Stafford L. Lightman

Relaxin levels in ovum donation pregnancies.

STUDY OBJECTIVE To investigate the relative contributions of the corpus luteum (CL) and the placenta to the circulating levels of relaxin during pregnancy. PATIENTS Forty-one patients in whom pregnancy had been achieved by ovum donation. RESULTS Relaxin was not detected in the serum of 36 patients; in the remaining 5, although it was detectable, the levels were markedly reduced when compared with those in normal pregnancies. CONCLUSION These results demonstrate that the CL is essential for the maintenance of normal circulating levels of relaxin during pregnancy.

Role of catecholamines in mediating messenger RNA and hormonal responses to stress

The role of catecholamines in regulating the neuroendocrine stress response is controversial. We have investigated the effects of unilateral ventral noradrenergic bundle (VNAB) lesions on corticotrophin-releasing factor (CRF) and proenkephalin A mRNA responses in the parvocellular paraventricular nucleus (pPVN) to both physical and psychological stresses. We have also determined the effects of direct bilateral PVN lesions on CRF mRNA, plasma ACTH and corticosterone responses to psychological stress. 6-OHDA lesions whether to the VNAB or direct to the PVN did not result in any change in basal levels of CRF mRNA. Depletion of endogenous noradrenaline following unilateral lesions of the VNAB did not affect the CRF mRNA or the proenkephalin A mRNA response to stress. These data suggest that noradrenergic pathways are not involved in maintaining basal levels of CRF mRNA and that the noradrenergic input through the VNAB does not mediate the accumulation of CRF and proenkephalin A mRNAs in response to these stressors. Direct bilateral lesions to the PVN prevented the accumulation of CRF mRNA but not the ACTH and corticosterone responses to restraint stress. This suggests that monoamines are involved in the regulation of CRF mRNA through a mechanism independent of CRF-41 secretion.

Many peptides that are present in the external zone of the median eminence are not secreted into the hypophysial portal blood of sheep

Apart from the well recognized factors that are produced by the hypothalamus and secreted into hypophysial portal blood to regulate pituitary function, there is a range of neuropeptides that are present in the median eminence and could be secreted to serve a modulatory function. In this study we have collected hypophysial portal blood and jugular venous blood from sheep in an attempt to identify which of these putative modulatory peptides might be secreted from the median eminence. We have measured neuropeptide Y (NPY), substance P (SP), galanin (GAL), neurokinin A (NKA), peptide histidine isoleucine (PHI), vasoactive intestinal peptide (VIP), neurotensin (NT) and cholecystokinin (CCK). We also examined the sheep median eminence using immunohistochemistry for NPY, SP and GAL and determined degradation profiles of NPY, SP, GAL and NKA in portal and jugular plasma. In no instance did we find that levels of the above peptides were consistently higher in portal blood than in peripheral blood. In some cases levels of peptide were lower in portal plasma e.g. for NPY (6/10 sheep). In one experimental series SP levels in portal plasma were significantly (p < 0.05) lower than levels in jugular plasma but this was not found in another experimental series. Galanin levels were significantly (p < 0.01) lower in portal plasma compared to levels in jugular plasma. We conducted in vitro studies to determine whether or not the above peptides are selectively degraded in portal blood but were unable to show any differences between the rates of degradation in portal and jugular plasma. Immunohistochemistry revealed projections into the external zone of the median eminence for NPY, GAL and SP. This study shows that none of the above peptides are secreted into the hypophysial portal blood of sheep. For some peptides e.g. GAL, enzymes from the endothelial cells of the portal vessels may enhance degradation. Projections into the external zone of the median eminence of neuronal systems containing these peptides may serve to modulate the secretion of the well recognized release and inhibiting factors by acting on the neurosecretory terminals.

Altered Adrenocorticotropin, Corticosterone and Oxytocin Responses to Stress during Chronic Salt Load

We have investigated the effects of stress on the plasma levels of adrenocorticotropin (ACTH), corticosterone and oxytocin (OT) in rats given 2% saline to drink for 12 days. Plasma ACTH levels were markedly decreased from 972 +/- 165 pg/ml in the control animals on water to 349 +/- 114 pg/ml in animals given 2% saline to drink. Stress-induced release of ACTH observed in animals on water (controls = 972 +/- 165 pg/ml and stressed animals = 1,439 +/- 105 pg/ml) was completely abolished following 2% saline treatment for 12 days (controls = 349 +/- 114 pg/ml and stressed animals = 205 +/- 27 pg/ml). After 2% saline, plasma corticosterone levels were unaltered in control and stressed animals as compared to control animals on water in which stress increased plasma corticosterone from 28.8 +/- 7.9 to 99.5 +/- 8 ng/ml. In contrast to ACTH, the OT response to stress was intact in animals on 2% saline despite raised plasma OT in the control group due to the osmotic stimulus. Removal of the source of circulating glucocorticoids by adrenalectomy partially restored both basal levels of ACTH and the response to stress in animals on 2% saline. Our results demonstrate that 2% saline treatment activates an inhibitory mechanism over the release of ACTH.

Response of pituitary and spleen pro-opiomelanocortin mRNA, and spleen and thymus interleukin-1β mRNA to adjuvant arthritis in the rat

During development of adjuvant-induced arthritis (AA) in the rat, pituitary pro-opiomelanocortin (POMC) mRNA expression was increased. Pituitary POMC mRNA was much higher following adrenalectomy and AA. Spleen POMC mRNA also increased with a similar time kinetics, although the levels in the spleen were much lower than those in the pituitary. In control animals, spleen interleukin-1 beta (IL-1 beta mRNA) was undetectable, whereas AA led to the accumulation of IL-1 beta mRNA and the highest levels were seen in the adrenalectomised AA group. Thymic IL-1 beta expression was also increased in AA animals. These results suggest that AA leads to the activation of both the neuroendocrine and the immune systems and the interaction between these systems may play a role in this disease state.

IGF-II mRNA expression in LI human glioblastoma cell line parallels cell growth.

A human glioblastoma cell line was found to express in vitro mRNA transcripts specific for insulin-like growth factor-II (IGF-II) and growth-hormone releasing-hormone (GHRH). In the absence of gross morphological changes, retinoic acid reduced the growth rate without major change of IGF-II mRNA expression, while alpha-difluoromethylornithine produced a complete growth arrest and a sharp decrease of IGF-II mRNA expression. Both reagents increased the expression of GHRH mRNA. Also in this glioblastoma cell line, like other neuroectodermal tumours, IGF-II mRNA is expressed independently from GHRH and seems to be parallel to growth rate.

Signals from the hypothalamus to the pituitary during chronic immune responses

The question of whether stress is good or bad is a pertinent one in relation to pathology. A major component of the host response to stress is the activation of the hypothalamo-pituitary-adrenal (HPA) axis. The end point of this activation is the release of glucocorticoid steroid hormones from the adrenal cortex (predominantly corticosterone in the rat and cortisol in man). The importance of this system to the integrity of the individual can be demonstrated by investigating the extreme situation where the adrenal is removed or no longer functioning. This situation can be created surgically in experimental animals and occasionally in humans, although more frequently it occurs as a result of defective function of the adrenals due to autoimmune disease or tuberculosis leading to adrenal insufficiency (Addison’s disease). In patients with Addison’s disease minor challenges which might only produce mild symptoms in a normal individual can be life threatening. An interesting parallel to this can be seen following injection of the cytokine interleukin-1 (IL-1) or the immune stimulant lipopolysaccharide (LPS) an agent which produces flu-like symptoms for a few hours when given to humans. Doses which only result in “sickness behaviour” in adrenal-intact animals prove fatal in the absence of adrenal glands. In a number of animal models of immune-mediated diseases such as experimental allergic encephalomyelitis (EAE) and adjuvant-induced arthritis (AA) it has been demonstrated that in adrenalectomized animals following induction of the disease there is an earlier onset, an increased severity and, if left untreated, a fatal outcome. In all these cases treatment with steroids diminishes the severity of disease and prevents the fatal outcome. This chapter will draw together our current understanding of the mechanisms, particularly at the central level, underlying the activation of the HPA axis following immune activation. Much of our understanding of these mechanisms comes from studies in experimental animal models and this chapter will concentrate principally but not exclusively on the evidence from these.

Mechanisms of Norepinephrine Mediated Corticotropin-Releasing Factor-41 Release from Cultured Fetal Hypothalamic Cells

Catecholamines have been shown to activate hypothalamic corticotropin-releasing factor-41 (CRF) synthesis and release. In order to study the mechanisms involved, fetal hypothalamic cells were cultured and CRF release was measured by radioimmunoassay. Norepinephrine (NE) induced CRF release in a dose-dependent manner. Further studies were performed with a protein kinase C inhibitor, H-7(1-(5-isoquinolinesulfonyl)-2-methylpiperazine) and a protein kinase A inhibitor, IP-20. NE-stimulated CRF release was reduced by H-7 (5 and 50 microM) in a dose-dependent fashion, while 5 microM IP-20 resulted in a small but significant inhibition. Pretreatment of the cells for 15 h with 20 and 200 nM 12-O-tetradecanoylphorbol-13-acetate, which down-regulates protein kinase C activity, blocked the release of CRF in response to NE (1 microM), further supporting protein kinase C as a mediator for NE-activated CRF release. Pretreatment with 50 and 500 ng/ml pertussis toxin (15 h) resulted in a dose-dependent inhibition of NE-activated CRF release. Both dexamethasone and aldosterone at the concentrations of 1 microM reduced NE-induced CRF release. These results suggest that CRF can be released from hypothalamic neurons in response to NE through both protein kinase C- and protein kinase A-dependent mechanisms, and that pertussis toxin-sensitive G-proteins are also involved in this response. Furthermore, glucocorticoids and mineralocorticoids can reduce NE-activated CRF release from cultured hypothalamic cells.

Dexamethasone and Aldosterone Modulate Corticotropin-Releasing Factor-41 Release from Cultured Rat Fetal Hypothalamic Cells through Type II and Type I Corticosteroid Receptors Respectively

Dexamethasone and aldosterone inhibit hypothalamic corticotropin releasing factor-41 (CRF) release. The possible receptors through which these adrenal steroids affect CRF release were studied using rat fetal hypothalamic cell cultures. Neither the antimineralocorticoid RU 28318 nor the antiglucocorticoid RU 38486 alone had any effect on forskolin-stimulated CRF release. RU 38486 and RU 28318 however suppressed dexamethasone (1 microM)- and aldosterone (1 microM)-induced inhibition of forskolin (20 microM)-stimulated CRF release, respectively, suggesting that dexamethasone and aldosterone reduce CRF release through type II and type I corticosteroid receptors, respectively. RU 38486 had no effect on aldosterone-induced inhibition of forskolin-stimulated CRF release, nor did RU 28318 have any effect on dexamethasone-induced inhibition of forskolin-stimulated CRF release, indicating specificity of the binding of aldosterone with type I receptors, and dexamethasone with type II receptors in the hypothalamic cell cultures.