ARTICLE |
Correspondence to: Marc Bulant, Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, 2 place Jussieu, 75251 Paris Cedex 05, France.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pro-thyrotropin-releasing hormone (pro-TRH) has been shown to be present throughout the central nervous system and in several peripheral tissues. In adrenals, TRH immunoreactivity has been reported but not characterized. We show here that two rat pro-TRH-derived peptides, TRH and prepro-TRH[160-169] (Ps4), were detected in extracts of rat adrenal glands by enzyme immunoassay. Endogenous TRH and Ps4 were purified by gel exclusion chromatography and reverse-phase HPLC. Structural identification of each peptide was achieved by chromatographic comparison with synthetic standards. By using the indirect immunofluorescence technique, TRH-immunoreactive cell bodies were found rather widely scattered outside the adrenal, in the brown adipose tissue in which the gland is embedded. These immunofluorescent cells have the typical appearance of mast cells and are metachromatic after histological staining with acidic Toluidine Blue. Our findings suggest that pro-TRH-derived peptides exist in rat mast cells. (J Histochem Cytochem 45:1623-1627, 1997)
Key Words: neuropeptide, mast cells, immunocytochemistry, HPLC
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thyrotropin-releasing hormone (TRH), originally isolated from hypothalamus, is a potent stimulator of thyroid-stimulating hormone (TSH) release after binding to its high-affinity receptor on the thyrotrope cells. TRH has since been identified throughout the central nervous system, including the retina and the spinal cord (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enzyme Immunoassay (EIA) Procedures
TRH EIA was performed as previously described (
Chromatographic Separation
Sixteen adult male Sprague-Dawley rats (Dépré; Saint Doulchard, France) weighing 300 g were sacrificed by decapitation and their whole adrenal glands were immediately removed. Tissues were immersed in 10% acetic acid at 95C for 10 min and then homogenized and extracted at 4C using a polytron homogeneizer. The resulting suspension was centrifuged at 3000 x g at 4C for 30 min and lyophilized. Crude adrenal extracts were first filtered through a Sephadex G-50 (fine) column at a flow rate of 8 ml/hr. For HPLC studies, the prepurified sample was dissolved in 200 µl of water plus 0.1% trifluoroacetic acid (TFA) and applied to 5-µm Lichrospher C-18 reverse-phase column (3.9 x 250 mm; Interchrom, Asnières, France) equilibrated with the same solvent at a flow rate of 0.75 ml/min. Three min after injection the bound material was eluted with a linear gradient (1%/3 min) of acetonitrile (0.07% TFA) in 0.1% TFA/water. Fractions of 750 µl were collected and lyophilized. Aliquots of each fraction were reconstituted in EIA buffer and assayed for TRH or Ps4 immunoreactivity.
Histology and Immunocytochemical Procedure
Two rats were perfused intracardiacally with heparinized saline (100 ml) and then with an infusion of 200 ml of McLean's fixative containing 2.5 mM sodium m-periodate, 18.7 mM L-lysine monohydrochloride, 0.1 M phosphate buffer (PB), pH 7.4, and 4% paraformaldehyde. The adrenal glands were removed and postfixed for 4 hr at 4C in the same fixative, washed in successive sucrose baths (5%, 15%, and 25%), embedded in Tissue Tek, frozen in liquid nitrogen, and cut on a cryostat at 8 µm. Sections were mounted on chromalum/gelatin-coated slides, air-dried, and then processed according to the indirect immunofluorescence method. Sections were rinsed in PB and incubated in 5% normal goat serum in 0.3% Triton X-100/PB for 1 hr at 25C. Visualization of the location for immobilized antigen was made using an incubation step with anti-TRH immunoglobulins (Igs F18, 1:400, 47 µg/ml, 12 hr at 4C) and a second incubation with a goat anti-rabbit immunoglobulin-fluorescein isothiocyanate (Sigma; St Louis, MO) (1:160, 1 hr at 25C) in PB containing 1% normal goat serum and 0.3% Triton X-100. The immunoglobulins F18 were purified from rabbit anti-TRH antiserum (4B18) by precipitation with 18% Na2SO4. For control, anti-TRH Igs were preabsorbed with TRH fixed on Sepharose beads as previously described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To detect the presence of peptides derived from pro-TRH in rat adrenal glands, tissue extract was subjected to size fractionation using Sephadex G-50 and reverse-phase HPLC analysis. Fractions were analyzed by EIA with two antisera directed against pro-TRH-derived products. By use of a TRH antiserum, a sharp immunoreactive peak was detected that emerged from the total volume of the column (Figure 1A). Application of the Ps4 EIA in the same procedure demonstrated the presence of immunoreactive material (Figure 1B). The predominant product co-eluted with synthetic Ps4 during gel filtration. To characterize more precisely the major pro-TRH-related products from adrenal glands, immunoreactive fractions 57-60 corresponding to the most immunoreactive fractions for either TRH or Ps4 EIA during gel filtration were pooled and subjected to reverse-phase HPLC on a 5-µm Lichrospher OD2 column with a very slow gradient of acetonitrile in water plus 0.1% TFA (Figure 1C). Under these conditions, the putative endogenous TRH from G-50 was fractionated into a major immunoreactive peak corresponding to TRH, which was accompanied by a minor trailing shoulder and two additional species. Aliquots of the fractions depicted in Figure 1C were also assayed for Ps4 immunoreactivity. Authentic Ps4 was recovered as a predominant immunoreactive species eluting in the same position as synthetic Ps4. Two additional and uncharacterized immunoreactive peaks were also detected in significant amounts.
|
Immunocytochemical localization of adrenal TRH-related peptides was examined in rats using specific anti-TRH immunoglobulins. Observation of the sections indicated that both the adrenal cortex and medulla were devoid of TRH immunoreactivity. At the light microscopic level, specific labeling was observed in scattered cells located in the brown adipose tissue (BAT) surrounding the adrenal glands (Figure 2A). These cells contained a distinguishable nucleus and were observed everywhere in BAT (Figure 2B), although most of them were proximal to the adrenal capsule. These cells were positively stained when sections were treated with Toluidine Blue (pH <2.5), which identifies mast cells. To determine if TRH-immunoreactive cells accounted for all of the adrenal mast cells, we first counted all of the fluorescent cells, then counterstained the sections with acidic Toluidine Blue. No additional metachromatic cells were evident within the tissue sections (Figure 2C-E). With an Alcian blue/Safranin mixture, mast cells stained red, indicating that they correspond to typical connective tissue mast cells (data not shown). No fluorescence was observed with TRH-treated anti-TRH immunoglobulins (Figure 2F).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several studies have indicated the presence of TRH in rat adrenal gland (
The level of TRH expressed in the mast cells is probably too low to exert endocrine hormonal effects on distant tissues, but it appears adequate to support local paracrine or autocrine regulatory effects. Interestingly, it has been reported that TRH receptor mRNA is widely distributed in rat peripheral tissues and that a substantial amount of mRNA was observed in adrenal glands (
![]() |
Acknowledgments |
---|
We are grateful to Dr G. Mory for his pertinent comments.
Received for publication March 31, 1997; accepted June 25, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bulant M, Delfour A, Vaudry H., Nicolas P (1988) Processing of thyrotropin-releasing hormone prohormone generates pro-TRH-connecting peptides. J Biol Chem 263:17189-17196
Cockle SM, Smyth DG (1987) Specific processing of the thyrotropin-releasing prohormone in rat brain and spinal cord. Eur J Biochem 165:693-698 [Abstract]
Faivre-Bauman A, Nemeskeri A, Tougard C, Tixier-Vidal A (1980) Immunological evidence for thyroliberin neurons in primary cultures of fetal mouse brain cells. Ontogenic aspects. Brain Res 185:289-304 [Medline]
Feng P, Gu J, Kim UJ, Carnell NE, Wilber JF (1993) Identification, localization and developmental studies of rat prepro-thyrotropin-releasing hormone mRNA in the testis. Neuropeptides 24:63-69 [Medline]
Fukusumi S, Ogi K, Onda H, Hinuma S (1995) Distribution of thyrotropin-releasing hormone receptor mRNA in rat peripheral tissues. Regul Peptides 57:115-121 [Medline]
Fuse Y, Polk DH, Lam RW, Fisher DA (1990) Distribution of thyrotropin-releasing hormone and precursor peptide in adult rat tissues. Endocrinology 127:2501-2505 [Abstract]
Gkonos PJ, Tavianini MA, Liu C-C, Roos BA (1989) Thyrotropin-releasing hormone gene expression in normal thyroid parafollicular cells. Mol Endocrinol 3:2101-2109 [Abstract]
Jackson IMD (1982) Thyrotropin-releasing hormone. N Engl J Med 306:145-155 [Medline]
Lechan RM, Wu P, Jackson IMD, Wolf H, Cooperman S, Mandel G, Goodman RH (1986) Thyrotropin-releasing hormone precursor: characterization in rat brain. Science 231:159-161 [Medline]
Leduque P, Aratan-Spire S, Wolf B, Dubois PM, Czernichow P (1987) Localization of thyrotropin-releasing hormone- and insulin-immunoreactivity in the pancreas of neonatal rats after injection of streptozotocin at birth. Cell Tissue Res 248:89-94 [Medline]
Montagne J-J, Ladram A, Grouselle D, Nicolas P, Bulant M (1996) Identification and cellular localization of thyrotropin-releasing hormone-related peptides in rat testis. Endocrinology 137:185-191 [Abstract]
Morley JE (1981) Neuroendocrine control of thyrotropin secretion. Endocrine Rev 2:396-436 [Medline]
Mory G, Combes-George M, Nechad M (1983) Localization of serotonin and dopamine in the brown adipose tissue of the rat and their variations during cold exposure. Biol Cell 48:159-166 [Medline]
Simard M, Pekary AE, Smith VP, Hershman JM (1989) Thyroid hormones modulate thyrotropin-releasing hormone biosynthesis in tissues outside the hypothalamic-pituitary axis of male rats. Endocrinology 125:524-531 [Abstract]
Wu P, Lechan RM, Jackson IMD (1987) Identification and characterization of thyrotropin-releasing hormone precursor peptides in rat brain. Endocrinology 121:108-115 [Abstract]
Xu LR, Carr MM, Bland AP, Hall GA (1993) Histochemistry and morphology of porcine mast cells. Histochem J 25:516-522 [Medline]