical studies have revealed a neuronal localization of N P Y - L I in the CNS of ratl,12A 4, h u m a n 6, m o n k e y 17 and cat ( D e Quidt, unpublished observations). The present study investigated the subcellular distribution of N P Y - L I in neocortex, using biochemical fractionation techniques and by comparing the NPY distribution with that of a variety of m a r k e r enzymes and two putative n e u r o t r a n s m i t t e r s - - somatostatin (SRIF) and n o r a d r e n a l i n e ( N A ) . A d u l t female guinea pigs (250-400 g) were stunned and decapitated. T h e brain was rapidly removed and the n e o c o r t e x p e e l e d off and placed on ice. Subcellular fractionation was carried out in two
stages. The first involved the p r e p a r a t i o n of a crude mitochondrial fraction (P2), largely by the m e t h o d of Gray and W h i t t a k e r 16 as described by Richardson et al. 25. The second stage of fractionation involved hypo-osmotic lysis of the s y n a p t o s o m e fraction P2 by suspension in 10 n M H e p e s p H 7.4 for 10 min at 4 °C. followed by fractionation of the lysate on a discontinuous sucrose density gradient 31, Samples of all fractions were assayed for total protein content 3 and a series of enzyme activities. 5'-Nucleotidase (EC 3.1.3.5) was used as a m a r k e r for plasma m e m b r a n e s and assayed by the m e t h o d of Stanley et al. 27. Lactate d e h y d r o g e n a s e ( L D H : EC 1.1.1.27), a cytoplasmic m a r k e r , and fumarase (EC 4.2.1.2), a mitochondrial m a r k e r , were assayed by the methods of Keiding et al.18 and Racker23. respectively. A d d i t i o n a l samples of all fractions were m a d e to 50 mM HCI, in o r d e r both to inhibit enzyme activity (proteases, catecholamine catabolising enzymes) and to extract both peptides and N A . This was carried out at 4 °C to minimise the oxidation of N A . After centrifugation (10,000 g, 4 min) to r e m o v e cell debris, the supernatants were assayed directly for NPYand S R I F - L I by radioimmunoassay. Both assays
Correspondence: M. E. de Quidt, MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Hills Road, Cambridge CB2 2QH, U.K. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
355 were able to tolerate up to one third total assay volume of concentrated (1 M) sucrose with 50 mM HC1 or 10 mM Hepes per 50 mM HC1 without non-specific interference in antiserum-tracer binding. The antisera crossreactivities and assay characteristics have been described previously (SRIF: C-terminal assayS; NPY: ref. 11, with modifications using Bolton-Hunter [125I]NPY as tracer (Amersham, S.A. - 2 0 0 0 Ci/mmol) and synthetic porcine NPY as standard (Bachem, U.S.A.), which did not alter antiserum crossreactivity characteristics (De Quidt, in preparation)). The assay buffer (50 mM phosphate/0.1% bovine serum albumin (BSA; R I A grade, Sigma)/50 mM E D T A , pH 7.4) contained trasylol (200 kIU/ml, Sigma) as an additional peptidase inhibitor, which did not alter the assay characteristics. Serial dilution of the subcellular fractions in both NPY and SRIF radioimmunoassays demonstrated that the extracted immunoreactivities diluted in parallel with the synthetic peptide standards. NA was measured by reverse-phase HPLC with electrochemical detection essentially as described by Reynolds 24. Fractions were diluted 1:3 to 1:5 with 1.5 Tris/0.2 mM E D T A and mixed with 20 mg activated alumina for 20 min. The HPLC buffer was 0.1 M phosphate pH 3.6, not phosphate-citrate as previously described 24. Recovery of internal standard (3,4-dihydroxybenzylamine) was 81.2 + 2.5% (n = 24) from homogenate and fractions P 1 - S 2 , and 71.5 _+ 1.6% (n = 40) from fractions W (supernatant)-I (mean _+ S.E.M.). Further characterization of the subcellular NPYLI was carried out by means of reverse-phase HPLC using a Waters Associates/~Bondapak C18 column (30 x 0.39 cm) with an acetonitrile gradient (2
ml/min) as shown in Fig. 2D. 0.1% trifluoroacetic acid (TFA) was present throughout. 2-ml fractions were collected, dried at 50 °C (12-18 h) and the remaining aqueous solvents then freeze-dried. Fractions were reconstituted in assay buffer for radioimmunoassay. The following NPY-like synthetic peptides were used to characterise the gradient: porcine NPY, porcine peptide YY (PYY, Bachem, U.S.A.) and avian pancreatic polypeptide (APP, Bachem, U.S.A.). Differential centrifugation. The separation of enzyme, peptide and NA activities are summarised in Table I. The absolute amounts of substances measured in the homogenate were as follows (mean _+ S.E.M. from 3 or 4 experiments unless otherwise stated): total protein 124.7 _+ 33.4 mg; 5'-nucleotidase 1.93 + 0.23 ymol/h/mg protein; L D H 1.98 + 0.60 k~mol/min/mg protein; fumarase 0.46 + 0.20 ¢tmol/min/mg protein; SRIF-LI 3.65 + 2.22 pmol/mg protein; NPY-LI 4.65 + 1.05 pmol/mg protein; NA 3.85, 3.42 pmol/mg protein (n = 2). The recovery of all activities in P1 with S 1 from the homogenate was > 95% and in P2 with $2, from Sl, was > 90% for protein, enzymes and NA, 82.0 + 7.3% for SRIF-LI and 65.5 _ 10.2% for NPY-LI. An enrichment (relative specific activity (RSA) > 1) of 5'-nucleotidase, fumarase, SRIF-LI, NPY-LI and NA was detected in P1 which has been shown to contain cell debris including cell bodies and nuclei 16. This represented between one third to one half of total homogenate enzyme and transmitter activity. Fraction P2 contained enriched levels of these enzymes and both peptide immunoreactivities. Isopycnic centrifugation. The fractionation of subcellular markers and putative neurotransmitters is
TABLE I
Fractionation of guinea pig neocortex homogenate by differential centrifugation Fractions were prepared by two centrifugation steps, the first at 1000 g, 10 min at 4 °C to produce P1 and S 1 from the homogenate, the second at 2,000 g for 20 rain at 4 °C to form P2 and S2 from S v RSA values of enzyme of neurochemical markers are given as means _+ S.E.M. for 3 - 4 separate experiments (NA two experiments). The RSA values are defined as (% of total recovered activity in fraction)/(% of total recovered protein in fraction).
Fraction
P1 S1 P2 S2
Relative specific activity 5'-Nucleotidase
Lactate dehydrogenase
Fumarase
SRIF-L1
NPY-LI
NA
1.16 0.95 1.23 0.59
0.94 1.05 0.62 1.89
1.66 0.78 1.38 0.31
1.47 0.82 1.45 0.26
1.54 0.78 1.37 0.44
1.95, 1.87 0.65, 0.62 0.6, 1.14 1.97, 1.09
_+ 0.09 + 0.05 + 0.09 + 0.09
+ 0.06 + 0.04 + 0.12 +_ 0.32
_+ 0.52 _+ 0.22 _+ 0.10 _+ 0.07
+ + + +
0.20 0.10 0.19 0.08
+ 0.18 + 0.07 + 0.19 _+ 0.13
356 Noradrenaline
5'- Nucleotidase
!!
o! 11
i
SRIF-like immunoreactivity
Lactate dehydrogenase _
-t-! ~m
(D ¢J
2-
2-
1
-t
(J
== >= 3
O
I
i
0
i
NPY-like irnmunoreactivity
Fumarase 3-
3-
2-
2--
1
S
+
--
O
O
O
5O
1
5'0
O
1OO
1OO
% recovered protein W
ODE
F
G
H
I
W
ODE
F
G
H
I
Fraction Fig. 1. Fractionation of guinea pig neocortex synaptosomes by isopycnic centrifugation. Fraction P2 was layered onto a discontinuous sucrose density gradient (0, 0.2 M Sucrose to I, 1.2 M sucrose) after hypo,osmotic shock. RSAs of enzymes and putative neurotransmitters were calculated as described in the legend to Table I. Values given are means _+S.E.M. for 3 or 4 separate experiments.
presented in Fig. t according to the scheme proposed by De Duve et al.7. The enrichment of L D H in W and the mitochondrial marker (fumarase) in fraction I indicated that the hypo-osmotic shock had successfully lysed the majority of the synaptosomes present in P2. The small peak of L D H in fractions F and G indicated that a small proportion of the synaptosomes was resistant to complete lysis. Membrane fragments and synaptic vesicles of various densities had been separated mainly into fractions E - H as shown by the distribution of 5'-nucleotidase. Further characterization of the density gradient
was provided by the distribution of NA. which was found significantly enriched (P < 0.05. paired Student's t-test) in fraction G but was also evenly distributed in W - F . In sympathetic nerves heterogeneity of adrenergic vesicles has been demonstrated by both morphological and biochemical techniques (see Zimmerman 33 for references). Our results are consistent with the localization of cortical N A stores within dense or 'heavy' vesicles (fraction G) but also within 'light' vesicles equilibrating at 0.4 M sucrose (fraction D). The recovery of 15% of the N A within W suggests either that this soluble NA was originally
357 free in the cytosol within synaptosomes, or that it was released from vesicles damaged by hypo-osmotic shock 26. In contrast to the NA distribution both peptide immunoreactivities concentrated at the inferface between 0.8 and 1.0 M sucrose, in the 'heavy' or densecored vesicle fraction, (SRIF, P < 0.005, NPY, P < 0.05; paired sample t-test). Only low levels of immunoreactivity were detected in the light vesicle fraction D. The enrichment of both peptides in fractions F and G indicated a location within dense vesicles and synaptic vesicles closely attached to presynaptic membranes respectively 31. The low levels of peptide recovered with mitochondria (fraction I) indicate that the majorities of both SRIF- and NPY-LI in the original P2 fraction were contained in synaptosomes and not in the mitochondrial contaminants of that fraction. The recoveries of all enzymes, protein, NA and NPY-LI on the sucrose density gradient were between 72 and 95%, but the recovery of SRIF-L! was lower, 45.3 +_ 6.3% (n = 3), perhaps indicating that a proportion (35%) of synaptosomal SRIF-LI was contained in the cytoplasm or released from vesicles damaged by hypo-osmotic shock and degraded during the preparation of the density gradient. NPY-LI, however, was recovered by 79.0 _+ 18.2% (n = 4), of which 75% was found in the dense vesicle fractions F-H. HPLC analysis of the NPY-LI extracted from the original homogenate and from the heavy vesicle fractions of the density gradient (G and H) indicated that the majority of the material was similar to the synthetic porcine NPY standard (Fig. 2), with a small proportion of material (9%), possibly proteinbound, failing to adhere to the column, eluting in the breakthrough peak (Fig. 2A). These results demonstrate that guinea-pig cortical NPY- and SRIF-LI are found in synaptosomes, particularly concentrated in a dense vesicular compartment and not in a light vesicular compartment. In addition, approximately 45% of the peptides may be found inside cell bodies as indicated by their concentration in P r Previous studies have shown the fractionation of SRIF-LI in synaptosomes prepared from rat brain 2,]3,28. However, these studies 2,28 did not demonstrate a convincing enrichment in vesicular fractions because peptide recovery was matched by protein recovery or no controls were included. To
PY~YspN 'Wsp APPI 5"0-
A
4-03"0>,
2"0"~
~ 1-0~ ~
o-~
I
- 1"°t ~ 0-5 0 t-O o-5
o
I
B
I i
C
j
20-~ I 0
[
10
20 30 Elution vol [ml~
4O
Fig. 2. Reverse phase HPLC purification of NPY-LI in subcellular fractions (0.1 ml). A, homogenate; B, fraction G; C, fraction H; D, acetonitrile gradient used; 0.1% TFA was included throughout. our knowledge this is the first biochemical demonstration of a heavy vesicular localization of peptide immunoreactivity in the CNS. In the periphery, a similar localization of neuronal vasoactive intestinal polypeptide-like immunoreactivity has been demonstrated biochemically 19. The storage of a range of CNS peptides within nerve endings or synaptic vesicles has been demonstrated in several studies (see Emsonl0 for references). Morphological studies have indicated that CNS peptide storage granules are large (80-120 nm) and dense-cored in most cases 22. However, peptide storage in peripheral nerves such as bovine splenic nerve may involve small vesicles 15. The role of differently sized vesicles is unclear but may be related to usage or vesicle recycling mechanisms. An interesting possibility would be the costorage of NPY with NA, or NPY with SRIF within large vesicles in the cortex, in a similar way to the costorage of
358 e n k e p h a l i n with N A in b o v i n e splenic n e r v e ~:. N P Y -
this p e p t i d e . F u r t h e r c o n f i r m a t i o n of such a role will
LI has b e e n s h o w n to coexist with N A in locus c o e r u -
r e q u i r e the d e v e l o p m e n t of a c o m p e t i t i v e p h a r m a c o -
leus cells t4 which m a y give rise to p r o j e c t i o n s to n e o -
logical a n t a g o n i s t to facilitate studies of its biological
cortex, and also to coexist with S R 1 F in cortical inter-
actions.
n e u r o n e s 17. F u r t h e r m o r e , N P Y is f o u n d in high conc e n t r a t i o n s in s y m p a t h e t i c ganglia > and a p p e a r s to
T h e w o r k was, in part, s u p p o r t e d
by grants to
be c o n t a i n e d within the s u p e r i o r cervical g a n g l i o n in-
P . J . R . f r o m t h e M u s c u l a r D y s t r o p h y G r o u p of G r e a t
n e r v a t i o n to c e r e b r a l a r t e r i e s s. A n i n t e r a c t i o n with
Britain and N o r t h e r n I r e l a n d and the S m i t h K l i n e
N A at the p o s t s y n a p t i c level in the c o n t r o l of b l o o d
F o u n d a t i o n . W e t h a n k G a v i n R e y n o l d s for the use of
flow s e e m s likely f r o m b o t h central and p e r i p h e r a l
c a t e c h o l a m i n e H P L C and d e t e c t o r , P e t e r H o r s f i e l d
studies (see E d v i n s s o n et al. 9 for r e f e r e n c e s ) .
for technical assistance and M a r y W y n n for typing
T h e p r e s e n t d e m o n s t r a t i o n of N P Y - L I in synaptic
the m a n u s c r i p t . M . E . de Q. is an M R C Scholar.
vesicles is consistent with a n e u r o t r a n s m i t t e r role for 1 Allen, Y. S., Adrian, T. E., Allen, J. M., Tatemoto, K., Crow, T. J., Bloom, S. R. and Polak, J. M., Neuropeptide Y distribution in the rat brain, Science, 221 (1983) 877-879. 2 Berelowitz, M., Hudson, A., Pimstone, B., Kronheim, S. and Bennett, G. W., Subcellular localization of growth hormone release inhibiting hormone in rat hypothalamus, cerebral cortex, striatum and thalamus, J. Neurochem., 31 (1978) 751-753. 3 Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem.. 72 (1976) 248-254. 4 Corder, R., Emson, P. C. and Lowry, P. J., Purification and characterization of human neuropeptide Y from adrenal medullary phaeochromocytoma tissue, Biochem. J., 219 (1984) 699-706. 5 Dawbarn, D., De Quidt, M. E. and Emson, P. C.. Survival of basal ganglia neuropeptide Y/somatostatin neurones in Huntington's disease, Brain Research, (in press). 6 Dawbarn, D., Hunt, S. P. and Emson, P. C., Neuropeptide Y: regional distribution, chromatographic characterization and immunohistochemical demonstration in post-mortem human brain, Brain Research, 296 (1984) 168-173. 7 De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R. and Appelmans, F., Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat liver tissue, Biochern. J., 60 (1955) 604-617. 8 Edvinsson, L., Emson, P. C., McCulloch, J., Tatemoto, K. and Uddman, R., Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat, Neurosci. Lett., 43 (1983) 79-84. 9 Edvinsson, L., Ekblad, E., Hfikanson, R. and Wahlestedt, C., Neuropeptide Y potentiates the effect of various vasoconstrictor agents on rabbit blood vessels, Brit. J. Pharmacol., 83 (1984) 519-525. 10 Emson, P. C., Peptides as neurotransmitter candidates in the mammalian CNS, Progr. Neurobiol., 13 (1979) 61-116. 11 Emson, P. C., Corder, R. and Lowry, P. J., Demonstration of a neuropeptide Y-like immunoreactivity in human phaeochromocytoma extracts, Regul. Pept., 8 (1984) 89-94. 12 Emson, P. C. and de Quidt, M. E., NPY - a new member of the pancreatic polypeptide family, Trends Neurosci., 7 (1984) 31-35. 13 Epelbaum, J., Brazeau, P., Tsang, D., Brawer, J. and Mar-
tin, J. B., Subcellular distribution of radimmmunoassayable somatostatin in rat brain. Brain Research, 126 (1977) 309-323. 14 Everitt, B. J., H6kfelt, T., Terenius, L., Tatemoto, K., Mutt, V. and Goldstein, M., Differential coexistence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat, Neuroscience, 11 (1984) 443-462. 15 Fried, G., Lundberg, J. M., H6kfelt, T., Lagercrantz, tt., Fahrenkrug, J., Lundgren, G., Holmstedt, B., Brodin, E., Efendic, S. and Terenius, L., Do peptides coexist with classical transmitters in the same neuronal storage vesicles? In L. L. Stjarne (Ed.), Chemical Transmission: 75 years, Academic Press, New York, 1981, pp. 105-Ill. 16 Gray, E. G. and Whittaker, V. P., The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79-87. 17 Hendry, S. H. C., Jones, E. G. and Emson, P. C., Morphology, distribution and synaptic relations of somatostatin and neuropeptide Y immunoreactive neurons in rat and monkey neocortex, J. Neurosci., 4 (1984) 2497-2517. 18 Keiding, R., Horder. M.. Gerhardt. W.. Pitkanen. E.. Tenhunen, R., Stromme. J. H.. Theordersen. L. Waldenstrom, J., Tryding, N. and Westlund. L., Recommended methods for the determination of four enzymes in blood. Scand. J. clin. Lab. Invest.. 33 (1974) 291-306 19 Lundberg, J. M., Fried, G.. Fahrenkrug, J.. Holmstedt, B.. HOkfelt, T., Lagercrantz. H.. Lundgren. G. and/~,ngghrd. A., Subcellular fractionation of cat submandibular gland: comparative studies on the distribution of acetylcholine and vasoactive intestinal polypeptide (VIPL Neuroscience (1981) 1001-1010. 20 Lundberg, J. M.. Terenius. L.. Htikfelt, T. and Goldstein. M., High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man, Neurosci. Lett., 42 (1983) 167-172 21 Minth, C. D., Bloom, S. R., Polak. J. M. and Dixon. J. E.. Cloning, characterization, and DNA sequence of a human cDNA encoding neuropeptide tyrosine. Proc. nat. Acad. Sci. U.S.A.. 81 (1984) 4577-4581. 22 Priestley, J. V. and Cuelto. A. C.. Electron nricroscoDc lmmunocytochemistry for CNS transmitters and transmitter markers. In A C. Cuello (Ed.), Methods in the Neurosctences, Vol. 3, IBRO (1983) pp. 273-322.
359 23 Racker, E., Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids, Biochim. biophys. Acta, 4 (1950) 211-214. 24 Reynolds, G. P., Increased concentrations and lateral asymmetry of amygdala dopamine in schizophrenia, Nature (Lond.), 305 (1983) 527-529. 25 Richardson, P. J., Siddle, K. and Luzio, J. P., Immunoaffinity purification of intact, metabolically active, cholinergic nerve terminals from mammalian brain, Biochem. J., 219 (1984) 647-654. 26 Smith, A. D., Subcellular localisation of noradrenaline in sympathetic neurons, Pharm Rev., 24 (1972) 435-457. 27 Stanley, K. K., Edwards, M. R, and Luzio, J. P., Subcellular distribution and movement of 5'-nucleotidase in rat cells, Biochem. J., 186 (1980) 59-69. 28 Styne, D. M., Goldsmith, P. C., Burstein, S. R., Kaplan, S. L. and Grumbach, M. M., Immunoreactive somatostatin and luteinizing hormone releasing hormone in median eminence synaptosomes of the rat: detection by immunohisto-
29
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chemistry and quantification by radioimmunoassay, Endocrinology, 101 (1977) 1099-1103. Tatemoto, K., Neuropeptide Y: complete amino acid sequence of the brain peptide, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 5485-5489. Tatemoto, K., Carlquist, M. and Mutt, V., Neuropeptide Y a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (Lond.). 296 (1982) 659-660. Von Schwarzenfeld, I., Origin of transmitters released by electrical stimulation from a small metabolically very active vesicular pool of cholinergic synapses in guinea-pig cerebral cortex, Neuroscience, 4 (1979) 477-493. Wilson, S. P., Klein, R. L., Chang, K.-J., Gasparis, M. S., Viveros, O. H. and Yang, W.-H., Are opioid peptides cotransmitters in noradrenergic vesicles of sympathetic nerves? Nature (Lond.), 288 (1980) 707-709. Zimmerman, H., Vesicle recycling and transmitter release, Neuroscience, 4 (1979) 1773-1804. -
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