3/1996

The Effect of Androgens on the Concentration of TRH-Like Peptides in Rat Prostate

R. Bílek, P. J. Gkonos*, M. Soutorová, L. Stárka, D. G. Smyth**

Institute of Endocrinology, Národni 8, 116 94 Prague 1, Czech Republic
* University of Miami Schoo/ of Medicine, Department of Medicine, Miami, USA
** Institute of Molecular Biology, Salzburg, Austria

 

Summary

TRH-like peptides are substances with immunochemical similarity to thyrotropin-releasing hormone (TRH), from which they differ in that the central basic aminoacid HIS, characteristic for thyroliberin, is exchanged in their molecules for the acidic aminoacid GLU or for neutral PHE, GLN or VAL. Pyroglutamyl-glutamyl-prolinamide (pEEPam) is the dominant TRH-like peptide in rat prostates; an attempt was made in the experiment to determine whether its concentration in this tissue is dependent on the steroid state of the animals. In the testosterone experiments, rats were divided to three groups. The first one was used as control, rats in second group were castrated, rats in third group were castrated and one week after castration they were next one week treated with testosterone cypionate (0.2 mg/day). In the dexamethasone experiments, saline (0. l 5 ml) was administered intraperitoneally daily for a period of 4 days to control rats and 0.15 ml of saline containing 0.2 mg dexamethasone was administered to other rats under the same conditions. After separation of the prostate extracts on ion- exchange minicolumn with subsequent analysis on a reverse-phase highperformance liquid chromatography the content of TRH immunoreactive peptides was determined using TRH specific radioimmunoassay.

 The concentration of TRH-like peptides rapidly increased in the prostates of castrated rats; this was especially apparent for pEEPam, whose concentration increased approximately 6x. Administration of testosterone decreased TRH immunoreactivity in the prostate, including pEEPam, to almost the same value as found for the control animals. In comparison with the control animals, there was no significant change in the TRH immunoreactivity in rats to which dexamethasone was administered and the effect of dexamethasone on the pEEPam concentration in rat prostate was not confirmed in this experiment.

Introduction

The occurrence of TRH (thyrotropinreleasing hormone) immunoreactivity has been described in a wide range of human and animal tissues [1,2,3,4,5,6] and this fact has led to the necessity of discovering more facts on the nature of substances that can crossreact with a TRH-specific antibody. The basic question from point of view of TRH expression in peripheral tissues of the organism is - whether this is a truly TRH or whether immunochemically similar TRH-like peptides occur there. The TRH-like peptides known at the present time differ from thyroliberin in that the central basic aminoacid histidine (HIS) characteristic for TRH is exchanged for glutamic acid [7], or neutral glutamine, phenylalanine [8, 10], or for a peptide whose retention position is very similar to tritium-labelled pyroglutamylvalinyl-prolinamide (pEVPam). The development of chromatographic methods has permitted good separation of neutral and acidic TRH-like peptides from basic authentic TRH and the use of TRH-specific radioimmunoassay is sufficiently sensitive (units of fmol) for quantification of TRH immunoreactivity in the individual fractíons collected after the chromatographic separation. Previous studies have shown [e.g 7,9, 11 ] that most of the TRH immunoreactivity in the prostate is concentrated in pyroglutamyl-glutamylprolinamide (pEEPam), whose primary structure (similar to the structures of the other TRH-like peptides) is not contained in the TRH-coding gene.

The growth and maintenance of the prostate secretory epithelium is highly dependent on the concentration of the circulating androgens, especially dihydrotestosterone and its biologically active precursor, testosterone. From this standpoint, the work describes the change in the prostatic TRH and pEEPam when the concentration of circulating androgens is altered in some signifcant manner, e. g. through castration, and whether this change is reversible over a certain period of time, i.e. whether the administration of exogenic testosterone eliminates the changes caused by a defciency of endogenous androgens.

 Glucocorticoids generally have catabolic role in living organisms. It has been stated in some earlier studies that dexamethasone increases the expression of TRH-coding gene in a cell culture of pituitary cells [12], in the hypothalamus [13] and also has an impact on the expression of TRH-specific receptors, whose number was tripled for GH3 cells when dexamethasone was added to a nutrient medium [14]. On the other hand, experiments have also been published in which dexamethasone tended rather to inhibit the biosynthesis of TRH. For example, the administration of dexamethasone decreased the formation of the direct precursor of TRH, i. e. glutamyl-histidyl- prolyl-glycinein rat hypothalamus [15]. It is possible that the effect of dexamethasone on the TRH or TRH-like peptides is tissue-dependent. This work describes the consequences of administration of dexamethasone for the concentration of TRH-immunoreactive peptides in rat prostate glands.

Material and methods

Experimental Animals

The experiment investigating the effect of androgens on the concentration of TRH immunoreactive peptides used 2 rats as control animals, 2 rats were castrated, and a further 2 rats were castrated and then were treated one week after castration for a period of one week by subcutaneous administration of 0.2 mg of testosterone cypionate. The dexamethasone experiment was carried out using 2 rats as control animals, to whom 0.15 ml of physiological solution was administered intraperitoneally for 4 days; 2 further rats were treated daily in the same manner with a physiological solution containing 0.2 mg of dexamethasone. After the end of the experiment the animals were killed, the prostate was immediately removed and was stored until further examination at a temperature of -80±C. All the rats used in the experiments were 3-month-old males of the Wistar species.

Analysis of TRH immunoreactive peptides in rat prostates

The prostates were homogenized in pairs for a period of 2 x 1 minute in 1 mol/1 acetic acid using an Ultra Turrax T25 homogenizer (Janke-Kunkel gmbH, FRG) at 20 000 rpm. To the homogenization mixture was added approx. 2000 cpm [125I]-TRH prepared by a laboratory procedure using Nal2SI (37 MBq, Amersham Int., UK), synthetic TRH (Novabiochem Ltd., UK) and chloramine T (Merck, FRG). After leaving to stand for 10 minutes at laboratory temperature, the homogenization mixture was placed in a boiling water bath for 10 minutes and, after cooling, was centrifuged at 4±C and 20 000 rpm for a period of 30 minutes. The supernatant was evaporated to dryness on a rotation vacuum evaporator, the vaporization residue was extracted with methanol and the methanol extract was again evaporated on a rotation vacuum evaporator. This was followed by dissolving of the residue in 1.5 ml of 50% acetic acid, measuring of the radioactivity of [125I]-TRH and subsequent calculation of the effciency of the isolation procedure. The separation of the dissolved residue was performed on a 1 x 6 cm SP Sephadex C25 minicolumn by step-wise elution with 8 x 0.5 ml of acetic acid (the unretained fractions of TRH-like peptides were eluted) and 12 x 0.5 ml of 0.4 mol/l pyridine in 50% acetic acid. The position of the [125I]-TRH marker was measured in the eluted fractions and the immunoreactivity of TRH-specific radioimmunoassay was determined in 50 µl aliquots.

 The unretained TRH immunoreactive fractions containing acidic to neutral TRH-like peptides and the retained fractions containing basic TRH were evaporated separately on a vacuum centrifuge (Jouan VX 100, France); the residue was dissolved in 0.9 ml of 10 mmol/l hydrochloric acid and applied to a 4 x 300 mm steel analytical reverse-phase high-performance chromatographic column (uBondapak C 18, particle diameter 10 µm, Millipore Waters, UK). The column was eluted by the linear gradient method using a mixture of 10 mmol/l hydrochloric acid and methanol (during the first 6 minutes, the concentration of methanol in the elution mixture increased by 0.5%/min; over the next 24 minutes this increase equalled 2%/min.). The mobile phase had a flow rate of 1.5 ml/min. Fractions were collected every 0.3 minutes, then they were evaporated on a vacuum centrifuge and the TRH immunoreactivity was determined by TRH- specific radioimmunoassay.

 Radioimmunoanalytical determination of TRH immunoreactive peptides was carried out by a laboratory procedure using sheep antiserum specific for TRH at a final dilution of 1:50 000, obtained from Dr. H. M. Fraser, Edinburgh University, UK. Compared with TRH, the cross reactions of pEEPam with respect to this antibody were about 50%. An amount of 200 µl of 0.05 mol/1 pH 7.6 phosphate buffer with 0.25% (w/v) bovine serum albumin, 50 µl of a dilute solution of the specific antibody and 50 µl of solution of radioligand with radioactivity of approximately 20 000 cpm were added to test tubes containing the residue from the HPLC separation of TRH immunoreactive peptides. An amount of 100 µl of heat-deactivated horse serum and 500 ul of a 20% solution of PEG 6000 were added to the mixture following incubation overnight at 4°C. The mixture was stirred, centrifuged (20 min. at 4000 rpm and 4°C) and the radioactivity was measured after withdrawal of the supernatant. The concentration of TRH immunoreactive peptides in the individual collected fractions was assessed on the basis of calibration curves using a standard solution of synthetic TRH.

Results

TRH-Like Peptides in Rat Prostates Testosterone Experiment

The level of TRH-like peptides in the prostates of control rats, rats following castration and rats following castration treated with testosterone was measured in this experiment. Fig 1 depicts the elution profile of TRH immunoreactive peptides in the extract of homogenized rat prostates after separation of TRH immunoreactivity by cation exchange chromatography on SP Sephadex C25 minicolumn.

A sharp, approximately six-fold increase in TRH immunoreactivity in castrated rats can be seen in the figure; after administration of testosterone, this value decreased almost to the normal value compared to the control experiments. Unretained fractions 1-8 eluted with 50% acetic acid, containing TRH-like peptides, and retained fractions 9-20 eluted with a mixture of 0.4 mol/1 pyridine in 50% acetic acid, containing basic TRH, were evaporated on a vacuum centrifuge. After dissolving the residue in 3 x 300 µl of 10 mmol/l HCl, the mixture was separated by RP HPLC and TRH immunoreactive peptides were quantified by TRH-specific RIA. The results are given in Fig. 3, from which it can be seen that the increase in the concentration of TRH-like peptides in unretained fractions 1-8 (see Fig. 1) is concentrated in an increase in the pEEPam peak, while these changes were not signifcant in retained fractions 9-20 (see Fig. 1). If the total TRH immunoreactivity after separation of TRH immunoreactive peptides on an SP Sephadex C25 column (Fig. 1) is expressed numerically as the TRH concentration in fmol/g of prostate tissue, then this concentration equals 1620 fmol/g for the control animals, increases to 7930 fmol/g for castrated animals and again decreases to 2330 fmol/g for castrated animals treated with testosterone one week after castration. In each run a blank was carried out together with the actual analysis, along with RP HPLC analysis of synthetic standards of TRH- like eptides, described in Fig. 2

View figure 1 (24 k)
View figure 2 (18 k)

TRH-Like Peptides in Rat Prostates Dexamethasone Experiment

The elution profiles of TRH immunoreactive peptides from the extract of homogenized rat prostate separated on a SP Sephadex C25 column is depicted in Fig. 4, from which it can be seen that, compared to the control animals, nonsignificant changes occurred in the concentration of TRH immunoreactivity in rats treated with dexamethasone. RP HPLC analysis of the retained (fractions 10-14, see Fig. 4) and unretained (fractions 2-6, see Fig. 4) TRH immunoreactive fractions from cation-exchange chromatography is described in Fig. 6 and once again it can be seen that there was no substantial change in the TRH immunoreactivity, especially when compared with the testosterone experiment (Fig. 3). Fig. 5 represents the separation of synthetic standards of TRH-like peptides carried out in parallel under the same conditions.

The dexamethasone experiment was controlled by determining corticosterone, total T3 and total T4 in the blood of the animals after completion of the experiment. The levels of these hormones in the serum varied for the control animals between 150 and 400 ng/ml (corticosterone), 60 to 70 ng/ 100 ml (T4) and 4 to 6 ng/ 100 ml (T3). The administration of dexamethasone led to a substantial change in the concentrations of T3 and T4, but the corticosterone level decreased to below the detection limit (<17 ng/ml). For the control animals, the total concentration of TRH immunoreactive peptides after separation on the SP Sephadex C25 column had a value of 620 fmol TRH/g of prostate. This value equalled 950 fmol TRH/g prostate for animals treated with dexamethasone (see Fig 4). RP HPLC separation of the TRH synthetic standards (Fig. 5) and immunoreactive fractions (Fig. 6) yielded the elution profile of TRH immunoreactive peptides, from which it can be seen that there was no substantial change either in the overall TRH immunoreactivity nor a substantial change in the concentration of pEEPam or pEVPam, in contrast to the testosterone experiments.

View figure 3 (43 k)
View figure 4 (19 k)
View figure 5 (16 k)
View figure 6 (41 k)

Discussion

It was found in this experiment that the main TRH-like peptide in rat prostate is pEEPam. This finding is in agreement with the formerly published study of the concentration of TRH-like peptides in rat prostate in dependence on the thyroid state of the experimental animals (9), in which authentic TRH was not found in the rat prostate. The concentration of TRH-like peptides in rat prostate was hormonally affected by androgens. The effect of androgens in prostatic tissue is signifcant. The decrease of testosterone after castration to nearly zero (the production of androgens in the testes is substituted to a certain degree by biosynthesis of androgens in the adrenal cortex) leads over a very few days to elimination of the prostatic epithelium and to substantial reduction in the prostate mass. Insuffcient androgens were manifested in this experiment by a massive increase in the TRH-specific immunoreactivity. Analysis of the unretained fractions by RP HPLC (see Fig. 3) demonstrated that this change in the level of TRH immunoreactive peptides in rat prostate is concentrated primarily in the pEEPam peak. The content of pEEPam in units of fmol TRH/g prostate is approximately 6 times greater for castrated animals than for control animals. The pEEPam value for castrated animals treated with testosterone decreased to the value found for the control animals. It is interesting that identical behaviour was exhibited by another TRH immunoreactive peptide, whose retention position (fractions 62-67, Part A, Fig. 3) is identical with synthetic [3H]-pEVPam. The analysis of TRH immunoreactivity in rats treated with dexamethasone was the same as in control. Results from dexamethasone experiment indicated that dexamethasone has not the important influence on prostatic TRH immunoreactivity.

In concluding, it can be stated that the presence of authentic TRH in rat prostate was not demonstrated in this experiment; in addition, the level of TRH- like peptides in rat prostate is as for the prostate alone affected by the level of circulating androgens in the sense that a deficiency increases the production of pEEPam and other TRH immunoreactive peptides with the same retention as pEVPam. In contrast, replacement of deficient endogenic testosterone by exogenic testosterone led to a decrease in the concentration of TRH immunoreactive peptides in the rat prostate.

Acknowledgements

We are grateful to Dr. H Fraser of Edinburgh University, Great Britain, for the provision of TRH specific antibody. The work was financed by funds from IGA R (Grant No. 0849-i), GA R (Grant No. 0994) and Wellcome Trust (UK).

Contact address:

 Ing. R. Bílek, CSc.
Institute of Endocrinology
Národní 8
116 94 Praha 1

 

References

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