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TRH immunoreactivity in rat hypothalamus and brain: assessment by gel filtration and thin-layer chromatography
Brain Research, 171 (1979) 161-165 O Elsevier/North-Holland Biomedical Press
161
TRH immunoreactivity in rat hypothalamus and brain: assessment by gel filtration and thin-layer chromatography
MARGARET SZPILA KREIDER, ANDREW WINOKUR* and ROBERT D. UTIGER Departments of Pharmacology, Psychiatry and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pa. (U.S.A.)
(Accepted March 29th, 1979)
Thyrotropin-releasing hormone (TRH) has a number of central nervous system neuropharmacological and behavioral actions 4. Interest in T R H as a possible endogenous neuromodulator was increased by the finding of T R H immunoreactivity (IR-TRH) throughout the central nervous system 2,z,5. Extrahypothalamic and hypothalamic I R - T R H and synthetic T R H have similar physiochemical and biological properties in a number of systems, including gel filtration, electrophoresis and bioassay. These findings provided indirect but strong support that hypothalamic and extrahypothalamic I R - T R H was indeed TRH. Recently, Youngblood et al, reported studies suggesting that I R - T R H differed from synthetic T R H 6. Specifically, I R - T R H from brain extracts did not co-migrate with synthetic T R H on thin-layer chromatography and could not be recovered by affinity chromatography of brain extracts. These data were the first suggesting that extrahypothalamic I R - T R H differed from synthetic TRH. We have repeated these studies, but were unable to discern differences in extrahypothalamic and hypothalamic I R - T R H and synthetic TRH. Some suggestions are offered for the differences between our findings and those of Youngblood et al. 6. Male Sprague-Dawley rats (180-200 g) were decapitated, the brains removed, and extracts of whole brain, extrahypothalamic brain, frontal cortex or hypothalamus were prepared in two ways. Whole brains were each homogenized in 2 M acetic acid (5 ml/g tissue), as described by Youngblood et al. 6. After centrifugation at 2000 r.p.m. for 10 min, the supernatants were extracted with an equal volume of diethyl ether, frozen, and thawed to break the interphase. The aqueous layer was lyophilized. The dried residue was extracted with 15 ml ethanol, centrifuged at 2000 r.p.m, and the supernatants dried overnight at 60 °C in an air stream. Hypothalami, frontal cortices, extrahypothalamic brains, and whole brains also were each homogenized in 2.0 ml 0.15 M NaCI, 0.01 M NaH2PO4, pH 7.5, and extracted with 10 ml methanol. The homogenates were centrifuged at 2000 r.p.m, for 10 min and the supernatants dried * To whom correspondence should be addressed at: Department of Psychiatry G-I, University of Pennsylvania, Philadelphia, Pa. 19104, U.S.A.
162 overnight at 60 °C in an air stream. All dried residues were redissolved in water and the dissolved residues from a particular region pooled for gel filtration. The pooled extracts of whole brain, extrahypothalamic brain, hypothalamus, or frontal cortex were applied to a 2.8 x 30 cm Sephadex G-25 column. The column was eluted with water and 50 4.0 ml fractions collected. Each fraction was assayed for T R H immunoreactivity. Those containing IR-TRH were pooled and lyophilized, the residues extracted with methanol or acetone-water (8:2), centrifuged at 2000 r.p.m, for l0 min, and the supernatants air-dried at 60 °C overnight. The resulting residues were dissolved in 100-200 ffl methanol or 200 ffl acetone-water (8:2). Aliquots of the methanol- or acetone-water-dissolved lyophilized G-25 column pools and synthetic T R H were applied to 20 x 20 cm silica gel plates. The plates were developed in methylene chloride-methanol (60:40) for 45 min at 25 °C, dried and divided into 17 one cm segments which were scraped off the plate and extracted with methanol. The residues obtained after evaporation of the methanol were dissolved in 0 . 2 5 ~ bovine serum albumin, 0.15 M NaCI, 0.01 M NaH~PO4, pH 7.5, for T R H assay. Two points should be made regarding the methodological aspects of these studies. First, two types of silica gel plates were used in the thin-layer chromatography, Bakerflex and Eastman. Since synthetic T R H migrated with different mobilities on these different plates, synthetic T R H was always chromatographed on the same plate with brain extracts. Second, elution of silica gel alone produced some interference in the T R H assay, equivalent to 0.05-0.13 ng TRH/segment, and immunoreactivity in this blank range was therefore not interpreted as being due to TRH. To compare the final extraction procedures, 5 hypothalami and 5 extrahypothalamic brains were each homogenized in 0.15 M NaC1, 0.01 M NaH2PO4, pH 7.5, extracted with methanol and subjected to Sephadex G-25 gel filtration as described above. The resulting fractions which contained I R - T R H were pooled, divided into 6 equal parts, and lyophilized. Three of both the hypothalamic and the extrahypothalamic pools were extracted with 4 ml methanol and 3 of each were extracted with 4 ml acetonewater (8:2). The methanol or acetone-water was evaporated and the resulting residues were redissolved in 100 ffl methanol or acetone-water respectively and aliquots removed for T R H assay. T R H was determined by a previously described radioimmunoassay, which has been shown to be highly specific for T R H 1. The sensitivity of the assay as used in these experiments was 0.01 rig/assay tube, and usually 100 or 200 ffl aliquots of extracts, column fractions or thin-layer fractions were assayed in duplicate. The gel filtration profiles of IR-TRH extracted from whole brain with acidethanol or with methanol and synthetic T R H are shown in Fig. IA. The I R - T R H from brains extracted with acid-ethanol was found in fractions 24-33 and totaled 19.5 ng. IR-TRH from brains extracted with methanol was found in fractions 25-32 and totaled 23.3 ng. Synthetic T R H was found in fractions 25-35 and totaled 105.1 ng. The gel filtration fractions from brains initially extracted with methanol were extracted and redissolved in methanol and subjected to thin-layer chromatography. IR-TRH was found in the same elution positions as synthetic T R H (Fig. 1B). 34 ~ of the I R - T R H present in the initial brain extract and 45~o of synthetic T R H subjected to all
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Fig. 1. A:g••fi•trati•n•fm•t•an••andacid--ethan••extracts•fwh•••ratbrainsandsyntheticTRH (100 rig). B: thin-layer chromatography of the IR-TRH recovered from the Sephadex columns (see text for sample preparation for the thin-layer chromatography).
extraction steps were recovered after thin-layer chromatography. The gel filtration fractions from the brains initially extracted with acid-ethanol, after lyophilization and acetone-water (8:2) solubilization6, were also subjected to thin-layer chromatography. No IR-TRH was recovered from the silica gel plate (Fig. 1B). IR-TRH from hypothalamus, frontal cortex and extrahypothalamic brain was found in the same fractions as described previously for whole brain TRH after Sephadex G-25 gel filtration. No IR-TRH was recovered from thin-layer chromatograms of lyophilized column fractions of any of the brain regions which were dissolved in acetone-water. When the acetone-water was evaporated and the residue dissolved in methanol, 66.6 i 9.5 ~ S.E.M. (39-85 ~) of the IR-TRH eluted from Sephadex G25 was recovered. Thin-layer chromatography of the re-extracted samples produced the elution profiles shown in Fig. 2. Both hypothalamic and frontal cortical IR-TRH co-eluted with synthetic TRH. Preparation of hypothalamic and frontal cortical extracts using methanol at all extraction steps resulted in elution profiles similar to those shown in Fig. 2.
164
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Fig. 2. Thin-layer chromatography of TRH immunoreactivity of hypothalamic and frontal cortical extracts and synthetic TRH.
The absence of IR-TRH after thin-layer chromatography of brain extracts subjected to gel filtration which were redissolved in acetone-water prompted a comparison of the solvent systems used to extract the lyophilized gel filtration IRT R H residues. In this study, hypothalami and extrahypothalamic brains were used. Methanol extraction resulted in a mean recovery of 98.6 4- 6 . 8 ~ S.E.M. of hypothalamic ]R-TRH and a mean recovery of 100.8 4- 3.6 ~ S.E.M. of extrahypothalamic IR-TRH (n = 3). Acetone-water extraction resulted in a mean recovery of 41.7 4- 0.68 ~ of hypothalamic IR-TRH and 13.0 4- 2.7 ~ of extrahypothalamic IRT R H (n -- 3). These results provide further evidence that extrahypothalamic IR-TRH is indistinguishable from synthetic TRH. The reasons for the discrepancies between our findings and those of Youngblood et al. are not completely clear, but some data have been obtained which bear on this issue. T R H appears to be variably soluble in acetone-water, the solvent system used in the studies of Youngblood et al., and the recovery was inversely related to the amount of brain tissue from which the I R - T R H was extracted. In contrast, methanol extraction resulted in good recovery of synthetic T R H and IR-TRH from both hypothalamic and extrahypothalamic brain tissue. In the sequence of steps involved in these studies, I R - T R H was recovered effectively after gel filtration on Sephadex G-25, but was not recovered after lyophilization followed by extraction with acetone-water. This result would explain why no IR-TRH was recovered after thin-layer chromatography of acetone-water dissolved column fractions. Re-extraction of the acetone-water extract with methanol resulted in substantial recovery of IR-TRH from both brain and hypothalamic residues. Another aspect of the discrepancy between our findings and those of Youngblood et al. involves their finding of small amounts of IR-TRH in extracts of extrahypothalamic brain tissue with mobility on thin-layer chromatography differing from that of synthetic TRH. In this study, no I R - T R H was found other than in positions corresponding to synthetic TRH. We found that extracts of silica gel alone resulted in small, and
165 variable, amounts of inhibition in the T R H radioimmunoassay. Thus, only fractions which contained 3 or more times that amount of assay inhibitory activity were considered to have I R - T R H . The I R - T R H not co-migrating with synthetic T R H found by Youngblood et al. may thus be non-specific. To summarize these findings, hypothalamic and extrahypothalamic brain tissue homogenates and synthetic T R H were fractioned by gel filtration and thin-layer chromatography. I R - T R H from all tissues and synthetic T R H were recovered from the same fractions in both systems, and no I R - T R H not co-migrating with synthetic T R H was found. These findings support the hypothesis that I R - T R H obtained from extrahypothalamic brain tissue is T R H . As noted previously, brain and hypothalamic I R - T R H have other properties similar to that of synthetic T R H . However, conclusive proof of the identity of extrahypothalamic T R H will require purification and amino acid sequencing. Supported by Research Scientist Development Award K01-MH00044 from the National Institute of Mental Health and Research Grant AM 14039 from the National Institute of Arthritis, Metabolic and Digestive Diseases. We thank Mrs. Elaine Paolini for expert secretarial help.
1 Bassiri, R. M. and Utiger, R. D., The preparation and specificity of antibody to thyrotropin releasing hormone, Endocrinology, 90 (1972) 722-727. 2 Jackson, I. and Reichlin, S., Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates. Endocrinology, 95 (1974) 854-862. 3 Oliver, C., Eskay, R. L., Ben-Jonathan, N. and Porter, J. C., Distribution and concentration of TRH in the rat brain. Endocrinology, 74 (1974) 540-546. 4 Prange, A. J., Jr., Nemeroff, C. B., Lipton, M. A., Breese, G. R. and Wilson, I. C., Peptides and the central nervous system. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 13, Plenum Press, New York, 1978, pp. 1-107. 5 Winokur, A. and Utiger, R. D., Thyrotropin-releasing hormone: regional distribution in rat brain, Science, 185 (1974) 265-267. 6 Youngblood, W. W., Lipton, M. A. and Kizer, J. S., TRH-like immunoreactivity in urine, serum, and extrahypothalamic brain: non-identity with synthetic pyroglu-hist-pro-NH~ (TRH), Brain Research, 151 (1978)99-116.
161
TRH immunoreactivity in rat hypothalamus and brain: assessment by gel filtration and thin-layer chromatography
MARGARET SZPILA KREIDER, ANDREW WINOKUR* and ROBERT D. UTIGER Departments of Pharmacology, Psychiatry and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pa. (U.S.A.)
(Accepted March 29th, 1979)
Thyrotropin-releasing hormone (TRH) has a number of central nervous system neuropharmacological and behavioral actions 4. Interest in T R H as a possible endogenous neuromodulator was increased by the finding of T R H immunoreactivity (IR-TRH) throughout the central nervous system 2,z,5. Extrahypothalamic and hypothalamic I R - T R H and synthetic T R H have similar physiochemical and biological properties in a number of systems, including gel filtration, electrophoresis and bioassay. These findings provided indirect but strong support that hypothalamic and extrahypothalamic I R - T R H was indeed TRH. Recently, Youngblood et al, reported studies suggesting that I R - T R H differed from synthetic T R H 6. Specifically, I R - T R H from brain extracts did not co-migrate with synthetic T R H on thin-layer chromatography and could not be recovered by affinity chromatography of brain extracts. These data were the first suggesting that extrahypothalamic I R - T R H differed from synthetic TRH. We have repeated these studies, but were unable to discern differences in extrahypothalamic and hypothalamic I R - T R H and synthetic TRH. Some suggestions are offered for the differences between our findings and those of Youngblood et al. 6. Male Sprague-Dawley rats (180-200 g) were decapitated, the brains removed, and extracts of whole brain, extrahypothalamic brain, frontal cortex or hypothalamus were prepared in two ways. Whole brains were each homogenized in 2 M acetic acid (5 ml/g tissue), as described by Youngblood et al. 6. After centrifugation at 2000 r.p.m. for 10 min, the supernatants were extracted with an equal volume of diethyl ether, frozen, and thawed to break the interphase. The aqueous layer was lyophilized. The dried residue was extracted with 15 ml ethanol, centrifuged at 2000 r.p.m, and the supernatants dried overnight at 60 °C in an air stream. Hypothalami, frontal cortices, extrahypothalamic brains, and whole brains also were each homogenized in 2.0 ml 0.15 M NaCI, 0.01 M NaH2PO4, pH 7.5, and extracted with 10 ml methanol. The homogenates were centrifuged at 2000 r.p.m, for 10 min and the supernatants dried * To whom correspondence should be addressed at: Department of Psychiatry G-I, University of Pennsylvania, Philadelphia, Pa. 19104, U.S.A.
162 overnight at 60 °C in an air stream. All dried residues were redissolved in water and the dissolved residues from a particular region pooled for gel filtration. The pooled extracts of whole brain, extrahypothalamic brain, hypothalamus, or frontal cortex were applied to a 2.8 x 30 cm Sephadex G-25 column. The column was eluted with water and 50 4.0 ml fractions collected. Each fraction was assayed for T R H immunoreactivity. Those containing IR-TRH were pooled and lyophilized, the residues extracted with methanol or acetone-water (8:2), centrifuged at 2000 r.p.m, for l0 min, and the supernatants air-dried at 60 °C overnight. The resulting residues were dissolved in 100-200 ffl methanol or 200 ffl acetone-water (8:2). Aliquots of the methanol- or acetone-water-dissolved lyophilized G-25 column pools and synthetic T R H were applied to 20 x 20 cm silica gel plates. The plates were developed in methylene chloride-methanol (60:40) for 45 min at 25 °C, dried and divided into 17 one cm segments which were scraped off the plate and extracted with methanol. The residues obtained after evaporation of the methanol were dissolved in 0 . 2 5 ~ bovine serum albumin, 0.15 M NaCI, 0.01 M NaH~PO4, pH 7.5, for T R H assay. Two points should be made regarding the methodological aspects of these studies. First, two types of silica gel plates were used in the thin-layer chromatography, Bakerflex and Eastman. Since synthetic T R H migrated with different mobilities on these different plates, synthetic T R H was always chromatographed on the same plate with brain extracts. Second, elution of silica gel alone produced some interference in the T R H assay, equivalent to 0.05-0.13 ng TRH/segment, and immunoreactivity in this blank range was therefore not interpreted as being due to TRH. To compare the final extraction procedures, 5 hypothalami and 5 extrahypothalamic brains were each homogenized in 0.15 M NaC1, 0.01 M NaH2PO4, pH 7.5, extracted with methanol and subjected to Sephadex G-25 gel filtration as described above. The resulting fractions which contained I R - T R H were pooled, divided into 6 equal parts, and lyophilized. Three of both the hypothalamic and the extrahypothalamic pools were extracted with 4 ml methanol and 3 of each were extracted with 4 ml acetonewater (8:2). The methanol or acetone-water was evaporated and the resulting residues were redissolved in 100 ffl methanol or acetone-water respectively and aliquots removed for T R H assay. T R H was determined by a previously described radioimmunoassay, which has been shown to be highly specific for T R H 1. The sensitivity of the assay as used in these experiments was 0.01 rig/assay tube, and usually 100 or 200 ffl aliquots of extracts, column fractions or thin-layer fractions were assayed in duplicate. The gel filtration profiles of IR-TRH extracted from whole brain with acidethanol or with methanol and synthetic T R H are shown in Fig. IA. The I R - T R H from brains extracted with acid-ethanol was found in fractions 24-33 and totaled 19.5 ng. IR-TRH from brains extracted with methanol was found in fractions 25-32 and totaled 23.3 ng. Synthetic T R H was found in fractions 25-35 and totaled 105.1 ng. The gel filtration fractions from brains initially extracted with methanol were extracted and redissolved in methanol and subjected to thin-layer chromatography. IR-TRH was found in the same elution positions as synthetic T R H (Fig. 1B). 34 ~ of the I R - T R H present in the initial brain extract and 45~o of synthetic T R H subjected to all
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Fig. 1. A:g••fi•trati•n•fm•t•an••andacid--ethan••extracts•fwh•••ratbrainsandsyntheticTRH (100 rig). B: thin-layer chromatography of the IR-TRH recovered from the Sephadex columns (see text for sample preparation for the thin-layer chromatography).
extraction steps were recovered after thin-layer chromatography. The gel filtration fractions from the brains initially extracted with acid-ethanol, after lyophilization and acetone-water (8:2) solubilization6, were also subjected to thin-layer chromatography. No IR-TRH was recovered from the silica gel plate (Fig. 1B). IR-TRH from hypothalamus, frontal cortex and extrahypothalamic brain was found in the same fractions as described previously for whole brain TRH after Sephadex G-25 gel filtration. No IR-TRH was recovered from thin-layer chromatograms of lyophilized column fractions of any of the brain regions which were dissolved in acetone-water. When the acetone-water was evaporated and the residue dissolved in methanol, 66.6 i 9.5 ~ S.E.M. (39-85 ~) of the IR-TRH eluted from Sephadex G25 was recovered. Thin-layer chromatography of the re-extracted samples produced the elution profiles shown in Fig. 2. Both hypothalamic and frontal cortical IR-TRH co-eluted with synthetic TRH. Preparation of hypothalamic and frontal cortical extracts using methanol at all extraction steps resulted in elution profiles similar to those shown in Fig. 2.
164
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Fig. 2. Thin-layer chromatography of TRH immunoreactivity of hypothalamic and frontal cortical extracts and synthetic TRH.
The absence of IR-TRH after thin-layer chromatography of brain extracts subjected to gel filtration which were redissolved in acetone-water prompted a comparison of the solvent systems used to extract the lyophilized gel filtration IRT R H residues. In this study, hypothalami and extrahypothalamic brains were used. Methanol extraction resulted in a mean recovery of 98.6 4- 6 . 8 ~ S.E.M. of hypothalamic ]R-TRH and a mean recovery of 100.8 4- 3.6 ~ S.E.M. of extrahypothalamic IR-TRH (n = 3). Acetone-water extraction resulted in a mean recovery of 41.7 4- 0.68 ~ of hypothalamic IR-TRH and 13.0 4- 2.7 ~ of extrahypothalamic IRT R H (n -- 3). These results provide further evidence that extrahypothalamic IR-TRH is indistinguishable from synthetic TRH. The reasons for the discrepancies between our findings and those of Youngblood et al. are not completely clear, but some data have been obtained which bear on this issue. T R H appears to be variably soluble in acetone-water, the solvent system used in the studies of Youngblood et al., and the recovery was inversely related to the amount of brain tissue from which the I R - T R H was extracted. In contrast, methanol extraction resulted in good recovery of synthetic T R H and IR-TRH from both hypothalamic and extrahypothalamic brain tissue. In the sequence of steps involved in these studies, I R - T R H was recovered effectively after gel filtration on Sephadex G-25, but was not recovered after lyophilization followed by extraction with acetone-water. This result would explain why no IR-TRH was recovered after thin-layer chromatography of acetone-water dissolved column fractions. Re-extraction of the acetone-water extract with methanol resulted in substantial recovery of IR-TRH from both brain and hypothalamic residues. Another aspect of the discrepancy between our findings and those of Youngblood et al. involves their finding of small amounts of IR-TRH in extracts of extrahypothalamic brain tissue with mobility on thin-layer chromatography differing from that of synthetic TRH. In this study, no I R - T R H was found other than in positions corresponding to synthetic TRH. We found that extracts of silica gel alone resulted in small, and
165 variable, amounts of inhibition in the T R H radioimmunoassay. Thus, only fractions which contained 3 or more times that amount of assay inhibitory activity were considered to have I R - T R H . The I R - T R H not co-migrating with synthetic T R H found by Youngblood et al. may thus be non-specific. To summarize these findings, hypothalamic and extrahypothalamic brain tissue homogenates and synthetic T R H were fractioned by gel filtration and thin-layer chromatography. I R - T R H from all tissues and synthetic T R H were recovered from the same fractions in both systems, and no I R - T R H not co-migrating with synthetic T R H was found. These findings support the hypothesis that I R - T R H obtained from extrahypothalamic brain tissue is T R H . As noted previously, brain and hypothalamic I R - T R H have other properties similar to that of synthetic T R H . However, conclusive proof of the identity of extrahypothalamic T R H will require purification and amino acid sequencing. Supported by Research Scientist Development Award K01-MH00044 from the National Institute of Mental Health and Research Grant AM 14039 from the National Institute of Arthritis, Metabolic and Digestive Diseases. We thank Mrs. Elaine Paolini for expert secretarial help.
1 Bassiri, R. M. and Utiger, R. D., The preparation and specificity of antibody to thyrotropin releasing hormone, Endocrinology, 90 (1972) 722-727. 2 Jackson, I. and Reichlin, S., Thyrotropin-releasing hormone (TRH): distribution in hypothalamic and extrahypothalamic brain tissues of mammalian and submammalian chordates. Endocrinology, 95 (1974) 854-862. 3 Oliver, C., Eskay, R. L., Ben-Jonathan, N. and Porter, J. C., Distribution and concentration of TRH in the rat brain. Endocrinology, 74 (1974) 540-546. 4 Prange, A. J., Jr., Nemeroff, C. B., Lipton, M. A., Breese, G. R. and Wilson, I. C., Peptides and the central nervous system. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 13, Plenum Press, New York, 1978, pp. 1-107. 5 Winokur, A. and Utiger, R. D., Thyrotropin-releasing hormone: regional distribution in rat brain, Science, 185 (1974) 265-267. 6 Youngblood, W. W., Lipton, M. A. and Kizer, J. S., TRH-like immunoreactivity in urine, serum, and extrahypothalamic brain: non-identity with synthetic pyroglu-hist-pro-NH~ (TRH), Brain Research, 151 (1978)99-116.