Greti Aguilera

Hormonal regulation of peptide receptors and target cell responses

Regulation of plasma membrane receptors for peptide hormones by the prevailing ligand concentration often causes altered target cell function. Receptor number is determined by hormone-induced changes in membrane conformation, irreversible ligand binding, and processing of ligand–receptor complexes during hormone action.

Regulation of Pituitary ACTH Secretion during Chronic Stress

Maintenance of adequate levels of response of the hypothalamic-pituitary-adrenal axis during chronic stress is important for survival. Three basic patterns of response can be identified depending on the type of stress: (a) desensitization of ACTH responses to the sustained stimulus, but hyperresponsiveness to a novel stress despite elevated plasma glucocorticoid levels, as occurs in physical-psychological paradigms; (b) no desensitization of ACTH response to the repeated stimulus and hyperresponsiveness to a novel stress, as occurs during repeated painful stress and insulin hypoglycemia; and (c) small and transient increases in ACTH, but sustained elevations of plasma corticosterone and diminished ACTH responses. The level of response of the pituitary corticotroph is determined by differential regulation of the hypothalamic regulators, corticotropin-releasing hormone (CRH) and vasopressin (VP), and the sensitivity of the negative glucocorticoid feedback. While osmotic stimulation increases VP expression in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei of the hypothalamus, chronic stress paradigms with high pituitary responsiveness are associated with activation of CRH and CRH/VP parvicellular neurons of the PVN, predominantly of the VP-containing population. While moderate increase of CRH output is important for stimulation of POMC transcription, the increase of the VP:CRH secretion ratio appears to be important in maintaining the secretory capacity of the pituitary corticotroph during chronic stimulation. Decreased sensitivity of the glucocorticoid feedback, probably due to interaction of glucocorticoid receptors with transcription factors induced by CRH and VP, is critical for the maintenance of ACTH responses in the presence of elevated plasma glucocorticoid levels during chronic stress. Although both CRH and VP receptors are activated and undergo regulatory variations during chronic stress, only the changes in VP receptor levels are parallel to the changes in pituitary ACTH responsiveness. The inhibitory effect of chronic osmotic stimulation on ACTH secretion in spite of high circulating levels of VP is probably the result of diminished activity of parvicellular PVN neurons and downregulation of pituitary VP receptors. Although the exact interaction between regulatory factors and the molecular mechanisms controlling the sensitivity of the corticotroph during adaptation to chronic stress remain to be determined, it is clear that regulation of the proportional secretion of CRH and VP in the PVN, modulation of pituitary VP receptors, and the sensitivity to feedback inhibition play a critical role.

Vasopressinergic regulation of the hypothalamic-pituitary-adrenal axis : implications for stress adaptation

In addition to its role on water conservation, vasopressin (VP) regulates pituitary ACTH secretion by potentiating the stimulatory effects of corticotropin releasing hormone (CRH). The pituitary actions of VP are mediated by plasma membrane receptors of the V1b subtype, coupled to calcium-phospholipid signaling systems. VP is critical for adaptation of the hypothalamic-pituitary-adrenal (HPA) axis to stress as indicated by preferential expression of VP over CRH in parvocellular neurons of the hypothalamic paraventricular nucleus, and the upregulation of pituitary VP receptors during stress paradigms associated with corticotroph hyperresponsiveness. V1b receptor mRNA levels and coupling of the receptor to phospolipase C are stimulated by glucocorticoids, effects which may contribute to the refractoriness of VP-stimulated ACTH secretion to glucocorticoid feedback. The data suggest that vasopressinergic regulation of the HPA axis is critical for sustaining corticotroph responsiveness in the presence of high circulating glucocorticoid levels during chronic stress.

Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy

Specific receptors for corticotropin releasing factor (CRF) were identified in two functionally distinct systems within the brain, the cortex and the limbic system. Autoradiographic mapping of the CRF receptors in the brain revealed high binding density throughout the neocortex and cerebellar cortex, subiculum, lateral septum, olfactory tract, bed nucleus of the stria terminalis, interpeduncular nucleus and superior colliculus. Moderate to low binding was found in the hippocampus, nucleus accumbens, claustrum, nucleus periventricularis thalamus, mammillary bodies, subthalamic nucleus, periaqueductal grey, locus coeruleus and nucleus of the spinal trigeminal tract. As in the anterior pituitary gland, CRF receptors in the brain were shown to be coupled to adenylate cyclase. However, in contrast to the marked decrease in CRF receptors observed after adrenalectomy in the anterior pituitary gland, CRF receptor concentration in the brain and pars intermedia of the pituitary was unchanged. The presence of CRF receptors in areas involved in the control of hypothalamic and autonomic nervous system functions is consistent with the major role of CRF in the integrated response to stress.

CRF receptor regulation and sensitization of ACTH responses to acute ether stress during chronic intermittent immobilization stress

The relationship between corticotropin releasing factor (CRF) receptors and pituitary-adrenal responses was determined after chronic intermittent immobilization (2.5 h restraint/day) to examine the hypothesis that CRF receptor regulation is involved in the sensitization of the pituitary-adrenocortical axis to novel stimuli during repeated stress. Following the 11-fold stimulation of ACTH secretion on the first day of restraint stress, a desensitization of the pituitary ACTH response to immobilization was observed over the next 9 days of chronic intermittent stress. In contrast, the magnitude of the restraint-stimulated release of corticosterone on the 2nd and 4th day of stress was similar to the day 1 adrenocortical response. Furthermore, the significant stimulation of corticosterone secretion by restraint stress persisted to the 16th day of immobilization (P less than 0.001), even though significant increases in plasma ACTH were absent. The concentration of anterior pituitary CRF receptors was unchanged after a single period of restraint; however, a down-regulation of anterior pituitary CRF receptors was observed following 4 days (P less than 0.001) and 10 days (P less than 0.005) of repeated immobilization stress. CRF receptors in the olfactory bulb were unchanged following acute or chronic restraint stress, consistent with previous observations that brain CRF receptors are neither changed by adrenalectomy, glucocorticoid administration, nor 18-48 h of continuous restraint stress. The concentration of CRF receptors in the intermediate lobe of the pituitary also was not influenced by immobilization stress.(ABSTRACT TRUNCATED AT 250 WORDS)

The Role of mPer2 Clock Gene in Glucocorticoid and Feeding Rhythms

The circadian clock synchronizes the activity level of an organism to the light-dark cycle of the environment. Energy intake, as well as energy metabolism, also has a diurnal rhythm. Although the role of the clock genes in the sleep-wake cycle is well characterized, their role in the generation of the metabolic rhythms is poorly understood. Here, we use mice deficient in the clock protein mPer2 to study how the circadian clock regulates two critical metabolic rhythms: glucocorticoid and food intake rhythms. Our findings indicate that mPer2-/- mice do not have a glucocorticoid rhythm even though the corticosterone response to hypoglycemia, ACTH, and restraint stress is intact. In addition, the diurnal feeding rhythm is absent in mPer2-/- mice. On high-fat diet, they eat as much during the light period as they do during the dark period and develop significant obesity. The diurnal rhythm of neuroendocrine peptide alphaMSH, a major effector of appetite control, is disrupted in the hypothalamus of mPer2-/- mice even though the diurnal rhythm of ACTH, the alphaMSH precursor, is intact. Peripheral injection of alphaMSH, which has been shown to enter the brain, restored the feeding rhythm and induced weight loss in mPer2-/- mice. These findings emphasize the requirement of mPer2 in appetite control during the inactive period and the potential role of peripherally administered alphaMSH in restoring night-day eating pattern in individuals with circadian eating disorders such as night-eating syndrome, which is also associated with obesity.

Stress‐Specific Regulation of Corticotropin Releasing Hormone Receptor Expression in the Paraventricular and Supraoptic Nuclei of the Hypothalamus in the Rat

Corticotropin releasing hormone (CRH), a major regulator of pituitary ACTH secretion, also acts as a neurotransmitter in the brain. To determine whether CRH is involved in the regulation of hypothalamic function during stress, CRH receptor binding and CRH receptor mRNA levels were studied in the hypothalamus of rats subjected to different stress paradigms: immobilization, a physical‐psychological model; water deprivation and 2% saline intake, osmotic models; and i.p. hypertonic saline injection, a combined physical‐psychological and osmotic model. In agreement with the distribution of CRH receptor binding in the brain, in situ hybridization studies using 35S‐labeled cRNA probes revealed low levels of CRH receptor mRNA in the anterior hypothalamic area, which were unaffected after acute or chronic exposure to any of the stress paradigms used. Under basal conditions, there was no CRH binding or CRH receptor mRNA in the supraoptic (SON) or paraventricular (PVN) nuclei. However, 2 h after the initiation of acute immobilization, CRH receptor mRNA hybridization became evident in the parvicellular division of the PVN, with levels substantially increasing from 2 to 4 h, decreasing at 8 h and disappearing by 24 h. Identical hybridization patterns of CRH receptor mRNA were found in the parvicellular PVN after repeated immobilization; levels were similar to those after 2 h single stress following immobilization at 8‐hourly intervals for 24 h (3 times), and very low, but clearly detectable 24 h after 8 or 14 days daily immobilization for 2 h. On the other hand, water deprivation for 24 or 60 h and intake of 2% NaCI for 12 days induced expression of CRH receptor mRNA in the SON and magnocellular PVN, but not in the parvicellular pars of the PVN. Both parvicellular and magnocellular hypothalamic areas showed CRH receptor mRNA following i.p. hypertonic saline injection, single (4 h after) or repeated at 8‐hourly intervals for 24 h (3 injections), or one injection daily for 8 or 14 days. Consistent with the expression of CRH receptor mRNA, autoradiographic studies showed binding of 125I‐Tyr‐oCRH in the parvicellular division of the PVN after immobilization; in the magnocellular division of the PVN after osmotic stimulation, and in the PVN and SON after i.p. hypertonic saline injection. The data show that stress‐specific activation of the parvicellular and magnocellular systems is associated with CRH receptor expression, and suggest a role for CRH in the autoregulation of hypothalamic function.

Properties and regulation of high-affinity pituitary receptors for corticotropin-releasing factor

Specific receptors for corticotropin-releasing factor (CRF) were identified in the rat anterior pituitary gland by binding studies with 125I-Tyr-CRF. Binding of the labeled CRF analog to pituitary particles was rapid and temperature-dependent, and reached steady state within 45 min at 22 degrees C. The CRF binding sites were saturable and of high affinity, with dissociation constant (Kd) of 0.76 X 10(-9) M. Pituitary binding of 125I-Tyr-CRF was inhibited by CRF, Tyr-CRF and the active 15-41 fragment of CRF, but not by the inactive 21-41 CRF fragment and unrelated peptides. The binding-inhibition potencies of the CRF peptides were similar to their activities as stimuli of adrenocorticotropic hormone (ACTH) release. The high-affinity CRF sites were markedly reduced in adrenalectomized rats, and this change was reversed by dexamethasone treatment. These data indicate that the high-affinity CRF sites demonstrated in the anterior pituitary are the functional receptors which mediate the stimulatory action of the peptide on ACTH release, and that CRF receptors are down-regulated during increased secretion of the hypothalamic hormone.

Direct Regulation of Hypothalamic Corticotropin-Releasing-Hormone Neurons by Angiotensin II

The possible role of angiotensin II (AII) in the control of the hypothalamic-pituitary-adrenal (HPA) axis was studied in the rat by examining the regulation and cellular localization of AII receptors in the paraventricular nucleus (PVN) of the hypothalamus and the effect of AII on corticotropin-releasing hormone (CRH) and vasopressin (VP) mRNA levels. In situ hybridization studies using cRNA 35S-labelled probes showed that while type 1 AII receptor (AT1) mRNA levels were high in the periventricular and parvicellular pars of the PVN, only very low levels were present in the magnocellular pars. A similar distribution of AT1 receptor binding in the periventricular, parvicellular and magnocellular divisions of the PVN was observed in autoradiographic studies in hypothalamic sections labelled with 125I[Sar1,Ile8]AII. In addition, AII receptor binding was clearly evident in nerve fibers adjacent to the PVN. Double-labelling hybridization using digoxigenin-labelled CRH, VP and oxytocin probes and 35S-labelled AT1 receptor cRNA probes showed AT1 receptor mRNA in cells stained for CRH mRNA, but not in VP or oxytocin cells. Four hours after a single intracerebroventricular (i.c.v.) injection of 50 ng AII in conscious rats, CRH mRNA levels in the PVN were increased by 43%, similar to the increases observed following acute stress by intraperitoneal (i.p.) injection of 1.5 M NaCl (76%). On the other hand, while i.p. hypertonic saline injection increased VP mRNA levels by 29% in the PVN and by 32% in the supraoptic nucleus, i.c.v. AII injection had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain angiotensin II modulates sympathoadrenal and hypothalamic pituitary adrenocortical activation during stress.

Angiotensin II (Ang II) type‐1 (AT1) receptors are present in areas of the brain controlling autonomic nervous activity and the hypothalamic‐pituitary‐adrenal (HPA) axis, including CRH cells in the hypothalamic paraventricular nucleus (PVN). To determine whether brain AT1 receptors are involved in the activation of the HPA axis and sympathetic system during stress, we studied the effects of acute immobilization stress on plasma catecholamines, ACTH and corticosterone, and mRNA levels of CRH and CRH receptors (CRH‐R) in the PVN in rats under central AT1 receptor blockade by the selective antagonist, Losartan. While basal levels of epinephrine, norepinephrine and dopamine in plasma were unaffected 30 min after icv injection of Losartan (10 μg), the increases after 5 and 20 min stress were blunted in Losartan treated rats (P<0.05 for norepinephrine, and P<0.01 for epinephrine and dopamine, vs controls). Basal or stress‐stimulated plasma ACTH and corticosterone levels were unaffected by icv Losartan treatment. Using in situ hybridization studies, basal levels of CRH mRNA and CRH‐R mRNA in the PVN were unchanged after icv Losartan. While Losartan had no effect on the increases in CRH‐R mRNA levels 2 or 3 h after 1 h immobilization, it prevented the increases in CRH mRNA. The blunted plasma catecholamine responses after central AT1 receptor blockade indicate that endogenous Ang II in the brain is required for sympathoadrenal activation during immobilization stress. While Ang II appears not to be involved in the acute secretory response of the HPA axis, it may play a role in regulating CRH expression in the PVN.