Retention of indoles in the pineal organ of the bird (Melopsittacus undulatus, Shaw) during its preparation for radioautographic study

Since high-resolution radioautography (dipping technique) might be very useful for the study of indole metabolism in the pineal cells, the retention of [3H]-indoles has to be examined during the preparation of specimens for electron microscopy (EM). The pineal organ of the parakeet (Melopsittacus undulatus) was used in the present work. 1) Indole metabolism: following uptake of [3H]-5-hydroxytryptophan ([3H]-HW) in vivo and [3H]-5-hydroxytryptamine ([3H]-HT) in vitro in similar seasonal and nycthemeral conditions--all the known pineal indolic metabolites were recovered by thin layer chromatography. [3H]-5-methoxyindoles were also formed from [3H]-melatonin ([3H]-aMT). 2) The radioactivity of fluids used in the processing of pineal organs in EM was determined by liquid scintillation counting: (a) no exogenous [3H]-indoles could be revealed in EM solutions after [3H]-HW in vivo uptake. (b) 8.8 to 13.4% of [3H]-indoles were washed out by glutaraldehyde after [3H]-HT in vitro uptake. (c) most of the 5-methoxyindoles after in vitro uptake of [3H]-aMT were lost in glutaraldehyde. Our chromatography procedures did not permit the identification of [3H]-indoles extracted by the glutaraldehyde fixative. In previous experiments, [3H]-HW and [3H]-HT uptake showed the presence of selective radioautographic reactions in the cells of the receptor line; however, silver grains were scarce and diffusely distributed in the pineal parenchyma after [3H]-aMT uptake.     

Retention of indoles in the pineal organ of the bird (Melopsittacus undulatus,Shaw) during its preparation for radioautographic study

Since high-resolution radioautography (dipping technique) might be very useful for the study of indole metabolism in the pineal cells, the retention of [3H]-indoles has to be examined during the preparation of specimens for electron microscopy (EM). The pineal organ of the parakeet (Melopsittacus undulatus) was used in the present work. 1) Indole metabolism: following uptake of [3H]-5-hydroxytryptophan ([3H]-HW) in vivo and [3H]-5-hydroxytryptamine ([3H]-HT) in vitro in similar seasonal and nycthemeral conditions—all the known pineal indolic metabolites were recovered by thin layer chromatography. [3H]-5-methoxyindoles were also formed from [3H]-melatonin ([3H]-aMT). 2) The radioactivity of fluids used in the processing of pineal organs in EM was determined by liquid scintillation counting: (a) no exogenous [3H]-indoles could be revealed in EM solutions after [3H]-HW in vivo uptake. (b) 8.8 to 13.4% of [3H]-indoles were washed out by glutaraldehyde after [3H]-HT in vitro uptake. (c) most of the 5-methoxyindoles after in vitro uptake of [3H]-aMT were lost in glutaraldehyde. Our chromatography procedures did not permit the identification of [3H]-indoles extracted by the glutaraldehyde fixative. In previous experiments, [3H]-HW and [3H]-HT uptake showed the presence of selective radioautographic reactions in the cells of the receptor line; however, silver grains were scarce and diffusely distributed in the pineal parenchyma after [3H]-aMT uptake.     

Metabolism of tryptopan and serotonin by the chick pineal gland in organ culture.

Marked differences were seen between the metabolism of L-[3-14C] tryptophan and of [2-14C]serotonin by the intact chick pineal gland in organ culture. The major metabolite of tryptophan recovered by our procedures was melatonin, which accounted for about half the radioactivity recovered as metabolic products. In contrast, the principal product of serotonin metabolism recovered was hydroxyindoleacetic acid, and the yield of products derived through monoamine oxidase (EC 1.4.3.4) activity vastly exceeded that of melatonin. Metabolism of tryptophan yielded a much larger proportion of methlated metabolites among the products recovered than did metabolism of serotonin. However, the yield of methoxyindoleacetic acid from serotonin was greater than that from tryptophan. Serotonin formed endogenously and serotonin supplied exogenously appear to enter two or more largely distinct metabolic pools.     

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.     

Identification of the factors affecting the rate of deactivation of hypochlorous acid by melatonin

It has been found that melatonin reacts rapidly with hypochlorous acid in phosphate-buffered, ethanol-water solutions to produce 2-hydroxymelatonin. The rate law, d[2 - HOMel]/dt - kHOCl[Mel][HOCl] - kOCl-[Mel][OCl-], was obtained. At 37 degrees C and at a water concentration of 23.5 M, kOCl- = 6.0 x 10(2) L. mol-1. s-1, and kHOCl was found to be a function of the water concentration, kHOCl = 11 +/- 3 L3. mol-3. s-1. [H2O]2, indicating that the availability of water at the site of the reaction plays a significant role. The part that the structural components of melatonin play in determining the reaction pathway was examined by comparing the rate of deactivation of HOCl by melatonin to that of the model compounds indole, 5-methoxyindole, and 3-methylindole. The relative reactivity is explained in terms of steric and electronic effects, and it was found that the presence of the substituent at the 3-position influences the nature of the oxidation product. Melatonin and 3-methylindole yielded hydroxylated products, whereas indole and 5-methoxyindole produce chlorinated products.     

Derivatives of [3H]serotonin in organ cultures of hamster pineal gland

The purpose of this study was to determine the viability of the hamster pineal gland in organ culture and to test the effect of norepinephrine (NE) on [3H]serotonin derivatives. In this study, elevated levels of melatonin (7-fold, p less than .05), 5- hydroxytrytophol (5-fold, p less than .001), 5-methoxytryptophol (1.78-fold, p less than .05), and depressed levels of 5-hydroxyindoleacetic acid (3.8-fold, p less than .02) and methoxyindoleacetic acid (1.78-fold, p less than .05) were detected in the glands following the addition of NE to the medium. In a separate experiment, melatonin concentration in the media was also periodically measured by radioimmunoassay to determine the viability of the organ culture over a four-day period. The melatonin level on day 2 (2321 +/- 106 pg/gland) was significantly higher (p less than 0.01) than on day 3 (1542 +/- 86 pg/gland) or day 4 (805 +/- 39 pg/gland). The results of these experiments verify the viability of the hamster pineal organ culture and show that the gland responds to NE in vitro.     

Coexistence of central and peripheral benzodiazepine binding sites in the human pineal gland

The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [³H] flunitrazepam and [³H] PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [³H] flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [³H] flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [³H] PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [³H] PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [³H] PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters melatonin metabolism in fish hepatocytes

Pineal hormone melatonin is an important regulator of endocrine and circadian rhythms in vertebrates. Since liver is assumed to be the major organ in the metabolism of this indole hormone, we investigated the effect of the known Ah-receptor agonist, 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on melatonin metabolism in fish hepatocytes as well as the in vitro effect of melatonin on trout hepatic microsomal cytochrome P4501A (CYP1A) catalyst. Primary cell cultures of rainbow trout hepatocytes were exposed to [3H]melatonin (1 nM to 1 microM) alone and in combination with TCDD (50 pM) at 15 degrees C for 24 or 48 h. Analysis of melatonin and its metabolites in the culture medium and hepatocytes by HPLC revealed that about 96% of the added [3H]melatonin was metabolised after 24 h in both control and TCDD treated cultures. 3H-radioactivity was found mainly in the culture medium and less than 5% of the total 3H-radioactivity retained inside hepatocytes. Of the HPLC separated metabolites, one coeluted with 6-hydroxymelatonin and one unknown metabolite eluted after 6-hydroxymelatonin. In addition, two other metabolites were more water-soluble than 6-hydroxymelatonin and were considered to be conjugated products. Treatment of the hepatocytes with TCDD increased the amount of the major oxidated product, 6-hydroxymelatonin, about 2.5-fold after 24 h and 1.2-fold after 48 h exposure, respectively when compared with the control cultures. Whereas the amount of the unknown metabolite eluting after 6-hydroxymelatonin decreased about 1.3-fold after 24 h and 1.2-fold after 48 h exposure, respectively. Melatonin alone did not affect P4501A associated EROD-activity or CYP1AmRNA levels in the primary hepatocyte cultures. TCDD-treatment increased EROD-activity 3 to 5-fold and respective CYP1AmRNA content 6 to 14-fold, when compared with the control or melatonin-treated cultures. Furthermore, melatonin competitively inhibited EROD-activity in liver microsomes with a Ki value of 62.06+/-3.78 microM. The results show that TCDD alters metabolic degradation of melatonin in hepatocytes and suggest that P4501A may be an important P450 isoenzyme involved in oxidative metabolism of melatonin in fish liver.     

Beta-adrenergic binding is increased by melatonin and alpha-adrenergic compounds

Binding of the beta-adrenergic ligands [3H]dihydroalprenolol and [125I]cyanopindolol to pineal particulate fractions was increased 1- to 3.5-fold by addition of low concentrations of melatonin, alpha-adrenergic agonists, or alpha-adrenergic antagonists. Minimum concentrations of melatonin or alpha-adrenergic compounds which increased beta-adrenergic binding were between 1 pM and 0.1 nM. The increased binding of [3H]dihydroalprenolol caused by melatonin (0.1 muM) was attributed to a major increase in Bmax, which persisted in protein fractions after removal of melatonin. Melatonin enhancement of [3H]dihydroalprenolol binding was apparent after 5 to 7 min (30(0], was was optimal between 20 and 40 min, and decreased at longer times. Alpha-Adrenergic receptors are unchanged during beta-receptor enhancement.     

Morphological and immunocytochemical features of the pineal organ of C3H and C57BL mice at different stages of postnatal development

Considerable progress is currently being made in elucidating the molecular basis of the circadian (photoneuroendocrine) system by use of transgenic mice generated from the inbred strains C57BL and C3H. As in all other vertebrate species, the pineal organ is an important component of the photoneuroendocrine system in these mouse strains, but very little is known about its morphological and immunocytochemical features. We therefore investigated the pineal organ and the adjacent epithalamic region of adult, 10-, and 5-day-old C57BL and C3H mice for S-antigen, serotonin, and dopamine-ß-hydroxylase (DBH) immunoreactions. In adult animals, the pineal organ was more than 2 times bigger in C3H than in C57BL mice. In younger animals, this difference was already evident, but less pronounced. The S-antigen immunoreactivity was more intense in adult C3H than in C57BL mice. This difference developed with increasing age; it was not yet detectable in 5-day-old animals. The intensity of the serotonin immunoreaction was similar in both strains at all stages investigated. However, the serotonin immunoreaction was more pronounced in adult than in young animals. The relative DBH-immunoreactive area (used as a marker for the sympathetic innervation of the pineal organ) was much bigger in C3H than in C57BL mice; within each strain it remained relatively constant during postnatal development. Adult individuals of both strains contained S-antigen- and serotonin-immunoreactive cells in the habenular complex. Their number increased with age, but they were always more numerous in C3H. In conclusion, the study has shown considerable differences in pineal morphology between C3H and C57BL, which may be related to the well-known differen- ces in melatonin formation between these two strains.     

Iodinated melatonin: preparation and characterization of the molecular structure by mass and 1H NMR spectroscopy

Synthetic melatonin was iodinated by treatment with potassium iodide in the presence of an oxidizing agent, Iodo-Gen. The iodination products of melatonin were extracted with chloroform and separated by HPLC. The fraction showing immunoreactivity with respect to melatonin antisera was characterized as iodomelatonin by mass spectrometry, so that the substitution of iodine had occurred at a ring carbon atom. 1H NMR spectra showed the iodine to be incorporated at the C-2 position of the indole moiety. The N-[2-(2-iodo-5-methoxy-1H-indol-3-yl)ethyl]acetamide (2-iodomelatonin) reported here is more useful than [3H]melatonin as a tracer in melatonin radioimmunoassay. This method offers also the possibility of preparing iodinated serotonin and other indoleamines for biological studies.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

Retinal ganglion cells are autonomous circadian oscillators synthesizing N-acetylserotonin during the day

Retinal ganglion cells send visual and circadian information to the brain regarding the environmental light-dark cycles. We investigated the capability of retinal ganglion cells of synthesizing melatonin, a highly reliable circadian marker that regulates retinal physiology, as well as the capacity of these cells to function as autonomous circadian oscillators. Chick retinal ganglion cells presented higher levels of melatonin assessed by radioimmunoassay during both the subjective day in constant darkness and the light phase of a light-dark cycle. Similar changes were observed in mRNA levels and activity of arylalkylamine N-acetyltransferase, a key enzyme in melatonin biosynthesis, with the highest levels of both parameters during the subjective day. These daily variations were preceded by the elevation of cyclic-AMP content, the second messenger involved in the regulation of melatonin biosynthesis. Moreover, cultures of immunopurified retinal ganglion cells at embryonic day 8 synchronized by medium exchange synthesized a [3H]melatonin-like indole from [3H]tryptophan. This [3H]indole was rapidly released to the culture medium and exhibited a daily variation, with levels peaking 8 h after synchronization, which declined a few hours later. Cultures of embryonic retinal ganglion cells also showed self-sustained daily rhythms in arylalkylamine N-acetyltransferase mRNA expression during at least three cycles with a period near 24 h. These rhythms were also observed after the application of glutamate. The results demonstrate that chick retinal ganglion cells may function as autonomous circadian oscillators synthesizing a melatonin-like indole during the day.     

Selective effect of melatonin on the proliferation of lymphoid cells

• 1.1. The present study was designed to investigate the effect of melatonin on the proliferation of normal lymphocytes and certain T-lymphomas and myelomas under in vitro conditions.
• 2.2. The results revealed that administration of 200 μM melatonin inhibited significantly the incorporation of [³H]thymidine into both normal mouse and human lymphocytes and T-lymphoblastoid cell lines.
• 3.3. On the contrary, melatonin provoked an increase of myeloma cell proliferation.
• 4.4. The influence of melatonin on hybridoma cell lines was negligible.
• 5.5. Collectively, these data demonstrated that the chief pineal indole affect selectively the processes of lymphoblastoid cell growth.

     

Synthesis of 1-acyl-2-[3H]acetyl-SN-glycero-3-phosphocholine, a structural analog of platelet activating factor, by vascular endothelial cells

Human umbilical vein endothelial cells (HUVECS) were challenged with thrombin in the presence of [3H]acetate to stimulate the production of radiolabeled platelet activating factor (PAF, 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine, 1-O-alkyl-2-[3H]acetyl-GPC). The 3H-product was isolated by thin-layer chromatography, and 1-radyl-2[3H],3- diacetylglycerols were prepared by phospholipase C digestion and subsequent acetylation at the sn-3 position. When the 1-radyl-2[3H],3-diacetylglycerols were analyzed by zonal thin-layer chromatography, 96-97% of the radiolabeled derivative migrated with 1-acyl-2,3-diacetylglycerol standard. Only minor amounts (3-4%) of 1-alkyl-2[3H],3-diacetylglycerol were observed, demonstrating that the predominant acetylated product synthesized by thrombin-stimulated HUVECS was 1-acyl-2-[3H]acetyl-GPC. This relative abundance of 1-acyl-2-[3H]-acetyl-GPC was not significantly affected by thrombin dose, incubation time, or cell passage, and was also observed in HUVECS challenged with ionophore A23187. In addition, the acetylated product from ionophore A23187- or bradykinin-stimulated bovine aortic endothelial cells contained 90% 1-acyl-2-[3H]acetyl-GPC, suggesting that the synthesis of the 1-acyl PAF analog is not unique to HUVECS. These findings demonstrate that PAF is a minor synthetic component of HUVECS and bovine aortic endothelial cells. In light of the integral role which the vascular endothelial cell plays in the regulation of thrombosis, these findings also suggest that the production of 1-acyl-2-acetyl-GPC may be biologically important.     

Inhibition by a nonmetabolized analogue of L-leucine of protein biosynthesis in tumoral pancreatic islet cells

The effect of L-leucine, its deaminated metabolite 2-ketoisocaproate and its nonmetabolized analogue b(+/-)2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) upon protein labelling was examined in tumoral islet cells (RINm5F line) exposed to L-[4-3H]phenylalanine or L-[3-3H]serine. The interpretation of the results, in terms of changes in biosynthetic activity, was obscured by a possible interference of the tested nutrients with the uptake and further metabolism of the tracer tritiated amino acids. Nevertheless, when the cells were preincubated with the nutrient secretagogues and then incubated in the sole presence of L-[3-3H]serine, BCH, but not L-leucine or 2-ketoisocaproate, still inhibited protein labelling, this coinciding with a decrease in the ratio between TCA-precipitable and total radioactivity in the RINm5F cells. The inhibitory action of BCH was antagonized, to a limited extent, by D-glucose. It is proposed that BCH could be used as a tool to interfere with the function and growth of insulinoma cells.     

Effects of diazepam and its metabolites on nocturnal melatonin secretion in the rat pineal and Harderian glands. A comparative in vivo and in vitro study

We investigated the effects of diazepam (DZP) and its three metabolites: nordiazepam (NZP), oxazepam (OZP), and temazepam (TZP) on pineal gland nocturnal melatonin secretion. We looked at the effects of benzodiazepines on pineal gland melatonin secretion both in vitro (using organ perifusion) and in vivo in male Wistar rats sacrificed in the middle of the dark phase. We also examined the effects of these benzodiazepines on in vivo melatonin secretion in the Harderian glands. Neither DZP (10^-5-10^-6 M) nor its metabolites (10^-4-10^-5 M) affected melatonin secretion by perifused rat pineal glands in vitro. In contrast, a 10^-4 M suprapharmacological concentration of DZP increased melatonin secretion of perifused pineal glands by 70%. In vivo, a single acute subcutaneous administration of DZP (3 mg/kg body weight) significantly affected pineal melatonin synthesis and plasma melatonin levels, while administration of the metabolites under the same conditions did not. DZP reduced pineal melatonin content (-40%), N-acetyltransferase activity (-70%), and plasma melatonin levels (-40%), but had no affects on pineal hydroxyindole-O-methyltransferase activity. Neither DZP nor its metabolites affected Harderian gland melatonin content. Our results indicate that the in vivo inhibitory effect of DZP on melatonin synthesis is not due to the metabolism of DZP. The results also show that the control of melatonin production in the Harderian glands differs from that observed in the pineal gland.     

Melatonin-induced stimulation of rat corpus epididymal epithelial cell proliferation.

Stimulation of rat epididymal epithelial cell proliferation by melatonin was demonstrated by thymidine incorporation and flow cytometric analyses. The stimulatory effect of melatonin was dependent on the hormone concentration and the duration of cell exposure to the hormone. Maximal stimulation of [3H]thymidine incorporation into epididymal epithelial cells by melatonin was observed at 1 x 10(-9) M 5alpha-dihydrotestosterone in medium, while lower or higher concentrations of androgen attenuated the stimulatory effect of melatonin. Interestingly, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxothiazolidine-2-ylidene]-4-methyl-thiosemi-carb azone, CGP 52608) induced opposite effect on epithelial cell proliferation to that produced by melatonin. Our data suggest that melatonin-induced stimulation of rat epididymal epithelial cell proliferation is not likely to be mediated by nuclear receptor. Furthermore, sequential changes of cell cycle distribution with melatonin treatment also supports a stimulatory action of melatonin on epididymal epithelial cell proliferation.     

Oxidation of melatonin by oxoferryl hemoglobin: a mechanistic study.

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H2O2, has been investigated at 37 degrees C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37 degrees C is 112 +/- 1.0 M(-1) s(-1). Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N'-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]:[melatonin]--the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hb(reduced):melatonin(consumed) is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.     

Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.