The origin of glucocorticoid hormone oscillations.

Oscillating levels of adrenal glucocorticoid hormones are essential for optimal gene expression, and for maintaining physiological and behavioural responsiveness to stress. The biological basis for these oscillations is not known, but a neuronal "pulse generator" within the hypothalamus has remained a popular hypothesis. We demonstrate that pulsatile hypothalamic activity is not required for generating ultradian glucocorticoid oscillations. We show that a constant level of corticotrophin-releasing hormone (CRH) can activate a dynamic pituitary-adrenal peripheral network to produce ultradian adrenocorticotrophic hormone and glucocorticoid oscillations with a physiological frequency. This oscillatory response to CRH is dose dependent and becomes disrupted for higher levels of CRH. These data suggest that glucocorticoid oscillations result from a sub-hypothalamic pituitary-adrenal system, which functions as a deterministic peripheral hormone oscillator with a characteristic ultradian frequency. This constitutes a novel mechanism by which the level, rather than the pattern, of CRH determines the dynamics of glucocorticoid hormone secretion.

Acute stress causes rapid synaptic insertion of Ca2+ -permeable AMPA receptors to facilitate long-term potentiation in the hippocampus.

The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca2+ -permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.

Ultradian corticosterone secretion is maintained in the absence of circadian cues.

Plasma levels of corticosterone exhibit both circadian and ultradian rhythms. The circadian component of these rhythms is regulated by the suprachiasmatic nucleus (SCN). Our studies investigate the importance of the SCN in regulating ultradian rhythmicity. Two approaches were used to dissociate the hypothalamic-pituitary-adrenal (HPA) axis from normal circadian input in rats: (i) exposure to a constant light (LL) environment and (ii) electrolytic lesioning of the SCN. Blood was sampled using an automated sampling system. As expected, both treatments resulted in a loss of the circadian pattern of corticosterone secretion. Ultradian pulsatile secretion of corticosterone however, was maintained across the 24 h in all animals. Furthermore, the loss of SCN input revealed an underlying relationship between locomotor and HPA activity. In control (LD) rats there was no clear correlation between ultradian locomotor activity and hormone secretion, whereas, in LL rats, episodes of ultradian activity were consistently followed by periods of increased pulsatile hormone secretion. These data clearly demonstrate that the ultradian rhythm of corticosterone secretion is generated through a mechanism independent of the SCN input, supporting recent evidence for a sub-hypothalamic pulse generator.

GHRH receptor-targeted botulinum neurotoxin selectively inhibits pulsatile GH secretion in male rats.

Botulinum neurotoxin is a potent inhibitor of acetylcholine secretion and acts by cleaving members of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor family, which are critical to exocytotic vesicular secretion. However, the potential of botulinum neurotoxin for treating secretory disease is limited both by its neural selectivity and the necessity for direct injection into the relevant target tissue. To circumvent these limitations, a technology platform called targeted secretion inhibitors (TSIs) is being developed. TSIs are derived from botulinum neurotoxin but are retargeted to specific cell types to inhibit aberrant secretion. A TSI called qGHRH-LHN/D, with a GHRH receptor targeting domain and designed to specifically inhibit pituitary somatotroph GH release through cleavage of the N-ethylmaleimide-sensitive factor-attachment protein receptor protein, vesicle-associated membrane protein (VAMP), has recently been described. Here we show this TSI activates GHRH receptors in primary cultured rat pituicytes is internalized into these cells, depletes VAMP-3, and inhibits phorbol-12-myristate-13-acetate-induced GH secretion. In vivo studies show that this TSI, but not one with an inactive catalytic unit, produces a dose-dependent inhibition of pulsatile GH secretion, thus confirming its mechanism of action through VAMP cleavage. Selectivity of action has been shown by the lack of effect of this TSI in vivo on secretion from thyrotrophs, corticotrophs, and gonadotrophs. In the absence of suitable in vivo models, these data provide proof of concept for the use of somatotroph-targeted TSIs in the treatment of acromegaly and moreover raise the potential that TSIs could be used to target other diseases characterized by hypersecretion.

ACTH-dependent ultradian rhythm of corticosterone secretion.

The activity of the hypothalamic-pituitary-adrenal axis is characterized by an ultradian pulsatile pattern of glucocorticoid secretion. Despite increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, little is known about the mechanism underlying the pulsatility of glucocorticoid synthesis and release. We tested the hypothesis that pulsatile ACTH release is critical for optimal adrenocortical function. Hypothalamic-pituitary-adrenal activity was suppressed by oral methylprednisolone, and ACTH (4 ng/h) was infused for 24h either as a constant infusion or in 5-min pulses at hourly intervals. Control methylprednisolone-treated rats had very low plasma corticosterone (CORT) levels with undetectable pulses and also had steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc) heteronuclear RNA levels reduced to approximately 50% of that seen in untreated animals. Pulsatile but not constant ACTH infusion restored pulsatile CORT secretion, and this was accompanied by parallel rises in StAR and P450scc heteronuclear RNA levels during the rising phase of the CORT pulse, which then fell during the falling phase. The pulsatile pattern of StAR and P450scc was paralleled by pulsatile transcription of the melanocortin 2 receptor accessory protein. Pulsatile ACTH activation of the adrenal cortex not only is critical for the secretion of CORT but also induces episodic transcription of the rate-limiting enzymes necessary for physiological steroidogenic responses. Because constant infusion of identical amounts of ACTH did not activate CORT secretion, pulsatility of ACTH provides a more effective signaling system for the activation of adrenocortical activity.

Different degrees of somatotroph ablation compromise pituitary growth hormone cell network structure and other pituitary endocrine cell types.

We have generated transgenic mice with somatotroph-specific expression of a modified influenza virus ion channel, (H37A)M2, leading to ablation of GH cells with three levels of severity, dependent on transgene copy number. GH-M2(low) mice grow normally and have normal-size pituitaries but 40-50% reduction in pituitary GH content in adult animals. GH-M2(med) mice have male-specific transient growth retardation and a reduction in pituitary GH content by 75% at 42 d and 97% by 100 d. GH-M2(high) mice are severely dwarfed with undetectable pituitary GH. The GH secretory response of GH-M2(low) and GH-M2(med) mice to GH-releasing peptide-6 and GHRH was markedly attenuated. The content of other pituitary hormones was affected depending on transgene copy number: no effect in GH-M2(low) mice, prolactin and TSH reduced in GH-M2(med) mice, and all hormones reduced in GH-M2(high) mice. The effect on non-GH hormone content was associated with increased macrophage invasion of the pituitary. Somatotroph ablation affected GH cell network organization with limited disruption in GH-M2(low) mice but more severe disruption in GH-M2(med) mice. The remaining somatotrophs formed tight clusters after puberty, which contrasts with GHRH-M2 mice with a secondary reduction in somatotrophs that do not form clusters. A reduction in pituitary beta-catenin staining was correlated with GH-M2 transgene copy number, suggesting M2 expression has an effect on cell-cell communication in somatotrophs and other pituitary cell types. GH-M2 transgenic mice demonstrate that differing degrees of somatotroph ablation lead to correlated secondary effects on cell populations and cellular network organization.

Stress responsiveness varies over the ultradian glucocorticoid cycle in a brain-region-specific manner.

Glucocorticoid hormones are released in rapid hourly hormone bursts by the adrenal gland. These ultradian oscillations are fundamental to hypothalamic-pituitary-adrenal activity and transcriptional regulation of glucocorticoid responsive genes. The physiological relevance of glucocorticoid pulsatility is however unknown. Using a novel automated infusion system, we artificially created different patterns (modulating pulse amplitude) of corticosterone (cort). Identical amounts of cort either in constant or in hourly pulses were infused into adrenalectomized rats. At the end of the infusion period, either during rising or falling concentrations of a cort pulse, animals were exposed to 99 dB noise stress (10 min). Pulsatile cort infusion led to a differential stress response, dependent on the phase of the pulse during which the stress was applied. Although constant administration of cort resulted in a blunted ACTH response to the stressor, a brisker response occurred during the rising phase of plasma cort than during the falling phase. This phase-dependent effect was also seen in the behavioral response to the stressor, which was again greater during the rising phase of each cort pulse. Within the brain itself, we found differential C-fos activation responses to noise stress in the pituitary, paraventricular nucleus, amygdala, and hippocampus. This effect was both glucocorticoid pulse amplitude and phase dependent, suggesting that different stress circuits are differentially responsive to the pattern of glucocorticoid exposure. Our data suggest that the oscillatory changes in plasma glucocorticoid levels are critical for the maintenance of normal physiological reactivity to a stressor and in addition modulate emotionality and exploratory behavior.

Reversal of the hypothalamo-pituitary-adrenal response to oestrogens around puberty.

The neuroendocrine gender dimorphism that begins during perinatal development is completed during puberty. We have previously described how the perinatal gonadal steroids programme hypothalamic-pituitary-adrenal (HPA) activity in adulthood and we now assess the role of peripubertal ovarian hormones. Prepubertal females were treated subcutaneously with either cholesterol or 17beta-oestradiol and their pituitary-adrenal activity was assessed 5 days later. Oestradiol suppressed the ACTH and corticosterone responses to restraint stress in the prepubertal female. Furthermore, groups of female rats were ovariectomised (OVX) either before or after puberty and adult animals were subsequently treated with subcutaneous implants containing either 17beta-oestradiol or cholesterol. Corticosterone pulsatility was assessed using an automated blood sampling system to collect blood from freely moving animals at 10 min intervals over 24 h. Oestradiol administered to adults that had been OVX either pre- or post-pubertally displayed a significantly higher mean corticosterone level as well as increased pulse frequency and pulse amplitude compared with cholesterol treated controls. These data demonstrate a reversal in the effect of oestrogens on HPA axis activity over the time of puberty with inhibitory effects prepubertally and stimulatory actions after puberty and imply an ovarian steroid-independent mechanism of pubertal maturation of HPA sensitivity to oestrogens.

Sequence and structural duality: designing peptides to adopt two stable conformations.

To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intramolecularly disulfide bridged state. Using an iterative process, we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally, for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.