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Gastrin-Releasing Peptide Receptors in Fetal Cortex Transplants
EXPERIMENTAL NEUROLOGY ARTICLE NO.
142, 195–201 (1996)
0190
Autoradiographic Distribution of Bombesin/Gastrin-Releasing Peptide Receptors in Fetal Cortex Transplants T. W. MOODY,* R. GETZ,*
AND
J. M. ROSENSTEIN†
*Department of Biochemistry and Molecular Biology and †Department of Anatomy and Cell Biology, George Washington University Medical Center, Washington, DC 20037
Bombesin/gastrin-releasing peptide (BB/GRP) receptors were characterized in fetal cortex transplants in the rat. Using in vitro autoradiography techniques, the density of (125I-Tyr4)BB grains was low 2 weeks after transplantation but increased 4 weeks after cortex transplantation into the adult rat fourth ventricle. Densitometry analysis of the autoradiograms indicated that (125I-Tyr4)BB bound with high affinity (Kd 5 5 nM) to a single class of sites in the transplant tissue. Specific (125I-Tyr4)BB binding was inhibited with high affinity by (Tyr4)BB but not by NMB (IC50 values of 3 and 100 nM, respectively). These data suggest that GRP may act as a novel growth factor and play a regulatory role in the development of fetal cortex transplants. r 1996 Academic Press, Inc.
INTRODUCTION
Bombesin (BB) represents one class of peptides biologically active in the mammalian brain. The 14-aminoacid BB was initially isolated from frog skin, which is enriched in peptides and biologically active amines (2). Subsequently, BB and C-terminal analogues were chemically synthesized and found to be biologically active when directly injected into the brain. Injection of microgram doses of BB into the rat ventricles resulted in hypothermia in cold-exposed rats (4), hyperglycemia in rats regardless of ambient temperature (5), increased grooming behavior (7, 14), and satiety (10). These data suggest that mammalian BB-like peptides are present and biologically active in the central nervous system (CNS). Subsequently, the 27-amino-acid mammalian derivative of BB, gastrin-releasing peptide (GRP), was isolated (13). GRP and BB have 9 of the same 10 Cterminal amino acid residues. Also, the 10-amino-acid neuromedin B (NMB) was characterized and has 7 of the same 10 C-terminal amino acid residues as BB (15). Endogenous GRP is present in high concentrations in certain hypothalamic areas such as the paraventricular and suprachiasmatic nuclei but not the cerebellum (18, 24). In contrast, NMB is present in high concentra-
tions in the olfactory bulb and the hippocampus (22). Also, using in situ hybridization techniques, it was found that GRP mRNA predominates in the hypothalamus and cortex whereas NMB mRNA predominates in the olfactory bulb, dentate gyrus, and dorsal root gangion (33). Because GRP and NMB have a different distribution in the rat brain, they may be derived from different precursor proteins. Subsequently, it was found that GRP is derived from a 148-amino-acid precursor protein, whereas NMB was derived from a 116-aminoacid precursor protein (11, 27, 29). Both GRP and NMB are localized to synaptosomes and are released by depolarizing stimuli in a Ca21-dependent manner (17, 21). When released, the peptides may interact with receptors for GRP and NMB. Distinct G-protein-coupled receptors exist for GRP and NMB. The GRP-preferring receptor (BB2) was cloned from Swiss 3T3 cells and is composed of 384 amino acids and contains seven hydrophobic domains (3, 30). The NMB-preferring receptor (BB1) was cloned from rat esophagus and contains 390 amino acid residues (32). There is a 53% amino acid sequence identity between the GRP and the NMB receptors. Nonetheless, the BB2 receptor binds GRP and BB with high affinity and NMB with moderate affinity. The BB1 receptor binds NMB with high affinity and GRP and BB with moderate affinity. After binding BB2 receptors, GRP stimulates phosphatidylinositol turnover, elevates cytosolic Ca21, increases c-fos gene expression, and stimulates the growth of normal and malignant cells (12, 23, 26, 28). Previously, we found that there was a moderate density of GRP receptors in the adult rat cortex (20). Here GRP receptors were localized to discrete regions in the rat brain using 125I-labeled ligands, and the developmental expression of these receptors was examined in neocortical transplants. METHODS
GRP receptors were localized to rat brain regions using in vitro autoradiographic techniques (35, 37). Intraventricular neocortical grafts were performed as
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0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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described previously (9). For the GRP receptors, 20-µmthick sections of fresh frozen rat brain were sliced on a cryostat and placed onto glass slides or coverslips. Sections were air dried and incubated with assay buffer composed of 130 mM NaCl, 5 mM MgCl2, 5 mM KCl, 1 mM MnCl2, 1 mM EGTA, 100 µg/ml of bacitracin, and 0.1% bovine serum albumin in 10 mM Hepes · NaOH (pH 7.4) plus radiolabeled peptide at 22°C in the presence or absence of competitor. After incubation, free (125I-Tyr4) BB was removed by two consecutive washes in buffer at 4°C, followed by a brief rinse in H2O. The sections on coverslips, which contained bound peptide, were then crushed and counted for radioactivity on a gamma counter or alternatively placed on
Amersham hyperfilm. After 2 weeks the film was developed and the grain density analyzed using a RAS 3000 densitometer (Amersham Corp.). Alternatively, the BB2 receptors could be visualized using emulsioncoated coverslips (36). RESULTS
GRP receptors in fetal cortex transplants were evaluated using in vitro autoradiographic techniques. Figure 1A shows that in sagittal sections from the host rat brain high (125I-Tyr4) BB grain densities were present in the anterior olfactory nucleus, nucleus accumbens, and olfactory tubercle. Moderate densities were pres-
FIG. 1. (125I-Tyr4)BB grain density in rat brain. Sagittal autoradiograms from Paxinos and Watson (25), coordinates lateral 2.1 mm, are shown. (125I-Tyr4)BB grains are present in the frontal cortex (Fr1), olfactory tubercle (Tu), anterior olfactory nucleus (Ao), nucleus acumbens (Acb), caudate putamen (CPu), and hippocampus (Hi). Radiolabeled (Tyr4)BB binding is shown in the implant (Imp) 2 (A), 4 (B), and 8 weeks (C) after implantation.
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ent in the cortex, caudate putamen, and hippocampus. Low densities were present in the nucleus of the solitary tract and the hypothalamus. Grains were absent from the cerebellum and corpus callosum. The GRP receptors in the adult host brain remained constant after implantation of the fetal cortex graft. In contrast, the grain density in the implant increased 4 (Fig. 1B) and 8 weeks (Fig. 1C) after grafting into the host brain relative to that at 2 weeks (Fig. 1A). This was further characterized by densitometry analysis of the autoradiograms. Figure 2 shows that the grain densities increased twofold 4 or 8 weeks after transplantation into the adult host brain relative to that at 2 weeks. The density of binding sites in the cortex prior to transplant (30 pM) was similar to that observed 2 weeks after cortex transplantation into the cisterna magnum (35 pM). The structure–activity relationship of binding was evaluated using coronal sections of rat hindbrain. Figure 3a shows that a high grain density was present in the transplant and that a low grain density was present in the nucleus of the solitary tract, nucleus ambiguis, and spinal tract of the trigeminal nerve of the host brain in the absence of competitor. Figures 3b and 3c show that the grain densities did not significantly decrease in the presence of 0.1 or 1 nM, respectively, of (Tyr4)BB. In the presence of 3 or 10 nM (Tyr4)BB the grain densities decreased dramatically in the transplant as well as host brain. Few grains remained after addition of 30 or 100 nM (Tyr4)BB (Figs. 3f and 3g), and grain density was negligible after addition of 400 nM unlabeled (Tyr4)BB (Fig. 3h). The grain densities decreased in both the host brain and the
implant. Figure 4 shows a Scatchard plot of the specific binding data. (125I-Tyr4)BB bound with high affinity (Kd 5 5 nM) to a single class of sites (Bmax 5 70 pM). Figure 5 shows that specific (125I-Tyr4)BB binding to the fetal transplant indicated that (Tyr4)BB and NMB inhibited specific (125I-Tyr4)BB binding with moderate affinity (IC50 values of 3 and 100 nM, respectively). DISCUSSION
Previously we found that (125I-Tyr4)BB binding sites develop on fetal cortex transplants (9). If fetal cortex grafts were placed in the cortex or spinal cord, high (Tyr4)BB grain densites developed on the transplants (16). These data suggest that the development of the (Tyr4)BB binding sites is a function of the graft and not the host tissue. In contrast, if superior cervical or cerebellar grafts were transplanted into the fourth ventricle, (Tyr4)BB binding sites did not develop. Because the adult cortex, but not superior cervical ganglia or cerebellum, has (Tyr4) BB binding sites, the graft tissue must be capable of expressing GRP receptors for them to develop. In this communication the (Tyr4)BB binding sites on the fetal cortex grafts were further characterized. Using in vitro autoradiographic techniques, (Tyr4)BB binding sites on autoradiograms were analyzed on a densitometer and compared to 125I standards. (Tyr4)BB binding sites developed on the graft during the first month after transplantation and were maximal after 4 weeks. One month after transplantation, (Tyr4)BB bound with high affinity to a single class of sites (Bmax 5 80 fmol/mg). This density of binding sites was
FIG. 2. Densitometry analysis of transplant. The (125I-Tyr4)BB grain densities in the implant were analyzed by a densitometer. The Bmax was calculated and is shown in the fetal cortex prior to transplantation (0) and in the implant 2, 4, and 8 weeks after transplantation. The mean value 6 SD of four experiments is shown.
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FIG. 3. (125I-Tyr4)BB grain density in implant as a function of competitor. Coronal autoradiograms from Paxinos and Watson (25), coordinates 25.3 mm, are shown. Radiolabeled (Tyr4)BB was incubated with the brain sections in the presence of 0 (a), 0.1 (b), 1 (c), 3 (d), 10 (e), 30 (f), 100 (g), and 400 nM (Tyr4)BB (h). (125I-Tyr4)BB binding to both the host brain and the implant is reduced with the addition of competitor.
GRP RECEPTORS IN TRANSPLANTS
FIG. 4. Scatchard analysis. The binding data were plotted and the best fit was drawn assuming a single class of sites.
approximately twice that in the normal adult cortex and 5 times that of the adult nucleus tractus solitarious. It is possible that these (Tyr4)BB binding sites develop to stimulate the growth and/or differentiation of the transplant. Bombesin, by interacting at GRP receptors, stimulates the growth of a number of normal tissues such as Swiss 3T3 cells, normal bronchial epithelial cells, and human endometrial stromal cells (8, 26, 34). Also, BB stimulates the growth of small cell lung cancer cells (6) and xenografts (1), and promotes gastric carcinogenesis induced by N-methyl-N8-nitro-Nnitrosoguanidine in Wistar rats (31). GRP receptors were present in the fetal cortex transplant. Specific (125I-Tyr4)BB binding was inhibited with high affinity by (Tyr4)BB and moderate affinity by NMB.
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Preliminary data (T. Moody, unpublished) indicate that (125I-Tyr4)BB binding is inhibited with high affinity by GRP and GRP receptor antagonists such as (Psi13,14Leu14)BB. (Psi13,14Leu14)BB is a specific antagonist for BB2 but not BB1 receptors (12). In order to further verify that GRP receptors were present on fetal cortex transplants, immunocytochemical techniques were used. Preliminary data (T. Moody, unpublished) indicate that BB2 receptor immunoreactivity is present in neurons of the fetal cortex transplant but not the host cerebellum. Currently we are investigating if NMB receptors are present in fetal cortex transplants. The fetal cortex GRP receptors may be activated by endogenous BB-like peptides. Previously, preproGRP and NMB mRNA were localized in the adult rat cortex (33). Also, GRP immunoreactivity was detected in the adult rat cortex by radioimmunoassay (19). By use of immunocytochemical techniques, immunoreactive preproGRP was localized to neurons in the fetal cortex transplant but not the adult cerebellum (T. Moody, unpublished). The neurons containing preproGRP were distinct from those containing immunoreactive GRP receptor. These data suggest that GRP may function as a novel growth factor in a paracrine manner in the fetal cortex transplant whereby one set of neurons synthesizes and releases peptide and another set of neurons has BB2 receptors. Alternatively, the fetal cortex GRP receptors may be activated by GRP which diffuses from host sites such as the nucleus tractus solitarious. In summary, GRP receptors develop in fetal cortex transplants in the adult rat brain. It remains to be
FIG. 5. Densitometry analysis of the implant as a function of competitor. The percentage specific (125I-Tyr4)BB binding is indicated as a function of (Tyr4)BB (W) and NMB (Q) concentration. The mean value 6 SE of four determinations is shown. The lines were drawn point to point.
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determined if the GRP receptors regulate the growth and/or differentiation of the transplant. 17.
ACKNOWLEDGMENT This research was supported in part by NIH Grant NS-17468.
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142, 195–201 (1996)
0190
Autoradiographic Distribution of Bombesin/Gastrin-Releasing Peptide Receptors in Fetal Cortex Transplants T. W. MOODY,* R. GETZ,*
AND
J. M. ROSENSTEIN†
*Department of Biochemistry and Molecular Biology and †Department of Anatomy and Cell Biology, George Washington University Medical Center, Washington, DC 20037
Bombesin/gastrin-releasing peptide (BB/GRP) receptors were characterized in fetal cortex transplants in the rat. Using in vitro autoradiography techniques, the density of (125I-Tyr4)BB grains was low 2 weeks after transplantation but increased 4 weeks after cortex transplantation into the adult rat fourth ventricle. Densitometry analysis of the autoradiograms indicated that (125I-Tyr4)BB bound with high affinity (Kd 5 5 nM) to a single class of sites in the transplant tissue. Specific (125I-Tyr4)BB binding was inhibited with high affinity by (Tyr4)BB but not by NMB (IC50 values of 3 and 100 nM, respectively). These data suggest that GRP may act as a novel growth factor and play a regulatory role in the development of fetal cortex transplants. r 1996 Academic Press, Inc.
INTRODUCTION
Bombesin (BB) represents one class of peptides biologically active in the mammalian brain. The 14-aminoacid BB was initially isolated from frog skin, which is enriched in peptides and biologically active amines (2). Subsequently, BB and C-terminal analogues were chemically synthesized and found to be biologically active when directly injected into the brain. Injection of microgram doses of BB into the rat ventricles resulted in hypothermia in cold-exposed rats (4), hyperglycemia in rats regardless of ambient temperature (5), increased grooming behavior (7, 14), and satiety (10). These data suggest that mammalian BB-like peptides are present and biologically active in the central nervous system (CNS). Subsequently, the 27-amino-acid mammalian derivative of BB, gastrin-releasing peptide (GRP), was isolated (13). GRP and BB have 9 of the same 10 Cterminal amino acid residues. Also, the 10-amino-acid neuromedin B (NMB) was characterized and has 7 of the same 10 C-terminal amino acid residues as BB (15). Endogenous GRP is present in high concentrations in certain hypothalamic areas such as the paraventricular and suprachiasmatic nuclei but not the cerebellum (18, 24). In contrast, NMB is present in high concentra-
tions in the olfactory bulb and the hippocampus (22). Also, using in situ hybridization techniques, it was found that GRP mRNA predominates in the hypothalamus and cortex whereas NMB mRNA predominates in the olfactory bulb, dentate gyrus, and dorsal root gangion (33). Because GRP and NMB have a different distribution in the rat brain, they may be derived from different precursor proteins. Subsequently, it was found that GRP is derived from a 148-amino-acid precursor protein, whereas NMB was derived from a 116-aminoacid precursor protein (11, 27, 29). Both GRP and NMB are localized to synaptosomes and are released by depolarizing stimuli in a Ca21-dependent manner (17, 21). When released, the peptides may interact with receptors for GRP and NMB. Distinct G-protein-coupled receptors exist for GRP and NMB. The GRP-preferring receptor (BB2) was cloned from Swiss 3T3 cells and is composed of 384 amino acids and contains seven hydrophobic domains (3, 30). The NMB-preferring receptor (BB1) was cloned from rat esophagus and contains 390 amino acid residues (32). There is a 53% amino acid sequence identity between the GRP and the NMB receptors. Nonetheless, the BB2 receptor binds GRP and BB with high affinity and NMB with moderate affinity. The BB1 receptor binds NMB with high affinity and GRP and BB with moderate affinity. After binding BB2 receptors, GRP stimulates phosphatidylinositol turnover, elevates cytosolic Ca21, increases c-fos gene expression, and stimulates the growth of normal and malignant cells (12, 23, 26, 28). Previously, we found that there was a moderate density of GRP receptors in the adult rat cortex (20). Here GRP receptors were localized to discrete regions in the rat brain using 125I-labeled ligands, and the developmental expression of these receptors was examined in neocortical transplants. METHODS
GRP receptors were localized to rat brain regions using in vitro autoradiographic techniques (35, 37). Intraventricular neocortical grafts were performed as
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0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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described previously (9). For the GRP receptors, 20-µmthick sections of fresh frozen rat brain were sliced on a cryostat and placed onto glass slides or coverslips. Sections were air dried and incubated with assay buffer composed of 130 mM NaCl, 5 mM MgCl2, 5 mM KCl, 1 mM MnCl2, 1 mM EGTA, 100 µg/ml of bacitracin, and 0.1% bovine serum albumin in 10 mM Hepes · NaOH (pH 7.4) plus radiolabeled peptide at 22°C in the presence or absence of competitor. After incubation, free (125I-Tyr4) BB was removed by two consecutive washes in buffer at 4°C, followed by a brief rinse in H2O. The sections on coverslips, which contained bound peptide, were then crushed and counted for radioactivity on a gamma counter or alternatively placed on
Amersham hyperfilm. After 2 weeks the film was developed and the grain density analyzed using a RAS 3000 densitometer (Amersham Corp.). Alternatively, the BB2 receptors could be visualized using emulsioncoated coverslips (36). RESULTS
GRP receptors in fetal cortex transplants were evaluated using in vitro autoradiographic techniques. Figure 1A shows that in sagittal sections from the host rat brain high (125I-Tyr4) BB grain densities were present in the anterior olfactory nucleus, nucleus accumbens, and olfactory tubercle. Moderate densities were pres-
FIG. 1. (125I-Tyr4)BB grain density in rat brain. Sagittal autoradiograms from Paxinos and Watson (25), coordinates lateral 2.1 mm, are shown. (125I-Tyr4)BB grains are present in the frontal cortex (Fr1), olfactory tubercle (Tu), anterior olfactory nucleus (Ao), nucleus acumbens (Acb), caudate putamen (CPu), and hippocampus (Hi). Radiolabeled (Tyr4)BB binding is shown in the implant (Imp) 2 (A), 4 (B), and 8 weeks (C) after implantation.
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ent in the cortex, caudate putamen, and hippocampus. Low densities were present in the nucleus of the solitary tract and the hypothalamus. Grains were absent from the cerebellum and corpus callosum. The GRP receptors in the adult host brain remained constant after implantation of the fetal cortex graft. In contrast, the grain density in the implant increased 4 (Fig. 1B) and 8 weeks (Fig. 1C) after grafting into the host brain relative to that at 2 weeks (Fig. 1A). This was further characterized by densitometry analysis of the autoradiograms. Figure 2 shows that the grain densities increased twofold 4 or 8 weeks after transplantation into the adult host brain relative to that at 2 weeks. The density of binding sites in the cortex prior to transplant (30 pM) was similar to that observed 2 weeks after cortex transplantation into the cisterna magnum (35 pM). The structure–activity relationship of binding was evaluated using coronal sections of rat hindbrain. Figure 3a shows that a high grain density was present in the transplant and that a low grain density was present in the nucleus of the solitary tract, nucleus ambiguis, and spinal tract of the trigeminal nerve of the host brain in the absence of competitor. Figures 3b and 3c show that the grain densities did not significantly decrease in the presence of 0.1 or 1 nM, respectively, of (Tyr4)BB. In the presence of 3 or 10 nM (Tyr4)BB the grain densities decreased dramatically in the transplant as well as host brain. Few grains remained after addition of 30 or 100 nM (Tyr4)BB (Figs. 3f and 3g), and grain density was negligible after addition of 400 nM unlabeled (Tyr4)BB (Fig. 3h). The grain densities decreased in both the host brain and the
implant. Figure 4 shows a Scatchard plot of the specific binding data. (125I-Tyr4)BB bound with high affinity (Kd 5 5 nM) to a single class of sites (Bmax 5 70 pM). Figure 5 shows that specific (125I-Tyr4)BB binding to the fetal transplant indicated that (Tyr4)BB and NMB inhibited specific (125I-Tyr4)BB binding with moderate affinity (IC50 values of 3 and 100 nM, respectively). DISCUSSION
Previously we found that (125I-Tyr4)BB binding sites develop on fetal cortex transplants (9). If fetal cortex grafts were placed in the cortex or spinal cord, high (Tyr4)BB grain densites developed on the transplants (16). These data suggest that the development of the (Tyr4)BB binding sites is a function of the graft and not the host tissue. In contrast, if superior cervical or cerebellar grafts were transplanted into the fourth ventricle, (Tyr4)BB binding sites did not develop. Because the adult cortex, but not superior cervical ganglia or cerebellum, has (Tyr4) BB binding sites, the graft tissue must be capable of expressing GRP receptors for them to develop. In this communication the (Tyr4)BB binding sites on the fetal cortex grafts were further characterized. Using in vitro autoradiographic techniques, (Tyr4)BB binding sites on autoradiograms were analyzed on a densitometer and compared to 125I standards. (Tyr4)BB binding sites developed on the graft during the first month after transplantation and were maximal after 4 weeks. One month after transplantation, (Tyr4)BB bound with high affinity to a single class of sites (Bmax 5 80 fmol/mg). This density of binding sites was
FIG. 2. Densitometry analysis of transplant. The (125I-Tyr4)BB grain densities in the implant were analyzed by a densitometer. The Bmax was calculated and is shown in the fetal cortex prior to transplantation (0) and in the implant 2, 4, and 8 weeks after transplantation. The mean value 6 SD of four experiments is shown.
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FIG. 3. (125I-Tyr4)BB grain density in implant as a function of competitor. Coronal autoradiograms from Paxinos and Watson (25), coordinates 25.3 mm, are shown. Radiolabeled (Tyr4)BB was incubated with the brain sections in the presence of 0 (a), 0.1 (b), 1 (c), 3 (d), 10 (e), 30 (f), 100 (g), and 400 nM (Tyr4)BB (h). (125I-Tyr4)BB binding to both the host brain and the implant is reduced with the addition of competitor.
GRP RECEPTORS IN TRANSPLANTS
FIG. 4. Scatchard analysis. The binding data were plotted and the best fit was drawn assuming a single class of sites.
approximately twice that in the normal adult cortex and 5 times that of the adult nucleus tractus solitarious. It is possible that these (Tyr4)BB binding sites develop to stimulate the growth and/or differentiation of the transplant. Bombesin, by interacting at GRP receptors, stimulates the growth of a number of normal tissues such as Swiss 3T3 cells, normal bronchial epithelial cells, and human endometrial stromal cells (8, 26, 34). Also, BB stimulates the growth of small cell lung cancer cells (6) and xenografts (1), and promotes gastric carcinogenesis induced by N-methyl-N8-nitro-Nnitrosoguanidine in Wistar rats (31). GRP receptors were present in the fetal cortex transplant. Specific (125I-Tyr4)BB binding was inhibited with high affinity by (Tyr4)BB and moderate affinity by NMB.
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Preliminary data (T. Moody, unpublished) indicate that (125I-Tyr4)BB binding is inhibited with high affinity by GRP and GRP receptor antagonists such as (Psi13,14Leu14)BB. (Psi13,14Leu14)BB is a specific antagonist for BB2 but not BB1 receptors (12). In order to further verify that GRP receptors were present on fetal cortex transplants, immunocytochemical techniques were used. Preliminary data (T. Moody, unpublished) indicate that BB2 receptor immunoreactivity is present in neurons of the fetal cortex transplant but not the host cerebellum. Currently we are investigating if NMB receptors are present in fetal cortex transplants. The fetal cortex GRP receptors may be activated by endogenous BB-like peptides. Previously, preproGRP and NMB mRNA were localized in the adult rat cortex (33). Also, GRP immunoreactivity was detected in the adult rat cortex by radioimmunoassay (19). By use of immunocytochemical techniques, immunoreactive preproGRP was localized to neurons in the fetal cortex transplant but not the adult cerebellum (T. Moody, unpublished). The neurons containing preproGRP were distinct from those containing immunoreactive GRP receptor. These data suggest that GRP may function as a novel growth factor in a paracrine manner in the fetal cortex transplant whereby one set of neurons synthesizes and releases peptide and another set of neurons has BB2 receptors. Alternatively, the fetal cortex GRP receptors may be activated by GRP which diffuses from host sites such as the nucleus tractus solitarious. In summary, GRP receptors develop in fetal cortex transplants in the adult rat brain. It remains to be
FIG. 5. Densitometry analysis of the implant as a function of competitor. The percentage specific (125I-Tyr4)BB binding is indicated as a function of (Tyr4)BB (W) and NMB (Q) concentration. The mean value 6 SE of four determinations is shown. The lines were drawn point to point.
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determined if the GRP receptors regulate the growth and/or differentiation of the transplant. 17.
ACKNOWLEDGMENT This research was supported in part by NIH Grant NS-17468.
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