Species-specificity of corticosteroid receptors in hamster and rat brains

In vitro cytosolic receptor binding assays and autoradiographical procedures have shown the localization and properties of two corticoid receptor types in the brain of the rat, a species in which corticosterone (B) is the predominant circulating glucocorticoid. The present study was designed to examine the localization, heterogeneity, and binding specificity of corticosteroid receptors in the brain of the hamster, a species which secretes B and cortisol (F), the latter being the predominantly circulating form. Our results show that two corticoid receptor systems can also be distinguished in the hamster brain. The type I receptor has an almost exclusive localization in the hippocampal region and the amounts measured in hypothalamic or whole brain (without the hippocampus) were negligible. The type II receptor, on the other hand, has a wider distribution in the brain. Scatchard and Woolf analyses of the binding data revealed that the hamster type I receptor has similar affinity to both F and B [dissociation constant (Kd) 0.9 nM]. In contrast the rat type I binds with higher affinity to B (Kd, 0.9 nM) than to F (Kd, 2.2 nM). The hamster type II binds to F with much higher affinity (Kd, 2.9 nM) than does the rat type II to F (Kd, 20.1 nM). This was similarly observed, although less pronounced in the binding of the hamster type II and the rat type II to B (Kd, 0.5 and 3.9 nM, respectively). Analysis of relative binding affinity of each receptor type gave the following results. Hamster type I: F greater than B much greater than aldosterone (ALDO) greater than dexamethasone (DEX); rat type I: B greater than F greater than ALDO greater than DEX; hamster type II: B greater than DEX greater than F much greater than ALDO; rat type II: DEX greater than B much greater than F much greater than ALDO. Graded doses of F or B given sc to adrenalectomized animals result in differential occupancy of the two receptor systems. In hamster, 1.0 microgram F vs. 1.0 mg B/100 g BW is required to occupy 80% of type I site. The rat shows the opposite (1.0 microgram B vs. 5.0 to 10.0 mg F/100 g BW to occupy type I to the same extent). The hamster type II is 80-90% occupied by an equal dose of F or B (1.0 mg/100 g BW) whereas in the rat, F at 5-10 mg/100 g BW (doses 5-10 times that of B) is required to achieve this same occupancy. This data demonstrate the     

Postnatal development of corticosteroid receptor immunoreactivity in the rat cerebellum and brain stem.

The postnatal development of corticosteroid receptor immunoreactivity in the rat cerebellum and related brainstem nuclei was studied using a type I receptor antiserum, MINREC4, and a type II receptor monoclonal antibody, BUGR2. Type I receptor immunoreactive (ir) Purkinje cells were first observed at postnatal day 5 (P5), and increased to adult levels by P20. Type I-ir cells, presumably migrating granule cells, were observed in the developing molecular layer of the cerebellum at P5. By P30, the density of type I-ir cells in the definitive molecular and granular layers was still less than adult levels. In contrast, type II-ir Purkinje cells were first observed at P15 and increased to adult levels by P20. No type II-ir cells were observed in the proliferative and migratory zones of the molecular layer. By P30, the density of type II-ir cells in the molecular and granular layers was far less than adult levels. In the deep cerebellar nuclei and most brain stem nuclei type I-ir was observed at P5 and developed to adult levels by P30. Type II-ir was observed in the deep cerebellar nuclei, red and medial vestibular nuclei by P15. The pontine and inferior olivary nuclei showed type II-ir cells by P10. Type II-ir in these regions developed to adult levels by P30. The earlier development of type I-ir suggests that the type I receptor may mediate the actions of corticosteroids in the cerebellum and related brain stem nuclei during early postnatal life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential corticosteroid regulation of type II glucocorticoid receptor-like immunoreactivity in the rat central nervous system: topography and implications

Neuronal type II glucocorticoid receptor-like immunoreactivity in the central nervous system shows heterogeneity in intensities and relative densities. This might predict variations in the regional responses of neuronal immunoreactivity to corticosteroids. We investigated changes in the intracellular location of immunoreactivity in the rat central nervous system after adrenalectomy and corticosteroid treatment, and carried out detailed statistical analysis of changes in neuronal nuclear immunoreactivity in the hippocampus and caudateputamen. Three types of responses were observed. The majority of neurons, classified type A, showed a predominant nuclear immunoreactivity in intact rats, lost nuclear and eventually cytoplasmic immunoreactivity after adrenalectomy, and regained nuclear immunoreactivity within 5 min of corticosterone and 2 h of aldosterone treatment, respectively. A subgroup of neurons in the hippocampus, striatum, septum, and habenula, classified type B, were not immunoreactive in intact rats, showed intense cytoplasmic immunoreactivity after adrenalectomy, and disappeared rapidly after corticosterone treatment and later in response to aldosterone. A subgroup of vermal cerebellar Purkinje neurons, classified type C, developed an intense cytoplasmic immunoreactivity after adrenalectomy, increased in number in response to corticosterone, and did not respond to aldosterone.     

Glucocorticoid and mineralocorticoid receptor mRNA expression in rat brain

In the rat brain, the binding of corticosterone is mediated through two receptor types, the type I receptor and the type II receptor, which are presumed to be encoded by genes designated as MR and GR, respectively. We have studied the regulation of these receptors by glucocorticoids, utilizing a cytosol receptor binding assay. In addition, we have employed molecular probes for the GR and the MR to measure receptor mRNAs. The level of type II receptor binding is uniform across several brain regions, as is the expression of GR (type II) mRNA. In contrast, type I receptor binding is concentrated in the hippocampus, and the MR (type I) mRNA similarly shows a higher level of expression in hippocampus than in the other brain regions studied. Removal of endogenous glucocorticoids by adrenalectomy (ADX) induces an increase, and corticosterone administration results in a decrease, in the level of type I and type II binding in the hippocampus; however, no significant changes in the MR (type I) or GR (type II) mRNA levels are seen with these treatments. The diurnal variation of serum corticosterone in intact rats is correlated with a circadian regulation of type I receptor binding in the hippocampus, while MR (type I) mRNA expression is unaffected. Thus, the changes in type I and type II receptor binding capacity elicited by differing steroid conditions cannot be attributed to modulation of the steady state levels of MR (type I) or GR (type II) mRNA.     

Properties and localization of beta-endorphin receptor in rat brain.

Stereospecific high-affinity binding sites for beta h-[3H]endorphin could be demonstrated in the P2 pellet of rat brain homogenate. Scatchard analysis of the binding data revealed binding sites with Kd values of 0.81 and 6.8 nM and density of 120 and 240 fmol/mg of protein. Distribution of beta h-[3H]endorphin binding in various brain regions parallels that of opiate receptor:striatum greater than thalamus greater than amygdala greater than hypothalamus, septum greater than cortex greater than midbrain, brainstem. Similar to their effect on 3H-labeled agonist binding, Na+ and other monovalent cations, GTP, trypsin, chymotrypsin, phospholipase A2, and N-ethylmaleimide all inhibited the specific binding of beta h-[3H]endorphin. In contrast to their action on alkaloid and enkephalin binding, Ca2+, Mg2+, and Mn2+ also inhibited beta h-[3H]endorphin binding. These data suggest a difference between beta h-endorphin and alkaloid/enkephalin binding sites.     

Activin receptor mRNA expression in rat testicular cell types.

cDNA encoding the extracellular domain of the rat activin receptor was cloned using the polymerase chain reaction (PCR). This cDNA is highly homologous to cDNA encoding the extracellular domain of the mouse activin receptor, whereas at the protein level the extracellular domains of both receptors are identical. Employing this cDNA as a probe in Northern blot analysis, expression of two activin receptor mRNAs (6 kb and 4 kb) was observed, in testes of immature and mature rats. Between day 21 and 28 of postnatal development, a large increase in testicular expression of the 4 kb mRNA was found, suggesting expression of this activin receptor mRNA in germ cells. The 4 kb mRNA was indeed present in isolated pachytene spermatocytes and round spermatids, but was absent in elongating spermatids. Sertoli cells obtained from immature and mature rats expressed both the 6 kb and 4 kb mRNAs, whereas the expression of these mRNAs in Leydig cell preparations was very low. These results may imply that activin has multiple actions in the control of testicular function.     

Sexual dimorphism in regulation of type II corticosteroid receptor immunoreactivity in the rat hippocampus.

To determine whether there are sex differences in the distribution of type II corticosteroid receptor-immunoreactive (type II-ir) cells in the rat hippocampus, we carried out a quantitative morphometric immunocytochemical study using a mouse monoclonal antibody, BUGR2. We report that in adrenally intact male and female rats, high densities of cells with nuclear type II-ir were observed in the pyramidal layer of field CA1 and the granular layer of the dentate gyrus. In intact males very few cells, presumably glia, in the stratum oriens showed type II-ir. In contrast, in females, interneurons with diffuse or cytoplasmic type II-ir were observed in the stratum oriens of CA1 and CA3. There were also sex differences in the regulation of type II-ir by corticosterone, the predominant glucocorticoid, and female sex steroids. In male rats the density of cells with nuclear type II-ir in all parts of Ammon's horn and the dentate gyrus was decreased significantly after adrenalectomy (adx). In contrast, in females such reductions were observed only in the pyramidal layer of CA1 and the granular layer of the dentate gyrus. In both sexes, cells with intense diffuse or mainly cytoplasmic type II-ir were observed in the pyramidal layer and stratum oriens after adx. The loss of nuclear type II-ir in the hippocampus of adx females was not affected significantly by ovariectomy. In adx males, nuclear Type II-ir was restored in CA1 and the dentate gyrus after treatment with corticosterone or progesterone. Cells in CA3 were, however, unresponsive to treatment with either hormone. In contrast, in adx females, treatment with either corticosterone or progesterone restored nuclear type II-ir to cells in all regions of the hippocampus. In both adx males and females, cytoplasmic type II-ir observed in some cells in the pyramidal layer and stratum oriens, was abolished completely by corticosterone, and partially by progesterone treatment. In both adx males and females, estradiol treatment did not affect significantly the pattern of type II-ir. Sex differences in the distribution of type II-ir interneurons in intact rats and the regulation of the intracellular location of type II-ir of adx rats by corticosterone and progesterone, may be important determinants of sex differences in the modulation of hippocampal function by glucocorticoids.     

Nerve growth factor receptor molecules in rat brain.

We have developed a method to immunoprecipitate rat nerve growth factor (NGF) receptor proteins and have applied the method to detect NGF receptor molecules in the rat brain. Crosslinking 125I-labeled NGF to either PC12 cells or cultured rat sympathetic neurons yielded two radiolabeled molecules (90 kDa and 220 kDa) that were immunoprecipitated by monoclonal antibody 192-IgG. Further, 192-IgG precipitated two radiolabeled proteins, with the expected sizes (80 kDa and 210 kDa) of noncrosslinked NGF receptor components, from among numerous surface-iodinated PC12 cell proteins. These results demonstrate the specific immunoprecipitation of NGF receptor molecules by 192-IgG. We applied the 125I-NGF crosslinking and 192-IgG-mediated immunoprecipitation procedures to plasma membrane preparations of the following areas of rat brain: medial septum, cerebellum, brainstem, hippocampus, cerebral cortex, thalamus, and olfactory bulb. NGF receptor molecules of the same molecular masses as the peripheral receptor components were consistently detected in all of these regions and in preparations from whole brains. Removal of the peripheral sympathetic innervation of the brain did not eliminate these NGF receptor proteins, indicating that the receptor is endogenous to central nervous system tissues. We also observed retrograde transport of 125I-labeled 192-IgG from the parietal cortex to the nucleus basalis and from the hippocampus to the nucleus of the diagonal band of Broca and the medial septal nucleus. These findings demonstrate the presence in brain of NGF receptor molecules indistinguishable from those of the peripheral nervous system.     

Opiate receptor: autoradiographic localization in rat brain.

Opiate receptor sites in rat brain can be labeled in vivo by [3H]diprenorphine, a potent opiate antagoinst. Using techniques to minimize diffusion in fresh, frozen, unfixed brain, we have localized [3H]diprenorphine by autoradiography to visualize the distribution of opiate receptors. Silver grains indicative of the binding of labeled [3H]diprenorphine are discretely localized in numerous areas of the brain with very high densities in the locus coeruleus, the substantia gelatinosa of the spinal cord, and in clusters within the caudate-putamen, amygdala, and parts of the periventricular gray matter.     

Androgen receptor localization in different cell types of the adult rat prostate

To better understand direct and indirect androgen action on rat prostatic growth and function, the various cell populations within the intact adult ventral, dorsal, and lateral prostate lobes were characterized for the presence or absence of androgen receptor (AR). Polyclonal rabbit antibodies raised against amino acids 1-21 of the rat AR (PG-21) were used in combination with a library of monoclonal antibodies directed against cell-specific antigens for positive cellular identification. Luminal epithelial cells were strongly AR positive, with an order of ventral greater than lateral greater than or equal to dorsal. In the lateral lobe, staining intensity was strongest in the peripheral regions, whereas a similar gradient was not apparent in the ventral and dorsal prostate. Basal epithelial cells were AR negative in all regions of the three lobes. Periacinar smooth muscle was strongly positive for AR, and this staining did not vary with the thickness of the muscle layer. Endothelial cells of the vasculature were AR negative, while the perivascular smooth muscle cells were AR positive. The majority of stromal fibroblasts were AR negative, although a number of AR-positive fibroblastic-appearing cells were observed within the ventral and dorsal lobes. Staining with ED2, a specific marker for tissue macrophages, revealed that fixed macrophages were present in significant quantities in the stroma of intact rat prostate lobes. Since these were frequently identified as AR positive, macrophages may partially account for the appearance of AR-positive stromal cells. Thus, the present findings indicate a complex pattern of AR expression among different cell types of the three prostate lobes. Cells types that express AR can potentially be considered as direct targets of androgen action, whereas those lacking AR should be considered as indirect targets or androgen-insensitive cells.     

Glucocorticoid receptor immunoreactivity in monoaminergic neurons of rat brain.

A monoclonal antibody against the rat liver glucocorticoid receptor was used in combination with rabbit antibodies against tyrosine hydroxylase, phenylethanolamine N-methyltransferase, and 5-hydroxytryptamine to demonstrate strong glucocorticoid receptor immunoreactivity in large numbers of central monoaminergic nerve cell bodies of the male rat. The receptor immunoreactivity was predominantly located in the nucleus, whereas the tyrosine hydroxylase, phenylethanolamine N-methyltransferase, and 5-hydroxytryptamine were detected mainly in the cytoplasm. The vast majority of the noradrenergic nerve cell bodies of groups A1-A7 and of the 5-hydroxytryptaminergic cell bodies of groups B1-B9 were found to contain strong glucocorticoid receptor immunoreactivity. The majority of the phenylethanolamine N-methyltransferase-immunoreactive nerve cells of the adrenergic cell groups C1-C3 and of the dorsal subnuclei of the nucleus tractus solitarius in the medulla oblongata were also strongly immunoreactive for glucocorticoid receptor. In the midbrain dopaminergic groups A8-A10, moderately (A8, A9) to strongly (A10) glucocorticoid receptor-immunoreactive cells were found, ranging from 40 to 75% of the total population. In the hypothalamic dopaminergic cell groups, all the cells of groups A12 and A14, as well as the majority of the dopaminergic cells of the zona incerta (A13), were found to contain moderate to strong glucocorticoid receptor immunoreactivity, but none of the large dopaminergic cells of the posterior hypothalamus (A11) showed such immunoreactivity.     

Differences in serotonin receptor types I and II binding in the brain regions of rats with 2 types of hereditary arterial hypertension

Serotonin C1- and C2-receptor binding was studied in various segments of the brain in two strains of rats with hereditary arterial hypertension--spontaneously hypertensive (SHR) and hereditary stress-induced hypertensive rats (HSIHR). In the hypothalamus, there was a higher C1-receptor binding in HSIHR than in Wistar rats. SHR showed a higher C2-receptor binding in the cortex and C1-receptor one in the pons than did HSIHR. The changes found are suggestive of both similarities and differences in the serotonin receptor system in the two strains of rats with hereditary arterial hypertension.     

Castration affects male rat brain opiate receptor content

We previously reported that saturable stereospecific binding of [3H]-naltrexone in rat brain homogenates prepared from castrated male rats was greater than the corresponding binding in intact animals. We now report that we have replicated these results and that the difficulty of other investigators in observing these differences is due to methodological factors. Specifically, when samples were filtered individually and rapidly, differences between castrated and intact rats were maintained. The increase in binding was also observed when tissues were washed to remove endogenous opioids prior to incubation, when [3H]-naloxone was used as the ligand, and when various antagonists were used as displacers in the radioreceptor assay.     

近视患者两种角膜地形图系统检测值的对比分析

目的 探讨不同程度近视患者两种角膜地形图系统检测值差异.方法 抽取2009-02-06在广西视光中心就诊的45例近视患者,其中男25例,女20例;年龄18～39岁,屈光度-0.50～-9.00DS(等效球镜);采用Bausch&Lomb公司生产的Orbscan-Ⅱz角膜地形图系统和Zeiss公司生产的Humphrey ALTAS角膜地形图系统对所有患者进行检测,收集每位患者的两种角膜地形图数据模拟K值(Sim K's)、角膜散光(Axis)、最大值(Max.)及最小值(Min.).使用SPSS13.0统计软件采用配对t检验分别比较各检测值的差异.结果 两种角膜地形图系统检测值Sim K's、Axis、Max.、Min.均不相同,P值分别为0.037、0.004、0.072、0.000.Humphrey ALTAS所得的陡峭K(Max.)和平坦K(Min.)及角膜散光轴向均大于Orbscan-Ⅱz的陡峭K(Max.)和平坦K(Min.)及角膜散光轴向(Axis),而前者所测得的角膜散光度数即Sim K's值则小于Orbscan-Ⅱz角膜地形图系统的Sim K's值.结论 Humphrey ALTAS和Orbscan-Ⅱz两种角膜地形图系统的一致性较好.     

Differential regulation of type II corticosteroid receptor messenger ribonucleic acid expression in the rat anterior pituitary and hippocampus

The present study was designed to characterize the regulation of the type II corticosteroid receptor (GR) mRNA in two tissues involved in the control of the hypothalamic-pituitary-adrenal axis. We have used a solution hybridization/S1 nuclease protection assay to quantitate GR mRNA levels in the rat hippocampus and anterior pituitary after CRF, dexamethasone (DEX), or corticosterone (CORT) treatment. In general, hippocampal GR mRNA levels increased after removal of endogenous corticosteroids by surgical adrenalectomy and decreased in response to glucocorticoid treatment. More specifically, in the hippocampus 1) GR mRNA expression was decreased when adrenalectomized (ADX) animals were replaced with a relatively low dose of CORT, but not with a low dose of DEX; 2) acutely, CRF was more effective than DEX in decreasing the levels of GR mRNA in intact animals; however, under the same paradigm in ADX animals, DEX decreased the level of GR mRNA, whereas CRF was ineffective; and 3) in contrast to the decrease in GR mRNA levels observed after acute and low doses of glucocorticoid treatment, chronic treatment with either DEX or CORT did not change the level of hippocampal GR mRNA. These results suggest that in the hippocampus the decrease in GR mRNA expression after CRF treatment is probably via the release of glucocorticoids, and that this tissue is more sensitive to endogenous glucocorticoids than DEX. Anterior pituitary GR mRNA was differentially regulated compared with that in the hippocampus. In marked contrast to Gr mRNA in the hippocampus, ADX did not alter anterior pituitary GR mRNA expression, and glucocorticoid treatment led to an increase in GR mRNA levels. In the anterior pituitary 1) glucocorticoid treatment led to an increase in GR mRNA expression, when replaced with a relatively low dose of DEX, but not when replaced with a low dose of CORT; 2) acutely, neither CRF nor DEX altered levels of GR mRNA in intact animals; however, under the same paradigm DEX increased levels in ADX animals; and 3) chronic DEX or CORT treatment of intact animals elevated levels of anterior pituitary GR mRNA. In summary, these data have demonstrated tissue-specific regulation of GR mRNA in the hippocampus and anterior pituitary, which is dependent on both the dose and length of treatment and, in addition, on the glucocorticoid itself.     

A sexually dimorphic distribution pattern of the novel estrogen receptor G-protein-coupled receptor 30 in some brain areas of the hamster

The isolation of the G-protein-coupled receptor 30 (GPR30), an orphan membrane receptor unrelated to nuclear estrogen receptors (ERs), has become a key factor towards the unraveling of rapid estrogen action. This membrane receptor together with cellular signaling intermediaries, i.e., extracellular signal-dependent kinases 1 and 2, may promote neuronal proliferation and differentiation activities. In the present study, an evident gene expression pattern of GPR30 characterized postnatal 7 (young) and 60 (adult) days of age hamsters as shown by its heterogeneous mRNA distribution in hypothalamic, amygdalar and cerebellar areas of both sexes. In particular, most of the brain areas considered in the adult hamster plus only the amygdala and cerebellum of young animals behaved in a sexually dimorphic fashion. This similar pattern was also detected for the ERalpha and beta, as shown by the latter receptor prevailing in young and adult females, while the former predominated in young females. Even for the two kinases, a sexually dimorphic distribution was featured above all for young hamsters. Overall, the findings of the present study established a distinct expression pattern of the novel ER (GPR30) that may operate differently in some brain areas of the hamster and this may provide interesting insights regarding its probable neuroprotective role during the execution of some hibernating states, which are typical of our rodent model.