Androgen receptors: relationship to growth response and to intracellular androgen transport in nine variant lines of the Shionogi mouse mammary carcinoma.

Aspects of the biological significance of androgen receptors have been studied in nine variant lines of the Shionogi carcinoma, two of which are androgen dependent and seven of which are autonomous. The dependent lines, and two of the seven autonomous lines, contain androgen receptors; this finding demonstrates that the presence of receptors is not an accurate marker of hormonal dependence in vivo. Since the ability to transport androgens into the nucleus, as judged from the relative maximal rates of transport, is virtually restricted to dependent and autonomous lines which possess cytoplasmic receptors, it is clear that such receptors may play a role in regulating the intranuclear concentration of androgens. The absence of cytoplasmic receptors and the comparative lack of perceptible transfer of androgens across the nuclear membrane are features peculiar to the autonomous condition.     

Developmental regulation of intracellular and surface androgen receptors in T cells

Increasing information indicates that testosterone actions on cells are mediated not only through the classical intracellular androgen receptor (iAR), but also through membrane androgen receptors (mAR) on cell surfaces. Here, we investigate the expression pattern of mAR and iAR in thymic T cells, which is compared with that of splenic T cells. Thymic T cells are testosterone-sensitive in vivo, i.e. treatment of female C57BL/10 mice with testosterone for 3 weeks decreased the total number of thymic T cells by approximately 90%. The percentage of CD4(-) CD8(-) T cells increased, whereas that of the subsequent CD4(+) CD8(+) T cells was diminished. Flow cytometry and confocal laser scanning microscopy (CLSM) with different anti-iAR antibodies localized iAR predominantly in the cytoplasm, but not on the surface of thymic T cells. The iAR are functionally active since the iAR are induced by testosterone to translocate from cytoplasm to nucleus, and they bind the testosterone analogue 3H-R1881 with high affinity (K(d) approximately 2.2 nM) and saturable capacity (approximately 10,000 binding sites per cell) as determined by Scatchard analysis. By contrast, the impeded ligand testosterone-BSA-FITC (T-BSA-FITC) did not bind to the surface of thymic T cells. In accordance, testosterone was unable to induce any rapid rise in the intracellular free Ca(2+) concentration of Fura-2 loaded thymocytes. This indicates that thymic T cells do not express any significant amounts of mAR. Conversely, splenic T cells express functionally active mAR, whereas their expressed iAR are not functional in the genomic pathway. Our results support the view of a delicately balanced developmental regulation of iAR and mAR in T cells.     

Androgen receptors: Relationship to growth response and to intracellular androgen transport in nine variant lines of the shionogi mouse mammary carcinoma

Aspects of the biological significance of androgen receptors have been studied in nine variant lines of the Shionogi carcinoma, two of which are androgen dependent and seven of which are autonomous. The dependent lines, and two of the seven autonomous lines, contain androgen receptors; this finding demonstrates that the presence of receptors is not an accurate marker of hormonal dependence in vivo. Since the ability to transport androgens into the nucleus, as judged from the relative maximal rates of transport, is virtually restricted to dependent and autonomous lines which possess cytoplasmic receptors, it is clear that such receptors may play a role in regulating the intranuclear concentration of androgens. The absence of cytoplasmic receptors and the comparative lack of perceptible transper of androgens across the nuclear membrane are features peculiar to the autonomous condition.     

Monitoring intracellular transport.

Ingested plastic scintillator spheres are shown to be a useful tool for investigating intracellular transport of material labelled by a weak beta emitter. It is found that-by numerically solving a linear integral equation with known kernel-the time evolution of the intracellular emitter density profile in the vicinity of the cell membrane is determinable from easily obtained experimental data. Applications to special biological systems are noted.     

Prostatic androgen receptor and plasma testosterone levels in streptozotocin-induced diabetic rats.

Diabetes was induced in male Sprague-Dawley (S-D) rats by streptozotocin (STZ) administration. Following STZ injection, plasma glucose levels in the treated rats were significantly elevated from values of untreated controls. Over the experimental period (140 days) plasma testosterone (T) levels, prostatic nuclear androgen receptor (AR) contents and prostatic weights declined with increasing age in the rats. The declines in both STZ-treated and untreated rats were similar in manner and no notable differences were discerned in the data obtained from the two groups. On the contrary, prostatic cytosolic AR contents in untreated rats remained unchanged with advancing age, but was reduced to 50% of normal control values in diabetic rats following STZ treatment. Correlation analyses revealed that prostatic nuclear AR contents correlated positively with plasma T levels while prostatic cytosolic AR contents correlated negatively with plasma glucose levels. These data support former claims that prostatic nuclear AR content is dependent on circulating T level and suggest a possible link between prostatic cytosolic AR content and plasma glucose concentrations.     

Immunoreactivity for intracellular androgen receptors in identified subpopulations of neurons, astrocytes and oligodendrocytes in primate prefrontal cortex.

Sex differences in and hormone malleability of a variety of cognitive and mnemonic functions suggest that the association cortices in human and nonhuman primates are targets of gonadal hormone stimulation. One mechanism involved in this stimulation may be genomic actions mediated by intracellular androgen receptors. To identify potential cellular targets of this influence, single- and double-labeling immunohistochemical methods were used to precisely localize androgen receptor proteins in the prefrontal association cortex of adult rhesus monkeys. In both the dorsolateral and orbitofrontal regions, receptor antibodies labeled substantial populations of small intensely immunoreactive nuclei, as well as much larger and less strongly immunoreactive nuclei in all major cellular layers and/or in underlying white matter. Double-labeling studies revealed that large and small immunolabeled nuclei were further distinguished by colocalization with different classes of cell-specific markers. Whereas the large, pale receptor-immunoreactive nuclei colocalized with immunomarkers for neurons, the small, strongly immunoreactive nuclei colocalized exclusively with glial markers. Among androgen receptor-immunoreactive glia, a majority were immunoreactive for astrocyte markers, with smaller numbers of nuclei colocalized with oligodendrocyte markers; immunolabels for microglia failed to colocalize with androgen receptor immunoreactivity. This discovery of an unexpectedly large population of androgen receptor bearing glia suggests that direct functional interactions between endocrine signaling pathways and glial cells such as those coming into view in studies in subcortical and allocortical structures may also take place in the cerebral cortex and contribute to gonadal hormone stimulation of cortical processing of cognitive information.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

Regulation of intracellular membrane transport.

A number of proteins that are necessary for membrane transport have been identified using cell-free assays and yeast genetics. Although our knowledge of transport mechanisms remains limited, common themes are clearly emerging. In particular, specific GTP-binding proteins appear to be involved, not only at all steps of membrane traffic but also at more than one check-point within each step. The ordered sequence of events occurring during vesicle formation, targeting and fusion may be regulated in a stepwise manner by specific GTP-dependent switches, which act as modular elements of the transport mechanism.     

Membrane tethering in intracellular transport.

Studies of various membrane trafficking steps over the past year indicate that membranes are tethered together prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction. The tethering proteins identified to date are quite large, being either fibrous proteins or multimeric protein complexes. The tethering factors employed at different steps are evolutionarily unrelated, yet their function seems to be closely tied to the more highly conserved Rab GTPases. Tethering factors may collaborate with Rabs and SNAREs to generate targeting specificity in the secretory pathway.     

Bidirectional intracellular transport: utility and mechanism

Bidirectional transport of intracellular cargo along microtubule tracks is the subject of intense debate in the motility field. In the present review, we provide an overview of the models describing the possible mechanisms driving intracellular saltatory transport, taking into account current experimental results that may at first seem contradictory. We examine the phenomenon of saltatory motion, in an attempt to interpret the mechanistic debate in terms of the utility of saltatory motion.     

Distribution of insulin receptors between plasmalemma and intracellular compartments: effect of amino acids

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.     

Steroid hormone receptors: intracellular distribution

Recent reports, both biochemical and morphological, have challenged the widely accepted two-step model of steroid hormone action. This model proposed that steroid hormone receptors existed under two different forms: the unliganded receptor in the cytoplasm and the hormone-bound receptor complex in the nucleus. A nuclear translocation mechanism was hypothesized as a necessary link between the two forms. In contradiction with this model, new studies have concluded to the absence of receptor in the cytoplasm and its presence in the nucleus under all hormonal conditions, thus rendering the hypothetical nuclear translocation unnecessary. In this review, we discuss how our concept of the mechanism of action of steroid hormone ought to be revised in the light of the new data.     

Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.     

Perforated MDCK cells support intracellular transport.

We have developed a method for perforating the plasma membrane of MDCK cells while retaining cellular functions. A nitrocellulose acetate filter was applied to the apical side of cells, grown on a glass coverslip, and allowed to dry. Segments of the apical plasma membrane adhered to the filter and were detached from the cell layer by shearing when the filter was peeled off. This allowed macromolecules such as antibodies and enzymes to diffuse into the cells. The cells were otherwise intact as judged by light and electron microscopy. The perforated cells maintained their capacity to support vesicular transport of proteins and lipids. Vesicular stomatitis virus infected cells readily incorporated [35S]methionine into G protein following permeabilization. This G protein was core-glycosylated during assembly in the endoplasmic reticulum, and was further transported to the trans Golgi with high efficiency. Experiments using lipid probes demonstrated that newly synthesized fluorescent sphingolipids were transported from the Golgi complex to the basolateral cell surface in perforated cells. Our results show that perforated cells provide a convenient and efficient alternative to cell-free assays for studying the molecular mechanism of intracellular transport.     

Cytoskeletal elements and intracellular transport

Recent advances in the understanding of the functions of various components of the cytoskeleton indicate that, besides serving a structural role, the cytoskeletal elements may regulate the transport of several proteins in the cell. Studies reveal that there are co-operative interactions between the actin and microtubule cytoskeletons including functional overlap in the transport influenced by different motor families. Multiple motors are probably involved in the control of the dynamics of many proteins and intriguing hints about how these motors are co-ordinated are appearing. It has been shown that some of the intermediate elements also participate in selected intracellular transport mechanisms. In view of the author's preoccupation with the steroid receptor systems, special attention has been given to the role of the cytoskeletal elements, particularly actin, in the intracellular transport of steroid receptors and receptor-related proteins.     

The enrichment of thymocytes bearing C3 receptors following cortisone involution.

Young CBA mice were injected with 2.5 mg of cortisone acetate, following which their spleen cells, thymocytes, and lymph node cells were tested for receptors for the third component of complement (C3) over a 20-day period. An erythrocyte-antibody-complement (EAC) rosette assay was used to detect C3 receptors. Cells bearing C3 receptors in the thymus emerged as early as 2 days after cortisone injection and peaked to a level of 18% at Day 7. This was followed by a decline to control levels by Day 14. There was no significant change in the percentage of C3 receptor-bearing cells in lymph node and spleen of the cortisone injected animals compared to appropriately matched uninjected animals. Evidence has been presented that cortisone-resistant thymocytes may bind EAC, and that these cells are surface Ig negative, contain no or very few macrophages, and bear thy-1 antigen. Complement receptor lymphocytes (CRLs) were separated from the nonrosetting thymus cells by sedimentation in an Isopaque-Ficoll gradient. These enriched C3 receptor-bearing cells were found to possess thy-1 antigens by indirect immunofluorescence. Specificity controls using antisera with thy-1.1 and thy-1.2 specificity and donor animals with thy-1.1 or thy-1.2 phenotypic expression indicated that C3 receptor-bearing cells appearing in the thymi of these respective donors following cortisone involution possessed the appropriate thy-1 phenotype.     

The effect of temperature on intracellular transport of asialo-glycoproteins in rainbow trout liver

The intracellular pathway of a endocytosed glycoprotein has been studied in rainbow trout ( Oncorhynchus mykiss ) liver by means of differential and isopycnic gradient centrifugation. When ¹²⁵I-tyramine-cellobiose-labelled asialoorosomucoid was injected intravenously, the protein was removed rapidly from the blood by hepatic endocytosis. The tracer was localized initially in a small, slowly sedimenting organelle (endosome) and transferred to the denser lysosomes where degradation took place. This process took several hours at the acclimated temperature of 10 ° C. The transport could be accelerated markedly or retarded by transfer of fish to higher or lower temperatures respectively. The intracellular transport steps were more susceptible to changing temperature than the internalization process. In particular, the degradation was temperature sensitive. These results may have implications for the effects of changing temperature on immune function in fishes. The impairment of transport upon a rapid temperature decrease, will reduce the rate of antigen processing and presentation in these animals.