Dihydrotestosterone regulation of thymocyte function in the rat mediation by serum factors.

We have previously reported that reticuloepithelial cells of rat thymic tissue contain specific, high-affinity estrogen (E) and dihydrotestosterone (DHT) receptors which are not present in thymocytes, and that stimulation of the concanavalin A (Con A) sensitive thymocyte population is by means of a thymic serum substance depressed by estradiol. To ascertain if a serum substance modulated by DHT could also effect a thymocyte population, we incubated thymocytes in vitro for 3 days in the presence of the mitogens Con A or phytohaemagglutinin (PHA) + tissue culture media + 20% specific rat serum preparations and pulse labelled with tritiated thymidine. Rat serum samples were prepared from control, castrate, thymectomized (Tx) and castrate-Tx animals as well as from similar groups of animals treated in vitro for 3 days with physiological doses of DHT. It was found that there was a significant enhancement of thymocyte blastogenesis in cultures incubated with castrate rat serum + Con A or PHA vs control serum cultures (P less than 0.001). Direct replacement of DHT either to normal or castrate sera in vitro at physiological concentrations did not alter the level of thymocyte blastogenesis from normal or castrate controls. Sera prepared from castrate animals treated with DHT at physiological concentrations did not depress the blastogenic response, but sera from animals treated with testosterone were successful in depressing the blastogenic response to noncastrate levels. Sera prepared from Tx animals were stimulatory on thymocyte blastogenesis when PHA were utilized (P less than 0.02) and this stimulation was depressed in sera from animals treated in vivo with DHT. Sera prepared from Tx or castrate-Tx animals were not able to stimulate thymocyte blastogenesis when Con A was utilized but were stimulatory when prepared from castrate-Tx animals pretreated in vivo with DHT. It is hypothesized that Con A sensitive thymocytes are depressed by a thymic serum factor under the control of E and that both Con A and PHA sensitive thymocytes are depressed by a serum factor possibly of extrathymic origin that is under the control of DHT. A tentative hypothesis for the regulation of thymocytes by gonadal steroid hormones is presented.

Characteristics of the cytoplasmic and nuclear dihydrotestosterone receptors of human thymic tissue.

Dihydrotestosterone receptor was prepared from both the cytoplasm and nucleus of human thymic tissue. Human cytoplasmic and nuclear dihydrotestosterone receptor possessed a KA greater than 1.0 and by competition assay was highly specific for dihydrotestosterone. Human thymic cytoplasmic dihydrotestosterone receptor prepared in standard buffer sedimented on 5-20% sucrose gradients in the 4s region but human thymic cytoplasmic dihydrotestosterone receptor prepared in buffer containing sodium molybdate (10 mM) sedimented in the 7s region on 5-20% sucrose gradients. It is concluded that the human thymic dihydrotestosterone receptor is similar to classic androgen receptor.

Estradiol regulation of thymic lymphocyte function in the rat: mediation by serum thymic factors.

Estradiol (E2) can depress the function of the thymic lymphocytes. To determine if this response to a gonadal steroid is regulated directly or indirectly, thymic lymphocytes were incubated in vitro for 3 days in the presence of the mitogens concanavalin A (Con A) or phytohaemagglutinin (PHA) + tissue culture media + 20% specific rat serum fractions and pulse labelled with tritiated thymidine. Rat serum fractions were prepared from control, castrate, thymectomized (Tx) and castrate-Tx animals as well as from similar groups of animals treated in vivo for three days with physiological doses of E2. It was found that there was a significant enhancement of thymocyte blastogenesis in cultures incubated with castrate rat serum + Con A or PHA vs. control serum cultures (P less than 0.001). Direct replacement of E2 to castrate sera in vitro at physiological concentrations failed to depress thymocyte blastogenesis to noncastrate levels. Sera prepared from castrate animals treated with E2 at physiological concentrations was successful in depressing the blastogenic response to noncastrate levels. Sera from Tx animals did not enhance Con A induced blastogenesis, but PHA response was significantly increased (P less than 0.01) This effect was lost utilizing sera from Tx animals treated with E2. It is concluded that thymocyte function is regulated by serum factors which have their origin in the thymus, and that these factors are modulated by a gonadal steroid estradiol.

Rat thymic dihydrotestosterone receptor: preparation, location and physiochemical properties.

Castration in the male rat has been shown to produce enlargement of the thymus gland while treatment with dihydrotestosterone (DHT) results in a decrease in thymic size in these animals. To determine if these changes might be receptor mediated, thymus tissue from castrate male rats was removed and homogenized in buffer and centrifuged to produce cytosol. By Scatchard plot analysis, it was shown that a specific DHT receptor was present at a concentration of 0.24 +/- 0.02 pmoles/g tissue and it possessed a KA of 2.51 +/- 0.45 x 10(9)M-1. This thymic DHT receptor sedimented on 5--20% sucrose gradients in the 8s region. By competition analysis it was found that testosterone only partially competed (25%) for this receptor, with virtually no binding noted for estradiol, progesterone, and cortisol. The receptor was found to be localized in the reticuloepithelial matrix of the thymus and was not present in the thymic lymphocyte fraction.

Progesterone receptor in the rat anterior pituitary: effect of estrogen priming and adrenalectomy.

The present study was done to determine if a progesterone receptor is present in rat pituitary. Cytosol was labeled with 3H-progesterone (3HP) or 3H-R5020 (3HR) and subjected to sucrose-glycerol density-gradient centrifugation. Serum progesterone was measured for correlation with progesterone receptor levels. Two 3HP-binding peaks (4S + 6S) were evident in uterine and pituitary cytosols. The 4S peak was eliminated by competition with unlabeled cortisol leaving a single 6S peak (progesterone receptor). Estradiol (E) priming of the male or female rat increased progesterone receptor levels in pituitary cytosol as demonstrated using 3HP and 3HR, and pituitary progesterone receptor bound 3HR with a higher affinity than 3HP. Following adrenalectomy of gonadectomized rats, progesterone receptor levels were increased in pituitary and uterine cytosol of both E-primed and unprimed groups. An inverse relationship was established between serum progesterone and progesterone receptor levels in the uterus and pituitary suggesting that stress-induced adrenal progesterone secretion significantly influences progesterone receptor levels in the rat. These results demonstrate an estrogen-inducible progesterone receptor in the rat pituitary with properties similar to those of the uterine progesterone receptor.

The effects of ephedrine and theophylline on dexamethasone metabolism in bronchial asthma.

The effect of ephedrine (nine patients) and theophylline (seven patients) on dexamethasone metabolism was studied before and after three weeks of drug therapy in 16 asthmatics. Five patients were studied similarily but treated with placebo. After treatment with ephedrine, there was a mean decrease in plasma dexamethasone half-life (t1/2) of 132 minutes, or 36 per cent (P less than 0.025), and mean increase in metabolic clearance rate (MCR) of 148 liters/day, or 42 per cent (P less than 0.001). Increase in the excretion of urinary radioactivity, predominantly in the conjugated fractions, was noted. In contrast, theophylline and placebo therapy resulted in no significant changes in dexamethasone t1/2 or MCR. The rate of urinary excretion of radioactivity was reduced after theophylline treatment. Since ephedrine accelerates labeled dexamethasone clearance while theophylline does not, caution is necessary when prescribing ephedrine for asthmatics requiring long-term therapy with dexamethasone and probably other corticosteroids. It would appear from the present investigation that theophylline is a more appropriate bronchodilator for these patients.

The effects of diethylstilbestrol and medroxyprogesterone acetate on kinetics and production of testosterone and dihydrotestosterone in patients with prostatic carcinoma.

Alterations in the metabolism of testosterone (T) and dihydrotestosterone (DHT) induced by diethylstilbestrol (DES) or medroxprogesterone acetate (MPA) could account for the beneficial therapeutic effect of these agents in prostatic carcinoma. To investigate this possibility we sutdied plasma kinetics of T and DHT in 17 elderly patients with prostatic carcinoma, before and after treatment with DES (1 or 5 mg/d) or MPA (10 or 30 mg/d) for 30 days. Metabolic clearance rates (MCR) were determined with the single injection technique and by use of two compartment model, plasma concentrations (PC) of T and DHT by radioimmunoassay, the per cent of T bound to plasma protein (T-binding) by charcoal adsorption of the unbound steroid. Production rate (PR) and PC of T were lower, PR and PC of DHT were higher in our patients than in normal men. With both DES regimens, PR, PC and MCR of either androgen declined; however, T was suppressed to a much greater extent than DHT. In either instance, the decrease may have been caused by direct suppression of testicular androgen synthesis and/or by decreased gonadotropin stimulation. Enhanced T-binding played an additional role in reducing the free testosterone index. High and low dose of DES were equally effective. The low dose regimen of MPA did not influence androgen metabolism. MPA in the higher dose suppressed PR and PC of T and DHT, possibly due to effects on testicular synthesis or by gonadotropin suppression as suggested for DES. In contrast to DES, MPA failed to cause profound changes in MCR of either androgen or in T-binding. When judged by its influence on the metabolism of T and DHT in prostatic carcinoma, MPA in higher doses is much less effective than either dose regimen of DES.

PIP: The effects of diethylstilbestrol(DES) and medroxyprogesterone acetate (MPA) on plasma kinetics and production of testosterone(T) and dihydrotestosterone (DHT), and on plasma protein binding of T were measured in 17 patients (50-93 years of age) suffering with metastatic carcinoma of the prostate both before and during treatment for this disease. Blood samples were obtained before injection of 30 mcCi tritiated T or tritiated DHT and at 10, 15, 20, 40, 55, 70, and 90 minutes after injection. T and DHT were measured by radioimmunoassay. Metabolic clearance rates were measured as well. Production rate (PR) and plasma concentrations (PCs) of T and DHT were higher in these patients than in normal men. When DES was given, PR, PC,and metabolic clearance rates of T and DHT declined, with T suppressed to a greater extent. There was also enhanced T-binding. In patients treated with MPA, 10 mg given for 30 enhanced T-binding. In patients treated with MPA, 10 mg given for 30 days, significantly different changes were seen in the kinetics of T and DHT in 8 patients. Patients treated with 30 mg of MPA for 30 days showed suppression of PR and PC of T and DHT. MPA failed to cause profound changes in the metabolic clearance rate of either androgen or in T-binding. These results indicate that MPA is less effective than DES.

The effects of disodium cromoglycate on dexamethasome metabolism.

The effect of disodium cromoglycate on dexamethasone metabolism was studied in 4 patients with bronchial asthma before and after 3 weeks of treatment. Labeled dexamethasome was injected intravenously and serial blood samples were collected during the next 6 hours. Transport kinetics of labeled dexamethasone were determined using a 2-compartment model. Baseline plasma equilibrated half-life values ranged from 133 to 258 min, and metabolic clearance rate ranged from 134 to 912 liter per day. After disodium cromoglycate therapy a small decrease in labeled dexamethasone clearance occurred. There was a mean increase in half-life of 20 min (9 per cent), being 201+/-51 (mean +/- SD) before therapy and 220+/-57 min after therapy. This was not significant. Metabolic clearance rate decreased 13 per cent (59 liter per day), being 441+/-334 liter per day before and 382+/-304 liter per day after treatment (P less than 0.05). Other transport kinetics of labeled dexamethasone were not significantly different with therapy. The results of this investigation demonstrate that disodium cromoglycate produces small but probably not significant changes in dexamethasone metabolism. The investigation supports the thesis that the steroid-sparing effect of disodium cromoglycate is not the result of depression of corticosteroid plasma clearance with longer duration of steroid action but most likely is the result of the effect of disodium cromoglycate on cellular mechanisms and inhibition of mediator release.

The in-vitro and in-vito effects of diethylstilbestrol on testicular synthesis of testosterone.

The effect of diethylstilbestrol (DES) on the percent conversion of a 14C-progesterone (14C-P) substrate to 14C-testosterone (14C-T) when added to incubates fo rat testicular homogenates has been measured. Similarly the effect of DES administered parenterally to intact adult rats at 9 dose regimens on such conversion has been evaluated. In addition, in the latter group serum luteinizing hormone (LH) levels were assessed simultaneously. DES added in-vitro significantly reduced T formation at every level. Only the large parenteral dose of DES affected T synthesis after 24 hours, while every parenteral dose significantly reduced T synthesis with 4 and 8 day regimens. Mean serum LH levels failed to reflect a significant decrease at the 1 and 4 day dose level, but did decrease significantly in 2 of 3 groups on the 8 day regimen. These results suggest that there is a direct effect of DES at a testicular level inhibiting T synthesis not associated with a simultaneous drop in mean serum LH levels. A measurable decrease in LH concentration does occur with continued treatment.

PIP: The effect of diethylstilbestrol (DES) on the conversion of carbon-14-progesterone to carbon-14-testosterone in incubates of rat testicular homogenates was studied, as well as the in vivo effect of DES, administered parenterally, on the conversion of the hormones and levels of luteinizing hormone (LH). In vitro, DES significantly diminished testosterone formation at every dose level (p.01). DES, administered in repeated daily doses for 4 or 8 days, caused a markedly decreasing conversion to testosterone. The 1- and 4-day dose levels of DES did not produce significant changes in LH levels (p .1), though an 8-day regimen of 5 mg and 50 mg DES significantly decreased LH values (p .02, p .02, respectively). It is suggested that DES has a direct effect, at the testicular level, in inhibiting the synthesis of testosterone, which is not associated with a drop in serum LH levels.

Plasma renin activity in juvenile diabetes mellitus and effect of diazoxide.

Low plasma renin activity (PRA) has been reported in patients with long-term diabetes mellitus complicated by hypertension and nephropathy. We have assayed PRA in twelve normal subjects and in eight age- and sex-matched juvenile diabetics of greater than twelve years' duration without hypertension and nephropathy under control conditions and following stimulation with diazoxide. During control conditions PRA did not decrease with time in the diabetics as it did in the normals. Following diazoxide infusion, PRA increased in both groups, and although the levels were often higher in diabetics than in normals, the mean differences were not statistically significant. The findings are consistent with the suggestion that PRA is normal or possibly elevated in clinically uncomplicated insulin-dependent diabetes mellitus and decreases with establishment of hypertension and nephropathy.