0013-7227/90/1275-2501I02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Distribution of Thyrotropin-Releasing Hormone (TRH) and Precursor Pep tide (TRH-Gly) in Adult Rat Tissues YOZEN FUSE, DANIEL H. POLK, ROBERT W. LAM, AND DELBERT A. FISHER Department of Pediatrics, University of California-Los Angeles, School of Medicine, Harbor-UCLA Medical Center, Torrance, California 90509; and King-Drew Medical Center, Los Angeles, California 90059
ABSTRACT. TRH (pGlu-His-Pro-NH2) arises from the posttranslational processing of a larger precursor peptide containing multiple copies of the TRH progenitor sequence, Gln-His-ProGly. Concentrations of TRH and its precursor peptide (TRHGly) were determined in serum and a variety of tissues of the rat using specific RIA systems. TRH and TRH-Gly immunoreactivities were detectable in almost all tissues studied. TRH was distributed mainly in neural tissues, with the highest mean concentration (126 pg/mg tissue) in hypothalamus. In extraneural tissues, mean TRH levels ranged from 0.6-4.8 pg/mg tissue; the mean serum concentration was 12.4 pg/ml. In contrast to the distribution of TRH, relatively higher mean TRH-Gly concentrations were observed in serum (76.5 pg/ml) and in
T
RH (L-pyroglutamyl-L-histidyl-L-prolinamide, pGlu-His-Pro-NH2) arises from the posttranslational cleavage of a large precursor molecule (1). Rat hypothalamic pro-TRH has a mol wt of approximately 29,000 and contains five progenitor sequences flanked by paired basic amino acid residues (2). In the formation of TRH, the sequence -Lys-Arg-Gln-His-Pro-Gly- is cleaved from the prohormone by trypsin-like enzymes at paired basic amino acid residues, followed by cyclization of the N-terminal glutamine (3) and amidation of the proline using the C-terminal glycine as the nitrogen donor (4). In this process, the glycine-extended form of TRH (TRH-Gly) is the immediate precursor peptide for TRH. TRH is distributed in extrahypothalamic brain regions and in extraneural tissues, including pancreas and gastrointestinal tissues (5). TRH-Gly has been reported in high concentration in rat prostate (6). However, there are limited data regarding the distribution of TRHGly and TRH in adult rat tissues (7). In the present study we measured the concentrations of both TRH-Gly and TRH in a wide variety of tissues using specific RIA systems. Received March 19, 1990. Address requests for reprints to: Daniel H. Polk, M.D., Martin Research Center, Harbor-University of California-Los Angeles Medical Center, 1000 West Carson Street, Torrance, California 90509.
extraneural tissues, including prostate (83.3 pg/mg tissue), spleen (19.0 pg/mg), adrenal (16.2 pg/mg), kidney (13.3 pg/mg), and gastrointestinal tract (6.3-19.8 pg/mg). Among brain tissues, the TRH-Gly concentration was highest in pituitary gland (13.1 pg/mg). The mean ratio of TRH-Gly/TRH concentrations was less than 1 in neural tissues and pancreas. The lowest ratio (0.04) was observed in hypothalamus, and the highest ratio (66) in prostate gland. Assuming that tissue TRH-Gly levels reflect TRH synthesis, these results suggest that 1) the processing of TRH-Gly to TRH varies among tissues, 2) TRH-Gly to TRH conversion occurs most efficiently in neural tissues, and 3) TRHGly to TRH conversion may be a rate-limiting step in TRH biosynthesis. (Endocrinology 127: 2501-2505, 1990)
Materials and Methods Subject and sampling Ten-week-old Wistar rats (220-320 g) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). A total of 10 animals (5 males and 5 females) were used for the study. Animals were housed individually in an air-conditioned environment (21 C; 55% humidity), with rat chow and water available ad libitum. Lights were on from 0600-1800 h. After 1 week of acclimation, rats were killed by decapitation between 09001200 h. Organs were immediately excised, blotted to remove excess blood, weighed, and placed in chilled 1 M acetic acid (20%; wt/vol; minimum, 1 ml). The whole brain was cut into cerebellum, hypothalamic region, and cerebellum with brain stem. The hypothalamic fragment was delimited in the sagittal plane by the posterior margin of the optic chiasm and the anterior portion of the mammilary body; the lateral margins were the hypothalamic sulci. The dorsal extent of the cut was 2 mm. Contents of digestive organs were washed out with cold distilled water after dissection. Extraction of TRH-Gly and TRH from tissues To inactivate tissue TRH-degrading enzymes, samples were immediately heated at 97 C for 15 min and then homogenized using a Polytron tissue grinder (Brinkmann Instruments, Inc., Westbury, NY). The homogenates were dried completely using a SVC200H Speed-Vac concentrator (Savant Instruments, Hicksville, NY). Dried residues were rehomogenized in 2-3 ml 100% methanol and centrifuged at 2500 X g for 20 min. Meth2501
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 04:39 For personal use only. No other uses without permission. . All rights reserved.
DISTRIBUTION OF TRH AND TRH-GLY IN RATS
2502
anol extraction was repeated, and two aliquots were pooled, dried, and stored at -20 C for later analysis. Trunk blood, collected in precooled glass tubes, was immediately placed on ice for 1 h, then centrifuged at 2500 X g for 20 min. One milliliter of serum with 0.1 ml 2 N acetic acid was applied to a Sep-Pak C18 cartridge (Waters Associates, Inc., Milford, MA) and eluted with 4 ml of an elution solvent (containing 0.8 ml 20 mm triethylamine, pH 4.0, and 3.2 ml 100% methanol). The sample was dried and stored at —20 C.
E n d o • 1990 Vol 127 • No 5
across-assay coefficients of variation were 3.4% (range, 3.75.9%) and 11.8% (range 5.7-15.4%), respectively. TRH-Gly and TRH immunoreactivities were determined in the same tissue extracts. Peptide recoveries from tissues were greater than 85% for TRH and 98% for TRH-Gly, as determined by adding exogenous synthetic peptides to samples. No correction was made for extraction. Synthetic TRH-Gly and TRH were obtained from Peninsula Laboratories, Inc. (San Carlos, CA). Goat antirabbit 7-globulin was purchased from Antibodies, Inc. (Davis, CA).
TRH-Gly RIA HPLC analysis
[125I]TRH-Gly was prepared as described by Butler et al. (8). TRH-Gly antiserum was obtained from rabbits after immunization with synthetic TRH-Gly conjugated with BSA according to Lombardi et al. (9). This antiserum to TRH-Gly showed no significant cross-reaction with TRH or TRH-related peptides (Table 1). The dried tissue or serum extracts were reconstituted with 0.5-2.0 ml assay buffer and centrifuged at 2000 X g for 20 min. The supernatants were used for assay. The incubation volume was 300 jul in a 10 x 75-mm plastic tube. Each assay tube contained 50-100 n\ sample, 50 ^1 TRH-Gly antiserum, 50 nl [125I]TRH-Gly (7000 cpm), and 0.01 M phosphate saline buffer, pH 7.4, containing 0.1% BSA, 0.01% EDTA and 0.01% polyoxyethylenesorbitan monolaurate (Tween-20). The TRH-Gly antiserum was used in a final dilution of 1:1000-3000. All samples were assayed in duplicate. Tubes were incubated at 4 C for 3 days. Separation of bound from free label was accomplished using goat antirabbit immunoglobulin G and normal rabbit serum. Radioactivity of each tube was measured using an Isoflex Automatic Gamma Counter (ICN Micromedic Systems, Inc., Huntsville, AL). The least detectable concentration of ligand in the assay system averaged 11 pg/assay tube. The average within-assay coefficient of variation was 4.5% (range, 2.2-8.7%); the value across assays was 10.3 (range, 8.4-14.9%).
The HPLC analysis was performed on an IBM 9533 liquid chromatograph with a Scientific Systems, Inc. (State College, PA) variable wavelength detector. Solvent A was 0.1% trifluoroacetic acid (Pierce Chemicals, Rockford, IL), and solvent B was 60% acetonitrile. All analyses were performed at a flow rate of 1 ml/min, with a linear gradient of 5-65% solvent B over 30 min. Initial separation of synthetic TRH and TRHGly standards was effected on a Analytichem International Sepralyte 4.6-mm X 25-cm C-28 reverse phase column, with postcolumn detection at 214 nM. Analysis of plasma samples was performed on a Vydac 4.6mm X 25-cm protein C-18 column. Plasma samples (1 ml) were extracted on Sep-Pak columns as described and dissolved in 0.1% trifluoroacetic acid, and the extracts from four animals combined. The combined extracts were filtered (0.22 jiM type Gs filters; Milipore Corp., Bedford, MA), and 200 ^1 were injected onto the column. One-minute samples of the eluate were collected with a fraction collector. Subsequently, 200 fA of a combined standard containing synthetic TRH (1 ng/ml) and TRH-Gly (2.5 ng/ml) was injected, and fractions were collected. Plasma and combined standard eluate fractions were dried and dissolved in assay buffer, and the TRH and TRH-Gly immunoreactivities were assessed in each fraction.
TRH RIA
Statistical analysis
The procedures for generating TRH antiserum and labeling TRH with 125I, and the TRH RIA system were identical to those described above for the TRH-Gly RIA except for substitution of TRH standards, [125I]TRH, and rabbit anti-TRH antibody (1:80,000-100,000 final concentration). The crossreactivities of TRH-Gly and TRH-COOH in this TRH assay system were less than 0.01%. The average detection limit for the assay was 7 pg/assay tube. The average within-assay and
Results were analyzed using one-way analysis of variance and expressed as picograms per mg wet tissue wt or picograms per ml serum. When samples contained undetectable ligand concentrations, the value for the sensitivity threshold of the assay was used to calculate the mean values. P < 0.05 was used as the limit of significance.
1. Relative reactivity of TRH-Gly antiserum with TRH and related peptides
HPLC analysis
TABLE
Materials TRH-Gly TRH(pGlu-His-Pro-NH2) pGlu-His-Pro-Gly-NH2 pGlu-His-Pro-Gly-Lys-Arg Arg-Glu-His-Pro-Gly Cyclo His-Pro The final dilution was 1:1500.
Cross-reactivity 100 10) were prostate, spleen, kidney (mean ratios, 66, 34, and 14, respectively), and duodenum (mean ratio, 16). In other tissues, mean TRH-Gly/TRH ratios ranged from 1.6-8.2. These included stomach, small gut, large gut, liver, adrenal, testis, ovary, and serum. Discussion TRH immunoreactivity has been described in a variety of extraneural tissues and biological fluids of the rat, including pancreas, gastrointestinal tissues (10), kidney, liver, lung (11, 12), adrenal (13), male reproductive organs (14), blood (15), and urine (16). In addition to confirming the presence of TRH in these tissues, we detected TRH immunoreactivity in ovary and spleen. In agreement with earlier reports we observed that TRH concentrations were low in all extraneural tissues (
Vol. 127, No. 5 Printed in U.S.A.
Distribution of Thyrotropin-Releasing Hormone (TRH) and Precursor Pep tide (TRH-Gly) in Adult Rat Tissues YOZEN FUSE, DANIEL H. POLK, ROBERT W. LAM, AND DELBERT A. FISHER Department of Pediatrics, University of California-Los Angeles, School of Medicine, Harbor-UCLA Medical Center, Torrance, California 90509; and King-Drew Medical Center, Los Angeles, California 90059
ABSTRACT. TRH (pGlu-His-Pro-NH2) arises from the posttranslational processing of a larger precursor peptide containing multiple copies of the TRH progenitor sequence, Gln-His-ProGly. Concentrations of TRH and its precursor peptide (TRHGly) were determined in serum and a variety of tissues of the rat using specific RIA systems. TRH and TRH-Gly immunoreactivities were detectable in almost all tissues studied. TRH was distributed mainly in neural tissues, with the highest mean concentration (126 pg/mg tissue) in hypothalamus. In extraneural tissues, mean TRH levels ranged from 0.6-4.8 pg/mg tissue; the mean serum concentration was 12.4 pg/ml. In contrast to the distribution of TRH, relatively higher mean TRH-Gly concentrations were observed in serum (76.5 pg/ml) and in
T
RH (L-pyroglutamyl-L-histidyl-L-prolinamide, pGlu-His-Pro-NH2) arises from the posttranslational cleavage of a large precursor molecule (1). Rat hypothalamic pro-TRH has a mol wt of approximately 29,000 and contains five progenitor sequences flanked by paired basic amino acid residues (2). In the formation of TRH, the sequence -Lys-Arg-Gln-His-Pro-Gly- is cleaved from the prohormone by trypsin-like enzymes at paired basic amino acid residues, followed by cyclization of the N-terminal glutamine (3) and amidation of the proline using the C-terminal glycine as the nitrogen donor (4). In this process, the glycine-extended form of TRH (TRH-Gly) is the immediate precursor peptide for TRH. TRH is distributed in extrahypothalamic brain regions and in extraneural tissues, including pancreas and gastrointestinal tissues (5). TRH-Gly has been reported in high concentration in rat prostate (6). However, there are limited data regarding the distribution of TRHGly and TRH in adult rat tissues (7). In the present study we measured the concentrations of both TRH-Gly and TRH in a wide variety of tissues using specific RIA systems. Received March 19, 1990. Address requests for reprints to: Daniel H. Polk, M.D., Martin Research Center, Harbor-University of California-Los Angeles Medical Center, 1000 West Carson Street, Torrance, California 90509.
extraneural tissues, including prostate (83.3 pg/mg tissue), spleen (19.0 pg/mg), adrenal (16.2 pg/mg), kidney (13.3 pg/mg), and gastrointestinal tract (6.3-19.8 pg/mg). Among brain tissues, the TRH-Gly concentration was highest in pituitary gland (13.1 pg/mg). The mean ratio of TRH-Gly/TRH concentrations was less than 1 in neural tissues and pancreas. The lowest ratio (0.04) was observed in hypothalamus, and the highest ratio (66) in prostate gland. Assuming that tissue TRH-Gly levels reflect TRH synthesis, these results suggest that 1) the processing of TRH-Gly to TRH varies among tissues, 2) TRH-Gly to TRH conversion occurs most efficiently in neural tissues, and 3) TRHGly to TRH conversion may be a rate-limiting step in TRH biosynthesis. (Endocrinology 127: 2501-2505, 1990)
Materials and Methods Subject and sampling Ten-week-old Wistar rats (220-320 g) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). A total of 10 animals (5 males and 5 females) were used for the study. Animals were housed individually in an air-conditioned environment (21 C; 55% humidity), with rat chow and water available ad libitum. Lights were on from 0600-1800 h. After 1 week of acclimation, rats were killed by decapitation between 09001200 h. Organs were immediately excised, blotted to remove excess blood, weighed, and placed in chilled 1 M acetic acid (20%; wt/vol; minimum, 1 ml). The whole brain was cut into cerebellum, hypothalamic region, and cerebellum with brain stem. The hypothalamic fragment was delimited in the sagittal plane by the posterior margin of the optic chiasm and the anterior portion of the mammilary body; the lateral margins were the hypothalamic sulci. The dorsal extent of the cut was 2 mm. Contents of digestive organs were washed out with cold distilled water after dissection. Extraction of TRH-Gly and TRH from tissues To inactivate tissue TRH-degrading enzymes, samples were immediately heated at 97 C for 15 min and then homogenized using a Polytron tissue grinder (Brinkmann Instruments, Inc., Westbury, NY). The homogenates were dried completely using a SVC200H Speed-Vac concentrator (Savant Instruments, Hicksville, NY). Dried residues were rehomogenized in 2-3 ml 100% methanol and centrifuged at 2500 X g for 20 min. Meth2501
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 04:39 For personal use only. No other uses without permission. . All rights reserved.
DISTRIBUTION OF TRH AND TRH-GLY IN RATS
2502
anol extraction was repeated, and two aliquots were pooled, dried, and stored at -20 C for later analysis. Trunk blood, collected in precooled glass tubes, was immediately placed on ice for 1 h, then centrifuged at 2500 X g for 20 min. One milliliter of serum with 0.1 ml 2 N acetic acid was applied to a Sep-Pak C18 cartridge (Waters Associates, Inc., Milford, MA) and eluted with 4 ml of an elution solvent (containing 0.8 ml 20 mm triethylamine, pH 4.0, and 3.2 ml 100% methanol). The sample was dried and stored at —20 C.
E n d o • 1990 Vol 127 • No 5
across-assay coefficients of variation were 3.4% (range, 3.75.9%) and 11.8% (range 5.7-15.4%), respectively. TRH-Gly and TRH immunoreactivities were determined in the same tissue extracts. Peptide recoveries from tissues were greater than 85% for TRH and 98% for TRH-Gly, as determined by adding exogenous synthetic peptides to samples. No correction was made for extraction. Synthetic TRH-Gly and TRH were obtained from Peninsula Laboratories, Inc. (San Carlos, CA). Goat antirabbit 7-globulin was purchased from Antibodies, Inc. (Davis, CA).
TRH-Gly RIA HPLC analysis
[125I]TRH-Gly was prepared as described by Butler et al. (8). TRH-Gly antiserum was obtained from rabbits after immunization with synthetic TRH-Gly conjugated with BSA according to Lombardi et al. (9). This antiserum to TRH-Gly showed no significant cross-reaction with TRH or TRH-related peptides (Table 1). The dried tissue or serum extracts were reconstituted with 0.5-2.0 ml assay buffer and centrifuged at 2000 X g for 20 min. The supernatants were used for assay. The incubation volume was 300 jul in a 10 x 75-mm plastic tube. Each assay tube contained 50-100 n\ sample, 50 ^1 TRH-Gly antiserum, 50 nl [125I]TRH-Gly (7000 cpm), and 0.01 M phosphate saline buffer, pH 7.4, containing 0.1% BSA, 0.01% EDTA and 0.01% polyoxyethylenesorbitan monolaurate (Tween-20). The TRH-Gly antiserum was used in a final dilution of 1:1000-3000. All samples were assayed in duplicate. Tubes were incubated at 4 C for 3 days. Separation of bound from free label was accomplished using goat antirabbit immunoglobulin G and normal rabbit serum. Radioactivity of each tube was measured using an Isoflex Automatic Gamma Counter (ICN Micromedic Systems, Inc., Huntsville, AL). The least detectable concentration of ligand in the assay system averaged 11 pg/assay tube. The average within-assay coefficient of variation was 4.5% (range, 2.2-8.7%); the value across assays was 10.3 (range, 8.4-14.9%).
The HPLC analysis was performed on an IBM 9533 liquid chromatograph with a Scientific Systems, Inc. (State College, PA) variable wavelength detector. Solvent A was 0.1% trifluoroacetic acid (Pierce Chemicals, Rockford, IL), and solvent B was 60% acetonitrile. All analyses were performed at a flow rate of 1 ml/min, with a linear gradient of 5-65% solvent B over 30 min. Initial separation of synthetic TRH and TRHGly standards was effected on a Analytichem International Sepralyte 4.6-mm X 25-cm C-28 reverse phase column, with postcolumn detection at 214 nM. Analysis of plasma samples was performed on a Vydac 4.6mm X 25-cm protein C-18 column. Plasma samples (1 ml) were extracted on Sep-Pak columns as described and dissolved in 0.1% trifluoroacetic acid, and the extracts from four animals combined. The combined extracts were filtered (0.22 jiM type Gs filters; Milipore Corp., Bedford, MA), and 200 ^1 were injected onto the column. One-minute samples of the eluate were collected with a fraction collector. Subsequently, 200 fA of a combined standard containing synthetic TRH (1 ng/ml) and TRH-Gly (2.5 ng/ml) was injected, and fractions were collected. Plasma and combined standard eluate fractions were dried and dissolved in assay buffer, and the TRH and TRH-Gly immunoreactivities were assessed in each fraction.
TRH RIA
Statistical analysis
The procedures for generating TRH antiserum and labeling TRH with 125I, and the TRH RIA system were identical to those described above for the TRH-Gly RIA except for substitution of TRH standards, [125I]TRH, and rabbit anti-TRH antibody (1:80,000-100,000 final concentration). The crossreactivities of TRH-Gly and TRH-COOH in this TRH assay system were less than 0.01%. The average detection limit for the assay was 7 pg/assay tube. The average within-assay and
Results were analyzed using one-way analysis of variance and expressed as picograms per mg wet tissue wt or picograms per ml serum. When samples contained undetectable ligand concentrations, the value for the sensitivity threshold of the assay was used to calculate the mean values. P < 0.05 was used as the limit of significance.
1. Relative reactivity of TRH-Gly antiserum with TRH and related peptides
HPLC analysis
TABLE
Materials TRH-Gly TRH(pGlu-His-Pro-NH2) pGlu-His-Pro-Gly-NH2 pGlu-His-Pro-Gly-Lys-Arg Arg-Glu-His-Pro-Gly Cyclo His-Pro The final dilution was 1:1500.
Cross-reactivity 100 10) were prostate, spleen, kidney (mean ratios, 66, 34, and 14, respectively), and duodenum (mean ratio, 16). In other tissues, mean TRH-Gly/TRH ratios ranged from 1.6-8.2. These included stomach, small gut, large gut, liver, adrenal, testis, ovary, and serum. Discussion TRH immunoreactivity has been described in a variety of extraneural tissues and biological fluids of the rat, including pancreas, gastrointestinal tissues (10), kidney, liver, lung (11, 12), adrenal (13), male reproductive organs (14), blood (15), and urine (16). In addition to confirming the presence of TRH in these tissues, we detected TRH immunoreactivity in ovary and spleen. In agreement with earlier reports we observed that TRH concentrations were low in all extraneural tissues (
