Neuroscience Letters, 133 (1991) 117-120
117
© 1991 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/91/$03.50 NSL 08208
Quantitative autoradiographic localisation of [125I]endothelin-1 binding sites in spinal cord and dorsal root ganglia of the rat S. K a r , J.-G. C h a b o t a n d R. Q u i r i o n Douglas Hospital Research Center and Department of Psychiatry, McGill University, Verdun, Que. (Canada)
(Received 5 June 1991;Revisedversion received20 August 1991;Accepted23 August 1991) Key words: Receptorautoradiography;Endothelin-l; Rat; Spinalcord; Dorsal root ganglia; Development
The autoradiographic distribution of [t25I]endothelin (ET)-1 binding sites was studied in the spinal cord and dorsal root ganglia of developing and adult rat. In the spinal cord, high density of [~25I]ET-1binding sites were diffuselydistributed throughout the grey matter whereas in the ganglia discrete silvergrains were localisedprimarily on the satellite cells. A variation in the density of binding sites was evident, particularly in the spinal cord, during development.Thesedata, in conjunctionwith other reports, suggest a possible neuromodu|atoryrole for ET-1 in spinal cord and dorsal root ganglia of the rat.
Endothelin (ET), originally isolated from porcine aortic endothelial cell culture, is a 21-amino acid peptide with potent vasoactive property [12]. Screening of a human genomic library has subsequently identified 3 forms of ET, i.e. ET- 1, ET-2 and ET-3, each possessing diverse pharmacological activities of varying potency on both vascular and non-vascular tissues [7, 13]. Recently, two distinct receptors, each coupled to a G-protein, have been cloned and their mRNAs have been detected in a wide variety of tissues including the central nervous system. One of these receptors shows high specificity for ET-1 whereas the other one is recognised equally well by all 3 ETs [1, 11]. The presence of ET-l-like immunoreactivity and its gene transcript has been demonstrated in neurones of the spinal cord and dorsal root ganglia [5, 14]. Furthermore, it has been shown that ET-1 induces ventral root depolarisation in vitro [14] and upon intrathecal injection, it causes complete paresis of the hind legs [6]. In the present study, using quantitative in vitro receptor autoradiography, we investigated the spatial distribution as well as the postnatal development of [125I]ET-1 binding sites in the spinal cord and dorsal root ganglia of the rat. Timed-pregnant Sprague-Dawley rats, obtained from Charles River Canada were individually housed and maintained on a 12 h light-dark cycle. After birth, pups
Correspondence: R. Quirion, Douglas Hospital Research Center, 6875 La Salle Blvd., Verdun, Que. H4H 1R3, Canada. Fax: (1) (514) 7662503.
at days 1, 4, 7, 14, 21 and 28 were decapitated and their spinal cords with attached ganglia were snap-frozen in 2-methylbutane at - 4 0 ° C . In addition, adult rats (200250 g) were sacrificed and the spinal cord and ganglia from cervical (C6-C8), thoracic (T6-T8), lumbar (L4-L6) and sacral ($2-$4) levels were removed rapidly and snap-frozen as mentioned above. The tissues were then serially cut (20 pm) on a cryostat and thaw-mounted on gelatin-coated slides. The sections were incubated with 25 pM [125I]ET-1 (spec. act. 2000 Ci/mmol, Amersham, Canada) in the presence or absence of 1 /zM unlabeled ET-1 (Peninsula Laboratories, Belmont, CA) for 2 h at room temperature. The incubation solution contained 50 m M Tris-HCl buffer (pH 7.4), 10 m M magnesium chloride, 1 m M phenylmethylsulfonyl fluoride, 0.4 mg/ml soybean trypsin inhibitor, 1 mM tetrasodium ethylenediamine tetraacetate, 100 U/ml aprotinin and 2 mg/ml bovine serum albumin. Following incubation, slides were rinsed in Tris-HCl buffer, rapidly air-dried and then juxtaposed against tritium-sensitive films for 36 h along with iodinated standard (Amersham, Canada). The autoradiograms were quantified densitometrically using a M C I D image analysis system (Imaging Research Inc., Canada). For cellular localisation of binding sites, a batch of slides after incubation were fixed in 5% glutaraldehyde solution in 50 m M phosphate buffer (pH 7.4) and then dehydrated in graded alcohols. Sections were then dipped in Kodak NTB-2 emulsion (diluted 1 : 1 in water) and developed after 6-8 days. The distribution of [125I]ET-1 binding sites demonstrated a characteristic pattern in the developing and
118
adult spinal cord, and in the dorsal root ganglia of the rat (Table IA,B; Fig. 1A~)). At postnatal day 1, [125I]ET-1 binding sites were observed throughout the grey matter as well as the white matter of the spinal cord. However, no discrete pattern of labelling was apparent between dorsal, ventral or central canal regions of the grey matter (Fig. 1A,F). During postnatal ontogeny, the density of [125I]ET-1 binding sites in the grey matter first decreases (days 1-7) (Fig. 1B,G) and then gradually increases (days 7-28) (Fig. 1C,H) with little change in its localisation (Table IA). [125I]ET-1 binding sites in the white matter, on the contrary, showed a gradual decrease during development (Table IA; Fig. 1A-C). In the adult spinal cord, the density of [125I]ET-1 binding sites in the white matter was relatively low, and grey matter did not exhibit much variation in the labelling between various laminae (Fig. 1D,I; Table IB). At higher resolution, the emulsion-processed material showed a uniform pattern of silver grain distribution in the grey matter of developing and adult spinal cord. In the ventral horn, particularly in laminae IX, perikarya of motoneurones were found to be relatively spared of silver grains. In the dorsal root ganglia, [125I]ET-1 binding sites were observed in postnatal day 1 (Fig. 1K). The density of [125I]ET-l binding sites, unlike the spinal cord, remained somewhat constant throughout development (Fig. 1L,M; Table IA). In the adult, ganglia pulled to-
gether from various segmental levels of the spinal cord showed little variation in the density of labelling (Fig. 1B). At higher resolution, silver grains were found to be concentrated primarily in the satellite cells and not in sensory neurones (Fig. IN,O). The present study demonstrates that [125I]ET-1 binding sites are widely distributed throughout the spinal cord and dorsal root ganglia of the developing and adult rat. It is likely that ET itself appears early during prenatal development since significant density of [125I]ET binding sites are already apparent in the spinal cord and dorsal root ganglia 1 day postnatally. Moreover, during the course of development, [LESI]ET-1 binding sites showed dramatic variation in their density, particularly in the spinal cord, suggesting an important role for the peptide in early maturation and organisation of the cord. However, the homogenous distribution of [tzsI]ET-1 binding sites in spinal grey matter does not correspond precisely to ET-like immunoreactivity [5, 14], which is primarily localised in some scattered fibers (laminae I-VI) and cell bodies (laminae IV-VI) in the dorsal horn and in the majority of motoneurones of the ventral horn. This apparent discrepancy is at present unclear, but given the evidence from other brain regions [3, 10], this could possibly be due to the presence of [12SI]ET-1 binding sites on some non-neuronal elements of the spinal cord. In many tissues of the organism, ET has been sug-
TABLE I QUANTITATIVE DISTRIBUTION OF [t25IlENDOTHELIN-I BINDING SITES IN POSTNATAL (A) AND ADULT (B) SPINAL CORD AND DORSAL ROOT GANGLIA OF THE RAT Each value represents the mean ± S.E.M. of 8-12 sections from 4 rats. The optical density values from different sections were converted to receptor density values by reference to iodinated standards after subtraction of non-specific binding and background film density. Binding densities are expressed as fmol/mg of tissue, wet weight. P, number of days after birth; DRG, dorsal root ganglia; CCR, central canal region; Lain, lamina(e). A Developmental stage
Dorsal horn
Ventral horn
CCR
White matter
DRG
P1 P4 P7 P14 P21 P28
6.1_+0.3 5.1_+0.7 3.74-0.6 5.8_+0.9 6±0.5 8.14-0.4
7.5±0.7 6_+0.6 4.2_+0.6 5.84-0.9 5.5_+0.6 6.2±0.5
6.8±0.8 5.84-0.8 4.7_+0.6 5.84-1.2 4.6_+0.6 6.5_+0.4
7.1±0.4 5.2_+0.8 4.2_+0.5 3.24-0.9 3.9_+0.5 2.5_+0.3
6.3_+0.5 5.8±0.5 4.9_+0.3 4.6_+0.2 4.64-0.8 6.9_+0.3
B
Spinal cord
Lam I-III
Lam IV-VI
Lam VII-VIII
Lam IX
Lam X
White matter
Cervical Thoracic Lumbar Sacral
9.8 + 0.5 11.94-0.5 12.94-0.6 164- 1
9.44- 0.6 10.54-0.5 11.4+_0.6 14.4+0.9
9.8 _ 0.6 10.7_+0.9 12.6_+0.7 13.7_+0.9
11 _ 0.8 11.9_+ 1.3 13.9_+ 1.1 16.2_+ 1.2
10.7 _+0.4 11.3 _+ 1.1 12.2_+0.6 13.8_+ 1.3
4.9 -+0.6 5.1 _+0.8 5.74-0.6 6.84- 1.3
Adudlt DRG 10.9_+ 1.2
119
Fig. 1. Photomicrographs showing the distribution of [nSI]Endothelin (ET)-I binding sites in the spinal cord (A-I) and dorsal root ganglia (J-O) of postnatal day 1 (A,F,K), day 7 (B,G,L), day 28 (C,H,M) and adult (D,I,N) rat. Note the overall decrease of [125I]ET-I binding sites in day 7 spinal cord. F, G, H, I and O are the brightfield representative of A, B, C, D and N respectively. E and J represent [m25I]ET-1 binding sites in presence of 1 gM ET-I in the spinal cord of postnatal day 28 (E) and adult dorsal root ganglia (J). DH, dorsal horn; VH, ventral horn; cc, central canal. A,B,F,G: x 45; C,E,H: x 25; D,I: x 12; K-M: x 760; J,N,O: x 525.
120 gested to act on s m o o t h muscle or other cells close to sites of its synthesis [2, 9]. M a c C u m b e r et al. [10] have recently provided evidence that ET-like materials in the cerebellum can be p r o d u c e d by glia and act u p o n b o t h glia and neurones in a paracrine/autocrine fashion. In the spinal cord, the diffuse pattern of [125I]ET-1 binding sites in dorsal, ventral and lateral horns suggests possible role(s) for ET in spinal synaptic transmission. It is also likely that ET-like materials released from interneurones which are present in the dorsal horn, m a y act in a autocrine and/or paracrine m a n n e r to regulate its own release or the release o f other n e u r o m o d u l a t o r s from adjacent cells. In the n e w b o r n rat spinal cord, ET-1 induces ventral root depolarisation by releasing substance P f r o m dorsal spinal cord [14]. This effect, which can be blocked by either nicardipine, a calcium channel blocker or by spantide, a substance P antagonist, provides evidence for a n e u r o m o d u l a t o r y role for E T in the spinal cord [14]. F u r t h e r m o r e , it has been shown that ET, administered intrathecally, causes complete paresis o f the hind legs along with loss of calcitonin gene-related peptide i m m u noreactivity f r o m the m o t o n e u r o n e s [6]. At present it is not clear whether this action o f ET is a direct one or mediated through other n e u r o m o d u l a t o r s but in all probability it represents the involvement o f ET in m o t o r functions of the spinal cord. Further studies are, however, necessary to explain the significance o f high density of [125I]ET-1 binding sites in areas associated particularly with sensory and a u t o n o m i c functions of the spinal cord. An interesting finding o f the present study relates to the localisation of [125I]ET-1 binding sites on the satellite cells o f the dorsal root ganglia. Satellite cells are considered to provide trophic s u p p o r t to the neurones they surround and regulate the passage o f materials between neurones and blood vessels [8]. The presence o f ET-like immunoreactivity and its gene transcript in the majority of neurones o f dorsal root ganglia and the localisation of [125I]ET-1 binding sites in the surrounding satellite cells, strongly lend support to the paracrine role of this peptide in this tissue. This perhaps also explains why ET, which is synthesised and stored in neurones o f dorsal root ganglia, is present in only a few fibers in the dorsal spinal cord [6] and remains unaltered following transection o f peripheral nerve or p r e t r e a t m e n t with a sensory neurotoxin capsaicin [4]. In s u m m a r y , the widespread distribution o f [125I]ET-1 binding sites in the spinal cord and dorsal root ganglia of developing and adult rat
suggests that this peptide m a y act as a m o d u l a t o r of neuronal function in these tissues. 1 Arai, H., Hori, S., Aramori, I., Ohkubo, H. and Nakanishi, S., Cloning and expression of a eDNA encoding an endothelin receptor, Nature, 348 (1990) 730-732. 2 de Nucci, G., Thomas, R., D'Orleans-Juste, P., Antunes, E., Walder, C., Warner, T.D. and Vane, J.R., Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyelin and endothelium-derived relaxing factor, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 9797-9800. 3 Ehrenreich, H., Kehrl, J.H., Anderson, R.W., Rieckmann, P., Vitkovic, L., Coligan, J.E. and Fauci, A.S., A vasoactive peptide, endothelin-3, is produced by and specifically binds to primary astrocytes, Brain Res., 538 (I991) 54-58. 4 Franco-Cereceda, A., Rydh, M., Lou, Y.P., Dalsgaard, C.J. and Lundberg, J.M., Endothelin as a putative sensory neuropeptide in the guinea-pig: different properties in comparison with calcitoningene-related peptide, Reg. Peptides, 32 (1991) 253-265. 5 Giaid, A., Gibson, S.J., Ibrahim, N.B.N., Legon, S., Bloom, S.R., Yanagisawa, M., Masaki, T., Varendell, 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. 6 H6kfelt, T., Post, C., Freedman, J., Lundberg, J.M. and Terenius, L., Endothelin induces spinal lesions after intrathecal administration, Acta Physiol. Seand., 137 (1989) 555-556. 7 Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. and Masaki, K., The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 2863-2867. 8 Lieberman, A.R., Sensory ganglia. In D.N. Landon (Ed.), The Peripheral Nerve, Chapmann and Hall, London, 1976, pp. 188-278. 9 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. 10 MacCumber, M.W., Ross, C.A. and Snyder, S.H., Endothelin in brain: receptors, mitogenesis, and biosynthesis in giial cells, Proc. Natl. Acad. Sci. U.S.A., 87 (1990) 2359-2363. 11 Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, K. and Masaki, T., Cloning of a eDNA encoding a nonisopeptide-selective subtype of the endothelin receptor, Nature, 348 (1990) 732-735. 12 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~,15. 13 Yanagisawa, M. and Masaki, T., Molecular biology and biochemistry of the endothelins, Trends Pharmaeol. Sci., 10 (1989) 374-378. 14 Yoshizawa, T., Kimura, S., Kanazawa, I., Uchiyama, Y., Yanagisawa, M. and Masaki, T., Endothelin localizes in the dorsal horn and acts on the spinal cord neurones: possible involvement of dihydropyridine-sensitive calcium channels and substance P release, Neurosci. Lett., 102 (1989) 179-184.
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© 1991 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/91/$03.50 NSL 08208
Quantitative autoradiographic localisation of [125I]endothelin-1 binding sites in spinal cord and dorsal root ganglia of the rat S. K a r , J.-G. C h a b o t a n d R. Q u i r i o n Douglas Hospital Research Center and Department of Psychiatry, McGill University, Verdun, Que. (Canada)
(Received 5 June 1991;Revisedversion received20 August 1991;Accepted23 August 1991) Key words: Receptorautoradiography;Endothelin-l; Rat; Spinalcord; Dorsal root ganglia; Development
The autoradiographic distribution of [t25I]endothelin (ET)-1 binding sites was studied in the spinal cord and dorsal root ganglia of developing and adult rat. In the spinal cord, high density of [~25I]ET-1binding sites were diffuselydistributed throughout the grey matter whereas in the ganglia discrete silvergrains were localisedprimarily on the satellite cells. A variation in the density of binding sites was evident, particularly in the spinal cord, during development.Thesedata, in conjunctionwith other reports, suggest a possible neuromodu|atoryrole for ET-1 in spinal cord and dorsal root ganglia of the rat.
Endothelin (ET), originally isolated from porcine aortic endothelial cell culture, is a 21-amino acid peptide with potent vasoactive property [12]. Screening of a human genomic library has subsequently identified 3 forms of ET, i.e. ET- 1, ET-2 and ET-3, each possessing diverse pharmacological activities of varying potency on both vascular and non-vascular tissues [7, 13]. Recently, two distinct receptors, each coupled to a G-protein, have been cloned and their mRNAs have been detected in a wide variety of tissues including the central nervous system. One of these receptors shows high specificity for ET-1 whereas the other one is recognised equally well by all 3 ETs [1, 11]. The presence of ET-l-like immunoreactivity and its gene transcript has been demonstrated in neurones of the spinal cord and dorsal root ganglia [5, 14]. Furthermore, it has been shown that ET-1 induces ventral root depolarisation in vitro [14] and upon intrathecal injection, it causes complete paresis of the hind legs [6]. In the present study, using quantitative in vitro receptor autoradiography, we investigated the spatial distribution as well as the postnatal development of [125I]ET-1 binding sites in the spinal cord and dorsal root ganglia of the rat. Timed-pregnant Sprague-Dawley rats, obtained from Charles River Canada were individually housed and maintained on a 12 h light-dark cycle. After birth, pups
Correspondence: R. Quirion, Douglas Hospital Research Center, 6875 La Salle Blvd., Verdun, Que. H4H 1R3, Canada. Fax: (1) (514) 7662503.
at days 1, 4, 7, 14, 21 and 28 were decapitated and their spinal cords with attached ganglia were snap-frozen in 2-methylbutane at - 4 0 ° C . In addition, adult rats (200250 g) were sacrificed and the spinal cord and ganglia from cervical (C6-C8), thoracic (T6-T8), lumbar (L4-L6) and sacral ($2-$4) levels were removed rapidly and snap-frozen as mentioned above. The tissues were then serially cut (20 pm) on a cryostat and thaw-mounted on gelatin-coated slides. The sections were incubated with 25 pM [125I]ET-1 (spec. act. 2000 Ci/mmol, Amersham, Canada) in the presence or absence of 1 /zM unlabeled ET-1 (Peninsula Laboratories, Belmont, CA) for 2 h at room temperature. The incubation solution contained 50 m M Tris-HCl buffer (pH 7.4), 10 m M magnesium chloride, 1 m M phenylmethylsulfonyl fluoride, 0.4 mg/ml soybean trypsin inhibitor, 1 mM tetrasodium ethylenediamine tetraacetate, 100 U/ml aprotinin and 2 mg/ml bovine serum albumin. Following incubation, slides were rinsed in Tris-HCl buffer, rapidly air-dried and then juxtaposed against tritium-sensitive films for 36 h along with iodinated standard (Amersham, Canada). The autoradiograms were quantified densitometrically using a M C I D image analysis system (Imaging Research Inc., Canada). For cellular localisation of binding sites, a batch of slides after incubation were fixed in 5% glutaraldehyde solution in 50 m M phosphate buffer (pH 7.4) and then dehydrated in graded alcohols. Sections were then dipped in Kodak NTB-2 emulsion (diluted 1 : 1 in water) and developed after 6-8 days. The distribution of [125I]ET-1 binding sites demonstrated a characteristic pattern in the developing and
118
adult spinal cord, and in the dorsal root ganglia of the rat (Table IA,B; Fig. 1A~)). At postnatal day 1, [125I]ET-1 binding sites were observed throughout the grey matter as well as the white matter of the spinal cord. However, no discrete pattern of labelling was apparent between dorsal, ventral or central canal regions of the grey matter (Fig. 1A,F). During postnatal ontogeny, the density of [125I]ET-1 binding sites in the grey matter first decreases (days 1-7) (Fig. 1B,G) and then gradually increases (days 7-28) (Fig. 1C,H) with little change in its localisation (Table IA). [125I]ET-1 binding sites in the white matter, on the contrary, showed a gradual decrease during development (Table IA; Fig. 1A-C). In the adult spinal cord, the density of [125I]ET-1 binding sites in the white matter was relatively low, and grey matter did not exhibit much variation in the labelling between various laminae (Fig. 1D,I; Table IB). At higher resolution, the emulsion-processed material showed a uniform pattern of silver grain distribution in the grey matter of developing and adult spinal cord. In the ventral horn, particularly in laminae IX, perikarya of motoneurones were found to be relatively spared of silver grains. In the dorsal root ganglia, [125I]ET-1 binding sites were observed in postnatal day 1 (Fig. 1K). The density of [125I]ET-l binding sites, unlike the spinal cord, remained somewhat constant throughout development (Fig. 1L,M; Table IA). In the adult, ganglia pulled to-
gether from various segmental levels of the spinal cord showed little variation in the density of labelling (Fig. 1B). At higher resolution, silver grains were found to be concentrated primarily in the satellite cells and not in sensory neurones (Fig. IN,O). The present study demonstrates that [125I]ET-1 binding sites are widely distributed throughout the spinal cord and dorsal root ganglia of the developing and adult rat. It is likely that ET itself appears early during prenatal development since significant density of [125I]ET binding sites are already apparent in the spinal cord and dorsal root ganglia 1 day postnatally. Moreover, during the course of development, [LESI]ET-1 binding sites showed dramatic variation in their density, particularly in the spinal cord, suggesting an important role for the peptide in early maturation and organisation of the cord. However, the homogenous distribution of [tzsI]ET-1 binding sites in spinal grey matter does not correspond precisely to ET-like immunoreactivity [5, 14], which is primarily localised in some scattered fibers (laminae I-VI) and cell bodies (laminae IV-VI) in the dorsal horn and in the majority of motoneurones of the ventral horn. This apparent discrepancy is at present unclear, but given the evidence from other brain regions [3, 10], this could possibly be due to the presence of [12SI]ET-1 binding sites on some non-neuronal elements of the spinal cord. In many tissues of the organism, ET has been sug-
TABLE I QUANTITATIVE DISTRIBUTION OF [t25IlENDOTHELIN-I BINDING SITES IN POSTNATAL (A) AND ADULT (B) SPINAL CORD AND DORSAL ROOT GANGLIA OF THE RAT Each value represents the mean ± S.E.M. of 8-12 sections from 4 rats. The optical density values from different sections were converted to receptor density values by reference to iodinated standards after subtraction of non-specific binding and background film density. Binding densities are expressed as fmol/mg of tissue, wet weight. P, number of days after birth; DRG, dorsal root ganglia; CCR, central canal region; Lain, lamina(e). A Developmental stage
Dorsal horn
Ventral horn
CCR
White matter
DRG
P1 P4 P7 P14 P21 P28
6.1_+0.3 5.1_+0.7 3.74-0.6 5.8_+0.9 6±0.5 8.14-0.4
7.5±0.7 6_+0.6 4.2_+0.6 5.84-0.9 5.5_+0.6 6.2±0.5
6.8±0.8 5.84-0.8 4.7_+0.6 5.84-1.2 4.6_+0.6 6.5_+0.4
7.1±0.4 5.2_+0.8 4.2_+0.5 3.24-0.9 3.9_+0.5 2.5_+0.3
6.3_+0.5 5.8±0.5 4.9_+0.3 4.6_+0.2 4.64-0.8 6.9_+0.3
B
Spinal cord
Lam I-III
Lam IV-VI
Lam VII-VIII
Lam IX
Lam X
White matter
Cervical Thoracic Lumbar Sacral
9.8 + 0.5 11.94-0.5 12.94-0.6 164- 1
9.44- 0.6 10.54-0.5 11.4+_0.6 14.4+0.9
9.8 _ 0.6 10.7_+0.9 12.6_+0.7 13.7_+0.9
11 _ 0.8 11.9_+ 1.3 13.9_+ 1.1 16.2_+ 1.2
10.7 _+0.4 11.3 _+ 1.1 12.2_+0.6 13.8_+ 1.3
4.9 -+0.6 5.1 _+0.8 5.74-0.6 6.84- 1.3
Adudlt DRG 10.9_+ 1.2
119
Fig. 1. Photomicrographs showing the distribution of [nSI]Endothelin (ET)-I binding sites in the spinal cord (A-I) and dorsal root ganglia (J-O) of postnatal day 1 (A,F,K), day 7 (B,G,L), day 28 (C,H,M) and adult (D,I,N) rat. Note the overall decrease of [125I]ET-I binding sites in day 7 spinal cord. F, G, H, I and O are the brightfield representative of A, B, C, D and N respectively. E and J represent [m25I]ET-1 binding sites in presence of 1 gM ET-I in the spinal cord of postnatal day 28 (E) and adult dorsal root ganglia (J). DH, dorsal horn; VH, ventral horn; cc, central canal. A,B,F,G: x 45; C,E,H: x 25; D,I: x 12; K-M: x 760; J,N,O: x 525.
120 gested to act on s m o o t h muscle or other cells close to sites of its synthesis [2, 9]. M a c C u m b e r et al. [10] have recently provided evidence that ET-like materials in the cerebellum can be p r o d u c e d by glia and act u p o n b o t h glia and neurones in a paracrine/autocrine fashion. In the spinal cord, the diffuse pattern of [125I]ET-1 binding sites in dorsal, ventral and lateral horns suggests possible role(s) for ET in spinal synaptic transmission. It is also likely that ET-like materials released from interneurones which are present in the dorsal horn, m a y act in a autocrine and/or paracrine m a n n e r to regulate its own release or the release o f other n e u r o m o d u l a t o r s from adjacent cells. In the n e w b o r n rat spinal cord, ET-1 induces ventral root depolarisation by releasing substance P f r o m dorsal spinal cord [14]. This effect, which can be blocked by either nicardipine, a calcium channel blocker or by spantide, a substance P antagonist, provides evidence for a n e u r o m o d u l a t o r y role for E T in the spinal cord [14]. F u r t h e r m o r e , it has been shown that ET, administered intrathecally, causes complete paresis o f the hind legs along with loss of calcitonin gene-related peptide i m m u noreactivity f r o m the m o t o n e u r o n e s [6]. At present it is not clear whether this action o f ET is a direct one or mediated through other n e u r o m o d u l a t o r s but in all probability it represents the involvement o f ET in m o t o r functions of the spinal cord. Further studies are, however, necessary to explain the significance o f high density of [125I]ET-1 binding sites in areas associated particularly with sensory and a u t o n o m i c functions of the spinal cord. An interesting finding o f the present study relates to the localisation of [125I]ET-1 binding sites on the satellite cells o f the dorsal root ganglia. Satellite cells are considered to provide trophic s u p p o r t to the neurones they surround and regulate the passage o f materials between neurones and blood vessels [8]. The presence o f ET-like immunoreactivity and its gene transcript in the majority of neurones o f dorsal root ganglia and the localisation of [125I]ET-1 binding sites in the surrounding satellite cells, strongly lend support to the paracrine role of this peptide in this tissue. This perhaps also explains why ET, which is synthesised and stored in neurones o f dorsal root ganglia, is present in only a few fibers in the dorsal spinal cord [6] and remains unaltered following transection o f peripheral nerve or p r e t r e a t m e n t with a sensory neurotoxin capsaicin [4]. In s u m m a r y , the widespread distribution o f [125I]ET-1 binding sites in the spinal cord and dorsal root ganglia of developing and adult rat
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