Life Sciences, Vol. 51, pp. 1715-1724 Printed in the USA
Pergamon Press
ONTOGENY OF ENDOTHELIN AND ITS RECEPTORS IN RAT BRAIN Anil Gulati, Sam Rebello, Gopal Chari" and Rama Bhat" Department of Pharmacodynamics and "Pediatrics, The University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612 USA (Received in final form September 21, 1992)
Summary The ontogeny of endothelin (ET) system in rats was studied in preterm (1 8 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (K~) and density (Bm,x) were determined using [~2Sl] ET-1 and [~2~1] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 _+ 3.1 8 pg/g protein in preterm, 47.37 + 5.31 pg/g protein in term and 48.30 + 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [12~1] ET-1 and [12~1] SRT 6b. The Kd values for [1251] ET-1 and [~2Sl] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [1251] ET-1 and [1251]SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bin,x values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density. Endothelin (ET), a 21 amino acid peptide and a potent endogenous vasoconstrictor (1), has also been considered to be a neuromodulator, endocrine or paracrine hormone or a trophic factor (2). ET receptors are widely distributed in the central nervous system (CNS) as well as cardiovascular system (1,2). There are at least three isoforms of ETs named; ET-1, ET-2 and ET-3 (1,3). All these three isoforms are found in the CNS. They are structurally similar to the snake (Atractapsis engaddensis) venom toxin, sarafotoxin 6b (SRT 6b) (4-6). The strong sequence similarities suggest that ET and SRT 6b have a common evolutionary origin, both can induce phosphoinositide hydrolysis that is in part Ca 2÷ independent (4), indicating that ET and SRT 6b may be sharing a common receptor. ET-1 binding sites on the neurons of rat and human brain have been demonstrated using autoradiographic visualization (7,8). It was also confirmed that ET binding sites were localized to nerve cell bodies (9). ET-1 and ET-3 like immunoreactivity have been demonstrated in rat (1 O, 1 1 ), porcine (1 2,1 3) and human brain (14). The localization of ET like immunoreactivity in both glial and neuronal cells of hippocampus has been shown (1 5). ET 0 0 2 4 - 3 2 0 5 / 9 2 $ 5 , 0 0 + .O0 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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mRNA has been demonstrated in porcine paraventricular nucleus (1 2), cultured rat cerebellar astroglia (1 6) and in human brain (1 7). ET binding sites as well as ETs themselves are present in the brain indicating that they are synthesized by the neurons of the CNS (1 8). Central ET mechanisms have been shown to be involved in blood pressure regulation (2,19,20). Intracerebroventricular (icv) injection of ET-1 increases blood pressure in conscious rats (21). ET-1 or ET-3 administration in area postrema, cisterna magna and ventral surface of medulla have also been shown to produce increase in blood pressure (22,23). Behavioral changes were also produced when ET-1 was given icy, the rats leaned to one side and often barrel rolling (24) and loss of righting reflex (25) was observed. ET-3, when administered in the third cerebral ventricle, has been found to produce a dose-related inhibitory effect on water intake (26). Central ET has also been found to regulate the secretion of posterior (1 2) and anterior (27) pituitary hormones. Intracisternal administration of ET-1 was found to affect respiratory function and produced expiratory apnea (28). It was speculated that central ET, like other peptides may be involved in sudden infant death in view of its high potency in producing apnea (28). Above studies strongly suggest that ET mechanisms in the CNS are involved the regulation of a wide variety of functions. The characteristics of ET receptors have been studied in aged rats (29) but no study has yet been performed regarding the characteristics of ET receptors in new born rats. The present study was carried out to investigate the characteristics of ET receptors using [12511 SRT 6b and [1251] ET-1 binding and the concentration of ET-like immunoreactivity in the brain of preterm, term and post term rats. Materials and methods Animals Pregnant Sprague Dawley rats were obtained and housed three to a cage, in a room with controlled temperature (23 + 1 °C), humidity (50 + 10%) and artificial light (06001800 hr). The animals were given food and water ad libitum. New born rats were delivered using cesarian section on 18 day of gestation (preterm), 21 days of gestation (term) and some rats were allowed to deliver naturally and new born rats were taken for study after 1 week of birth (post term). Drugs The [~251]SRT 6b (specific activity 2000 Ci/mmol) and [1251] ET-1 (specific activity 2000 Ci/mmol) were purchased from Amersham Corporation, Arlington Heights, IL. ET-1 was purchased from Sigma Chemical Co., St. Louis, MO. SRT 6b and radioimmunoassay (RIA) kit for ET-1 were purchased from Peninsula Laboratories Inc., Belmont, CA. Determination of the binding of [lz51] SRT 6b and |1251] ET-1 to ET receptors in neuronal membranes. The rats were sacrificed by decapitation, and brains were dissected out in cold. The tissue was homogenized in 30 volumes of ice-cold Tris-HCI buffer (0.05 M, pH 7.4) using a Polytron homogenizer (setting 5 for 20 sec). The homogenate was centrifuged at 4 9 , 0 0 0 X g for 15 rain and the pellet resuspended in 50 mM Tris-HCI (pH 7.4) buffer. After a second centrifugation at 4 9 , 0 0 0 x g for 1 5 min, the pellet was resuspended in 50 mM Tris-HCI buffer (pH 7.4) containing aprotinin (5 U/ml), pepstatin A (5 pg/ml), phenylmethylsulfonylfluoride (PMSF) (0.1 mM), ethylenediaminetetraacetic acid (EDTA) (3 mM) and ethylene glycol-bis (i&amino ethyl ether) N,N,N',N'-tetraacetic acid (EGTA) (1 mM) and used for the binding studies. The binding of [1251] SRT 6b was performed according to the procedure of Gulati, (19) and Gulatiand Rebello,(20) using 100-1 5 0 p g protein in each sample. The binding of [1251] SRT 6b was carried out in a total volume of 0.3 ml which contained 50 mM Tris-HCI buffer (pH 7.4) containing aprotinin (5 U/ml), pepstatin A (5 pg/ml), PMSF (0.1 raM), EDTA (3 mM)
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and EGTA (1 raM) and 0.1 ml of the homogenate. All binding assays were carried out in triplicate at 25 °C for 60 rain. Binding was terminated by rapidly filtering the content of the incubation tubes under reduced pressure using a Brandel M-24R cell harvester and Whatman GF/B glass fiber filters. The filters were washed twice with 5 ml of the same ice-cold buffer used for the binding assay. The filters were transferred to vials and the radioactivity in the samples was determined in a Packard Minaxi Gamma Counter with a 74% counting efficiency. To determine the binding of [12~1] ET-1, the experiment was carried out essentially as described above. The specific binding was defined as the difference in binding observed in the absence and presence of 0.1 pM SRT 6b or ET-1. The concentration for the binding of [1251] SRT 6b and [12sI] ET-1 was 1.0 nM. The concentration of protein in the samples was determined by the method of Lowry et al. (30). The maximal binding capacity (Bmax) and apparent dissociation constant (K~) values were determined using varying concentration of cold SRT 6b or ET-1. The amount of specific binding of the ligand was expressed as fmol/mg protein (mean ± S.E.M.). The saturation curves and the Scatchard plots were drawn using computer program EBDA and final estimates of Bm,, and K~ values were obtained using LIGAND (31). The data was subjected to analysis of variance followed by the Scheffe's S-test. A value of p < 0.05 was considered to be significant. Five determinations one in each animal, were made to compute the means and their standard errors. Determination of ET-1 in brain by radioimmunoassay. Brains were dissected out in cold as described above and homogenized in 4 ml of O. 1 % trifluoroacetic acid using a Polytron homogenizer (setting 5 for 20 sec). Protein estimations in the homogenate were done (31). The extraction of ET was carried out by activating the SEP-PAK C18 Column (Waters, Milford, MA) containing 300 mg of material by washing with 60 % acetonitrile in 0 . 1 % trifluoroacetic acid (4 x 1 ml) followed by washing with 0 . 1 % trifluoroacetic acid (4 x 5 ml). The sample solution (4 ml) was slowly applied on the column and then washed with 0 . 1 % trifluoroacetic acid (4 x 5 ml). ET like materials were eluted with 60 % acetonitrile in 0 . 1 % trifluoroacetic acid (3 x 1 ml) into polypropylene tubes. The eluant was evaporated to dryness in a centrifugal concentrator and the residue was dissolved in RIA buffer. The recovery was found to be 77 % by this method. RIA was performed using RIA kit for ET-1 (Peninsula Lab. Inc, Belmont, CA). This kit has only 7 % cross reactivity with ET-2 or ET-3. The incubation mixture for RIA consisted of 100 pl of standards or samples and 100 pl of rabbit anti-endothelin-1 serum. The tubes were vortexed and incubated for 16 hours at 4°C. Next day, [1251] ET-1 (100pl) was added and the mixture vortexed and incubated for 24 hours. On day 3, goat anti-rabbit IgG serum (100 pl) and normal rabbit serum (100 pl) was added to all the tubes, vortexed and incubated for 2 hours at room temperature. RIA buffer (500 pl) was added and the tubes vortexed and then centrifuged at 1,700 g for 20 min. The supernatant was carefully aspirated and the radioactivity in the pellet was determined using Packard Gamma Counter (Model Cobra 5005). The concentration of ET like material was determined from the standard plots of Iogit B/Bo versus log dose (pg) and expressed as pg/g protein. T w o rats were used for one experiment and each experiment was performed 4 times for each group. The data was subjected to analysis of variance followed by the Scheffe's Stest. A value of P < 0.05 was considered to be significant. Results [1251] SRT 6b and [':'Sl] ET-1 binding to neuronal membranes of preterm, term and post term rats. [1251] SRT 6b bound to a single high affinity binding site in the neuronal membranes. In the preterm rats the Ka value was 0.43 + 0.07 nM and the Bma. value was 1435 _ 124 fmol/mg protein. The binding of SRT 6b was found to be significantly higher in post term rats as compared to preterm and term rats. There was no difference in the K~ values between preterm, term and post term rats (F(2,1 2) = 0.492; P = O. 109). The Bm~, values were found
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SARAFOTOXIN
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1992
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Ontogeny of Endothelin Receptors
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to be significantly (F(2,12) = 5 . 6 1 0 ; P = 0 . 0 1 9 ) different b e t w e e n the groups. The B ~ , values w e r e found to be similar in preterm and term rats, but t h e y w e r e found to be significantly higher (P = 0 . 0 2 9 ) in post term rats as c o m p a r e d to preterm and t e r m rats (Figure 1 ). The saturation curve and Scatchard plot for SRT 6b binding is s h o w n in Figure 3. [~2~1] ET-1 also bound to a single high affinity binding site w i t h the Kd value of 0 . 4 9 + 0 . 0 4 nM and the Bm,x value of 1 5 4 0 + 92 f m o l / m g protein, in the brain of p r e t e r m rats. The Kd values of ET-1 binding to ET receptors w e r e found to be similar (F(2,12) = 0 . 0 0 6 ; P = 0 . 9 9 4 ) in p r e t e r m , t e r m and post t e r m rats. The Bm~, values w e r e found to be significantly (F(2,12) = 7 . 2 3 8 ; P = 0 . 0 0 9 ) different b e t w e e n the groups. The B=,, values w e r e found to be similar (P = 0 . 8 0 8 ) in preterm and term rats, but they w e r e significantly higher (P = 0 . 0 1 2 ) in post t e r m rats as c o m p a r e d to preterm and term rats (Figure 2). The saturation curve and Scatchard plot for ET-1 binding is s h o w n in Figure 4. In the adult (4 m o n t h old) rat brain, [12Sl) SRT 6b bound w i t h a Kd value of 0 . 4 3 + 0 . 0 2 nM and Bin,, of 1 4 7 1 . 0 4 : 1 : 5 5 . 8 8 f m o l / m g protein, while [12~1] ET-1 bound w i t h a K d value o f 0 . 3 7 + 0 . 0 3 nM and Bin8, value of 1 2 0 5 . 7 5 + 5 0 . 9 4 f m o l / m g protein. ET-1 concentrations in the brain of preterm, term and post term rats. ET-1 like i m m u n o r e a c t i v i t y w a s found to be present in l o w c o n c e n t r a t i o n in the brains of p r e t e r m , t e r m and post t e r m rats. ET-1 concentrations w e r e found to be significantly different b e t w e e n the groups (F(2,9) = 1 1 . 8 2 0 ; P = 0 . 0 0 3 ) . ET-1 c o n c e n t r a t i o n s w e r e similar in term and post t e r m rats, but they w e r e significantly l o w e r (P = 0 . 0 0 5 ) in preterm as c o m p a r e d to t e r m and post t e r m rats (Figure 5).
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Discussion In the present study t w o different ligands, SRT 6b and ET-1 were used to label brain ET receptors. The characteristics of ET receptors in new born rats appear to be similar to those of adult rats. The affinity of [1251] SRT 6b and [1251] ET-1 binding are similar in neonatal and adult rats. In neonatal rats, ET-1 and SRT 6b showed that there was no difference in the affinity of ET receptors in preterm, term and post term rats. The density of both ET-1 and SRT 6b binding were found to be similar in preterm and term rats. However, the density of ET-1 and SRT 6b binding were found to be significantly increased in post term rats as compared to preterm and term rats. Thus both ligands showed similar results. It has been also suggested that SRT 6b and ET share a common receptor (4-6). ET-1 like immunoreactivity could be detected in the brain of preterm, term and post term rats. Since only 7% cross reactivity is present with ET-2 and ET-3, most of the ET detected is of ET-1 type. ET-1 levels were found to be significantly increased in term and post term rats as compared to preterm rats. ET-1 levels were similar in term and post term rats. Thus, the concentration of ET-1 increases significantly in term rats followed by significant increase in the density of ET receptors in post term rats. Many neurotransmitters have been shown to act as developmental signals or regulators like serotonin (32), dopamine (33), noradrenaline (34), acetylcholine (35) and endorphins (36). It could be possible that the initial increase in the concentration of ET-1 provides a stimulus for the rapid multiplication of ET receptors. The development of receptors depends upon the level of stimulation at critical times (37). This development is important and could explain many findings of altered receptor densities in disease states. Although the role of central ET in the regulation of body functions is not established, it appears to be involved in regulation of blood pressure (1 9-23). A possibility exists that during development if the expression of central ET receptors is altered then it could lead to development of a hypertensive state. The Bin,x values for ET-1 and SRT 6b in the post term rats are much higher than those for the whole brain of adult rats. Thus, it appears that ET receptors are over expressed in the developmental phase. Several studies have demonstrated that over expression of receptors occurs in the development phase. It has been observed that serotonergic receptors in rats attain peak levels in fetal or early neonatal life and then decrease to adult levels (38,39). Dopamine receptors are also found in high numbers in immature brain of rat (40). Similarly, a peak in receptor number of immature brain has been found for substance P (41) and opiate (42) receptors. The functional significance of over expression of ET receptors in neonatal rats is not known. It could be possible that the receptors simply over express initially and those which are not required are eliminated during subsequent course of development. These over expressed receptors may themselves play a role in development through the production of second messengers, which in turn regulate the expression of these receptors (35). Few studies have been conducted indicating the role of ET system in neonatology. The plasma ET-1 like immunoreactive levels were found to be t w o fold higher in infants younger than 3 months of age as compared to older children or adults. The physiological significance of higher ET levels in early infancy is not known (43). The role of ET in perinatal period appears to be vital in parturition, lactation, pulmonary circulation and closure of the fetal ductus arteriosus (44-46). Central ET has been found to regulate several body functions (2), but their importance from perinatal point of view is not known at present. In summary the present study shows that (a) the density of ET receptors in the brain is similar in preterm and term rats while it is significantly increased in post term rats and (b) the endogenous concentration of ET in the brain is significantly lower in preterm rats as compared to term and post term rats. The role of endogenous ET in the regulation of ET receptor density is a possibility. Although the importance of central ET system in perinatology is not known, the possibility of its role in the pathophysiology of certain disease states, particularly, hypertension exists.
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1 9 3 2 6 5 - 2 7 5 (1951). P.J. MUNSON AND D. RODBARD, Analyt. Biochem. 1 0 7 2 2 0 - 2 3 9 (1980). J.M. LAUDER, Psychoneuroendocrinol. 8 1 2 1 - 1 5 5 (1983). A. KALSBEEK, R.M. BUIJS, M.A. HOFMAN, M.A.H. MATTHIJEESN, C.W. POOL AND H.B.M. UYLINGS, Dev. Brain Res. 3__22123-132 (1987). J.G. PARNAVELAS AND M.E. BLUE, Dev. Brain Res. 3 1 4 0 - 1 4 4 (1982). H.A. NAVARRO, F.J. SEIDLER, J.P. EYLERS, F.E. BAKER, S.S. DOBBINS, S.E. LAPPI, T.A. SLOTKIN, J. Pharmacol. Exp. Ther. 2 5 1 8 9 4 - 9 0 0 (1989). I.S. ZAGON AND P.J. MCLAUGHLIN, In, Developmental Neuroscience: Physiological, Pharmacological and Clinical Aspects, Eds. F. Caciagli, E. Giacobini and R. Paoletti, Elsevier Science Publishers, New York, 67-70 (1984). P.M. WHITAKER-AZMITIA, Pharmacol. Rev. 4:3553-561 (1991). P.M. WHITAKER-AZMITIA, D. QUARTERMAIN D AND A.V. SHEMER, Dev. Brain Res. 5"/ 181-185 (1990). G. DAVAL, D. VERGE, A. BECERRIL, H. GOZLAN, U. SPAMPINATO AND M. HAMON, Int. J. Dev. Neurosci. 5 1 7 1 - 1 8 9 (1987). J.C. MILLER AND A.J. FRIEDHOFF, Int. J. Dev. Neurosci. 4 2 1 - 2 6 (1986). R. QUIRION AND T. DAM, J. Neurosci. 6 2 1 8 7 - 2 1 9 9 (1986). J. BARG AND R. SIMANTOV, Dev. Neurosci. 1_j_1428-434 (1989). M. YOSHIBAYASHI, K. NISHIOKA, K. NAKAO, Y. SAITO, S. TEMMA, M. MATSUMURA, T. UEDA, G. SHIRAKAMI, H. IMURA AND H. MIKAWA, J. Cardiovasc. Pharmacol. 17 (Suppl 7) S404-S405 (1991). T. MASAKI, S. USUKI, M. YANAGISAWA AND N. SUZUKI, Seminars Perinatol. 1.52729 (1991). R. KRZESKI, W. LONG, H. K A T A Y A M A AND W. HENRY, J. Cardiovasc. Pharmacol. 17 (Suppl 7) S322-S325 (1991). M.D. MITCHELL, Seminars Perinatol. 1.579-85 (1991).
Pergamon Press
ONTOGENY OF ENDOTHELIN AND ITS RECEPTORS IN RAT BRAIN Anil Gulati, Sam Rebello, Gopal Chari" and Rama Bhat" Department of Pharmacodynamics and "Pediatrics, The University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612 USA (Received in final form September 21, 1992)
Summary The ontogeny of endothelin (ET) system in rats was studied in preterm (1 8 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (K~) and density (Bm,x) were determined using [~2Sl] ET-1 and [~2~1] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 _+ 3.1 8 pg/g protein in preterm, 47.37 + 5.31 pg/g protein in term and 48.30 + 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [12~1] ET-1 and [12~1] SRT 6b. The Kd values for [1251] ET-1 and [~2Sl] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [1251] ET-1 and [1251]SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bin,x values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density. Endothelin (ET), a 21 amino acid peptide and a potent endogenous vasoconstrictor (1), has also been considered to be a neuromodulator, endocrine or paracrine hormone or a trophic factor (2). ET receptors are widely distributed in the central nervous system (CNS) as well as cardiovascular system (1,2). There are at least three isoforms of ETs named; ET-1, ET-2 and ET-3 (1,3). All these three isoforms are found in the CNS. They are structurally similar to the snake (Atractapsis engaddensis) venom toxin, sarafotoxin 6b (SRT 6b) (4-6). The strong sequence similarities suggest that ET and SRT 6b have a common evolutionary origin, both can induce phosphoinositide hydrolysis that is in part Ca 2÷ independent (4), indicating that ET and SRT 6b may be sharing a common receptor. ET-1 binding sites on the neurons of rat and human brain have been demonstrated using autoradiographic visualization (7,8). It was also confirmed that ET binding sites were localized to nerve cell bodies (9). ET-1 and ET-3 like immunoreactivity have been demonstrated in rat (1 O, 1 1 ), porcine (1 2,1 3) and human brain (14). The localization of ET like immunoreactivity in both glial and neuronal cells of hippocampus has been shown (1 5). ET 0 0 2 4 - 3 2 0 5 / 9 2 $ 5 , 0 0 + .O0 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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mRNA has been demonstrated in porcine paraventricular nucleus (1 2), cultured rat cerebellar astroglia (1 6) and in human brain (1 7). ET binding sites as well as ETs themselves are present in the brain indicating that they are synthesized by the neurons of the CNS (1 8). Central ET mechanisms have been shown to be involved in blood pressure regulation (2,19,20). Intracerebroventricular (icv) injection of ET-1 increases blood pressure in conscious rats (21). ET-1 or ET-3 administration in area postrema, cisterna magna and ventral surface of medulla have also been shown to produce increase in blood pressure (22,23). Behavioral changes were also produced when ET-1 was given icy, the rats leaned to one side and often barrel rolling (24) and loss of righting reflex (25) was observed. ET-3, when administered in the third cerebral ventricle, has been found to produce a dose-related inhibitory effect on water intake (26). Central ET has also been found to regulate the secretion of posterior (1 2) and anterior (27) pituitary hormones. Intracisternal administration of ET-1 was found to affect respiratory function and produced expiratory apnea (28). It was speculated that central ET, like other peptides may be involved in sudden infant death in view of its high potency in producing apnea (28). Above studies strongly suggest that ET mechanisms in the CNS are involved the regulation of a wide variety of functions. The characteristics of ET receptors have been studied in aged rats (29) but no study has yet been performed regarding the characteristics of ET receptors in new born rats. The present study was carried out to investigate the characteristics of ET receptors using [12511 SRT 6b and [1251] ET-1 binding and the concentration of ET-like immunoreactivity in the brain of preterm, term and post term rats. Materials and methods Animals Pregnant Sprague Dawley rats were obtained and housed three to a cage, in a room with controlled temperature (23 + 1 °C), humidity (50 + 10%) and artificial light (06001800 hr). The animals were given food and water ad libitum. New born rats were delivered using cesarian section on 18 day of gestation (preterm), 21 days of gestation (term) and some rats were allowed to deliver naturally and new born rats were taken for study after 1 week of birth (post term). Drugs The [~251]SRT 6b (specific activity 2000 Ci/mmol) and [1251] ET-1 (specific activity 2000 Ci/mmol) were purchased from Amersham Corporation, Arlington Heights, IL. ET-1 was purchased from Sigma Chemical Co., St. Louis, MO. SRT 6b and radioimmunoassay (RIA) kit for ET-1 were purchased from Peninsula Laboratories Inc., Belmont, CA. Determination of the binding of [lz51] SRT 6b and |1251] ET-1 to ET receptors in neuronal membranes. The rats were sacrificed by decapitation, and brains were dissected out in cold. The tissue was homogenized in 30 volumes of ice-cold Tris-HCI buffer (0.05 M, pH 7.4) using a Polytron homogenizer (setting 5 for 20 sec). The homogenate was centrifuged at 4 9 , 0 0 0 X g for 15 rain and the pellet resuspended in 50 mM Tris-HCI (pH 7.4) buffer. After a second centrifugation at 4 9 , 0 0 0 x g for 1 5 min, the pellet was resuspended in 50 mM Tris-HCI buffer (pH 7.4) containing aprotinin (5 U/ml), pepstatin A (5 pg/ml), phenylmethylsulfonylfluoride (PMSF) (0.1 mM), ethylenediaminetetraacetic acid (EDTA) (3 mM) and ethylene glycol-bis (i&amino ethyl ether) N,N,N',N'-tetraacetic acid (EGTA) (1 mM) and used for the binding studies. The binding of [1251] SRT 6b was performed according to the procedure of Gulati, (19) and Gulatiand Rebello,(20) using 100-1 5 0 p g protein in each sample. The binding of [1251] SRT 6b was carried out in a total volume of 0.3 ml which contained 50 mM Tris-HCI buffer (pH 7.4) containing aprotinin (5 U/ml), pepstatin A (5 pg/ml), PMSF (0.1 raM), EDTA (3 mM)
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and EGTA (1 raM) and 0.1 ml of the homogenate. All binding assays were carried out in triplicate at 25 °C for 60 rain. Binding was terminated by rapidly filtering the content of the incubation tubes under reduced pressure using a Brandel M-24R cell harvester and Whatman GF/B glass fiber filters. The filters were washed twice with 5 ml of the same ice-cold buffer used for the binding assay. The filters were transferred to vials and the radioactivity in the samples was determined in a Packard Minaxi Gamma Counter with a 74% counting efficiency. To determine the binding of [12~1] ET-1, the experiment was carried out essentially as described above. The specific binding was defined as the difference in binding observed in the absence and presence of 0.1 pM SRT 6b or ET-1. The concentration for the binding of [1251] SRT 6b and [12sI] ET-1 was 1.0 nM. The concentration of protein in the samples was determined by the method of Lowry et al. (30). The maximal binding capacity (Bmax) and apparent dissociation constant (K~) values were determined using varying concentration of cold SRT 6b or ET-1. The amount of specific binding of the ligand was expressed as fmol/mg protein (mean ± S.E.M.). The saturation curves and the Scatchard plots were drawn using computer program EBDA and final estimates of Bm,, and K~ values were obtained using LIGAND (31). The data was subjected to analysis of variance followed by the Scheffe's S-test. A value of p < 0.05 was considered to be significant. Five determinations one in each animal, were made to compute the means and their standard errors. Determination of ET-1 in brain by radioimmunoassay. Brains were dissected out in cold as described above and homogenized in 4 ml of O. 1 % trifluoroacetic acid using a Polytron homogenizer (setting 5 for 20 sec). Protein estimations in the homogenate were done (31). The extraction of ET was carried out by activating the SEP-PAK C18 Column (Waters, Milford, MA) containing 300 mg of material by washing with 60 % acetonitrile in 0 . 1 % trifluoroacetic acid (4 x 1 ml) followed by washing with 0 . 1 % trifluoroacetic acid (4 x 5 ml). The sample solution (4 ml) was slowly applied on the column and then washed with 0 . 1 % trifluoroacetic acid (4 x 5 ml). ET like materials were eluted with 60 % acetonitrile in 0 . 1 % trifluoroacetic acid (3 x 1 ml) into polypropylene tubes. The eluant was evaporated to dryness in a centrifugal concentrator and the residue was dissolved in RIA buffer. The recovery was found to be 77 % by this method. RIA was performed using RIA kit for ET-1 (Peninsula Lab. Inc, Belmont, CA). This kit has only 7 % cross reactivity with ET-2 or ET-3. The incubation mixture for RIA consisted of 100 pl of standards or samples and 100 pl of rabbit anti-endothelin-1 serum. The tubes were vortexed and incubated for 16 hours at 4°C. Next day, [1251] ET-1 (100pl) was added and the mixture vortexed and incubated for 24 hours. On day 3, goat anti-rabbit IgG serum (100 pl) and normal rabbit serum (100 pl) was added to all the tubes, vortexed and incubated for 2 hours at room temperature. RIA buffer (500 pl) was added and the tubes vortexed and then centrifuged at 1,700 g for 20 min. The supernatant was carefully aspirated and the radioactivity in the pellet was determined using Packard Gamma Counter (Model Cobra 5005). The concentration of ET like material was determined from the standard plots of Iogit B/Bo versus log dose (pg) and expressed as pg/g protein. T w o rats were used for one experiment and each experiment was performed 4 times for each group. The data was subjected to analysis of variance followed by the Scheffe's Stest. A value of P < 0.05 was considered to be significant. Results [1251] SRT 6b and [':'Sl] ET-1 binding to neuronal membranes of preterm, term and post term rats. [1251] SRT 6b bound to a single high affinity binding site in the neuronal membranes. In the preterm rats the Ka value was 0.43 + 0.07 nM and the Bma. value was 1435 _ 124 fmol/mg protein. The binding of SRT 6b was found to be significantly higher in post term rats as compared to preterm and term rats. There was no difference in the K~ values between preterm, term and post term rats (F(2,1 2) = 0.492; P = O. 109). The Bm~, values were found
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SARAFOTOXIN
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1992
Vol. 51, No. 22, 1992
Ontogeny of Endothelin Receptors
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to be significantly (F(2,12) = 5 . 6 1 0 ; P = 0 . 0 1 9 ) different b e t w e e n the groups. The B ~ , values w e r e found to be similar in preterm and term rats, but t h e y w e r e found to be significantly higher (P = 0 . 0 2 9 ) in post term rats as c o m p a r e d to preterm and t e r m rats (Figure 1 ). The saturation curve and Scatchard plot for SRT 6b binding is s h o w n in Figure 3. [~2~1] ET-1 also bound to a single high affinity binding site w i t h the Kd value of 0 . 4 9 + 0 . 0 4 nM and the Bm,x value of 1 5 4 0 + 92 f m o l / m g protein, in the brain of p r e t e r m rats. The Kd values of ET-1 binding to ET receptors w e r e found to be similar (F(2,12) = 0 . 0 0 6 ; P = 0 . 9 9 4 ) in p r e t e r m , t e r m and post t e r m rats. The Bm~, values w e r e found to be significantly (F(2,12) = 7 . 2 3 8 ; P = 0 . 0 0 9 ) different b e t w e e n the groups. The B=,, values w e r e found to be similar (P = 0 . 8 0 8 ) in preterm and term rats, but they w e r e significantly higher (P = 0 . 0 1 2 ) in post t e r m rats as c o m p a r e d to preterm and term rats (Figure 2). The saturation curve and Scatchard plot for ET-1 binding is s h o w n in Figure 4. In the adult (4 m o n t h old) rat brain, [12Sl) SRT 6b bound w i t h a Kd value of 0 . 4 3 + 0 . 0 2 nM and Bin,, of 1 4 7 1 . 0 4 : 1 : 5 5 . 8 8 f m o l / m g protein, while [12~1] ET-1 bound w i t h a K d value o f 0 . 3 7 + 0 . 0 3 nM and Bin8, value of 1 2 0 5 . 7 5 + 5 0 . 9 4 f m o l / m g protein. ET-1 concentrations in the brain of preterm, term and post term rats. ET-1 like i m m u n o r e a c t i v i t y w a s found to be present in l o w c o n c e n t r a t i o n in the brains of p r e t e r m , t e r m and post t e r m rats. ET-1 concentrations w e r e found to be significantly different b e t w e e n the groups (F(2,9) = 1 1 . 8 2 0 ; P = 0 . 0 0 3 ) . ET-1 c o n c e n t r a t i o n s w e r e similar in term and post t e r m rats, but they w e r e significantly l o w e r (P = 0 . 0 0 5 ) in preterm as c o m p a r e d to t e r m and post t e r m rats (Figure 5).
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Discussion In the present study t w o different ligands, SRT 6b and ET-1 were used to label brain ET receptors. The characteristics of ET receptors in new born rats appear to be similar to those of adult rats. The affinity of [1251] SRT 6b and [1251] ET-1 binding are similar in neonatal and adult rats. In neonatal rats, ET-1 and SRT 6b showed that there was no difference in the affinity of ET receptors in preterm, term and post term rats. The density of both ET-1 and SRT 6b binding were found to be similar in preterm and term rats. However, the density of ET-1 and SRT 6b binding were found to be significantly increased in post term rats as compared to preterm and term rats. Thus both ligands showed similar results. It has been also suggested that SRT 6b and ET share a common receptor (4-6). ET-1 like immunoreactivity could be detected in the brain of preterm, term and post term rats. Since only 7% cross reactivity is present with ET-2 and ET-3, most of the ET detected is of ET-1 type. ET-1 levels were found to be significantly increased in term and post term rats as compared to preterm rats. ET-1 levels were similar in term and post term rats. Thus, the concentration of ET-1 increases significantly in term rats followed by significant increase in the density of ET receptors in post term rats. Many neurotransmitters have been shown to act as developmental signals or regulators like serotonin (32), dopamine (33), noradrenaline (34), acetylcholine (35) and endorphins (36). It could be possible that the initial increase in the concentration of ET-1 provides a stimulus for the rapid multiplication of ET receptors. The development of receptors depends upon the level of stimulation at critical times (37). This development is important and could explain many findings of altered receptor densities in disease states. Although the role of central ET in the regulation of body functions is not established, it appears to be involved in regulation of blood pressure (1 9-23). A possibility exists that during development if the expression of central ET receptors is altered then it could lead to development of a hypertensive state. The Bin,x values for ET-1 and SRT 6b in the post term rats are much higher than those for the whole brain of adult rats. Thus, it appears that ET receptors are over expressed in the developmental phase. Several studies have demonstrated that over expression of receptors occurs in the development phase. It has been observed that serotonergic receptors in rats attain peak levels in fetal or early neonatal life and then decrease to adult levels (38,39). Dopamine receptors are also found in high numbers in immature brain of rat (40). Similarly, a peak in receptor number of immature brain has been found for substance P (41) and opiate (42) receptors. The functional significance of over expression of ET receptors in neonatal rats is not known. It could be possible that the receptors simply over express initially and those which are not required are eliminated during subsequent course of development. These over expressed receptors may themselves play a role in development through the production of second messengers, which in turn regulate the expression of these receptors (35). Few studies have been conducted indicating the role of ET system in neonatology. The plasma ET-1 like immunoreactive levels were found to be t w o fold higher in infants younger than 3 months of age as compared to older children or adults. The physiological significance of higher ET levels in early infancy is not known (43). The role of ET in perinatal period appears to be vital in parturition, lactation, pulmonary circulation and closure of the fetal ductus arteriosus (44-46). Central ET has been found to regulate several body functions (2), but their importance from perinatal point of view is not known at present. In summary the present study shows that (a) the density of ET receptors in the brain is similar in preterm and term rats while it is significantly increased in post term rats and (b) the endogenous concentration of ET in the brain is significantly lower in preterm rats as compared to term and post term rats. The role of endogenous ET in the regulation of ET receptor density is a possibility. Although the importance of central ET system in perinatology is not known, the possibility of its role in the pathophysiology of certain disease states, particularly, hypertension exists.
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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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