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Binding sites for125I ET-1, ET-2, ET-3 and vasoactive intestinal contractor are present in adult rat brain and neurone-enriched primary cultures of embryonic brain cells
278
Brain Research, 554 (L991) 278-285 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391168091
BRES 16809
Binding sites for 125I ET-1, ET-2, ET-3 and vasoactive intestinal contractor are present in adult rat brain and neurone-enriched primary cultures of embryonic brain cells A n t h o n y P. Davenport I and A. Jennifer Morton 2 t Clinical Pharmacology Unit, Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge ( U. K.) and eAFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U. K.) (Accepted 19 February 1991)
Key words: Endothelin; Sarafotoxin S6b; Mouse vasoactive intestinal contractor; Neuron; Primary culture; Receptor; Quantitative autoradiography
Binding sites for iodinated endothelin (ET)-2, ET-3 and vasoactive intestinal contractor (VIC) were visualised in the adult rat brain using quantitative autoradiography and have a similar anatomical distribution to that of ET-1 and sarafotoxin S6b. Highest densities of binding sites for all 5 labelled peptides were present in the granular layer of the cerebellum. Cross-competition experiments show that at a concentration of 1/~M, unlabelled ET-I, ET-2, ET-3, VIC and sarafotoxin S6b were able to compete for the binding sites detected by each of the iodinated peptides. Binding sites for the ET isoforms were also present after 7-14 days in vitro in neurone-enriched primary cultures derived from embryonic rat cerebellum (16-18 days gestation) in which more than 90% of cells stained with an anti-neurofilament antibody. Using micro-autoradiography to detect the binding sites, an average of 14% of cells in these cultures with a diameter of 9.2 + 0.6/~m were associated with high silver grain densities (> 400 grains/100/AmZ).With some of these cells, silver grains were Iocalised over cell bodies and branching processes characteristic of a neuronal phenotype. A second group of cells with high grain densities were more difficult to classify using morphological criteria and may be non-neuronal. The density of silver grains over the remaining cells was low (< 20 grains/100/zm2) and was similar to that measured in nuclear emulsion overlying cultures used to assess non-specific binding. These results indicate that binding sites for all ET peptides are present in both adult rat brain and embryonic cerebellar cultures. In these cultures, binding does not appear to be confined to a single class of cells but may be present on both neuronal and non-neuronal cells. The results suggest that one or more of the ET isoforms could have actions in the rat CNS in addition to the proposed vasoactive role in the peripheral circulation. INTRODUCTION
h u m a n vascular smooth muscle 5'2t'~e but there is increasing evidence that these actions are not confined to
The endothelins (ET) are a family of related isopeptides, ET-1, ET-2 and ET-3, derived from 3 distinct genes found in h u m a n s and rats 22'23'45-47. A fourth E T peptide, vasoactive intestinal contractor (VIC) has been predicted from sequencing mouse genomic D N A and the synthetic peptide has been shown to have pharmacological activity41. Vasoactive snake venom peptides, the sarafotoxins I-2"25-2s, have a high degree of sequence similarity to the ETs and one of these, sarafotoxin S6b, is thought to share a c o m m o n receptor in neuronal tissues 2"26. ET-1 is synthesised by vascular endothelial cells 45"47 and ET-1 m R N A is widely expressed in h u m a n tissue 3' 38,39. We have previously demonstrated that binding sites for ET-1 are present in m a m m a l i a n vascular smooth muscle, and that their distribution parallels that of endothelial cells ll'a2 suggesting that ET-1 may function as a locally released factor TM. ET-1 is a potent constrictor of
the cardiovascular system. O n e or more of the E T isoforms may also function in the CNS 7'33'19'34'48'49. The presence of ET-1 m R N A , ET-1 and ET-3-1ike i m m u n o reactivity has b e e n reported in the brain and spinal cord of the rat 6'17'33"36"42A3"50. Binding sites for 1251 ET-1 have also been detected in these tissues 2,12,16,20,24.26,29,30. We have found an extensive distribution of binding sites for 125I ET-2, ET-3 and VIC in rat vascular tissues 4"J3. However, their anatomical localisation in the CNS has not been established and it is unclear whether binding sites for E T peptides are confined to the vascular tissue or if they are also present on n e u r o n e s and glial cells. The aim of the present study was to determine whether binding sites for the E T isoforms are present in neuronerich cultures derived from embryonic rat cerebellum and to compare their distribution with those of ET-1 and sarafotoxin S6b in the adult rat brain.
Correspondence: A.P. Davenport, Clinical Pharmacology Unit, University of Cambridge, Level 2, F and G Block, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, U.K. Fax: (44) (223) 216893.
279 MATERIALS AND METHODS The following materials were used with the name of the supplier given in parentheses. Dulbecco's Modified Eagles Medium (DMEM, Flow, U.K.); penicillin, streptomycin, glutamine (Gibco, Paisley, U.K.); cytosine arabinoside, Trypan blue, diaminobenzidine, poly-L-lysine (Sigma Chemical, Poole, U.K.); mouse mono-
Total
clonai anti-neurofilament 200 kDa (NF) antibody, horse radish peroxidase (HRP)-conjugated anti-mouse IgG (Vector Laboratories, Peterborough, U.K.); Hyperfilm flmax, (3-[125I]iodotyrosyl13)ET-1, (3-[125I]iodotyrosyl13)ET-2, (3-[lzsI]iodotyrosyl6)ET-3, (3-[125I]iodotyrosyl13)VIC and (3-[l~I]iodotyrosyl13)sarafotoxin S6b, spec. act. 2000 Ci/mmoi (Amersham International, Amersham, U.K.); K2 nuclear emulsion (IIford, Cheshire, U. K.); unlabelled ET-1 and ET-3 (Novabiochem,
NSB
Specific (amol/mm2)
W'--
I
t--W
O4 I
4
~i~ •
~:;.
LLi
I
t--t.l.!
if3 (D
f]3
O >
Fig. 1. Autoradiographical localisation of '~I-ET-1, ET-2 and ET-3 to saggital sections of rat brain. Sections (10/~m thick) were incubated with 20 pM solutions of the labelled peptides at 23 °C for 120 rain followed by washing, drying and apposition to Hyperfilm flmax. After development, the resulting film was used as a negative to produce the black and white autoradiogram for total binding. The second image was digitally subtracted from the first in order to produce a new image showing the amount of specific binding of each peptide coded according to the grey levels shown in the scale bar by comparison with a calibrated radioactive scale co-exposed with the sections of radiolabelled tissue. Lateral 2.40 mm4°, Cx, cortex; St, striatum; Co, cerebellum. Bar = 1 mm.
280 TABLE I Density o f binding sites (pmol/mm 2) in selected areas of adult rat brain incubated with 20 p M of iodinated peptide Each value represents the mean of at least four sections from six animals. ET-1 Cortex Corpus callosum Striatum Hippocampus CA1 CA3 Cerebellum Granular Molecular Spinal cord Grey matter
ET-2
ET-3
VIC
S6b
2.5 ± 0.2 4.3 ± 0.2 4.9 ± 0.3
3.0±0.3 4.5±0.1 5.6±0.3
1.7±0.2" 2.8±0.3* 2.9±0.1"
3.4±0.1 5.0±0.2 5.6±0.1
6.7±0.3* 8.9±0.4* 7.0±0.2*
2.2 ± 0.1 5.2 ± 0.2
2.7±0.1 5.3±0.2
0.8±0.1" 3.2±0.6*
2.5±0.2 5.4±0.9
8.4±0.1" 12.9±0.8"
18.5 ± 0.4 10.5 ± 0.5
19.0±0.5 9.2±1.1
5.3±0.7* 3.6±0.2*
20.3±0.2 13.1±0.2
24.2±1.7" 13.4±1.0"
9.1 ± 1.9
8.3±0.6
4.9±0.2*
8.0±0.1
13.2±0.4"
* Significant difference compared to the density of ET-1 binding (P < 0.05, Tukey's Studentized range for multiple comparison).
Nottingham, U.K.); preproendothelin-(110-130) (Peninsula, St Helens, U.K.); ET-2, VIC and sarafotoxin S6b (Peptide Institute, Osaka, Japan). ET-l-(3-9) and preproendothelin (124-130) were synthesised using solid-phase t-Boc chemistry, purified by size exclusion chromatography and the sequence confirmed by aminoacid analysis. All other reagents were from Sigma Chemical or Fisons (Loughborough, U.K.). Neurone-rich primary cultures Cerebellar cortices were dissected from Wistar rat embryos (16th-18th day of gestation) and dissociated mechanically by trituration with a heat-polished, narrowed Pasteur pipette in sterile DMEM. Two million viable cells (viability > 80% observed by exclusion of Trypan blue) were plated onto sterile poly-L-lysinetreated coverslips in 35 mm Petri dishes. Cells were grown in DMEM containing 20% FCS, 20 IU/ml penicillin, 100 ~g/ml streptomycin and glutamine (0.365 g/l). Three to 5 days after plating, the culture medium was replaced with fresh medium containing 5 /~M cytosine arabinoside to control non-neuronal proliferation. Thereafter, the medium was replaced at intervals of 3 days. The cultures were kept at 37 °C in an humidified atmosphere of 5% CO 2 and 95% air. Cells were used in the receptor binding and immunocytochemistry assays after 7-14 days in culture.
1oo i5
H ET-a
IN v ET-2
/,~..,.~/ET-3~_ ET-1
vie
S6b
[1251] peptide (200 pM) Fig. 2. Comparison of the ability of unlabelled ET and sarafotoxin S6b to compete for binding of the iodinated peptides to the granular layer of the adult cerebellum. The results are expressed as percentage inhibition of the total binding. Each value is the mean of at least 3 sections from 3 animals.
Fibroblast-enriched primary cultures Cerebellar cortices were prepared as for the neurone-rich cultures except that the cells were seeded onto plastic dishes without coverslips. The culture medium was DMEM, 10% FCS, 20 U/ml penicillin, 20/~g/ml streptomycin and glutamine (0.365 g/l). Cells were grown to confluence, and passaged twice before plating onto poly-L-lysine treated coverslips. These cultures were used for autoradiography after 14-21 days in culture. Quantitative in vitro receptor autoradiography Binding sites for 125I-ET-1, 125I-ET-2, lzSI-ET-3, 125I-VIC and 125I-sarafotoxin S6b, were visualised using a method modified from Davenport 1L12. Adult male Wistar rats (150-200 g) were killed by cervical dislocation and decapitation. Brains were removed rapidly and frozen onto cryostat chucks before cutting 10-pm-thiek sections cryostat sections which were thaw-mounted onto gelatin-subbed slides. After a 15 min pre-incubation period in 10 mM HEPES containing MgC12 (5 mM), sections were incubated for 2 h at 23 °C with 20 pM 125I-ET-I, ET-2, ET-3, VIC or sarafotoxin S6b in 10 mM HEPES containing MgCi 2 (5 mM) and bovine serum albumin (0.3%). Non-specific binding was defined by co-incubating adjacent sections with 1 pM of the corresponding unlabeiled peptide in addition to the labelled peptide. At the end of the incubation period, sections were rinsed in 3 successive 5 min washes of ice cold Tris buffer, (pH 7.4) and dried under a stream of cold air. Sections were exposed to radiation sensitive film (Hyperfilm flmax) together with a calibrated radioactive scale s. The resulting autoradiograms were analysed using computer-assisted densitometry (Quantimet 970, Cambridge Instruments, Cambridge, U.K.) by the method of Davenport 9'1° using calibrated radioactive standards 8. At least 4 sections were analysed from 6 rat brains. Coverslips bearing cultured cells were rinsed in 100 ml of 10 mM HEPES buffer (pH 7.4) and then incubated for 1 h at 23 °C in the incubation buffer as previously described containing 200 pM of the radiolabelled peptides. Non-specific binding was defined by coincubating coverslips from the same culture with 1 /~M of the corresponding unlabelled peptide. Each assay was performed in duplicate using 3 separate preparations of neurone-enriched cultures. At the end of the incubation period, coverslips were processed as described for tissue sections. For macro-autoradiography, the dry coverslips were mounted onto microscope slides and apposed to radiation sensitive film. For micro-autoradiography, cells were fixed by exposing coverslips to formaldehyde at 80 °C for 1 h. The coverslips were coated with Ilford K2 emulsion and exposed for 3 weeks. After developing the emulsion, the cultured cells were stained with Methylene blue. Silver grains were visualised using dark-field illumination.
281
lmmunohistochemistry Cerebellar cortical cultures grown on coverslips were fixed for 30 min with 3.7% formaldehyde in 100 mM phosphate-buffered saline (PBS). Cultures were rinsed twice with PBS, incubated for 2-5 min with 0.2% Triton X-100 in PBS and blocked for 30 min at room temperature with PBS containing 3% normal sheep serum and 0.02% Triton X-100. They were then incubated overnight at 4 °C with the mouse monoclonal anti-NF antibody (diluted 1:500). The cultures were washed with PBS and incubated for 1 h at 37 °C with rabbit polyclonal HRP-conjugated second antibody (diluted 1:500 in PBS containing 3% normal sheep serum and 0.02% Triton X-100). After further washes the HRP-antibody complex was visualised with diaminobenzidine, activated with 0.025% H202 in 50 mM Tris-HCl, pH 7.5. The cells on the coverslips were then rinsed in PBS, dehydrated, cleared in xylene and mounted (DPX) onto slides. Specificity of the second antibody was tested by omission of the primary antibody. The number and size of cells per unit area were estimated using the image analyser equipped with a programme written for this purpose. Images of the stained cells were digitised into an array of 600,000 pixels each with a grey value of 0-255. The threshold was set to detect the cells and the number and area of cells within a live frame area of 0.4 mm 2 was measured. An area 100/~m from the perimeter of the coverslip was excluded in order to minimise edge effects.
RESULTS
Tissue sections of adult brain Binding sites for the 4 E T peptides were visualised in
the adult rat brain (Fig. 1, Table I). T h e p a t t e r n was similar for each p e p t i d e , with an increasing binding site density in a rostro-caudal direction. Highest densities were m e a s u r e d in the granular layer of the cerebellum with lower amounts present in the m o l e c u l a r layer. Binding was also d e t e c t e d in the cortex, striatum, h i p p o c a m p u s and spinal cord. A l t h o u g h the p a t t e r n was similar, the density of binding was always higher for sarafotoxin S6b and lower for ET-3 in these regions c o m p a r e d to the o t h e r peptides, even though the specific activity of the labelled p e p t i d e s used in these assays was the same. T h e level of non-specific binding to tissue sections was higher using 125I-ET-3 than that of the other peptides, but was consistently lower using 125I sarafotoxin S6b. A similar p a t t e r n of binding was o b s e r v e d when the labelled p e p t i d e s were i n c u b a t e d with tissue sections at concentrations up to 1 riM. In cross-competition experiments, unlabelled ET-1, ET-2, ET-3, sarafotoxin S6b and V I C (1 p M ) c o m p e t e d for the binding sites of all the i o d i n a t e d p e p t i d e s (Fig. 2) in the cerebellum. H o w e v e r , the h e p t a p e p t i d e fragment, E T - 1 - ( 3 - 9 ) , p r e p r o e n d o t h e l i n - ( l l 0 - 1 3 0 ) (ET-like peptide) and p r e p r o e n d o t h e l i n - ( 1 2 4 - 1 3 0 ) tested at the same concentration did not c o m p e t e for either iodinated E T or sarafotoxin sites in the rat brain.
Fig. 3. Brightfield photomicrograph of a neurone-enriched culture grown in parallel with those used for the receptor binding assay. Cells are stained with anti-NF antibody in combination with a polyclonal HRP-conjugated second antibody and visualised with diaminobenzidine. The cells show long branching processes characteristic of neurnnaMike phenotypes. Bar = 50 ~m.
282
Neurone-rich primary cultures of embryonic brain Cells in the neurone-rich cultures stained with anti-NF antibody are shown in Fig. 3. Positive cells had small cell bodies ( - 1 0 ~m in diameter) and long branching processes characteristic of neurones. Flat and stellate cells did not stain with anti-NF antibody. Binding sites for iodinated ET-1, ET-2 and ET-3 were detected in neurone-rich cultures (Fig. 4) using macro-autoradiography, but binding of the iodinated peptides to fibroblast beds was below the level for detection (< 0.01 ~mol/mm2). The image analyser was used to classify the cells by size. Under our culture conditions, 93% of the cells were estimated to have an average cell diameter of 9.2 _+ 0.6 /~m (n = 9 separate plates + S.E.M.). Using microautoradiography to detect the binding of the 4 iodinated ETs, high densities ( > 400 grains/100~m 2) of silver grains were associated with an average of 14% of cells in these particular neurone-enriched cultures. Many of the cells on which binding sites were present had a characteristic neuronal phenotype. Examples of high grain densities localised over a proportion of cell bodies and along the length of cell processes using labelled ET-2 and ET-3 are shown in Fig. 5b,d). The clustering of cells with high
Total
NSB
ET-1
ET-2
ET-3 if!if
¸ i~i ~i!~ m
Fig. 4. Autoradiographical localisation of 12sIET-1, ET-2 and ET-3 binding to neurone-rich cultures growing on coverslips. Coverslips were incubated with 20 pM solutions of the labelled peptides at 23 °C for 120 rain followed by washing, drying and apposing to Hypgrfilm flmax. Non-specific binding (NSB) was determined by incubating an adjacent coverslip in the presence of the corresponding unlabeUed peptide (1/~M). Bar = 2 mm.
densities of binding sites may reflect the pattern following dispersion of the dissociated cells onto the culture plate and the subsequent inclusion of cytosine arabinoside to prevent non-neuronal cell proliferation leaving groups of neurone-enriched cells. Similar results were obtained using labelled ET-1 and VIC. A second group of labelled cells were more difficult to classify and may be nonneuronal. Low numbers of silver grains ( < 20 grains/100 /~m2) were measured in the emulsion overlying the remaining cells and was similar to those seen in adjacent cultures used to assess non-specific binding. Some of these cells were distinctively non-neuronal (Fig. 5c,d), indicating that binding of ETs was absent or below the level for detection. DISCUSSION We have detected binding sites for 125I-ET-2, ET-3, and VIC in tissue sections from adult rat brain. The pattern was similar to that of ET-112"24and sarafotoxin S6b. The main difference was in the higher density of binding obtained using labelled sarafotoxin S6b, compared with the similar levels of ET-1, ET-2 and VIC. Non-specific binding was consistently higher using ~251ET-3, resulting in lower levels of specific binding with this peptide. Cross-competition experiments show that each unlabelled peptide tested at high concentration was able to compete for the binding of each of the other iodinated compounds. One explanation for these results is that all 5 peptides may be acting via the same population of binding sites. The techniques used in the present study cannot exclude the possibility that multiple receptor sub-types may exist in rat brain which differ in their rank order of affinity for the E T isoforms and sarafotoxin S6b, as proposed in non-neuronal tissue 35'44. Using macroautoradiography and a fixed concentration of iodinated peptide, our studies have not revealed discrete areas of the brain in which there is a differential distribution of one of the labelled peptides compared to the other isoforms. These results support the evidence from functional studies that sarafotoxin S6b and ET-1 share common binding sites and mechanism of action in the brain 2. It is not yet known whether ET-2 has any action in the CNS but ET-3 has been shown to be equipment to ET-1 in stimulating phosphoinositide hydrolysis in guinea-pig cerebellum and there was no evidence for receptor heterogeneity 7. This study has demonstrated that 125I-ETs bind to neurone-enriched cultures with a high density of sites present on a proportion of cell bodies and processes which show a clear neuronal phenotype. Fibroblasts and glia may also be present but we were unable to detect
283 binding of the E T isoforms to fibroblast-enriched cultures, suggesting that these sites were below the level for
a n o n - n e u r o n a l phenotype, but confirmation that these
detection or were not expressed in cultured cells. High
are glia requires the use of antibodies to specific marker proteins for these cells 31. O u r results are consistent with
densities of binding sites were present on some cells with
evidence from previous studies that ET-1, ET-3 and VIC
'
~i!~
!-
~:~
~,!?ii~iiii~ii~
~i:!i~i~ "!
Fig. 5. Localisation of ET binding sites in neurone-rich cultures as the pattern of developed silver grains in nuclear emulsion, revealed under dark-field illumination (b,d,f) lying above cells stained with Methylene blue visualised under bright-field illumination (a,c,e). An example of the high density of binding sites for ET isoforms associated with cells having a neuronal phenotype, is illustrated by the binding of I~I-ET-2, indicated by the arrows in a. Binding (illustrated with ~25I-ET-3)was also detected to some cells with an intermediate phenotype (indicated by arrow heads in c and d) although binding of the iodinated peptides to other cells which were clearly non-neuronal was absent or below the level for detection. Examples of non-specific binding for ET-3 are shown in (f) with the corresponding stained cells shown in (e). Bars = 50/~m.
284 can m o d u l a t e neuronal activity 4s:9 and stimulate the formation of second messengers 2'7'15'26,32:s, suggesting that binding sites for these peptides may be functional receptors The source of endogenous peptides which could be binding to these sites in the adult brain is unclear. R a d i o l a b e l l e d ET-1 does not a p p e a r to cross the b l o o d brain barrier 37 which suggests that release of E T from the endothelial cells lining the vessel walls into the systemic circulation is an unlikely source. ET-like immunoreactivity has been m e a s u r e d in cerebrospinal fluid TM and has been d e t e c t e d in epithelial cells including those from the rat choroid plexus ( D a v e n p o r t and van P a p e n d o r p , unpublished observations). A third possible source of E T peptides are neurones and glia. ET-like immunoreactivity and ET-1 m R N A have been visualised in both types of cell 6'17'34'43'50. ET-3-1ike immunoreactivity has been mea-
munoassay 36'42 but the identities of cells synthesising ET-2 and VIC have not been established. However, using antibodies directed to the N-terminals of ET-1 and ET-3 we have found immunoreactive cell bodies and processes in the adult brain as well as neurone-enriched cultures. We have also m e a s u r e d release of ET-like immunoreactivity into the m e d i a from these cells using a r a d i o i m m u n o a s s a y which can distinguish between ET-1 and ET-313. The results of this study d e m o n s t r a t e the presence of binding sites for the 125I-ETs in the rat CNS and provide further evidence in support of the suggestion that one or m o r e of the E T isoforms could have central actions in modulating neuronal activity in the rat.
by radioim-
Acknowledgements. We thank Caroline Purdy for excellent technical assistance and the Wellcome Trust, the Isaac Newton Trust and the British Heart Foundation for support (A.P.D.).
1 Ambar, I., Kloog, Y., Kochva, E., Wollberg, Z., Bdolah, A., Oron, U. and Sokolovsky, M., Characterization and localization of a novel neuroreceptor for the peptide sarafotoxin, Biochem. Biophys. Res. Commun., 157 (1988) 1104-1110. 2 Ambar, I., Kloog, Y., Schvartz, I., Hazum, E. and Sokolovsky, M., Competitive interaction between endothelin and sarafotoxin: binding and phosphoinositides hydrolysis in rat atria and brain, Biochem. Biophys. Res. Commun., 158 (1989) 195-201. 3 Bloch, K.D., Eddy, R.L., Shows, T.B. and Quertermous, T., cDNA cloning and chromosomal assignment of the gene encoding endothelin 3, J. Biol. Chem., 264 (1989) 18156--18161. 4 Brown, M. J., Davenport, A. P. and Nunez, D. J., Localisation of binding sites for [125]endothelin-1, endothelin-2 and endothelin-3 in rat, pig and human kidneys, J. Physiol., 417 (1989) 35P. 5 Brain, S.D., Crossman, D.C., Buckley, T.L. and Williams, T.J., Endothelin-l: demonstration of potent effects on the microcirculation of humans and other species, J. Cardiovasc. Pharmacol., 13 (1989) S147-149. 6 Cintra, A., Fuxe. K., Anggard, E, Tinner. B., Staines, W. and Agnati, L.F., Increased endothelin-like immunoreactivity in ibotenic acid-lesioned hippocampal formation of the rat brain, Acta Physiol. Scand., 137 (1989) 557-558. 7 Crawford, M.L.A., Hiley, C.R. and Young, J.M., Characteristics of endothelin-1 and endothelin-3 stimulation of phosphoinositide breakdown differ between regions of guinea-pig and rat brain, Naunyn-Schmiedeberg's Arch. Pharmaeol., 341 (1990) 268-271. 8 Davenport, A.P. and Hall, M.D., Comparison between brain paste and polymer [~25I] standards for quantitative receptor autoradiography, J. Neurosci. Methods, 25 (1988) 75-82. 9 Davenport, A.P., Beresford, I.J.M., Hall, M.D., Hill R.G. and Hughes, J., Quantitative autoradiography in neuroscience. In EW. van Leeuwen, R.M. Buijs, C.W. Pool and O. Pach (Eds.), Techniques in the Behaviour Science, Vol. III, Elsevier, Amsterdam, 1988, pp. 121-145. 10 Davenport, A.P., Hill R.G. and Hughes J., Quantitative analysis of autoradiograms. In J.M. Polak (Ed.), Regulatory Peptides, Birkhauser, Basle, 1989, pp. 137-153. 11 Davenport, A.P, Nunez, D.J. and Brown, M.J., Binding sites for 125I-labelledendothelin-1 in the kidneys: differential distribution in rat, pig and man demonstrated by using quantitative autoradiography, Clin. Sci., 77 (1989) 129-131. 12 Davenport, A.P., Nunez, D.J., Hall, J.A., Kaumann, A.J. and
Brown, M.J., Autoradiographical localization of binding sites for porcine [125I]endothelin-1 in humans, pigs, and rats: functional relevance in humans, J. Cardiovasc. Pharmacol., 13 (1989) S166-170. Davenport, A.P., Nunez, D.J. and Brown, M.J., Localisation of binding sites for iodinated endothelin and sarafotoxin peptides in mammalian tissues using quantitative autoradiography, Eur. J. Pharmacol., 183 (1990) 2153. Davenport, A.P., Ashby, M.J., Easton, P., Ella, S., Bedford, J., Dickerson, C., Nunez, D. J., Capper, S. J. and Brown, M.J., A sensitive radioimmunoassay measuring endothelin-like immunoreactivity in human plasma: comparison of levels in essential hypertension and normotensive controls, Clin. Sci., 78 (1990) 261-264. Fu, T., Chang, W., Ishida, N., Saida, K., Mitsui, Y., Okano, Y. and Nozawa, Y., Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells, FEBS Lett., 257 (1989) 351-353. Fuxe, K., Anggard, E., Lundgren, K., Cintra, A., Agnati, L.E, Galton, S. and Vane, J., Localization of [~25I]endothelin-1 and [125I]endothelin-3 binding sites in the rat brain, Acta. Physiol. Scand., 137 (1989) 563-564. Giaid, A., Gibson, S.J., lbrahim, B.N., Legon, S., Bloom, S.R., Yanagisawa, M., Masaki.,T., Varndeli, I.M. and Polak, J.M. Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7634-7638. Hoffman, A., Keiser, H.R., Grossman, E., Goldstein D.S., Gold, P.W. and Kling, M., Endothelin concentrations in cerebrospinal fluid in depressive patients, Lancet, 2 (1989) 1519. Hokfelt, T., Post, C., Freedman, J., Lundberg, J.M. and Terenius, L., Endothelin induces spinal lesions after intrathecal administration, Acta. Physiol. Scand., 137 (1989) 555-556. Hoyer, D., Waeber, C. and Palacios, J.M., [125I]Endothelin-1 binding sites: autoradiographic studies in the brain and periphery of various species including humans, J. Cardiovasc. Pharmacol., 13 (1989) S162-165. Hughes, A.D., Thorn, S.A., Woodall, N., Schachter, M., Hair, W.M., Martin, G.N. and Sever, P.S., Human vascular responses to endothelin-l: observations in vivo and in vitro, J. Cardiovasc. Pharmacol., 13 (1989) $225-228. Inoue, A., Yanagisawa, M, Kimura, S., Kasuya, Y., Miyauchi T., Goto, K. and Masaki, T., The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes, Proc. Natl. Acad. Sci.
sured
in h o m o g e n a t e s
of brain
tissue
REFERENCES
13
14
15
16
17
18 19 20
21
22
285 U.S.A., 86 (1989) 2863-2867. 23 Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Inoue, A., Ishida, N., Mitsui, Y., Onda, H., Fujino, M. et al., Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin, FEBS Lett., 231 (1988) 440-444. 24 Jones, C.R., Hiley, C.R., Pelton, J.T. and Mohr, M., Autoradiographic visualization of the binding sites for [125I]endothelin in rat and human brain, Neurosci. Lett., 97 (1989) 276-279. 25 Kloog, Y., Ambar, I., Sokolovsky, M., Kochva, E., Wollberg, Z. and Bdolah, A., Sarafotoxin, a novel vasoconstrictor peptide: phosphoinositide hydrolysis in rat heart and brain, Science, 242 (1988) 268-270. 26 Kloog,Y. and Sokolovsky, M., Similarities in mode and sites of action of sarafotoxins and endothelins, Trends. Pharmacol. Sci., 10 (1989) 212-214. 27 Kloog, Y., Ambar, I., Kochva, E., Wollberg, Z., Bdolah, A. and Sokolovsky, M., Sarafotoxin receptors mediate phosphoinositide hydrolysis in various rat brain regions, FEBS Len., 242 (1989) 387-390. 28 Kloog, Y., Bousso-Mittler, D., Bdolah, A. and Sokolovsky, M., Three apparent receptor subtypes for the endothelin/sarafotoxin family, FEBS Lea., 253 (1989) 199-202. 29 Koseki, C., Imai, M., Hirata, Y., Yanagisawa, M. and Masaki, T., Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide?, Am. J. Physiol., 256 (1989) R858-66. 30 Koseki, C., Imai, M., Hirata, Y., Yanagisawa, M. and Masaki, T., Autoradiographic localization of [12sI]-endothelin-1 binding sites in rat brain, Neurosci. Res., 6 (1989) 581-585. 31 Loffner, F., Lohmann, S.M., Walckhoff, B., Walter, U. and Hamprecht, B., Immunocytochemical characterization of neuron-rich primary cultures of embryonic rat brain cells by established neuronaland gliai markers and monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein synapsin, Brain Research, 363 (1986) 205-221. 32 Lin, W.W., Lee, C.Y. and Chuang, D.M., Cross-desensitization of endothelin- and sarafotoxin-induced phosphoinositide turnover in neurons, Eur. J. Pharmacol., 166 (1989) 581-582. 33 MacCumber, M.W., Ross, C.A., Glaser, B.M. and Snyder, S.H., Endothelin: visualization of mRNAs by in situ hybridization provides evidence for local action, Proc. Natl. Acad. Sci. U. S. A., 86 (1989) 7285-7289. 34 MacCumber, M.W., Ross, C.A. and Snyder, S.H., Endothelin in brain: Receptors, mitogenesis, and biosynthesis in glial cells, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 2359-2363. 35 Masuda, Y., Miyazaki, H., Kondoh, M., Watanabe, H., Yanagisawa, M., Masaki, T. and Murakami, K., Two different forms of endothelin receptors in rat lung, FEBS Lett., 257 (1989) 208-210. 36 Matsumoto, H., Suzuki, N., Onda, H. and Fujino, M., Abundance of endothelin-3 in rat intestine, pituitary gland and brain, Biochem. Biophys. Res. Commun., 164 (1989) 74-80. 37 Neuser, D., Steinke, W., Theiss, G. and Stasch, J.-P. Autoradiographic localization of [125I] endothelin-1 and [125I] atrial
natriuretic peptide in rat tissue: a comparative study, J. Cardiovasc. Pharmacol., 13 (5)(1989) 67-73. 38 Nunez, D.J., Davenport, A.P., Brown, M.J., Neylon, C.B., and Wyse, R., Distribution of endothelin-1 messenger RNA using complementary DNA amplification by the polymerase chain reaction, J. Hypertens., 7 (1989) 920-921. 39 Nunez, D.J., Brown, M.J., Davenport, A.P., Neylon, C.B., Schofield, J.P. and Wyse, R.K., Endothelin-1 mRNA is widely expressed in porcine and human tissues, J. Clin. Invest., 85 (1990) 1537-1541. 40 Paxinos, G. and Watson, C., The Rat Brain in Sterotaxic Coordinates, Academic Press, Sidney, 1986. 41 Saida, K., Mitsui, Y. and Ishida, N., A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity, J. Biol. Chem., 264 (1989) 14613-14616. 42 Shinmi, O., Kimura, S., Sawamura, T., Sugita, Y., Yoshizawa, T., Uchiyama, Y., Yanagisawa, M., Goto, K., Masaki, T. and Kanazawa, I., Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain, Biochem. Biophys. Res. Commun., 164 (1989) 587-593. 43 Shinmi, O., Kimura, S., Yoshizawa, T., Sawamura, T., Uchiyama, Y., Sugita. Y., Kanazawa, I., Yanagisawa, M., Goto, K. and Masaki, T., Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination, Biochem. Biophys. Res. Commun., 162 (1989) 340-346. 44 Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, Y., Kimura, S., Yanagisawa, M., Masaki, T. and Murakami, K., Two distinct types of endothelin receptors are present on chick cardiac membranes, Biochem. Biophys. Res. Commun., 161 (1989) 1252-1259. 45 Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T., A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature, 332 (1988) 411-415. 46 Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya Y., Kimura, S., Kumagaye, S., Nakajima K., Watanabe T.X., Sakakibara, S., Goto, K. et al., Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6964-6967. 47 Yanagisawa, M. and Masaki, T., Molecular biology and biochemistry of the endothelins, Trends Pharmacol. Sci., 10 (1989) 374-378. 48 Yoshizawa, T., Kimura, S., Kanazawa, I., Uchiyama, Y., Yanagisawa, M. and Masaki, T., Endothelin localizes in the dorsal horn and acts on the spinal neurones: possible involvement of dihydropyridine-sensitive calcium channels and substance P release, Neurosci. Lett., 102 (1989) 179-184. 49 Yoshizawa, T., Kimura, S., Kanazawa, I., Yanagisawa, M. and Masaki, T., Endothelin-1 depolarizes a ventral root potential in the newborn rat spinal cord, J. Cardiovasc. Pharmacol., 13, Suppl. 5 (1989) $216-217. 50 Yoshizawa, T., Shinmi, O., Giaid, A., Yanagisawa, M. Gibson, S.J., Kimura, S., Uchiyama, Y., Polak, J.M., Masaki, T. and Kanazawa, I., Endothelin: a novel peptide in the posterior pituitary system, Science, 247 (1990) 462-464.
Brain Research, 554 (L991) 278-285 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391168091
BRES 16809
Binding sites for 125I ET-1, ET-2, ET-3 and vasoactive intestinal contractor are present in adult rat brain and neurone-enriched primary cultures of embryonic brain cells A n t h o n y P. Davenport I and A. Jennifer Morton 2 t Clinical Pharmacology Unit, Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge ( U. K.) and eAFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U. K.) (Accepted 19 February 1991)
Key words: Endothelin; Sarafotoxin S6b; Mouse vasoactive intestinal contractor; Neuron; Primary culture; Receptor; Quantitative autoradiography
Binding sites for iodinated endothelin (ET)-2, ET-3 and vasoactive intestinal contractor (VIC) were visualised in the adult rat brain using quantitative autoradiography and have a similar anatomical distribution to that of ET-1 and sarafotoxin S6b. Highest densities of binding sites for all 5 labelled peptides were present in the granular layer of the cerebellum. Cross-competition experiments show that at a concentration of 1/~M, unlabelled ET-I, ET-2, ET-3, VIC and sarafotoxin S6b were able to compete for the binding sites detected by each of the iodinated peptides. Binding sites for the ET isoforms were also present after 7-14 days in vitro in neurone-enriched primary cultures derived from embryonic rat cerebellum (16-18 days gestation) in which more than 90% of cells stained with an anti-neurofilament antibody. Using micro-autoradiography to detect the binding sites, an average of 14% of cells in these cultures with a diameter of 9.2 + 0.6/~m were associated with high silver grain densities (> 400 grains/100/AmZ).With some of these cells, silver grains were Iocalised over cell bodies and branching processes characteristic of a neuronal phenotype. A second group of cells with high grain densities were more difficult to classify using morphological criteria and may be non-neuronal. The density of silver grains over the remaining cells was low (< 20 grains/100/zm2) and was similar to that measured in nuclear emulsion overlying cultures used to assess non-specific binding. These results indicate that binding sites for all ET peptides are present in both adult rat brain and embryonic cerebellar cultures. In these cultures, binding does not appear to be confined to a single class of cells but may be present on both neuronal and non-neuronal cells. The results suggest that one or more of the ET isoforms could have actions in the rat CNS in addition to the proposed vasoactive role in the peripheral circulation. INTRODUCTION
h u m a n vascular smooth muscle 5'2t'~e but there is increasing evidence that these actions are not confined to
The endothelins (ET) are a family of related isopeptides, ET-1, ET-2 and ET-3, derived from 3 distinct genes found in h u m a n s and rats 22'23'45-47. A fourth E T peptide, vasoactive intestinal contractor (VIC) has been predicted from sequencing mouse genomic D N A and the synthetic peptide has been shown to have pharmacological activity41. Vasoactive snake venom peptides, the sarafotoxins I-2"25-2s, have a high degree of sequence similarity to the ETs and one of these, sarafotoxin S6b, is thought to share a c o m m o n receptor in neuronal tissues 2"26. ET-1 is synthesised by vascular endothelial cells 45"47 and ET-1 m R N A is widely expressed in h u m a n tissue 3' 38,39. We have previously demonstrated that binding sites for ET-1 are present in m a m m a l i a n vascular smooth muscle, and that their distribution parallels that of endothelial cells ll'a2 suggesting that ET-1 may function as a locally released factor TM. ET-1 is a potent constrictor of
the cardiovascular system. O n e or more of the E T isoforms may also function in the CNS 7'33'19'34'48'49. The presence of ET-1 m R N A , ET-1 and ET-3-1ike i m m u n o reactivity has b e e n reported in the brain and spinal cord of the rat 6'17'33"36"42A3"50. Binding sites for 1251 ET-1 have also been detected in these tissues 2,12,16,20,24.26,29,30. We have found an extensive distribution of binding sites for 125I ET-2, ET-3 and VIC in rat vascular tissues 4"J3. However, their anatomical localisation in the CNS has not been established and it is unclear whether binding sites for E T peptides are confined to the vascular tissue or if they are also present on n e u r o n e s and glial cells. The aim of the present study was to determine whether binding sites for the E T isoforms are present in neuronerich cultures derived from embryonic rat cerebellum and to compare their distribution with those of ET-1 and sarafotoxin S6b in the adult rat brain.
Correspondence: A.P. Davenport, Clinical Pharmacology Unit, University of Cambridge, Level 2, F and G Block, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, U.K. Fax: (44) (223) 216893.
279 MATERIALS AND METHODS The following materials were used with the name of the supplier given in parentheses. Dulbecco's Modified Eagles Medium (DMEM, Flow, U.K.); penicillin, streptomycin, glutamine (Gibco, Paisley, U.K.); cytosine arabinoside, Trypan blue, diaminobenzidine, poly-L-lysine (Sigma Chemical, Poole, U.K.); mouse mono-
Total
clonai anti-neurofilament 200 kDa (NF) antibody, horse radish peroxidase (HRP)-conjugated anti-mouse IgG (Vector Laboratories, Peterborough, U.K.); Hyperfilm flmax, (3-[125I]iodotyrosyl13)ET-1, (3-[125I]iodotyrosyl13)ET-2, (3-[lzsI]iodotyrosyl6)ET-3, (3-[125I]iodotyrosyl13)VIC and (3-[l~I]iodotyrosyl13)sarafotoxin S6b, spec. act. 2000 Ci/mmoi (Amersham International, Amersham, U.K.); K2 nuclear emulsion (IIford, Cheshire, U. K.); unlabelled ET-1 and ET-3 (Novabiochem,
NSB
Specific (amol/mm2)
W'--
I
t--W
O4 I
4
~i~ •
~:;.
LLi
I
t--t.l.!
if3 (D
f]3
O >
Fig. 1. Autoradiographical localisation of '~I-ET-1, ET-2 and ET-3 to saggital sections of rat brain. Sections (10/~m thick) were incubated with 20 pM solutions of the labelled peptides at 23 °C for 120 rain followed by washing, drying and apposition to Hyperfilm flmax. After development, the resulting film was used as a negative to produce the black and white autoradiogram for total binding. The second image was digitally subtracted from the first in order to produce a new image showing the amount of specific binding of each peptide coded according to the grey levels shown in the scale bar by comparison with a calibrated radioactive scale co-exposed with the sections of radiolabelled tissue. Lateral 2.40 mm4°, Cx, cortex; St, striatum; Co, cerebellum. Bar = 1 mm.
280 TABLE I Density o f binding sites (pmol/mm 2) in selected areas of adult rat brain incubated with 20 p M of iodinated peptide Each value represents the mean of at least four sections from six animals. ET-1 Cortex Corpus callosum Striatum Hippocampus CA1 CA3 Cerebellum Granular Molecular Spinal cord Grey matter
ET-2
ET-3
VIC
S6b
2.5 ± 0.2 4.3 ± 0.2 4.9 ± 0.3
3.0±0.3 4.5±0.1 5.6±0.3
1.7±0.2" 2.8±0.3* 2.9±0.1"
3.4±0.1 5.0±0.2 5.6±0.1
6.7±0.3* 8.9±0.4* 7.0±0.2*
2.2 ± 0.1 5.2 ± 0.2
2.7±0.1 5.3±0.2
0.8±0.1" 3.2±0.6*
2.5±0.2 5.4±0.9
8.4±0.1" 12.9±0.8"
18.5 ± 0.4 10.5 ± 0.5
19.0±0.5 9.2±1.1
5.3±0.7* 3.6±0.2*
20.3±0.2 13.1±0.2
24.2±1.7" 13.4±1.0"
9.1 ± 1.9
8.3±0.6
4.9±0.2*
8.0±0.1
13.2±0.4"
* Significant difference compared to the density of ET-1 binding (P < 0.05, Tukey's Studentized range for multiple comparison).
Nottingham, U.K.); preproendothelin-(110-130) (Peninsula, St Helens, U.K.); ET-2, VIC and sarafotoxin S6b (Peptide Institute, Osaka, Japan). ET-l-(3-9) and preproendothelin (124-130) were synthesised using solid-phase t-Boc chemistry, purified by size exclusion chromatography and the sequence confirmed by aminoacid analysis. All other reagents were from Sigma Chemical or Fisons (Loughborough, U.K.). Neurone-rich primary cultures Cerebellar cortices were dissected from Wistar rat embryos (16th-18th day of gestation) and dissociated mechanically by trituration with a heat-polished, narrowed Pasteur pipette in sterile DMEM. Two million viable cells (viability > 80% observed by exclusion of Trypan blue) were plated onto sterile poly-L-lysinetreated coverslips in 35 mm Petri dishes. Cells were grown in DMEM containing 20% FCS, 20 IU/ml penicillin, 100 ~g/ml streptomycin and glutamine (0.365 g/l). Three to 5 days after plating, the culture medium was replaced with fresh medium containing 5 /~M cytosine arabinoside to control non-neuronal proliferation. Thereafter, the medium was replaced at intervals of 3 days. The cultures were kept at 37 °C in an humidified atmosphere of 5% CO 2 and 95% air. Cells were used in the receptor binding and immunocytochemistry assays after 7-14 days in culture.
1oo i5
H ET-a
IN v ET-2
/,~..,.~/ET-3~_ ET-1
vie
S6b
[1251] peptide (200 pM) Fig. 2. Comparison of the ability of unlabelled ET and sarafotoxin S6b to compete for binding of the iodinated peptides to the granular layer of the adult cerebellum. The results are expressed as percentage inhibition of the total binding. Each value is the mean of at least 3 sections from 3 animals.
Fibroblast-enriched primary cultures Cerebellar cortices were prepared as for the neurone-rich cultures except that the cells were seeded onto plastic dishes without coverslips. The culture medium was DMEM, 10% FCS, 20 U/ml penicillin, 20/~g/ml streptomycin and glutamine (0.365 g/l). Cells were grown to confluence, and passaged twice before plating onto poly-L-lysine treated coverslips. These cultures were used for autoradiography after 14-21 days in culture. Quantitative in vitro receptor autoradiography Binding sites for 125I-ET-1, 125I-ET-2, lzSI-ET-3, 125I-VIC and 125I-sarafotoxin S6b, were visualised using a method modified from Davenport 1L12. Adult male Wistar rats (150-200 g) were killed by cervical dislocation and decapitation. Brains were removed rapidly and frozen onto cryostat chucks before cutting 10-pm-thiek sections cryostat sections which were thaw-mounted onto gelatin-subbed slides. After a 15 min pre-incubation period in 10 mM HEPES containing MgC12 (5 mM), sections were incubated for 2 h at 23 °C with 20 pM 125I-ET-I, ET-2, ET-3, VIC or sarafotoxin S6b in 10 mM HEPES containing MgCi 2 (5 mM) and bovine serum albumin (0.3%). Non-specific binding was defined by co-incubating adjacent sections with 1 pM of the corresponding unlabeiled peptide in addition to the labelled peptide. At the end of the incubation period, sections were rinsed in 3 successive 5 min washes of ice cold Tris buffer, (pH 7.4) and dried under a stream of cold air. Sections were exposed to radiation sensitive film (Hyperfilm flmax) together with a calibrated radioactive scale s. The resulting autoradiograms were analysed using computer-assisted densitometry (Quantimet 970, Cambridge Instruments, Cambridge, U.K.) by the method of Davenport 9'1° using calibrated radioactive standards 8. At least 4 sections were analysed from 6 rat brains. Coverslips bearing cultured cells were rinsed in 100 ml of 10 mM HEPES buffer (pH 7.4) and then incubated for 1 h at 23 °C in the incubation buffer as previously described containing 200 pM of the radiolabelled peptides. Non-specific binding was defined by coincubating coverslips from the same culture with 1 /~M of the corresponding unlabelled peptide. Each assay was performed in duplicate using 3 separate preparations of neurone-enriched cultures. At the end of the incubation period, coverslips were processed as described for tissue sections. For macro-autoradiography, the dry coverslips were mounted onto microscope slides and apposed to radiation sensitive film. For micro-autoradiography, cells were fixed by exposing coverslips to formaldehyde at 80 °C for 1 h. The coverslips were coated with Ilford K2 emulsion and exposed for 3 weeks. After developing the emulsion, the cultured cells were stained with Methylene blue. Silver grains were visualised using dark-field illumination.
281
lmmunohistochemistry Cerebellar cortical cultures grown on coverslips were fixed for 30 min with 3.7% formaldehyde in 100 mM phosphate-buffered saline (PBS). Cultures were rinsed twice with PBS, incubated for 2-5 min with 0.2% Triton X-100 in PBS and blocked for 30 min at room temperature with PBS containing 3% normal sheep serum and 0.02% Triton X-100. They were then incubated overnight at 4 °C with the mouse monoclonal anti-NF antibody (diluted 1:500). The cultures were washed with PBS and incubated for 1 h at 37 °C with rabbit polyclonal HRP-conjugated second antibody (diluted 1:500 in PBS containing 3% normal sheep serum and 0.02% Triton X-100). After further washes the HRP-antibody complex was visualised with diaminobenzidine, activated with 0.025% H202 in 50 mM Tris-HCl, pH 7.5. The cells on the coverslips were then rinsed in PBS, dehydrated, cleared in xylene and mounted (DPX) onto slides. Specificity of the second antibody was tested by omission of the primary antibody. The number and size of cells per unit area were estimated using the image analyser equipped with a programme written for this purpose. Images of the stained cells were digitised into an array of 600,000 pixels each with a grey value of 0-255. The threshold was set to detect the cells and the number and area of cells within a live frame area of 0.4 mm 2 was measured. An area 100/~m from the perimeter of the coverslip was excluded in order to minimise edge effects.
RESULTS
Tissue sections of adult brain Binding sites for the 4 E T peptides were visualised in
the adult rat brain (Fig. 1, Table I). T h e p a t t e r n was similar for each p e p t i d e , with an increasing binding site density in a rostro-caudal direction. Highest densities were m e a s u r e d in the granular layer of the cerebellum with lower amounts present in the m o l e c u l a r layer. Binding was also d e t e c t e d in the cortex, striatum, h i p p o c a m p u s and spinal cord. A l t h o u g h the p a t t e r n was similar, the density of binding was always higher for sarafotoxin S6b and lower for ET-3 in these regions c o m p a r e d to the o t h e r peptides, even though the specific activity of the labelled p e p t i d e s used in these assays was the same. T h e level of non-specific binding to tissue sections was higher using 125I-ET-3 than that of the other peptides, but was consistently lower using 125I sarafotoxin S6b. A similar p a t t e r n of binding was o b s e r v e d when the labelled p e p t i d e s were i n c u b a t e d with tissue sections at concentrations up to 1 riM. In cross-competition experiments, unlabelled ET-1, ET-2, ET-3, sarafotoxin S6b and V I C (1 p M ) c o m p e t e d for the binding sites of all the i o d i n a t e d p e p t i d e s (Fig. 2) in the cerebellum. H o w e v e r , the h e p t a p e p t i d e fragment, E T - 1 - ( 3 - 9 ) , p r e p r o e n d o t h e l i n - ( l l 0 - 1 3 0 ) (ET-like peptide) and p r e p r o e n d o t h e l i n - ( 1 2 4 - 1 3 0 ) tested at the same concentration did not c o m p e t e for either iodinated E T or sarafotoxin sites in the rat brain.
Fig. 3. Brightfield photomicrograph of a neurone-enriched culture grown in parallel with those used for the receptor binding assay. Cells are stained with anti-NF antibody in combination with a polyclonal HRP-conjugated second antibody and visualised with diaminobenzidine. The cells show long branching processes characteristic of neurnnaMike phenotypes. Bar = 50 ~m.
282
Neurone-rich primary cultures of embryonic brain Cells in the neurone-rich cultures stained with anti-NF antibody are shown in Fig. 3. Positive cells had small cell bodies ( - 1 0 ~m in diameter) and long branching processes characteristic of neurones. Flat and stellate cells did not stain with anti-NF antibody. Binding sites for iodinated ET-1, ET-2 and ET-3 were detected in neurone-rich cultures (Fig. 4) using macro-autoradiography, but binding of the iodinated peptides to fibroblast beds was below the level for detection (< 0.01 ~mol/mm2). The image analyser was used to classify the cells by size. Under our culture conditions, 93% of the cells were estimated to have an average cell diameter of 9.2 _+ 0.6 /~m (n = 9 separate plates + S.E.M.). Using microautoradiography to detect the binding of the 4 iodinated ETs, high densities ( > 400 grains/100~m 2) of silver grains were associated with an average of 14% of cells in these particular neurone-enriched cultures. Many of the cells on which binding sites were present had a characteristic neuronal phenotype. Examples of high grain densities localised over a proportion of cell bodies and along the length of cell processes using labelled ET-2 and ET-3 are shown in Fig. 5b,d). The clustering of cells with high
Total
NSB
ET-1
ET-2
ET-3 if!if
¸ i~i ~i!~ m
Fig. 4. Autoradiographical localisation of 12sIET-1, ET-2 and ET-3 binding to neurone-rich cultures growing on coverslips. Coverslips were incubated with 20 pM solutions of the labelled peptides at 23 °C for 120 rain followed by washing, drying and apposing to Hypgrfilm flmax. Non-specific binding (NSB) was determined by incubating an adjacent coverslip in the presence of the corresponding unlabeUed peptide (1/~M). Bar = 2 mm.
densities of binding sites may reflect the pattern following dispersion of the dissociated cells onto the culture plate and the subsequent inclusion of cytosine arabinoside to prevent non-neuronal cell proliferation leaving groups of neurone-enriched cells. Similar results were obtained using labelled ET-1 and VIC. A second group of labelled cells were more difficult to classify and may be nonneuronal. Low numbers of silver grains ( < 20 grains/100 /~m2) were measured in the emulsion overlying the remaining cells and was similar to those seen in adjacent cultures used to assess non-specific binding. Some of these cells were distinctively non-neuronal (Fig. 5c,d), indicating that binding of ETs was absent or below the level for detection. DISCUSSION We have detected binding sites for 125I-ET-2, ET-3, and VIC in tissue sections from adult rat brain. The pattern was similar to that of ET-112"24and sarafotoxin S6b. The main difference was in the higher density of binding obtained using labelled sarafotoxin S6b, compared with the similar levels of ET-1, ET-2 and VIC. Non-specific binding was consistently higher using ~251ET-3, resulting in lower levels of specific binding with this peptide. Cross-competition experiments show that each unlabelled peptide tested at high concentration was able to compete for the binding of each of the other iodinated compounds. One explanation for these results is that all 5 peptides may be acting via the same population of binding sites. The techniques used in the present study cannot exclude the possibility that multiple receptor sub-types may exist in rat brain which differ in their rank order of affinity for the E T isoforms and sarafotoxin S6b, as proposed in non-neuronal tissue 35'44. Using macroautoradiography and a fixed concentration of iodinated peptide, our studies have not revealed discrete areas of the brain in which there is a differential distribution of one of the labelled peptides compared to the other isoforms. These results support the evidence from functional studies that sarafotoxin S6b and ET-1 share common binding sites and mechanism of action in the brain 2. It is not yet known whether ET-2 has any action in the CNS but ET-3 has been shown to be equipment to ET-1 in stimulating phosphoinositide hydrolysis in guinea-pig cerebellum and there was no evidence for receptor heterogeneity 7. This study has demonstrated that 125I-ETs bind to neurone-enriched cultures with a high density of sites present on a proportion of cell bodies and processes which show a clear neuronal phenotype. Fibroblasts and glia may also be present but we were unable to detect
283 binding of the E T isoforms to fibroblast-enriched cultures, suggesting that these sites were below the level for
a n o n - n e u r o n a l phenotype, but confirmation that these
detection or were not expressed in cultured cells. High
are glia requires the use of antibodies to specific marker proteins for these cells 31. O u r results are consistent with
densities of binding sites were present on some cells with
evidence from previous studies that ET-1, ET-3 and VIC
'
~i!~
!-
~:~
~,!?ii~iiii~ii~
~i:!i~i~ "!
Fig. 5. Localisation of ET binding sites in neurone-rich cultures as the pattern of developed silver grains in nuclear emulsion, revealed under dark-field illumination (b,d,f) lying above cells stained with Methylene blue visualised under bright-field illumination (a,c,e). An example of the high density of binding sites for ET isoforms associated with cells having a neuronal phenotype, is illustrated by the binding of I~I-ET-2, indicated by the arrows in a. Binding (illustrated with ~25I-ET-3)was also detected to some cells with an intermediate phenotype (indicated by arrow heads in c and d) although binding of the iodinated peptides to other cells which were clearly non-neuronal was absent or below the level for detection. Examples of non-specific binding for ET-3 are shown in (f) with the corresponding stained cells shown in (e). Bars = 50/~m.
284 can m o d u l a t e neuronal activity 4s:9 and stimulate the formation of second messengers 2'7'15'26,32:s, suggesting that binding sites for these peptides may be functional receptors The source of endogenous peptides which could be binding to these sites in the adult brain is unclear. R a d i o l a b e l l e d ET-1 does not a p p e a r to cross the b l o o d brain barrier 37 which suggests that release of E T from the endothelial cells lining the vessel walls into the systemic circulation is an unlikely source. ET-like immunoreactivity has been m e a s u r e d in cerebrospinal fluid TM and has been d e t e c t e d in epithelial cells including those from the rat choroid plexus ( D a v e n p o r t and van P a p e n d o r p , unpublished observations). A third possible source of E T peptides are neurones and glia. ET-like immunoreactivity and ET-1 m R N A have been visualised in both types of cell 6'17'34'43'50. ET-3-1ike immunoreactivity has been mea-
munoassay 36'42 but the identities of cells synthesising ET-2 and VIC have not been established. However, using antibodies directed to the N-terminals of ET-1 and ET-3 we have found immunoreactive cell bodies and processes in the adult brain as well as neurone-enriched cultures. We have also m e a s u r e d release of ET-like immunoreactivity into the m e d i a from these cells using a r a d i o i m m u n o a s s a y which can distinguish between ET-1 and ET-313. The results of this study d e m o n s t r a t e the presence of binding sites for the 125I-ETs in the rat CNS and provide further evidence in support of the suggestion that one or m o r e of the E T isoforms could have central actions in modulating neuronal activity in the rat.
by radioim-
Acknowledgements. We thank Caroline Purdy for excellent technical assistance and the Wellcome Trust, the Isaac Newton Trust and the British Heart Foundation for support (A.P.D.).
1 Ambar, I., Kloog, Y., Kochva, E., Wollberg, Z., Bdolah, A., Oron, U. and Sokolovsky, M., Characterization and localization of a novel neuroreceptor for the peptide sarafotoxin, Biochem. Biophys. Res. Commun., 157 (1988) 1104-1110. 2 Ambar, I., Kloog, Y., Schvartz, I., Hazum, E. and Sokolovsky, M., Competitive interaction between endothelin and sarafotoxin: binding and phosphoinositides hydrolysis in rat atria and brain, Biochem. Biophys. Res. Commun., 158 (1989) 195-201. 3 Bloch, K.D., Eddy, R.L., Shows, T.B. and Quertermous, T., cDNA cloning and chromosomal assignment of the gene encoding endothelin 3, J. Biol. Chem., 264 (1989) 18156--18161. 4 Brown, M. J., Davenport, A. P. and Nunez, D. J., Localisation of binding sites for [125]endothelin-1, endothelin-2 and endothelin-3 in rat, pig and human kidneys, J. Physiol., 417 (1989) 35P. 5 Brain, S.D., Crossman, D.C., Buckley, T.L. and Williams, T.J., Endothelin-l: demonstration of potent effects on the microcirculation of humans and other species, J. Cardiovasc. Pharmacol., 13 (1989) S147-149. 6 Cintra, A., Fuxe. K., Anggard, E, Tinner. B., Staines, W. and Agnati, L.F., Increased endothelin-like immunoreactivity in ibotenic acid-lesioned hippocampal formation of the rat brain, Acta Physiol. Scand., 137 (1989) 557-558. 7 Crawford, M.L.A., Hiley, C.R. and Young, J.M., Characteristics of endothelin-1 and endothelin-3 stimulation of phosphoinositide breakdown differ between regions of guinea-pig and rat brain, Naunyn-Schmiedeberg's Arch. Pharmaeol., 341 (1990) 268-271. 8 Davenport, A.P. and Hall, M.D., Comparison between brain paste and polymer [~25I] standards for quantitative receptor autoradiography, J. Neurosci. Methods, 25 (1988) 75-82. 9 Davenport, A.P., Beresford, I.J.M., Hall, M.D., Hill R.G. and Hughes, J., Quantitative autoradiography in neuroscience. In EW. van Leeuwen, R.M. Buijs, C.W. Pool and O. Pach (Eds.), Techniques in the Behaviour Science, Vol. III, Elsevier, Amsterdam, 1988, pp. 121-145. 10 Davenport, A.P., Hill R.G. and Hughes J., Quantitative analysis of autoradiograms. In J.M. Polak (Ed.), Regulatory Peptides, Birkhauser, Basle, 1989, pp. 137-153. 11 Davenport, A.P, Nunez, D.J. and Brown, M.J., Binding sites for 125I-labelledendothelin-1 in the kidneys: differential distribution in rat, pig and man demonstrated by using quantitative autoradiography, Clin. Sci., 77 (1989) 129-131. 12 Davenport, A.P., Nunez, D.J., Hall, J.A., Kaumann, A.J. and
Brown, M.J., Autoradiographical localization of binding sites for porcine [125I]endothelin-1 in humans, pigs, and rats: functional relevance in humans, J. Cardiovasc. Pharmacol., 13 (1989) S166-170. Davenport, A.P., Nunez, D.J. and Brown, M.J., Localisation of binding sites for iodinated endothelin and sarafotoxin peptides in mammalian tissues using quantitative autoradiography, Eur. J. Pharmacol., 183 (1990) 2153. Davenport, A.P., Ashby, M.J., Easton, P., Ella, S., Bedford, J., Dickerson, C., Nunez, D. J., Capper, S. J. and Brown, M.J., A sensitive radioimmunoassay measuring endothelin-like immunoreactivity in human plasma: comparison of levels in essential hypertension and normotensive controls, Clin. Sci., 78 (1990) 261-264. Fu, T., Chang, W., Ishida, N., Saida, K., Mitsui, Y., Okano, Y. and Nozawa, Y., Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells, FEBS Lett., 257 (1989) 351-353. Fuxe, K., Anggard, E., Lundgren, K., Cintra, A., Agnati, L.E, Galton, S. and Vane, J., Localization of [~25I]endothelin-1 and [125I]endothelin-3 binding sites in the rat brain, Acta. Physiol. Scand., 137 (1989) 563-564. Giaid, A., Gibson, S.J., lbrahim, B.N., Legon, S., Bloom, S.R., Yanagisawa, M., Masaki.,T., Varndeli, I.M. and Polak, J.M. Endothelin 1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7634-7638. Hoffman, A., Keiser, H.R., Grossman, E., Goldstein D.S., Gold, P.W. and Kling, M., Endothelin concentrations in cerebrospinal fluid in depressive patients, Lancet, 2 (1989) 1519. Hokfelt, T., Post, C., Freedman, J., Lundberg, J.M. and Terenius, L., Endothelin induces spinal lesions after intrathecal administration, Acta. Physiol. Scand., 137 (1989) 555-556. Hoyer, D., Waeber, C. and Palacios, J.M., [125I]Endothelin-1 binding sites: autoradiographic studies in the brain and periphery of various species including humans, J. Cardiovasc. Pharmacol., 13 (1989) S162-165. Hughes, A.D., Thorn, S.A., Woodall, N., Schachter, M., Hair, W.M., Martin, G.N. and Sever, P.S., Human vascular responses to endothelin-l: observations in vivo and in vitro, J. Cardiovasc. Pharmacol., 13 (1989) $225-228. Inoue, A., Yanagisawa, M, Kimura, S., Kasuya, Y., Miyauchi T., Goto, K. and Masaki, T., The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes, Proc. Natl. Acad. Sci.
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REFERENCES
13
14
15
16
17
18 19 20
21
22
285 U.S.A., 86 (1989) 2863-2867. 23 Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Inoue, A., Ishida, N., Mitsui, Y., Onda, H., Fujino, M. et al., Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin, FEBS Lett., 231 (1988) 440-444. 24 Jones, C.R., Hiley, C.R., Pelton, J.T. and Mohr, M., Autoradiographic visualization of the binding sites for [125I]endothelin in rat and human brain, Neurosci. Lett., 97 (1989) 276-279. 25 Kloog, Y., Ambar, I., Sokolovsky, M., Kochva, E., Wollberg, Z. and Bdolah, A., Sarafotoxin, a novel vasoconstrictor peptide: phosphoinositide hydrolysis in rat heart and brain, Science, 242 (1988) 268-270. 26 Kloog,Y. and Sokolovsky, M., Similarities in mode and sites of action of sarafotoxins and endothelins, Trends. Pharmacol. Sci., 10 (1989) 212-214. 27 Kloog, Y., Ambar, I., Kochva, E., Wollberg, Z., Bdolah, A. and Sokolovsky, M., Sarafotoxin receptors mediate phosphoinositide hydrolysis in various rat brain regions, FEBS Len., 242 (1989) 387-390. 28 Kloog, Y., Bousso-Mittler, D., Bdolah, A. and Sokolovsky, M., Three apparent receptor subtypes for the endothelin/sarafotoxin family, FEBS Lea., 253 (1989) 199-202. 29 Koseki, C., Imai, M., Hirata, Y., Yanagisawa, M. and Masaki, T., Autoradiographic distribution in rat tissues of binding sites for endothelin: a neuropeptide?, Am. J. Physiol., 256 (1989) R858-66. 30 Koseki, C., Imai, M., Hirata, Y., Yanagisawa, M. and Masaki, T., Autoradiographic localization of [12sI]-endothelin-1 binding sites in rat brain, Neurosci. Res., 6 (1989) 581-585. 31 Loffner, F., Lohmann, S.M., Walckhoff, B., Walter, U. and Hamprecht, B., Immunocytochemical characterization of neuron-rich primary cultures of embryonic rat brain cells by established neuronaland gliai markers and monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein synapsin, Brain Research, 363 (1986) 205-221. 32 Lin, W.W., Lee, C.Y. and Chuang, D.M., Cross-desensitization of endothelin- and sarafotoxin-induced phosphoinositide turnover in neurons, Eur. J. Pharmacol., 166 (1989) 581-582. 33 MacCumber, M.W., Ross, C.A., Glaser, B.M. and Snyder, S.H., Endothelin: visualization of mRNAs by in situ hybridization provides evidence for local action, Proc. Natl. Acad. Sci. U. S. A., 86 (1989) 7285-7289. 34 MacCumber, M.W., Ross, C.A. and Snyder, S.H., Endothelin in brain: Receptors, mitogenesis, and biosynthesis in glial cells, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 2359-2363. 35 Masuda, Y., Miyazaki, H., Kondoh, M., Watanabe, H., Yanagisawa, M., Masaki, T. and Murakami, K., Two different forms of endothelin receptors in rat lung, FEBS Lett., 257 (1989) 208-210. 36 Matsumoto, H., Suzuki, N., Onda, H. and Fujino, M., Abundance of endothelin-3 in rat intestine, pituitary gland and brain, Biochem. Biophys. Res. Commun., 164 (1989) 74-80. 37 Neuser, D., Steinke, W., Theiss, G. and Stasch, J.-P. Autoradiographic localization of [125I] endothelin-1 and [125I] atrial
natriuretic peptide in rat tissue: a comparative study, J. Cardiovasc. Pharmacol., 13 (5)(1989) 67-73. 38 Nunez, D.J., Davenport, A.P., Brown, M.J., Neylon, C.B., and Wyse, R., Distribution of endothelin-1 messenger RNA using complementary DNA amplification by the polymerase chain reaction, J. Hypertens., 7 (1989) 920-921. 39 Nunez, D.J., Brown, M.J., Davenport, A.P., Neylon, C.B., Schofield, J.P. and Wyse, R.K., Endothelin-1 mRNA is widely expressed in porcine and human tissues, J. Clin. Invest., 85 (1990) 1537-1541. 40 Paxinos, G. and Watson, C., The Rat Brain in Sterotaxic Coordinates, Academic Press, Sidney, 1986. 41 Saida, K., Mitsui, Y. and Ishida, N., A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity, J. Biol. Chem., 264 (1989) 14613-14616. 42 Shinmi, O., Kimura, S., Sawamura, T., Sugita, Y., Yoshizawa, T., Uchiyama, Y., Yanagisawa, M., Goto, K., Masaki, T. and Kanazawa, I., Endothelin-3 is a novel neuropeptide: isolation and sequence determination of endothelin-1 and endothelin-3 in porcine brain, Biochem. Biophys. Res. Commun., 164 (1989) 587-593. 43 Shinmi, O., Kimura, S., Yoshizawa, T., Sawamura, T., Uchiyama, Y., Sugita. Y., Kanazawa, I., Yanagisawa, M., Goto, K. and Masaki, T., Presence of endothelin-1 in porcine spinal cord: isolation and sequence determination, Biochem. Biophys. Res. Commun., 162 (1989) 340-346. 44 Watanabe, H., Miyazaki, H., Kondoh, M., Masuda, Y., Kimura, S., Yanagisawa, M., Masaki, T. and Murakami, K., Two distinct types of endothelin receptors are present on chick cardiac membranes, Biochem. Biophys. Res. Commun., 161 (1989) 1252-1259. 45 Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T., A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature, 332 (1988) 411-415. 46 Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya Y., Kimura, S., Kumagaye, S., Nakajima K., Watanabe T.X., Sakakibara, S., Goto, K. et al., Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6964-6967. 47 Yanagisawa, M. and Masaki, T., Molecular biology and biochemistry of the endothelins, Trends Pharmacol. Sci., 10 (1989) 374-378. 48 Yoshizawa, T., Kimura, S., Kanazawa, I., Uchiyama, Y., Yanagisawa, M. and Masaki, T., Endothelin localizes in the dorsal horn and acts on the spinal neurones: possible involvement of dihydropyridine-sensitive calcium channels and substance P release, Neurosci. Lett., 102 (1989) 179-184. 49 Yoshizawa, T., Kimura, S., Kanazawa, I., Yanagisawa, M. and Masaki, T., Endothelin-1 depolarizes a ventral root potential in the newborn rat spinal cord, J. Cardiovasc. Pharmacol., 13, Suppl. 5 (1989) $216-217. 50 Yoshizawa, T., Shinmi, O., Giaid, A., Yanagisawa, M. Gibson, S.J., Kimura, S., Uchiyama, Y., Polak, J.M., Masaki, T. and Kanazawa, I., Endothelin: a novel peptide in the posterior pituitary system, Science, 247 (1990) 462-464.