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FMRFamide-like immunoreactivity in rat brain: Development of a radioimmunoassay and its application in studies of distribution and chromatographic properties
Brain Research, 266 (1983) 295-303 Elsevier Biomedical Press
295
FMRFamide-Like Immunoreactivity in Rat Brain: Development of a Radioimmunoassay and its Application in Studies of Distribution and Chromatographic Properties GRAHAM J. DOCKRAY and R. G. WILLIAMS Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool L69 3BX (U.K.) (Accepted October 12th, 1982) Key words: FMRFamide - - radioimmunoassay - - neuropeptides - - pancreatic polypeptide
A radioimmunoassay is described for the molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide). The antibody used is C-terminal-specificand shows slight but significant (1-2 ~) cross-reactivitywith chicken pancreatic polypeptide (APP). The assay has been used to identify in rat brain extracts a pair of molecules that may represent mammalian counterparts of FMRFamide. Their concentrations were highest in spinal cord and hypothalamus (> 10 pmol'g 1) and lowest in cerebellum and striatum (<3.5 pmol'g-1). The two immunoreactive peptides were separated on CM ion-exchangechromatography where they appeared to be less basic than FMRFamide. On Sephadex G50 gel filtration one eluted in a similar position to FMRFamide and the other slightly earlier suggesting it may be of higher molecular weight. The rat immunoreactive components do not correspond to previously described neuropeptides or hormones, and may be members of a new group of mammalian neuropeptides with transmitter or modulatory functions. INTRODUCTION The tetrapeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide), was first isolated from ganglia of the clam, Macrocallista nimbosa, by Price and Greenberg in 197723. This peptide and its N-terminally extended analogues such as T y r - G l y - G I y FMRFamide (i.e. Y G G F M R F a m i d e , in the single letter notation for amino acids), have been shown to possess potent actions on many molluscan tissues, e.g. cardio-excitatory and cardio-inhibitory effects in different species, contraction of various noncardiovascular muscles, and direct effects on certain neurones4,9,12,1z, ~°. In addition, they evoke excitation of neurones in the brain stem of the rat 10. Recently, antibodies specific for FMRFamide have been used in immunohistochemical studies to reveal material in the nervous systems of other invertebrate groups, e.g. coelenterates, arthropods, and in the central nervous system and gut endocrine cells of a variety of vertebrate species such as fish, amphibia, birds and mammals 1,~-8,14,~1. Preliminary studies using a radioimmunoassay for FMRFamide have 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
also suggested the presence of immunoreactive material in extracts of the brain of amphibia, birds and mammals which is larger and less basic than F M R F amide 6-s. The primary sequence of F M R F a m i d e occurs at the C-terminus of the enkephalin-related heptapeptide, Tyr-Gly-Gly-Phe-Met-Arg-Phe, which has been isolated from bovine adrenal and striatum 24,27. However, on present evidence this peptide exists only in a form terminating in a free carboxyl terminus (Fig. 1). The antisera currently used to detect F M R F a m i d e require a C-terminal amide group and do not react with the enkephalinlike heptapeptide 6. The present paper describes the development of a radioimmunoassay (RIA) for FMRFamide and the results obtained when this assay was used to study the chromatographic properties and distribution of material in extracts of rat CNS. The data indicate the existence in rat CNS of two FMRFamide-like peptides which occur in highest concentrations in hypothalamus and spinal cord. A subsequent paper will describe the distribution of FMRFamide-like immunoreactivity in rat brain studied by immunohistochemistry.
296 MATERIALS AND METHODS
Peptides The following peptides were obtained from Peninsula: F M R F a m i d e , T y d F M R F a m i d e ( Y F M R F amide), Y G G F M R F a m i d e , and its free acid, Y G G F M R F . The C-terminal hexapeptide of bovine pancreatic polypeptide, designated BPP6, was obtained from UCB (Bruxelles, Belgium). Dr. Joe Kimmel donated avian pancreatic polypeptide (APP) 20. Dr. K. Tatemoto and Professor V. Mutt donated the porcine peptides N P Y and P Y Y (it should be noted that APP, BPP, N P Y and PYY all share a common penultimate Arg residue with FMRFamide, see Fig. 1)28. The remaining peptides used for the specificity studies presented in Table I were donated by Dr. J. Morley.
Antisera For production of antisera, rabbits were immunized with F M R F a m i d e coupled to thyroglobulin. Conjugation was by glutaraldehyde which crosslinks amino groups and so would be predicted to couple the primary amino group of F M R F a m i d e to the carrier protein thereby leaving the C-terminus
FMRFamide Met-enk Arg 6Phe 7 CCK8
free to promote antibody formation. F M R F a m i d e (500 nmol) was dissolved in 0.5 ml of phosphate buffer (0.1 M, p H 7.4) and added to 2 mg thyroglobulin dissolved in 0.5 ml of the same buffer. Glutaraldehyde (20 #1, 5 ~ ) was added slowly to the mixture which was then incubated at 22 °C for 30 min, and finally transfered to a dialysis sac and dialyzed against distilled water (4 1) for 24 h. The efficiency of coupling of peptide to carrier protein was assessed by the incorporation of a small amount of [lzSI]labelled Y F M R F a m i d e to the reaction mixture; in 5 conjugations the incorporation of peptide was 63 4- 6 ~ (mean 4- S.E.). Rabbits (n = 6) were immunized with the equivalent of 75 nmol of tetrapeptide emulsified in Freund's complete adjuvant and administered by multiple intradermal injections over the back. The rabbits were boosted at 6-10 week intervals and bleedings taken from an ear vein 7-10 days after each immunization. All rabbits produced antisera but only two (L134 and L155) were suitable for radioimmunoassay. Since the titre and sensitivity of assays with L134 were greater than with L155 only the former antiserum was used for the studies described here. The dilution of L134
Phe-Met-Arg-Phe-NH 2 Tyr-Gly-Gly-Phe-Met-Arg-Phe Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH 2
APP
-VaI-Thr-Arg-His-Arg-Tyr-NH 2
BPP
-Leu-Thr-Arg-Pro-Arg-Tyr-NH 2
PYY
-VaI-Thr-Arg-GIn-Arg-Tyr-NH 2
NPY
-Arg-Gin-Arg-Tyr-NH 2
Fig. 1. Amino acid sequences of FMRFamide and several structurally related peptides. Met-enkephalin Arg6Phe7 is the opioid heptapeptide isolated from bovine adrenal and striatum. CCK8 is the C-terminal octapeptide of cholecystokinin and has been isolated from sheep brain. APP is avian (chicken) pancreatic polypeptide, and BPP is bovine pancreatic polypeptide. PYY is a newly isolated gut peptide with Tyr at the N-terminus and Tyr-NH~ at the C-terminus; likewise the structurally related molecule, NPY - - isolated from pig brain - - also has Tyr at the N-terminus and Tyr-NH2 at the C-terminus.
297 needed to bind 50 ~ of a trace amount of Y F M R F amide label was 1:30,000 after the second immunization and thereafter decreased with repeated immunization with fresh conjugate, so that after the fourth immunization the titre was only 1:5000. However, there was no apparent change in either specificity or sensitivity of assays made with different bleedings of this antiserum. The results described here were made with serum obtained from the third immunization (titre 1:20,000).
Label A pentapeptide variant of FMRFamide with Tyr in the first position, i.e. YFMRFamide, was used for preparation of radio-iodinated peptide by the chloramine-T method. In a siliconized glass tube containing an aliquot of YFMRFamide (1.0 nmol in 7 #1 of ammonium bicarbonate 0.05 M) there was added in order - - 15 #1 phosphate buffer (0.25 M, pH 7.4), 5 #1 Na125I (18.5 MBq), 5 #1 chloramine-T (0.5 mg.ml-1) and finally 20 #1 of sodium metabisulphite (1.0 mg.m1-1) to stop the reaction. The reaction time was 20 s. The reaction mixture was purified by cation exchange chromatography on CM-Sephadex. The column (1 × 10 cm) was equilibrated with 0.05 M ammonium acetate (pH 4.5) and eluted with a gradient to 1.0 M ammonium acetate (pH 7.5); the acetate buffers contained 5 ~ dimethyl formamide and 0.1 ~ w/v bovine serum albumin (BSA). Unincorporated 125I was not retained by the column and emerged in the first 10 ml, a single peak of iodinated peptide then eluted at about 90 ml. Unsubstituted peptide emerged just before the labelled peptide, so that the specific activity of label on the ascending slope of the peak was less than on the descending slope. The specific activity of the latter was estimated by auto-inhibition curves to be 1388 cpm.fmol-~; allowing for counting efficiency this specific activity is close to the theoretical maximum for mono-iodo label. As might be expected, the later eluting label gave more sensitive assays than the earlier eluting tubes and was used routinely in the work described here. In some experiments the heptapeptide, YGGFMRFamide, was iodinated and used as label in place of YFMRFamide. The results obtained with the two labels were indistinguishable. Assay system The conditions for incubation and separation of
assays were systematically examined, and the following system adopted because it gave: (a) minimum non-specific binding, defined as apparant binding of label in the absence of antibody; (b) highest working dilution of antiserum in routine assays; and (c) the most sensitive assays, expressed in terms of the concentration of standard needed for 50 ~ inhibition of binding of label to antiserum. All assays were carried out in glass tubes pretreated with Sigmacoat to minimize non-specific adsorption of label to glass surfaces. The assay diluent consisted of phosphate buffer (0.05 M, pH 7.4), sodium chloride (0.14 M), sodium azide (3 mM), and BSA (0.05 ~, w/v); volume in each tube was 1.0 ml. Assays were incubated for 24 h at 4 °C and separated by addition of charcoal pre-coated with dextran and plasma, followed by centrifugation at 2000 g for 10 min. The separation step was found to be very temperaturesensitive and care was taken to maintain temperature at 4 °C throughout this step. All samples and standards were prepared in duplicate, and were added to the assay tube in volumes of 10-100 #1. Samples prepared in acetic acid caused non-specific interference when added directly to the assay in volumes greater than 20 #1; routinely therefore, these samples were lyophilized in the assay tube to remove acid prior to addition of diluent, antibody and label. Synthetic FMRFamide was used as a radioimmunoassay standard throughout this study. The specificity of the assay was established by examination of the relative concentrations of different peptides needed to produce 50 inhibition of binding of label. In these experiments each peptide was added in a range of concentrations to ensure that at least 4 points fell within the dynamic range of the assay. The results described below indicate that APP reacts slightly with antiserum L134, and some samples were therefore examined in a RIA for APP. Antiserum to APP was kindly donated by Dr. J. R. Kimmel and was used with natural APP as label. The APP assay showed no significant cross-reactivity ( < 0.0001) with FMRFamide and only slight cross-reactivity with BPP6 (0.0003). In addition, in a few experiments, samples were examined with an RIA using antiserum raised to BPP6 (antibody S 11, kindly donated by Dr. I. L. Taylor). This antiserum was used with BPP6 as label and standard; neither APP, nor
298 FMRFamide showed significant cross-reactivity in this assay ( < 0.0001).
Extracts Three extraction methods were examined. (1) Rat brain tissue (generally 0.1-0.2 g) was quickly weighed and added directly to boiling water (0.1 g.m1-1) and briefly boiled (2 rain). The extract was homogenized (Ultra-Turrax, 30 s) and then centrifuged (3000 g, 10 min). (2) The pellet after boiling water extraction was re-extracted in acid by suspending it in 0.5 M acetic acid in a volume equivalent to the original one. After standing for 30 min at 4 °C the acid extract was centrifuged as before. (3) Tissue was added directly to boiling 0.5 M acetic acid and processed along the lines described for method (1) above. When applied to the extraction of FMRFamide-like immunoreactivity in rat brain stem (n = 4), method (1) gave 6.7 4- 3.2 pmol.g -1, method (2) - - 2.4 4- 0.16 pmol.g -1, and method (3) - - 6.8 41.0 pmol.g -1. Since the sum of the activity recovered by methods (1) and (2) combined (8.8 4- 3.2 pmol.g -1) is greater than that of (3), the final procedure adopted was to extract first in boiling water and then re-extract the pellet in acetic acid.
Distribution studies Under deep halothane anaesthesia, rats were perfused transcardially with an ice-cold solution of 0.14 M NaC1 and the brain and spinal cord were removed. Brains were then dissected according to a modification of the method of Glowinski and Iversen (1966)11 to give the following regions: cerebellum, medulla and pons, amygdala and pyriform cortex, hypothalamus, cortex, hippocampus, striatum, olfactory bulb, thalamus, and mid brain; the spinal cord from cervical to sacral regions was also removed. The tissue samples were rapidly frozen on dry-ice, weighed and extracted by the method described above. In a few experiments samples were also taken from adrenal gland, and from stomach, intestine and pancreas.
Chromatographic studies In initial experiments crude tissue extracts were applied directly to gel filtration and ion-exchange columns. Generally, however, the resolution was poor in these experiments and in the later studies
which are described below all samples were first purified by adsorption on Sep-pak cartridges (Waters Associates) before column chromatography. The Sep-pak cartridges were primed with 2 ml of 80 ~ methanol, 19 ~ water and 1 ~ formic acid, and then washed with 10 ml of 1 ~ formic acid. The tissue extracts were acidified by addition of formic acid to give 1 ~ (any insoluble material was removed by centrifugation) and then passed twice through the cartridge. With extract volumes up to 80 ml the uptake of FMRFamide immunoreactivity on the first pass was 74 4- 3 ~ (n = 4), and after two passes the total uptake was 91 4- 1 ~ (n = 4). The cartridge was then briefly washed with 5 ml 1 ~ formic acid, and the FMRFamide-immunoreactivity eluted with 2 ml of the methanol solution already described. The eluates were dried in a Speedi-Vac Concentrator (Savant) and reconstituted in assay buffer. The recovery of retained immunoreactive material was 97 4- 15 ~. The product was fractionated on CM Sephadex using the same system as that described for the purification of YFMRFamide label. The recovery of material from these columns was 71 421 ~ (n = 5). Tubes corresponding to the peaks of immunoreactivity in the CM Sephadex eluates were pooled and desalted by passage through a Sep-pak as already described, and then separated by gel filtration on Sephadex G50 or G25. The latter columns (superfine grade, 1 × 100 cm) were equilibrated and run in 0.5 M acetic acid. RESULTS
Antibody specificity In a sequence of 13 typical assays the concentration of FMRFamide needed to inhibit the binding of label by 50~ was 29.4 ~ 1.6 pmol.1-1. It was previously noted that YFMRFamide and YGGFMRFamide react virtually equally with the tetrapeptide, but deletion of the C-terminal amide, e.g. FMRF-OH and YGGFMRF-OH, markedly reduced immunoreactivity6. A surprising finding in the present study was that APP showed slight, but significant cross-reactivity (0.017) with antiserum L134 (Fig. 2 and Table I). Aside from the shared penultimate residue, Arg, there are no other identical amino acids in the two peptides (Fig. 1). The Cterminal hexapeptide of BPP i.e. BPP6, which differs
299 100-
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40-
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~
ype
O-
1000
PEPTIDE
o.1
; RAT
lb
EXTRACT
10000 100000 1000000
(pmole I-!)
~o (ul mF1)
Fig. 2. Radioimmunoassay dilution curves of FMRFamide, APP, NPY, PYY and BPP6 (synthetic C-terminal hexapeptide of BPP), together with the two components found in rat brain extracts. The latter preparations were obtained after ion-exchange and gel filtration chromatography (see Figs. 3 and 4), their concentrations are expressed in terms of the volume of sample added to the assay tube. Concentrations of the other peptides are expressed in terms of pmol'l 1 in the assay tube. See text for details of the assay incubation and separation conditions.
from the C-terminus of APP in the substitution of Pro for His had only very low activity in the FMRFamide assay (Fig. 2). Two other naturally-occurring peptides which belong to the PP family, namely PYY and NPY, contain Gln in the third position from the C-terminus, and these peptides - - like BPP6 - - had only very low immunoreactivity with L134 (0.001 or less). It was of interest that although APP inhibited binding of labelled YFMRFamide to L134, the antiserum showed no significant binding to [~25I]-labelled APP (B/F 0.01 at a titre of 1:1000); presumably substitution of ~25I on the C-terminal Tyr residue of APP abolished immunoreactivity with L134. In order to characterize more precisely the specificity of antiserum L134, a number of tetrapeptide analogues of FMRFamide were studied (Table I). The substitution of Nle for Met was well tolerated. In analogues with the Nle substitution, further substitution in the first position had rela14,
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6.
o_ TABLE I
Immunochemical potencies of FMRFamide, PP and gastrinrelated peptides compared with standard FMRFamide Peptide
Immunochemical potency relative to FMRFamide
Phe-Met-Arg-Phe-NH2 Phe-Nle-Arg-Phe-NH~ Trp-Nle-Arg-Phe-NH~ Nle-Arg-Phe-NH2 Phe-Met-Lys-Phe-NHz Phe-Met-Orn-Phe-NH2 Phe-Nle-Arg (NO2) Phe-NH2 Trp-Met-Asp-Phe-NHz Trp-Met-Ser-Phe-NH2 Trp-Met-Cys-Phe-HN~ Trp-Met-Glu-Phe-NH2
1.000 0.35 0.31 0.21 0.0018 0.0022 0.636 0.00023 0.00018 0.00015 0.00024
APP BPP6 NPY PYY
0.017 0.0003 0.0010 0.0008
Z LU Z 0
L 1
i
I
4'
(3
2" 09
1110
0
w
m
~ ..J
O
Fig. 3. Distribution of FMRFamide-like immunoreactivity in rat brain. See text for the method of extraction. Values are mean ± S.E. for 6 rats.
300
Distribution of rat CNS immunoreactivity
FMRFamide
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100 (ml)
Fig. 4. Separation on CM Sephadex of an extract of rat spinal cord. The extract was first partially purified using a Sep-pak cartridge. The column (1 x 10 cm) was developed with a gradient from 0.05 M ammonium acetate (pH 4.5) to 1.0 M ammonium acetate (pH 7.5). The horizontal bar shows the elution position of standard FMRFarnide.
tively little effect on immunoreactivity. However, analogues with substitutions in the Arg position had markedly diminished immunoreactivity. This presumably explains why gastrin and CCK, which share with FMRFamide two residues in the C-terminal tri-peptide (i.e. Met-Asp-Phe-NH2 compared with Met-Arg-Phe-NH2), do not react significantly with L134.
FMRFamide '
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Fig. 5. Separation on Sephadex G50 of the two rat brain components with FMRFamide immunoreactivity obtained from Sephadex. The columns (1 × 100 cm) were eluted with 0.5 M acetic acid. V0 shows the elution position of bovine serum albumin and Vt the elution position of NaCI. Also indicated by vertical arrows at the top are the elution positions of standard APP, substance P (SP) and FMRFamide.
Extracts of rat brain and spinal cord produced graded inhibition of binding of label to antiserum L134 which was parallel to that of the standard peptide (Fig. 2). Similar results were obtained with crude extracts of rat brain and spinal cord, and with the two components obtained after gel filtration and ion-exchange chromatography (see below). The distribution of immunoreactivity in the rat CNS was not uniform, Concentrations were highest in the hypothalamus, brain stem and spinal cord, and were lowest in the cerebellum and cortex (Fig. 3). Within the spinal cord concentrations were about two-fold higher in the dorsal cord compared with the ventral cord. Outside the CNS, e.g. in adrenal gland, gastrointestinal tract and pancreas, the concentrations of FMRFamide-like immunoreactivity were less than 2 pmol.g -1. However, when extracts of pancreas were examined with an RIA using antisera specific for the C-terminus of BPP high concentrations of immunoreactivity were found (322 ~ 33 pmol.g-1). The results suggest that rat PP does not react significantly with L134.
Chromatographic properties Fractionation of spinal cord extracts on CM Sephadex resolved two peaks of immunoreactivity, designated I and II in order of emergence. Both peaks eluted significantly before standard F M R F amide, suggesting they were less basic. In 6 column runs the ratio of peak I:II varied from 39:61 to 60:40, and the mean was 49:51. Tubes corresponding to the two peaks were pooled, desalted by Seppak cartridges and the eluates run on Sephadex gel filtration columns. Peak I emerged from Sephadex G50 in a similar position to the tetrapeptide F M R F amide, i.e. in or just after the salt region. In contrast, peak II emerged slightly earlier which would be consistent with a higher molecular weight. In the same gel filtration system, natural APP emerged significantly earlier than the rat brain FMRFamidelike components. Neither of the rat brain components purified by ion-exchange and gel filtration chromatography reacted in the APP radioimrnunoassay. DISCUSSION Antibodies specific for the C-terminus of the
301 molluscan neuropeptide, FMRFamide, have been shown here to react in RIA with two molecules in extracts of rat brain. There are obvious structural similarities between FMRFamide and two other established mammalian neuropeptides, namely the opioid heptapeptide, Met-enk Arg6phe 7 (refs. 24, 27), and the CCK C-terminal octapeptide (CCK8) 5. However, neither of the latter peptides reacts significantly with the FMRFamide antibody, L1346. There are also structural similarities, albeit more subtle ones, between FMRFamide and the pancreatic polypeptide (PP) group of molecules, in the form of common penultimate Arg residues and an aromatic amino acid amide at the C-terminus. The known mammalian representatives of the PP family correspond to PP itself from pancreas, PYY from gut, and NPY from brain. These 3 peptides show very low immunoreactivity ( < 0.001) in the FMRFamide RIA, but chicken PP, i.e. APP, was found to have significant immunochemical potency (1-2 ~) in the present assay. For present purposes, the crucial difference between the 4 members of the PP group may be seen to lie in the residue at position 3, counting from the C-terminus: it would appear that antibody L134 tolerates the substitution of Met in FMRFamide for His, as in APP, rather better than that for Gln (PYY and NPY) or Pro (BPP). There have been several reports that antibodies to APP reveal material in rat CNS when used in immunohistochemistry~6,~s,2L The identity of the material is not known, but several lines of evidence indicate that the FMRFamide-like material we have described is distinct from APP and from rat PP. (1) The FMRFamide-like activity purified by ion-exchange and gel filtrationchromatographydid not react in an RIA for APP. (2) The chromatographic properties of rat FMRFamide-like material were clearly distinct from those of APP. (3) Extracts of rat pancreas which contained abundant material reacting in an RIA using C-terminal-specific BPP antisera, did not react significantly with L134. Although the structure of rat PP has not been reported it would appear reasonable to suppose that the FMRFamide-like material we have described is not the same as rat PP. The two FMRFamide-like molecules we have described eluted from cation exchange columns before standard FMRFamide and so are apparently
less basic. One of the immunoreactive forms also eluted before FMRFamide on gel filtration and might therefore be of higher molecular weight. In other species we have also found molecules that react with L134 but are larger and less basic than FMRFamide; thus in chicken brain there are at least 6 immunoreactive components of which two have now been isolated to homogeneity and their Cterminal dipeptides tentatively identified as -Arg-Phe-NH~ in both cases (G. J. Dockray and J. R. Reeve, unpublished observations). At first sight it is attractive to suppose that the rat brain peptides might correspond to C-terminallyamidated forms of Met-enk Arg6Phe 7 or its Nterminally extended variants z6. However, recent developments in our understanding of the biosynthetic mechanisms which are involved in the production of C-terminally amidated peptides makes this possibility unlikely. In the case of several peptides, including gastrin and calcitonin, it is probable that the C-terminal amide is generated from a glycine residue which in the large biosynthetic precursor protein immediately follows the putative C-terminal amide residue19,az. Moreover, an enzyme which converts peptides with the general structure -X-Gly to the corresponding amide, -X-NH2 (where X may be Phe, Val, or Gly, but not Lys or Asp), has been identified in pituitary homogenatesz. Structural studies on the mRNA coding for Met-enkephalin in bovine adrenal medulla and human phaeochromocytomas indicate that Met-enk Arg6phe 7 constitutes the final heptapeptide sequence of the precursora,15,2z; since there is no Gly following the Phe the possibility of generating a C-terminal amide by mechanisms similar to those presently understood can be excluded. The biological role of the rat brain material we have described is as yet unknown. However, it is clear from immunohistochemical studies that FMRFamide-like peptides in rat brain have a neuronal origin, and occur in nerve terminalsS,3L Since there is a wealth of evidence pointing to neurotransmitter or neuromodulatory roles for other neuropeptidesa7, 25, it would not seem unreasonable to suggest that the two molecules described here belong to this general class of regulatory substances. Furthermore, it is of interest that FMRFamide itself has direct excitatory actions on neurones in the brain
302 stem o f the rat when a p p l i e d b y i o n t o p h o r e s i s 1°. These actions are p l a i n l y d i s t i n g u i s h a b l e f r o m those o f M e t - e n k Arg6Phe 7 which has typical o p i o i d - l i k e i n h i b i t o r y effects on m a n y o f the cells t h a t are excited by F M R F a m i d e ; m o r e o v e r , n a l o x o n e blocks the a c t i o n o f M e t - e n k A r g 6 p h e 7 b u t n o t F M R F a m i d e 1°. Evidently m a m m a l i a n neurones b e a r receptors which recognize F M R F a m i d e , a n d it is t e m p t i n g to speculate t h a t the molecules we have described here are the n a t u r a l ligands for these receptors. T h e relatively high c o n c e n t r a t i o n s o f F M R F a m i d e - l i k e p e p t i d e s in spinal cord, b r a i n stem a n d h y p o t h a l a m u s w o u l d therefore p o i n t to a role in these regions as a n e u r o t r a n s m i t t e r - l i k e sub-
stance. C h e m i c a l i s o l a t i o n a n d e l u c i d a t i o n o f structure o f the r a t p e p t i d e s are n o w n e e d e d to establish their i d e n t i t y and to m a k e p o s s i b l e the study o f their p h y s i o l o g i c a l roles.
REFERENCES
12 Greenberg, M. J., Painter, S. D. and Price, D. A., The amide of the naturally occurring opioid (Met) enkephalinArg6-Phe7 is a potent analog of the molluscan neuropeptide FMRFamide, Neuropeptides, 1 (1981) 309-317. 13 Greenberg, M. J. and Price, D. A., Cardioregulatory peptides in molluscs. In F. R. Bloom (Ed.), Peptides: Integrators of Cell and Tissue Function, Raven Press, New York, t980, 107-126. 14 Grimmelikhuijzen, C. J. P., Dockray, G. J. and Schot, L. P. C., FMRFamide-like immunoreactivity in the nervous system of hydra, Histochemistry, 74 (1982) 499-508. 15 Gubler, U., Seeburg, P., Hoffman, B. J., Gage, L. P. and Udenfriend, S., Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides, Nature (Lond.), 295 (1982) 206-208. 16 H6kfelt, T., Lundberg, J. M., Terenius, L., Jancso, G. and Kimmel, J., Avian pancreatic polypeptide (APP) irnmunoreactive neurons in the spinal cord and spinal trigeminal nucleus, Peptides, 2 (1981) 81-87. 17 H6kfelt, T., Johansson, O., Ljungdahl, A., Lundberg, J. M. and Schultzberg, M., Peptidergic neurones, Nature (Lond.), 284 (1980) 515-521. 18 Hunt, S. P., Emson, P. C., Gilbert, R., Goldstein, M. and Kimmel, J. R., Presence of avian pancreatic polypeptide-like immunoreactivity in catecholamine and methionine-enkephalin-containing neurones within the central nervous system, Neurosci. Lett., 21 (1981) 125-130. 19 Jacobs, J. W., Goodman, R. H., Chin, W. W., Dee, P. C., Habener, J. F., Bell, N. H. and Potts, J. R., Jr., Calcitonin messenger RNA encodes multiple polypeptides in a single precursor, Science, 213 (1981) 457-459. 20 Kimmel, J. R., Hayden, J. and Pollock, H. G., Isolation and characterization of a new pancreatic polypeptide hormone, J. biol. Chem., 250 (1975) 9369-9376. 21 Lor6n, I., Alumets, J., Hhkanson, R. and Sundler, F., Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: preliminary immunocytochemical observations, Cell Tissue Res., 200 (1979) 179-186. 22 Noda, M., Furutani, Y., Takahashi, H., Toyosato, M., Hirose, T., Inayama, S., Nakanishi, S. and Numa, S.,
1 Boer, H. H., Schot, L. P. C., Veenstra, J. A. and Riechelt, D., Immunocytochemical identification of neural elements in the central nervous systems of a snail, some insects, a fish, and a mammal with an antiserum to the molluscan cardio-e×citatory tetrapeptide FMRFamide, Cell Tissue Res., 213 (1980) 21-27. 2 Bradbury, A. F., Finnie, M. D. A. and Smyth, D. G., Mechanism of C-terminal amide formation by pituitary enzymes, Nature (Lond.), 298 (1982) 686-688. 3 Comb, M., Seeburg, P. H., Adelman, J., Eiden, L. and Herbert, E., Primary structure of the human Met- and Leu-enkephalin precursor and its mRNA, Nature (Lond.), 295 (1982) 663-666. 4 Cottrell, G. A., FMRFamide neuropeptides simultaneously increase and decrease K + currents in an identified neurone, Nature (Lond.), 296 (1982) 87-89. 5 Dockray, G. J., Gregory, R.A., Hutchison, J. B., Harris, J. I. and Runswick, M. J., Isolation, structure and biological activity of two cholecystokinin octapeptides from sheep brain, Nature (Lond.), 274 (1978) 711-713. 6 Dockray, G. J., Vaillant, C. and Williams, R. G., New vertebrate brain-gut peptide related to a molluscan neuropeptide and an opioid peptide, Nature (Lond.), 293 (1981) 656-657. 7 Dockray, G. J., Vaillant, C., Williams, R. G., Gayton, R. J. and Osborne, N. N., Vertebrate brain-gut peptides related to FMRFamide and Met-enkephalin Arg6Phe7, Peptides, 2, Suppl. 2 (1981) 25-30. 8 Dockray, G. J. and Williams, R. G., FMRFamide immunoreactivity in rat brain: a new mammalian neuropeptide, Neurosci. Lett., Suppl. 7 (1981) $452. 9 Doble, K. E. and Greenberg, M. J., The clam rectum is sensitive to FMRFamide, the enkephalins and their common analogs, Neuropeptides, 2 (1982) 157-167. 10 Gayton, R. J., Mammalian neuronal actions of FMRFamide and the structurally related opioid Met-enkephalinArg6-Phe7, Nature (Lond.), 298 (1982) 275-276. ] 1 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in rat brain, J. Neuroehem., 13 (1966) 655-659.
ACKNOWLEDGEMENTS W e are grateful to the M R C for financial s u p p o r t . A s ever, it is a p l e a s u r e to a c k n o w l e d g e the skilled technical assistance o f C a r o l Higgins, H i l a r y S m i t h a n d Lesley C h a r l e s w o r t h . W e are also grateful to Drs. J. K i m m e l , J. M o r l e y , V. M u t t , K. T a t e m o t o a n d I. L. T a y l o r for gifts o f a n t i s e r u m a n d peptides.
303
23 24
25 26
27
Cloning and sequence analysis of eDNA for bovine adrenal preproenkephalin, Nature (Lond.), 295 (1982) 202-206. Price, D. A. and Greenberg, M. J., Structure of a molluscan cardioexcitatory neuropeptide, Science, 197 (1977) 670-671. Rossier, J., Audigier, Y., Ling, N., Cros, J. and Udenfriend, S., Met-enkephalin-Argn-Phe7, present in high amounts in brain of rat, cattle and man, is an opioid agonist, Nature (Lond.), 288 (1980) 88-90. Snyder, S.H., Brain peptides as neurotransmitters, Science, 209 (1980) 976-983. Stern, A. S., Jones, B. N., Shively, J. E., Stein, S. and Udenfriend, S., Two adrenal polypeptides: proposed intermediates in the processing of pro-enkephalin, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 1962-1966. Stern, A., Lewis, R. V., Kimura, S., Rossier, J., Gerber, L. D., Brink, L., Stein, S. and Udenfriend, S., Isolation of the opioid heptapeptide Met-enkephalin (Arg 6, Phe 7)
28
29 30 31 32
from bovine adrenal medullary granules and striatum, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 6680-6683. 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. Vaillant, C. and Taylor, I. L., Demonstration of carboxylterminal PP-like peptides in endocrine cells and nerves, Peptides, 2, Suppl. 2 (1981) 31-35. Voigt, K. H., Keihling, C., Frosch, D., Schieber, M. and Martin, R., Enkephalin-related peptides: direct action on the octopus heart, Neurosci. Lett., 27 (1981) 25-30. Weber, E., Evans, C. J., Samuelsson, S. J. and Barchas, J. D., Novel peptide neuronal system in rat brain and pituitary, Science, 214 (1981) 1248-1251, Yoo, O. J., Powell, C. T.and Agarwal, K. L., Molecular cloning and nucleotide sequence of full-length cDNA coding for procine gastrin, Proc. nat. Acad. Sci. U.S.A., 79 (1982) 1049-1053.
295
FMRFamide-Like Immunoreactivity in Rat Brain: Development of a Radioimmunoassay and its Application in Studies of Distribution and Chromatographic Properties GRAHAM J. DOCKRAY and R. G. WILLIAMS Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool L69 3BX (U.K.) (Accepted October 12th, 1982) Key words: FMRFamide - - radioimmunoassay - - neuropeptides - - pancreatic polypeptide
A radioimmunoassay is described for the molluscan neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide). The antibody used is C-terminal-specificand shows slight but significant (1-2 ~) cross-reactivitywith chicken pancreatic polypeptide (APP). The assay has been used to identify in rat brain extracts a pair of molecules that may represent mammalian counterparts of FMRFamide. Their concentrations were highest in spinal cord and hypothalamus (> 10 pmol'g 1) and lowest in cerebellum and striatum (<3.5 pmol'g-1). The two immunoreactive peptides were separated on CM ion-exchangechromatography where they appeared to be less basic than FMRFamide. On Sephadex G50 gel filtration one eluted in a similar position to FMRFamide and the other slightly earlier suggesting it may be of higher molecular weight. The rat immunoreactive components do not correspond to previously described neuropeptides or hormones, and may be members of a new group of mammalian neuropeptides with transmitter or modulatory functions. INTRODUCTION The tetrapeptide, Phe-Met-Arg-Phe-NH2 (FMRFamide), was first isolated from ganglia of the clam, Macrocallista nimbosa, by Price and Greenberg in 197723. This peptide and its N-terminally extended analogues such as T y r - G l y - G I y FMRFamide (i.e. Y G G F M R F a m i d e , in the single letter notation for amino acids), have been shown to possess potent actions on many molluscan tissues, e.g. cardio-excitatory and cardio-inhibitory effects in different species, contraction of various noncardiovascular muscles, and direct effects on certain neurones4,9,12,1z, ~°. In addition, they evoke excitation of neurones in the brain stem of the rat 10. Recently, antibodies specific for FMRFamide have been used in immunohistochemical studies to reveal material in the nervous systems of other invertebrate groups, e.g. coelenterates, arthropods, and in the central nervous system and gut endocrine cells of a variety of vertebrate species such as fish, amphibia, birds and mammals 1,~-8,14,~1. Preliminary studies using a radioimmunoassay for FMRFamide have 0006-8993/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
also suggested the presence of immunoreactive material in extracts of the brain of amphibia, birds and mammals which is larger and less basic than F M R F amide 6-s. The primary sequence of F M R F a m i d e occurs at the C-terminus of the enkephalin-related heptapeptide, Tyr-Gly-Gly-Phe-Met-Arg-Phe, which has been isolated from bovine adrenal and striatum 24,27. However, on present evidence this peptide exists only in a form terminating in a free carboxyl terminus (Fig. 1). The antisera currently used to detect F M R F a m i d e require a C-terminal amide group and do not react with the enkephalinlike heptapeptide 6. The present paper describes the development of a radioimmunoassay (RIA) for FMRFamide and the results obtained when this assay was used to study the chromatographic properties and distribution of material in extracts of rat CNS. The data indicate the existence in rat CNS of two FMRFamide-like peptides which occur in highest concentrations in hypothalamus and spinal cord. A subsequent paper will describe the distribution of FMRFamide-like immunoreactivity in rat brain studied by immunohistochemistry.
296 MATERIALS AND METHODS
Peptides The following peptides were obtained from Peninsula: F M R F a m i d e , T y d F M R F a m i d e ( Y F M R F amide), Y G G F M R F a m i d e , and its free acid, Y G G F M R F . The C-terminal hexapeptide of bovine pancreatic polypeptide, designated BPP6, was obtained from UCB (Bruxelles, Belgium). Dr. Joe Kimmel donated avian pancreatic polypeptide (APP) 20. Dr. K. Tatemoto and Professor V. Mutt donated the porcine peptides N P Y and P Y Y (it should be noted that APP, BPP, N P Y and PYY all share a common penultimate Arg residue with FMRFamide, see Fig. 1)28. The remaining peptides used for the specificity studies presented in Table I were donated by Dr. J. Morley.
Antisera For production of antisera, rabbits were immunized with F M R F a m i d e coupled to thyroglobulin. Conjugation was by glutaraldehyde which crosslinks amino groups and so would be predicted to couple the primary amino group of F M R F a m i d e to the carrier protein thereby leaving the C-terminus
FMRFamide Met-enk Arg 6Phe 7 CCK8
free to promote antibody formation. F M R F a m i d e (500 nmol) was dissolved in 0.5 ml of phosphate buffer (0.1 M, p H 7.4) and added to 2 mg thyroglobulin dissolved in 0.5 ml of the same buffer. Glutaraldehyde (20 #1, 5 ~ ) was added slowly to the mixture which was then incubated at 22 °C for 30 min, and finally transfered to a dialysis sac and dialyzed against distilled water (4 1) for 24 h. The efficiency of coupling of peptide to carrier protein was assessed by the incorporation of a small amount of [lzSI]labelled Y F M R F a m i d e to the reaction mixture; in 5 conjugations the incorporation of peptide was 63 4- 6 ~ (mean 4- S.E.). Rabbits (n = 6) were immunized with the equivalent of 75 nmol of tetrapeptide emulsified in Freund's complete adjuvant and administered by multiple intradermal injections over the back. The rabbits were boosted at 6-10 week intervals and bleedings taken from an ear vein 7-10 days after each immunization. All rabbits produced antisera but only two (L134 and L155) were suitable for radioimmunoassay. Since the titre and sensitivity of assays with L134 were greater than with L155 only the former antiserum was used for the studies described here. The dilution of L134
Phe-Met-Arg-Phe-NH 2 Tyr-Gly-Gly-Phe-Met-Arg-Phe Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH 2
APP
-VaI-Thr-Arg-His-Arg-Tyr-NH 2
BPP
-Leu-Thr-Arg-Pro-Arg-Tyr-NH 2
PYY
-VaI-Thr-Arg-GIn-Arg-Tyr-NH 2
NPY
-Arg-Gin-Arg-Tyr-NH 2
Fig. 1. Amino acid sequences of FMRFamide and several structurally related peptides. Met-enkephalin Arg6Phe7 is the opioid heptapeptide isolated from bovine adrenal and striatum. CCK8 is the C-terminal octapeptide of cholecystokinin and has been isolated from sheep brain. APP is avian (chicken) pancreatic polypeptide, and BPP is bovine pancreatic polypeptide. PYY is a newly isolated gut peptide with Tyr at the N-terminus and Tyr-NH~ at the C-terminus; likewise the structurally related molecule, NPY - - isolated from pig brain - - also has Tyr at the N-terminus and Tyr-NH2 at the C-terminus.
297 needed to bind 50 ~ of a trace amount of Y F M R F amide label was 1:30,000 after the second immunization and thereafter decreased with repeated immunization with fresh conjugate, so that after the fourth immunization the titre was only 1:5000. However, there was no apparent change in either specificity or sensitivity of assays made with different bleedings of this antiserum. The results described here were made with serum obtained from the third immunization (titre 1:20,000).
Label A pentapeptide variant of FMRFamide with Tyr in the first position, i.e. YFMRFamide, was used for preparation of radio-iodinated peptide by the chloramine-T method. In a siliconized glass tube containing an aliquot of YFMRFamide (1.0 nmol in 7 #1 of ammonium bicarbonate 0.05 M) there was added in order - - 15 #1 phosphate buffer (0.25 M, pH 7.4), 5 #1 Na125I (18.5 MBq), 5 #1 chloramine-T (0.5 mg.ml-1) and finally 20 #1 of sodium metabisulphite (1.0 mg.m1-1) to stop the reaction. The reaction time was 20 s. The reaction mixture was purified by cation exchange chromatography on CM-Sephadex. The column (1 × 10 cm) was equilibrated with 0.05 M ammonium acetate (pH 4.5) and eluted with a gradient to 1.0 M ammonium acetate (pH 7.5); the acetate buffers contained 5 ~ dimethyl formamide and 0.1 ~ w/v bovine serum albumin (BSA). Unincorporated 125I was not retained by the column and emerged in the first 10 ml, a single peak of iodinated peptide then eluted at about 90 ml. Unsubstituted peptide emerged just before the labelled peptide, so that the specific activity of label on the ascending slope of the peak was less than on the descending slope. The specific activity of the latter was estimated by auto-inhibition curves to be 1388 cpm.fmol-~; allowing for counting efficiency this specific activity is close to the theoretical maximum for mono-iodo label. As might be expected, the later eluting label gave more sensitive assays than the earlier eluting tubes and was used routinely in the work described here. In some experiments the heptapeptide, YGGFMRFamide, was iodinated and used as label in place of YFMRFamide. The results obtained with the two labels were indistinguishable. Assay system The conditions for incubation and separation of
assays were systematically examined, and the following system adopted because it gave: (a) minimum non-specific binding, defined as apparant binding of label in the absence of antibody; (b) highest working dilution of antiserum in routine assays; and (c) the most sensitive assays, expressed in terms of the concentration of standard needed for 50 ~ inhibition of binding of label to antiserum. All assays were carried out in glass tubes pretreated with Sigmacoat to minimize non-specific adsorption of label to glass surfaces. The assay diluent consisted of phosphate buffer (0.05 M, pH 7.4), sodium chloride (0.14 M), sodium azide (3 mM), and BSA (0.05 ~, w/v); volume in each tube was 1.0 ml. Assays were incubated for 24 h at 4 °C and separated by addition of charcoal pre-coated with dextran and plasma, followed by centrifugation at 2000 g for 10 min. The separation step was found to be very temperaturesensitive and care was taken to maintain temperature at 4 °C throughout this step. All samples and standards were prepared in duplicate, and were added to the assay tube in volumes of 10-100 #1. Samples prepared in acetic acid caused non-specific interference when added directly to the assay in volumes greater than 20 #1; routinely therefore, these samples were lyophilized in the assay tube to remove acid prior to addition of diluent, antibody and label. Synthetic FMRFamide was used as a radioimmunoassay standard throughout this study. The specificity of the assay was established by examination of the relative concentrations of different peptides needed to produce 50 inhibition of binding of label. In these experiments each peptide was added in a range of concentrations to ensure that at least 4 points fell within the dynamic range of the assay. The results described below indicate that APP reacts slightly with antiserum L134, and some samples were therefore examined in a RIA for APP. Antiserum to APP was kindly donated by Dr. J. R. Kimmel and was used with natural APP as label. The APP assay showed no significant cross-reactivity ( < 0.0001) with FMRFamide and only slight cross-reactivity with BPP6 (0.0003). In addition, in a few experiments, samples were examined with an RIA using antiserum raised to BPP6 (antibody S 11, kindly donated by Dr. I. L. Taylor). This antiserum was used with BPP6 as label and standard; neither APP, nor
298 FMRFamide showed significant cross-reactivity in this assay ( < 0.0001).
Extracts Three extraction methods were examined. (1) Rat brain tissue (generally 0.1-0.2 g) was quickly weighed and added directly to boiling water (0.1 g.m1-1) and briefly boiled (2 rain). The extract was homogenized (Ultra-Turrax, 30 s) and then centrifuged (3000 g, 10 min). (2) The pellet after boiling water extraction was re-extracted in acid by suspending it in 0.5 M acetic acid in a volume equivalent to the original one. After standing for 30 min at 4 °C the acid extract was centrifuged as before. (3) Tissue was added directly to boiling 0.5 M acetic acid and processed along the lines described for method (1) above. When applied to the extraction of FMRFamide-like immunoreactivity in rat brain stem (n = 4), method (1) gave 6.7 4- 3.2 pmol.g -1, method (2) - - 2.4 4- 0.16 pmol.g -1, and method (3) - - 6.8 41.0 pmol.g -1. Since the sum of the activity recovered by methods (1) and (2) combined (8.8 4- 3.2 pmol.g -1) is greater than that of (3), the final procedure adopted was to extract first in boiling water and then re-extract the pellet in acetic acid.
Distribution studies Under deep halothane anaesthesia, rats were perfused transcardially with an ice-cold solution of 0.14 M NaC1 and the brain and spinal cord were removed. Brains were then dissected according to a modification of the method of Glowinski and Iversen (1966)11 to give the following regions: cerebellum, medulla and pons, amygdala and pyriform cortex, hypothalamus, cortex, hippocampus, striatum, olfactory bulb, thalamus, and mid brain; the spinal cord from cervical to sacral regions was also removed. The tissue samples were rapidly frozen on dry-ice, weighed and extracted by the method described above. In a few experiments samples were also taken from adrenal gland, and from stomach, intestine and pancreas.
Chromatographic studies In initial experiments crude tissue extracts were applied directly to gel filtration and ion-exchange columns. Generally, however, the resolution was poor in these experiments and in the later studies
which are described below all samples were first purified by adsorption on Sep-pak cartridges (Waters Associates) before column chromatography. The Sep-pak cartridges were primed with 2 ml of 80 ~ methanol, 19 ~ water and 1 ~ formic acid, and then washed with 10 ml of 1 ~ formic acid. The tissue extracts were acidified by addition of formic acid to give 1 ~ (any insoluble material was removed by centrifugation) and then passed twice through the cartridge. With extract volumes up to 80 ml the uptake of FMRFamide immunoreactivity on the first pass was 74 4- 3 ~ (n = 4), and after two passes the total uptake was 91 4- 1 ~ (n = 4). The cartridge was then briefly washed with 5 ml 1 ~ formic acid, and the FMRFamide-immunoreactivity eluted with 2 ml of the methanol solution already described. The eluates were dried in a Speedi-Vac Concentrator (Savant) and reconstituted in assay buffer. The recovery of retained immunoreactive material was 97 4- 15 ~. The product was fractionated on CM Sephadex using the same system as that described for the purification of YFMRFamide label. The recovery of material from these columns was 71 421 ~ (n = 5). Tubes corresponding to the peaks of immunoreactivity in the CM Sephadex eluates were pooled and desalted by passage through a Sep-pak as already described, and then separated by gel filtration on Sephadex G50 or G25. The latter columns (superfine grade, 1 × 100 cm) were equilibrated and run in 0.5 M acetic acid. RESULTS
Antibody specificity In a sequence of 13 typical assays the concentration of FMRFamide needed to inhibit the binding of label by 50~ was 29.4 ~ 1.6 pmol.1-1. It was previously noted that YFMRFamide and YGGFMRFamide react virtually equally with the tetrapeptide, but deletion of the C-terminal amide, e.g. FMRF-OH and YGGFMRF-OH, markedly reduced immunoreactivity6. A surprising finding in the present study was that APP showed slight, but significant cross-reactivity (0.017) with antiserum L134 (Fig. 2 and Table I). Aside from the shared penultimate residue, Arg, there are no other identical amino acids in the two peptides (Fig. 1). The Cterminal hexapeptide of BPP i.e. BPP6, which differs
299 100-
80-
60-
40-
FMRFamicle
20-
~
~
ype
O-
1000
PEPTIDE
o.1
; RAT
lb
EXTRACT
10000 100000 1000000
(pmole I-!)
~o (ul mF1)
Fig. 2. Radioimmunoassay dilution curves of FMRFamide, APP, NPY, PYY and BPP6 (synthetic C-terminal hexapeptide of BPP), together with the two components found in rat brain extracts. The latter preparations were obtained after ion-exchange and gel filtration chromatography (see Figs. 3 and 4), their concentrations are expressed in terms of the volume of sample added to the assay tube. Concentrations of the other peptides are expressed in terms of pmol'l 1 in the assay tube. See text for details of the assay incubation and separation conditions.
from the C-terminus of APP in the substitution of Pro for His had only very low activity in the FMRFamide assay (Fig. 2). Two other naturally-occurring peptides which belong to the PP family, namely PYY and NPY, contain Gln in the third position from the C-terminus, and these peptides - - like BPP6 - - had only very low immunoreactivity with L134 (0.001 or less). It was of interest that although APP inhibited binding of labelled YFMRFamide to L134, the antiserum showed no significant binding to [~25I]-labelled APP (B/F 0.01 at a titre of 1:1000); presumably substitution of ~25I on the C-terminal Tyr residue of APP abolished immunoreactivity with L134. In order to characterize more precisely the specificity of antiserum L134, a number of tetrapeptide analogues of FMRFamide were studied (Table I). The substitution of Nle for Met was well tolerated. In analogues with the Nle substitution, further substitution in the first position had rela14,
12"
,T i
z
8.
OE I--
6.
o_ TABLE I
Immunochemical potencies of FMRFamide, PP and gastrinrelated peptides compared with standard FMRFamide Peptide
Immunochemical potency relative to FMRFamide
Phe-Met-Arg-Phe-NH2 Phe-Nle-Arg-Phe-NH~ Trp-Nle-Arg-Phe-NH~ Nle-Arg-Phe-NH2 Phe-Met-Lys-Phe-NHz Phe-Met-Orn-Phe-NH2 Phe-Nle-Arg (NO2) Phe-NH2 Trp-Met-Asp-Phe-NHz Trp-Met-Ser-Phe-NH2 Trp-Met-Cys-Phe-HN~ Trp-Met-Glu-Phe-NH2
1.000 0.35 0.31 0.21 0.0018 0.0022 0.636 0.00023 0.00018 0.00015 0.00024
APP BPP6 NPY PYY
0.017 0.0003 0.0010 0.0008
Z LU Z 0
L 1
i
I
4'
(3
2" 09
1110
0
w
m
~ ..J
O
Fig. 3. Distribution of FMRFamide-like immunoreactivity in rat brain. See text for the method of extraction. Values are mean ± S.E. for 6 rats.
300
Distribution of rat CNS immunoreactivity
FMRFamide
i 300 E n.-
i._
=E It.
E
u,I
o
200
/
FMRFamide-L
100
ill 0 z
0
20
40
ELUTION
60
80
VOLUME
100 (ml)
Fig. 4. Separation on CM Sephadex of an extract of rat spinal cord. The extract was first partially purified using a Sep-pak cartridge. The column (1 x 10 cm) was developed with a gradient from 0.05 M ammonium acetate (pH 4.5) to 1.0 M ammonium acetate (pH 7.5). The horizontal bar shows the elution position of standard FMRFarnide.
tively little effect on immunoreactivity. However, analogues with substitutions in the Arg position had markedly diminished immunoreactivity. This presumably explains why gastrin and CCK, which share with FMRFamide two residues in the C-terminal tri-peptide (i.e. Met-Asp-Phe-NH2 compared with Met-Arg-Phe-NH2), do not react significantly with L134.
FMRFamide '
®
Ii
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01
,-, 3001
2001 0Z =~ =E
1000 ~ i 0
i 0.2
r 0.4
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~ 0.6
= 0.8
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Fig. 5. Separation on Sephadex G50 of the two rat brain components with FMRFamide immunoreactivity obtained from Sephadex. The columns (1 × 100 cm) were eluted with 0.5 M acetic acid. V0 shows the elution position of bovine serum albumin and Vt the elution position of NaCI. Also indicated by vertical arrows at the top are the elution positions of standard APP, substance P (SP) and FMRFamide.
Extracts of rat brain and spinal cord produced graded inhibition of binding of label to antiserum L134 which was parallel to that of the standard peptide (Fig. 2). Similar results were obtained with crude extracts of rat brain and spinal cord, and with the two components obtained after gel filtration and ion-exchange chromatography (see below). The distribution of immunoreactivity in the rat CNS was not uniform, Concentrations were highest in the hypothalamus, brain stem and spinal cord, and were lowest in the cerebellum and cortex (Fig. 3). Within the spinal cord concentrations were about two-fold higher in the dorsal cord compared with the ventral cord. Outside the CNS, e.g. in adrenal gland, gastrointestinal tract and pancreas, the concentrations of FMRFamide-like immunoreactivity were less than 2 pmol.g -1. However, when extracts of pancreas were examined with an RIA using antisera specific for the C-terminus of BPP high concentrations of immunoreactivity were found (322 ~ 33 pmol.g-1). The results suggest that rat PP does not react significantly with L134.
Chromatographic properties Fractionation of spinal cord extracts on CM Sephadex resolved two peaks of immunoreactivity, designated I and II in order of emergence. Both peaks eluted significantly before standard F M R F amide, suggesting they were less basic. In 6 column runs the ratio of peak I:II varied from 39:61 to 60:40, and the mean was 49:51. Tubes corresponding to the two peaks were pooled, desalted by Seppak cartridges and the eluates run on Sephadex gel filtration columns. Peak I emerged from Sephadex G50 in a similar position to the tetrapeptide F M R F amide, i.e. in or just after the salt region. In contrast, peak II emerged slightly earlier which would be consistent with a higher molecular weight. In the same gel filtration system, natural APP emerged significantly earlier than the rat brain FMRFamidelike components. Neither of the rat brain components purified by ion-exchange and gel filtration chromatography reacted in the APP radioimrnunoassay. DISCUSSION Antibodies specific for the C-terminus of the
301 molluscan neuropeptide, FMRFamide, have been shown here to react in RIA with two molecules in extracts of rat brain. There are obvious structural similarities between FMRFamide and two other established mammalian neuropeptides, namely the opioid heptapeptide, Met-enk Arg6phe 7 (refs. 24, 27), and the CCK C-terminal octapeptide (CCK8) 5. However, neither of the latter peptides reacts significantly with the FMRFamide antibody, L1346. There are also structural similarities, albeit more subtle ones, between FMRFamide and the pancreatic polypeptide (PP) group of molecules, in the form of common penultimate Arg residues and an aromatic amino acid amide at the C-terminus. The known mammalian representatives of the PP family correspond to PP itself from pancreas, PYY from gut, and NPY from brain. These 3 peptides show very low immunoreactivity ( < 0.001) in the FMRFamide RIA, but chicken PP, i.e. APP, was found to have significant immunochemical potency (1-2 ~) in the present assay. For present purposes, the crucial difference between the 4 members of the PP group may be seen to lie in the residue at position 3, counting from the C-terminus: it would appear that antibody L134 tolerates the substitution of Met in FMRFamide for His, as in APP, rather better than that for Gln (PYY and NPY) or Pro (BPP). There have been several reports that antibodies to APP reveal material in rat CNS when used in immunohistochemistry~6,~s,2L The identity of the material is not known, but several lines of evidence indicate that the FMRFamide-like material we have described is distinct from APP and from rat PP. (1) The FMRFamide-like activity purified by ion-exchange and gel filtrationchromatographydid not react in an RIA for APP. (2) The chromatographic properties of rat FMRFamide-like material were clearly distinct from those of APP. (3) Extracts of rat pancreas which contained abundant material reacting in an RIA using C-terminal-specific BPP antisera, did not react significantly with L134. Although the structure of rat PP has not been reported it would appear reasonable to suppose that the FMRFamide-like material we have described is not the same as rat PP. The two FMRFamide-like molecules we have described eluted from cation exchange columns before standard FMRFamide and so are apparently
less basic. One of the immunoreactive forms also eluted before FMRFamide on gel filtration and might therefore be of higher molecular weight. In other species we have also found molecules that react with L134 but are larger and less basic than FMRFamide; thus in chicken brain there are at least 6 immunoreactive components of which two have now been isolated to homogeneity and their Cterminal dipeptides tentatively identified as -Arg-Phe-NH~ in both cases (G. J. Dockray and J. R. Reeve, unpublished observations). At first sight it is attractive to suppose that the rat brain peptides might correspond to C-terminallyamidated forms of Met-enk Arg6Phe 7 or its Nterminally extended variants z6. However, recent developments in our understanding of the biosynthetic mechanisms which are involved in the production of C-terminally amidated peptides makes this possibility unlikely. In the case of several peptides, including gastrin and calcitonin, it is probable that the C-terminal amide is generated from a glycine residue which in the large biosynthetic precursor protein immediately follows the putative C-terminal amide residue19,az. Moreover, an enzyme which converts peptides with the general structure -X-Gly to the corresponding amide, -X-NH2 (where X may be Phe, Val, or Gly, but not Lys or Asp), has been identified in pituitary homogenatesz. Structural studies on the mRNA coding for Met-enkephalin in bovine adrenal medulla and human phaeochromocytomas indicate that Met-enk Arg6phe 7 constitutes the final heptapeptide sequence of the precursora,15,2z; since there is no Gly following the Phe the possibility of generating a C-terminal amide by mechanisms similar to those presently understood can be excluded. The biological role of the rat brain material we have described is as yet unknown. However, it is clear from immunohistochemical studies that FMRFamide-like peptides in rat brain have a neuronal origin, and occur in nerve terminalsS,3L Since there is a wealth of evidence pointing to neurotransmitter or neuromodulatory roles for other neuropeptidesa7, 25, it would not seem unreasonable to suggest that the two molecules described here belong to this general class of regulatory substances. Furthermore, it is of interest that FMRFamide itself has direct excitatory actions on neurones in the brain
302 stem o f the rat when a p p l i e d b y i o n t o p h o r e s i s 1°. These actions are p l a i n l y d i s t i n g u i s h a b l e f r o m those o f M e t - e n k Arg6Phe 7 which has typical o p i o i d - l i k e i n h i b i t o r y effects on m a n y o f the cells t h a t are excited by F M R F a m i d e ; m o r e o v e r , n a l o x o n e blocks the a c t i o n o f M e t - e n k A r g 6 p h e 7 b u t n o t F M R F a m i d e 1°. Evidently m a m m a l i a n neurones b e a r receptors which recognize F M R F a m i d e , a n d it is t e m p t i n g to speculate t h a t the molecules we have described here are the n a t u r a l ligands for these receptors. T h e relatively high c o n c e n t r a t i o n s o f F M R F a m i d e - l i k e p e p t i d e s in spinal cord, b r a i n stem a n d h y p o t h a l a m u s w o u l d therefore p o i n t to a role in these regions as a n e u r o t r a n s m i t t e r - l i k e sub-
stance. C h e m i c a l i s o l a t i o n a n d e l u c i d a t i o n o f structure o f the r a t p e p t i d e s are n o w n e e d e d to establish their i d e n t i t y and to m a k e p o s s i b l e the study o f their p h y s i o l o g i c a l roles.
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ACKNOWLEDGEMENTS W e are grateful to the M R C for financial s u p p o r t . A s ever, it is a p l e a s u r e to a c k n o w l e d g e the skilled technical assistance o f C a r o l Higgins, H i l a r y S m i t h a n d Lesley C h a r l e s w o r t h . W e are also grateful to Drs. J. K i m m e l , J. M o r l e y , V. M u t t , K. T a t e m o t o a n d I. L. T a y l o r for gifts o f a n t i s e r u m a n d peptides.
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