The effects of fetal adrenalectomy at 110 days gestational age on AVP and CRH mRNA expression in the hypothalamic paraventricular nucleus of the ovine fetus.

AVP and CRH produced in the parvocellular neurons of the paraventricular nucleus (PVN) have both been implicated in the regulation of anterior pituitary ACTH synthesis and secretion. In sheep, fetal ACTH secretion increases around 120 days gestational age (dGA). Little is known about adrenal regulation of AVP and CRH immediately prior to this critical period. We investigated the effects of adrenalectomy and subsequent cortisol (F) administration on PVN AVP and CRH mRNA in the fetal sheep PVN at 109-125 dGA. At 109-113 dGA, fetal sheep adrenals were removed (ADX)(n = 8); or sham surgery performed (CONT)(n = 4). From day 6 post ADX, maternal plasma cortisol and fetal plasma ACTH and cortisol levels were determined daily by radioimmunoassays. From day 7 post ADX, cortisol (4 micrograms/min) was continuously infused intravenously to four ADX fetuses (ADX + F). Fetal hypothalami were collected at 123-125 dGA, and studied by in-situ hybridization and quantitative autoradiography for AVP and CRH mRNA. Plasma cortisol levels remained low in CONT and ADX fetuses (< 4.9 ng/ml), while during cortisol infusion to ADX + F fetuses, plasma F increased to 16.4 +/- 2.2 and 22.3 +/- 3.2 ng/ml (mean +/- S.E.M.) on day 10 and 13, respectively. Plasma ACTH levels increased significantly in ADX compared with CONT fetuses. This ACTH increase was completely suppressed in ADX + F fetuses. AVP mRNA abundance in the whole PVN was the same in all three groups, however, a separate analysis of AVP mRNA abundance in parvocellular and magnocellular regions of the PVN revealed that AVP mRNA in the parvocellular PVN showed a significant increase in ADX and suppression in ADX + F fetuses when compared to CONT. AVP mRNA in the magnocellular PVN remained unchanged. PVN CRH mRNA expression was augmented in ADX and suppressed in ADX + F when compared to CONT fetuses. We conclude that in fetal sheep at 109-125 dGA: AVP and CRH mRNA abundance in the parvocellular region of the PVN are increased by adrenalectomy and that cortisol inhibits this increase.

Changes in adrenocorticotropin and cortisol responsiveness after repeated partial umbilical cord occlusions in the late gestation ovine fetus.

Despite many studies reporting fetal ACTH and cortisol (F) responses to acute fetal hypoxemia induced by several methods, effects of repeated short-term fetal hypoxia produced by umbilical cord occlusion (UCO) on ACTH and F are unknown. We examined fetal ACTH and F responses to repeated, controlled, 50% reductions in common umbilical arterial blood flow (CUBF) produced by an inflatable cord occluder. Ten sheep fetuses were instrumented at 123-128 days gestation (dGA) with arterial, venous, and amniotic catheters. A common umbilical artery transit-time ultrasound flow probe was implanted to measure CUBF. An inflatable occluder was placed around the proximal portion of the umbilicus. In five fetuses (group I) at 131 +/- 1 dGA (mean +/- SEM), 12 UCOs (CUBF reduced by 50%), each lasting 5 min separated by 15 min recovery, were performed. Changes in fetal arterial blood gases, pH and plasma ACTH, and F concentrations were determined before, during, and after the 1st, 6th, and 12th UCOs. Sham experiments were conducted on the other five fetuses at 130 +/- 1 dGA (group II). In group I, CUBF decreased to 49 +/- 1% (mean +/- SEM of 12 UCOs). After each UCO, CUBF returned to baseline within 5 min. A modest fall in fetal arterial PO2 and arterial pH (21.2 +/- 0.2 to 16.8 +/- 0.2 mmHg and 7.33 +/- 0 to 7.29 +/- 0, respectively) and a mild increase in fetal PaCO2 (49.9 +/- 0.5 to 54.9 +/- 0.4 mmHg; mean +/- SEM of 12 UCOs) occurred with each UCO. Whereas preocclusion fetal ACTH concentrations increased by the 12th UCO, F remained unchanged. Fetal ACTH increased after the 1st, 6th, and 12th UCOs. Fetal F increased after the 1st and 6th UCOs but not after the 12th UCO. Fetal plasma ACTH and F remained unchanged throughout the experiments in group II fetuses. We conclude that: 1) partial reductions in CUBF induce significant activation of the fetal anterior pituitary-adrenocortical axis in late-gestation fetal sheep; 2) after repeated UCOs, fetal ACTH responsiveness is maintained, but fetal F responses become attenuated.

Implication of retinoic acid receptor gamma in squamous differentiation and response to retinoic acid in head and neck SqCC/Y1 squamous carcinoma cells.

Nuclear retinoic acid receptors are considered to be the mediators of most of the effects of retinoic acid (RA) on gene expression. To explore the role of RA receptor gamma (RARgamma) in the growth and differentiation of SqCC/Y1 head and neck squamous carcinoma cells, they were transfected with RARgamma sense and antisense expression vectors and stable clones in which RARgamma expression was either increased or blocked were isolated. The growth inhibitory effect of RA in monolayer culture was enhanced in the sense transfectants and decreased in the antisense ones. The ability to form colonies in semisolid medium was abolished by RA in the sense transfectants, while the antisense transfected clones exhibited heterogeneous responses. The expression the squamous differentiation markers cytokeratin K1 transglutaminase type I, and involucrin was increased in the absence of exogenous retinoid in a sense transfected clone and decreased in an antisense transfected clone. RA suppressed squamous differentiation in both types of transfectant. The expression of epidermal growth factor receptor (EGFR) was higher in the antisense and lower in the sense transfectant than in the parental cells and RA decreased EGFR mRNA level in the parental and the sense transfectant but not in the antisense transfectant. In addition activator protein-1 (AP-1) binding activity was decreased by the RA treatment in the sense clones, but not in the antisense ones. These results suggest that RARgamma mediates the effects of RA on the cell growth both in monolayer culture and in semisolid medium possibly through AP-1 suppression.

Appearance of inducible nitric oxide synthase in the rat central nervous system after rabies virus infection and during experimental allergic encephalomyelitis but not after peripheral administration of endotoxin.

The cellular localization of inducible (iNOS) and constitutive (cNOS) nitric oxide synthase was studied in rats by immunocytochemical techniques involving specific iNOS and cNOS directed antibodies and by NADPH-diaphorase histochemistry. Paraformaldehyde-fixed vibratome sections of brains and cryostat sections of peripheral lymph nodes were studied of rats treated with endotoxin (2.5 micrograms/kg or 2.5 mg/kg i.v.), rats infected with rabies virus, and rats exposed to experimental allergic encephalomyelitis (EAE). Endotoxin-treated animals showed no appearance of immunoreactive iNOS (ir-iNOS) cells in the brain with the exception of a few microglial cells near the median eminence and some meningeal macrophages. In the same animals however, iNOS-immunoreactive cells were found in peripheral lymph nodes. Neurons that stain positive for cNOS and for NADPH-diaphorase could be observed in brains of control as well as of endotoxin-treated animals with a similar distribution and staining intensity. In contrast, animals that had been infected with rabies virus or subjected to EAE, showed the appearance of ir-iNOS-positive cells in several brain areas. These cells are located near blood vessels and lesion sites. The majority of these cells are GSA-I-B4 isolectin-positive and therefore are likely to represent macrophages. Our data suggest that increased production of nitric oxide may play a role in the altered brain functions in rabies-infected and EAE rats. On the contrary, increased nitric oxide production is probably not involved in the non-specific symptoms of sickness induced by endotoxin.

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice.

The presence of cytokines, prostaglandins and lipocortin-1 was investigated in terminally affected mice in two models of scrapie. There was marked induction of glial interleukin-1 beta, tumour necrosis factor alpha, prostaglandin E2, prostaglandin F2 alpha and lipocortin-1 immunoreactivity in those areas of the brain showing the characteristic vacuolation of scrapie. A comparison of these staining patterns with those of GFAP and F4/80 showed that their expression occurred predominantly in astrocytes. It is possible that cytokines play a significant role in the pathogenesis of neurodegeneration in scrapie.