Splanchnic Nerve Transection Abolishes the Age-Dependent Increase of Neuropeptide Y-Like Immunoreactivity in Rat Adrenal Gland

Using an antiserum directed against porcine neuropeptide Y (NPY), a high concentration of NPY immunoreactivity (NPY-IR) was detected in rat adrenal gland. The level of NPY-IR in the adrenal gland was found to increase considerably with age. Biochemical characterization by reverse-phase HPLC indicated that this increase was due to accumulations of NPY and another molecular form of NPY-like immunoreactive peptide. Chronic denervation of the splanchnic nerve abolished this age-dependent increase of NPY-IR rat adrenal gland.     

Glucocorticoid receptor polymorphisms and haplotypes associated with chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is a significant public health problem of unknown etiology, the pathophysiology has not been elucidated, and there are no characteristic physical signs or laboratory abnormalities. Some studies have indicated an association of CFS with deregulation of immune functions and hypothalamic-pituitary-adrenal (HPA) axis activity. In this study, we examined the association of sequence variations in the glucocorticoid receptor gene (NR3C1) with CFS because NR3C1 is a major effector of the HPA axis. There were 137 study participants (40 with CFS, 55 with insufficient symptoms or fatigue, termed as ISF, and 42 non-fatigued controls) who were clinically evaluated and identified from the general population of Wichita, KS. Nine single nucleotide polymorphisms (SNPs) in NR3C1 were tested for association of polymorphisms and haplotypes with CFS. We observed an association of multiple SNPs with chronic fatigue compared to non-fatigued (NF) subjects (P < 0.05) and found similar associations with quantitative assessments of functional impairment (by the SF-36), with fatigue (by the Multidimensional Fatigue Inventory) and with symptoms (assessed by the Centers for Disease Control Symptom Inventory). Subjects homozygous for the major allele of all associated SNPs were at increased risk for CFS with odds ratios ranging from 2.61 (CI 1.05-6.45) to 3.00 (CI 1.12-8.05). Five SNPs, covering a region of approximately 80 kb, demonstrated high linkage disequilibrium (LD) in CFS, but LD gradually declined in ISF to NF subjects. Furthermore, haplotype analysis of the region in LD identified two associated haplotypes with opposite alleles: one protective and the other conferring risk of CFS. These results demonstrate NR3C1 as a potential mediator of chronic fatigue, and implicate variations in the 5' region of NR3C1 as a possible mechanism through which the alterations in HPA axis regulation and behavioural characteristics of CFS may manifest.     

Detection of disease-associated prion protein in the optic nerve and the adrenal gland of cattle with bovine spongiform encephalopathy by using highly sensitive immunolabeling procedures

A sensitive immunohistochemical procedure, the tyramide signal amplification (TSA) system, was applied to detect the localization of immunolabeled disease-associated prion protein (PrP(Sc)) in cattle affected with bovine spongiform encephalopathy (BSE). In this procedure, immunolabeling could be visualized in the optic nerve and the adrenal medulla. In the optic nerve, the dual immunofluorescent technique showed that the granular PrP(Sc) was occasionally detected in the astrocytes, microglia, and myelin sheath adjacent to the axon. Clustered PrP(Sc) was also scattered in association with microglial cells and astrocytes of the optic nerve. In the adrenal gland, PrP(Sc) immunolabeling was confined within the sympathetic nerve fibers and endings. The results suggest that (1) PrP(Sc) might centrifugally spread within and between glial cells and/or the non-axonal (also known as ad-axonal) region of nerve fibers, rather than the axonal and/or extracellular space pathway in the optic nerve, and (2) the sympathetic innervations might be important for the trafficking of BSE agent in the adrenal glands of cattle. This study also suggests that tyramide-based immunochemical analysis should be performed to detect immunolabeled PrP(Sc) in the extracerebral tissues of BSE-affected cattle.     

Structural motif-based homology modeling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation

Human CYP27A1 is a mitochondrial cytochrome P450, which is principally found in the liver and plays important roles in the biological activation of vitamin D(3) and in the biosynthesis of bile acids. We have applied a systematic analysis of hydrogen bonding patterns in 11 prokaryotic and mammalian CYP crystal structures to construct a homology-based model of CYP27A1. Docking of vitamin D(3) structures into the active site of this model identified potential substrate contact residues in the F-helix, the beta-3 sheet, and the beta-5 sheet. Site-directed mutagenesis and expression in COS-1 cells confirmed that these positions affect enzymatic activity, in some cases shifting metabolism of 1alpha-hydroxyvitamin D(3) to favor 25- or 27-hydroxylation. The results suggest that conserved hydrophobic residues in the beta-5 hairpin help define the shape of the substrate binding cavity and that this structure interacts with Phe-248 in the F-helix. Mutations directed toward the beta-3a strand suggested a possible heme-binding interaction centered on Asn-403 and a structural role for substrate contact residues Thr-402 and Ser-404.     

Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism

We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.     

Regulatory mechanism of the arginine vasopressin-enhanced green fluorescent protein fusion gene expression in acute and chronic stress

Various kinds of stress cause neuroendocrine responses such as corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) release from parvocellular division of the paraventricular nucleus (PVN) and activation of the hypothalamo-pituitary adrenal (HPA) axis. We examined the effects of acute and chronic stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using chronic salt loading as an osmotic stimulation, intraperitoneal administration of lipopolysaccharide (LPS) as acute inflammatory stress and adjuvant arthritis (AA) as chronic inflammatory/nociceptive stress. Salt loading caused a marked increase in the eGFP gene expression and eGFP fluorescence in the supraoptic nucleus, magnocellular division of the PVN and internal layer of the median eminence (ME). Administration of LPS caused increased fluorescence in parvocellular division of the PVN and external layer of the ME. AA rats revealed an increased expression of the eGFP gene and eGFP fluorescence in both magnocellular and parvocellular divisions of the PVN and both internal and external layers of the ME. On the other hand, the levels of the CRH gene expression in parvocellular division of the PVN were significantly decreased as AA developed, though plasma concentrations of corticosterone were significantly increased. These results indicate that AVP-eGFP transgenic rats enable the detection of changes in AVP expression more easily than by using procedures such as immunohistochemistry. We propose that AVP-eGFP transgenic rats represent a useful animal model for further understanding of the physiology of AVP expression in the hypothalamo-pituitary system under various physiological conditions, including various kinds of stress.     

Stress-protective effect of the KKRR synthetic peptide corresponding to the 15–18 sequence of human adrenocorticotropic hormone

The activity of the KKRR synthetic peptide corresponding to the 15-18 sequence of human adrenocorticotropic hormone (ACTH) and its analogues: KKKK, RRRR, RRKK, kKRR, KkRR, KKrR, and KKRr (amino acid residues of the D configuration are designated by small letters), was studied in vivo on rats under cold and heat shock. Intranasal administration of the KKRR peptide at doses of 2–10 μg/animal 1 day before the shock was found to prevent a dramatic increase in the level of corticosterone in rat adrenal glands and blood plasma caused by the temperature effect. Amino acid substitutions in the KKRR peptide were shown to result in abrupt decrease in its activity. The peptide analogues exhibit a low stress-protective activity and had a low affinity for the ACTH receptor.     

Development of a steroidogenic factor 1/Cre transgenic mouse line

The Cre-loxP strategy provides an approach to disrupt genes in specific tissues and/or cell types, circumventing lethality associated with global knockouts or secondary effects due to gene inactivation at other sites. A critical component is the development of transgenes that target Cre expression to specific cell types. Here, we describe the use of bacterial artificial chromosome (BAC) transgenesis to target Cre expression to tissues that express steroidogenic factor 1 (SF-1, officially designated Nr5a1). Consistent with the SF-1 expression pattern, the SF-1 BAC directed Cre expression to the somatic cells of the gonads, the adrenal cortex, the anterior pituitary, the spleen, and the ventromedial hypothalamic nucleus. This transgene provides a powerful tool to inactivate genes of interest in these tissues.     

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

beta-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid beta-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of non-opioid beta-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 +/- 2.0 nM, Bmax1 = 40.7 +/- 2.3 pmol/mg protein; Kd2 = 0.25 +/- 0.01 micro M, Bmax2 = 187.8 +/- 9.4 pmol/mg protein). beta-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 +/- 9.2 nM); naloxone, [Met5]enkephalin, and alpha- and gamma-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10(-9)-10(-6) M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 micro g/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

Molecular pathogenesis of lipoid adrenal hyperplasia and adrenal hypoplasia congenita

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty.

X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.