促肾上腺皮质激素释放激素受体在应激反应中的作用研究

促肾上腺皮质激素释放激素(CRH)是应激反应中的关键调节因子,协调应激过程中内分泌、自主神经、免疫和行为反应。CRH的作用是由其受体所介导的。目前已知的CRH受体有CRH-R1、CRH-R2、CRH-R3 3种。应激时,CRH主要通过CRH-R1、CRH-R2产生一系列生理、病理效应。近年来通过对转基因动物、选择性CRH受体拮抗剂和特异性CRH受体激动剂等的应用,对CRH受体在应激中作用有了更深的了解,也进一步揭示了应激机制。     

Sexually dimorphic responses of the brain norepinephrine system to stress and corticotropin-releasing factor.

Stress-related psychiatric disorders are more prevalent in females than males, and this has been attributed to differences in stress sensitivity. As activation of the locus coeruleus (LC)-norepinephrine (NE) system is an important component of the stress response, this study compared LC responses to stress in female and male rats under different hormonal conditions in the halothane-anesthetized state. The mean basal LC discharge rate was similar between groups. However, the magnitude of LC activation elicited by hypotensive stress was substantially greater in females, regardless of hormonal status. The difference in stress sensitivity could be attributed to the differential postsynaptic sensitivity of LC neurons to corticotropin-releasing factor (CRF), which mediates LC activation by hypotension. CRF was 10-30 times more potent in activating LC neurons in female vs male rats. Interestingly, previous exposure to swim stress differentially regulated LC responses to CRF by sensitizing LC neurons of male, but not female, rats to CRF. The net effect of this was to abolish sex differences in LC sensitivity. Finally, CRF receptor (CRF-R) protein levels in the LC were greater in ovarectomized female vs male rats. This is the first study to demonstrate sex differences in the stress responsiveness of the brain noradrenergic system. Substantial sex differences were apparent in postsynaptic sensitivity to CRF and stress-induced regulation of postsynaptic sensitivity to CRF. These sex differences in the CRF regulation of the LC-NE system translate to a differential response to stress and may play a role in the increased vulnerability of females to stress-related psychiatric disorders.     

Early life stress changes concentrations of neuropeptide Y and corticotropin-releasing hormone in adult rat brain. Lithium treatment modifies these changes.

Experiences of early life stress are more prevalent among depressed patients than healthy controls. Neuropeptide Y (NPY) was suggested to play a role in the pathophysiology of depression. Consequently, we investigated in adult rats the effects of maternal deprivation for 3 h/day during postnatal days (PND) 2-14 and of dietary lithium during PND 50-83 on brain levels of NPY-like immunoreactivity (LI). Brain levels of corticotropin-releasing hormone (CRH) and serum corticosterone were also measured. Maternal deprivation reduced NPY-LI levels in the hippocampus and the striatum but increased NPY-LI and CRH-LI levels in the hypothalamus. Lithium treatment counteracted the effect of maternal deprivation in the hippocampus and striatum by increasing NPY-LI levels. In the hypothalamus, lithium tended to decrease CRH-LI but further increased levels of NPY-LI; it also increased serum corticosterone levels. The results suggest that early life stress has long-term effects on brain NPY with implications for the development of depression/vulnerability to stress, and that one therapeutic mechanism of action of lithium is to increase brain NPY.     

Molecular mechanisms of the brain reward system

The brain possesses a reward system which produces positive emotion. To reveal the mechanisms of the brain reward system, investigation of mechanisms underlying actions of substances of abuse can be one of the promising research approaches. Various behavioral tests using animals and methods in genomic science are also useful for these studies. I introduce our findings obtained by these ideas and techniques as follows: (i) Inhibition of methamphetamine preference by G-protein activated inwardly rectifying potassium (GIRK) channel inhibitors. (ii) Essential role of NMDA receptor channel GluN2D subunit in phencyclidine effects on animal behavior. (iii) Association of polymorphisms in the mu-opioid receptor and GIRK genes with opioid sensitivity.     

Hydrophobically induced conformation in ovine corticotropin-releasing hormone

Multiple peptide synthesis has been applied for the simultaneous synthesis of systematic replacement sets of model peptides which varied in length from 18 to 36 residues and ovine corticotropin-releasing hormone (oCRH), a 41-residue receptor-binding peptide. The peptides were utilized to analyze the capability of the stationary phase during RP-HPLC to induce secondary structure in long-chain linear peptides. Double D-amino acid replacement studies demonstrate that nonamphipathic helical domains can be recognized, even in the presence of highly amphipathic domains. On the other hand, systematic alteration of hydrophobicity at each residue along the sequence by methionine and methionine sulfoxide replacements results in characteristic pattern of HPLC retention-time differences, which is shown to provide a useful method to probe hydrophobic surface regions in helical peptides. Both amino acid replacement strategies were successfully applied to characterize the hydrophobically induced structure of oCRH. Although an a-helix is formed from residues 6 to 32, the N-terminal residues 1–5 and the C-terminal region 33–41 do not show any regular structure. The helical domain from residues 12 to 20 is highly amphipathic.     

Endogenous corticotropin-releasing hormone inhibits conditioned-fear-induced vagal activation in the rat

The role of the endogenous corticotropin-releasing hormone (CRH) system in the regulation of heart rate, PQ interval (a measure of vagal activity), gross activity and release of adrenocorticotropic hormone (ACTH), noradrenaline and adrenaline into the blood during conditioned fear was studied in freely moving rats. Intracerebroventricular (i.c.v.) infusion of alpha-helical CRH-(9-41) (10 microgram/3 microliter), a non-selective CRH receptor antagonist, under resting conditions had no significant effect on gross activity, heart rate and PQ interval, indicating that alpha-helical CRH at this dose was devoid of agonist effects. Conditioned fear was induced by 10 min forced exposure to a cage in which the rat had experienced footshocks (5x0.5 mAx3 s) 1 day before. Conditioned-fear rats showed freezing behaviour, associated with an increase in heart rate, PQ interval, noradrenaline and adrenaline, indicating that the conditioned-fear-induced cardiac effects were the result of coactivation of the sympathetic and parasympathetic nervous system. The i.c.v. pre-treatment of rats with alpha-helical CRH significantly reduced the conditioned-fear-induced tachycardiac and ACTH response, and enhanced the increase in PQ interval, without affecting the noradrenaline and adrenaline response. These results suggest that endogenous CRH reduces the vagal response to conditioned-fear stress in rats. To test this, rats were pre-treated with atropine methyl nitrate (0.3 mg/kg, subcutaneously; s.c.), a peripherally acting cholinergic receptor antagonist. This resulted in a complete blockade of the alpha-helical CRH-induced decrease in heart rate response and increase in PQ interval. From these findings, it is concluded that endogenous CRH in the brain inhibits vagal outflow induced by emotional stress.     

Involvement of corticotropin-releasing factor receptor subtype 1 in morphine withdrawal regulation of the brain noradrenergic system

Effects of pretreatment with the selective corticotropin-releasing factor (CRF) subtype 1 (CRF₁) receptor antagonist, 2-(N-(2-methylthio-4-isopropylphenyl)-N-ethyl-amino-4-(4-(3-fluorophenyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine (CRA1000) on the behavioral and biochemical changes after naloxone-precipitated morphine withdrawal were examined in ICR mice. Mice were chronically treated with morphine (8–45 mg/kg) for 5 days. Naloxone (3 mg/kg, s.c.) precipitated jumping, diarrhea, and body weight loss in morphine-dependent mice. In addition, 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and noradrenaline turnover (MHPG/noradrenaline) levels in the cerebral cortex were increased following naloxone challenge in morphine-dependent mice. However, 5-hydroxytriptamine turnover did not alter the increase following naloxone challenge in morphine-dependent mice. Pretreatment with CRA1000 (20 mg/kg, i.p.) attenuated the incidence of withdrawal signs and naloxone-precipitated increases in noradrenaline turnover. These results suggest that the activation of CRF₁ receptor may play an important role in the elevation of noradrenaline transmission, but not in 5-hydroxytriptamine transmission, in the cerebral cortex, which projects from the locus coeruleus during morphine withdrawal.     

Cholecystokinin peptides and receptors in the rat brain during stress

Cholecystokinin (CCK) has been implicated in stress and anxiety disorders. We have studied the levels of different molecular forms of CCK and CCK receptor characteristics in rats kept for 1 h in individual cages and exposed to decapitation of conspecifics, and a control group which was decapitated immediately. Total CCK concentration was found to be increased in the hippocampus of stressed animals in the first experiment: this finding was not confirmed in further studies. No effect of stress was found on total CCK levels in the frontal cortex, hypothalamus, striatum, and septum. CCK-8-sulphated, CCK-8-nonsulphated, CCK-5, and CCK-4 were separated by HPLC and measured with two antibodies with different selectivity: no effect of stress was found on the levels of any of these molecular forms of CCK. Injection procedure and diazepam (5 mg/kg) administration had no effect on total CCK levels. Exposition of rats to the decapitation procedure increased [³H]-CCK-8 binding in the frontal and cerebral (whole - frontal) cortex. This effect could not be blocked by diazepam pretreatment. Injection procedure itself increased CCK receptor binding in the cerebral cortex, but the effect of this type of stress was smaller in magnitude. The upregulation of CCK receptors in stressed animals was due to the increased binding of radioligand on CCK_B receptor subtype.     

Chronic treatment with iprindole reduces immobility of rats in the behavioural 'despair' test by activating dopaminergic mechanisms in the brain

Iprindole, 10 mg kg-1 i.p., once daily for 21 days, enhanced the metabolism of dopamine in the frontal cortex and striatum of rats with no effect in the nucleus accumbens 1 h after the last injection. Noradrenaline metabolism in the brainstem and telencephalon was also increased in these conditions. No effect on dopamine or noradrenaline metabolism was seen 24 h after the last injection. The same repeated treatment schedule with iprindole markedly reduced the immobility of rats in the behavioural 'despair' test 1 h after the last injection and the effect was prevented by 0.5 mg kg-1 i.p. haloperidol and 100 mg kg-1 i.p. sulpiride but not by 3 mg kg-1 s.c. prazosin or 5 mg kg-1 i.p. (+/-)-propranolol. The data show that enhanced metabolism of brain dopamine and noradrenaline is associated with the presence of iprindole during repeated treatment and the effect on dopamine mechanism is important in iprindole's ability to reduce rats' immobility in the behavioural 'despair' test.     

Bisphenol A induces corticotropin-releasing hormone expression in the placental cells JEG-3

Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproduction. Placental corticotrophin-releasing hormone (CRH) is a peptide hormone which is involved in fetal development. Increased plasma CRH is associated with elevated risk of premature delivery. In the present study, we demonstrated that bisphenol A increased CRH mRNA expression in the placental JEG-3 cells at or above 25μM. Reporter gene assay also demonstrated that bisphenol A could induce CRH gene transactivity. Since cyclic AMP response element (CRE) is a major regulatory element located in CRH promoter, the sequence-specific binding activity was investigated by using electrophoretic mobility shift assay. Our data indicated that bisphenol A increased the CRE binding activity. Western analysis further illustrated that PKA could be the signal triggering the CRE binding and CRH gene transactivation. In summary, the present study demonstrated that bisphenol A could induce CRH expression in placental cells and the underlying signal transduction pathway was also described.     

Melanin-concentrating hormone functions in the nervous system: food intake and stress

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, which centrally regulates food intake and stress. MCH induces food intake in rodents and, more generally, acts as an anabolic signal in energy regulation. In addition, MCH seems to be activatory on the stress axis. Two receptors for MCH in humans have very recently been characterised, namely, MCH-R1 and MCH-R2. MCH-R1 has received considerable attention, as potent and selective antagonists acting at that receptor display anxiolytic, antidepressant and/or anorectic properties. Feeding and affective disorders are both debilitating conditions that have become serious worldwide health threats. There are as yet no efficient and/or safe cures that could contain the near-pandemia phenomen of both diseases. Thus, the discovery of MCH-R1 antagonists may lead to the development of valuable drugs to treat obesity, anxiety and depressive syndromes. In addition, it opens wide avenues to probe additional functions of the peptide, both in the brain and in the peripheral nervous system.     

Melanin-concentrating hormone functions in the nervous system: food intake and stress

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, which centrally regulates food intake and stress. MCH induces food intake in rodents and, more generally, acts as an anabolic signal in energy regulation. In addition, MCH seems to be activatory on the stress axis. Two receptors for MCH in humans have very recently been characterised, namely, MCH-R1 and MCH-R2. MCH-R1 has received considerable attention, as potent and selective antagonists acting at that receptor display anxiolytic, antidepressant and/or anorectic properties. Feeding and affective disorders are both debilitating conditions that have become serious worldwide health threats. There are as yet no efficient and/or safe cures that could contain the near-pandemia phenomen of both diseases. Thus, the discovery of MCH-R1 antagonists may lead to the development of valuable drugs to treat obesity, anxiety and depressive syndromes. In addition, it opens wide avenues to probe additional functions of the peptide, both in the brain and in the peripheral nervous system.     

Formation mechanisms of stress adaptation: role of functional coupling of glucocorticoids and brain serotonergic nervous system]

It is known that the incompetence of stress adaptation mechanisms is a primary factor affecting disorders such as anxiety and depression. Increased clinical evidence indicates that hyperactivity of the hypothalamo-pituitary-adrenal (HPA) axis and dysfunction of the brain serotonin (5-HT) nervous system are risk factors associated with these disorders. Given the high sensitivity of hippocampal 5-HT responses to glucocorticoids, functional changes in the hippocampal 5-HT nervous system induced by chronic hypercorticism are attracting more attention. Repeated exposure to stress stimuli or chronic administration of corticosterone produces hippocampal 5-HT1A receptor dysfunction as well as an imbalance in mineralocorticoid and glucocorticoid receptors. We recently demonstrated that activation of the 5-HT1A receptors facilitates the adaptive responses to stress stimuli by acting on the HPA axis. These findings suggest that abnormal coupling of glucocorticoid-mineralocorticoid/glucocorticoid receptors-5-HT1A receptors in the hippocampus may be one factor disrupting adaptation to stress situations. Moreover, it is shown that activation of the coupling system affects learning and memory processes associated with stress stimuli. These findings suggest that the functional coupling of glucocorticoid-mineralocorticoid/glucocorticoid receptors and serotonergic neurons in the brain may play a significant role in the recognition of stress stimuli and induction of stress adaptation, and dysfunction of this coupling system may be related to the onset of affective disorders.     

The role of corticotropin-releasing hormone in the pathophysiology of depression: therapeutic implications

Stress responses have been posited to be a key component of mental health and disease by playing essential roles both in normal adaptive processes and maladaptive physiological responses that in part underlie the pathogenesis of certain subtypes of mood and anxiety disorders. Early research focused on delineating the function of the hypothalamic-pituitary-adrenal (HPA) axis and subsequently examined its role in mediating the mammalian stress responses and its hyperactivity in depression. Much evidence now supports an important function of the biological mediators of this system in relation to not only depression, but also anxiety, substance abuse, and psychotic disorders, and implicates several components of this system as areas of intervention for novel pharmacotherapy. Perhaps the best studied central nervous system (CNS) component of this system is corticotropin-releasing factor (CRF), and considerable research has focused on its role in the HPA axis, as well in extrahypothalamic brain regions.     

Hemodynamic actions of corticotropin-releasing hormone and proopiomelanocortin derivatives in septic patients

Proopiomelanocortin (POMC) derivatives and mRNA of POMC have been detected in cardiomyocytes and vascular smooth muscle cells. Increased plasma levels of POMC derivatives have been found in septic patients during cardiovascular deregulation; therefore, we evaluated whether corticotroph-type (ACTH, β-endorphin, β-lipotropin) or melanotroph-type (α-melanocyte-stimulating hormone and N-acetyl-β-END) POMC derivatives have influences on patients' hemodynamics during sepsis. Seventeen septic patients were monitored by pulmonary artery catheter and corticotropin-releasing hormone (CRH) tests were performed by intravenous administration of 100 μg CRH. Before, 15, 30, 45, and 60 minutes after CRH administration, hemodynamic variables were measured, and plasma concentrations of POMC derivatives were determined. After CRH administration, heart rate, cardiac index, and stroke index increased, and the systemic vascular resistance index decreased; moreover, a correlation between ACTH concentration and stroke index as well as an inverse correlation between (α-melanocyte-stimulating hormone concentration and systemic vascular resistance index was observed. CRH and ACTH may have opposite effects on the blood pressure (mean arterial pressure). Immediately after CRH injection mean arterial pressure decreased. ACTH (in contrast to β-endorphin or β-lipotropin), released into the cardiovascular compartment 15 minutes after CRH injection, might have raised mean arterial pressure as compatible with the correlation between ACTH levels and stroke index. (α-melanocyte-stimulating hormone appears to have a vasodilative effect during sepsis.     

Thyrotropin-releasing hormone increased heat production without the involvement of corticotropin-releasing factor in neonatal chicks

Thyrotropin-releasing hormone (TRH) is a hypothalamic signal in the hypothalamic-pituitary-thyroid (HPT) axis, and is well known as a hyperthermic hormone in the brain of chicks. The thermogenetic effect leads to the hypothesis that central TRH increases heat production (HP) in chicks. The purpose of the present study was to clarify whether central TRH affects HP of neonatal chicks, and if such an effect is mediated by corticotropin-releasing factor (CRF) since the thermogenetic effect of TRH is mediated by CRF. Intracerebroventricular (ICV) injection of TRH (14 and 55 nmol) dose-dependently increased oxygen consumption, carbon dioxide production and HP, and a similar effect was also observed with CRF (2.1 and 21 pmol). The TRH-induced increase in HP could not be attenuated by astressin, a CRF receptor antagonist, while the effect of CRF was completely diminished by astressin. The present study demonstrates that central TRH increases HP in chicks but the effect was not related to CRF.     

Barrier mechanisms in the brain, II. Immature brain

1. It is widely believed that 'the' blood-brain barrier is immature in foetuses and newborns. 2. Much evidence in support of this belief is based on experiments that were unphysiological and likely to have disrupted fragile blood vessels of the developing brain. Some confusion about barrier development arises from insufficient recognition that the term 'blood-brain barrier' describes a complex series of mechanisms controlling the internal environment of the brain. 3. We present evidence showing that the brain develops within an environment that, particularly with respect to protein, is different from that of the rest of the body and that possesses a number of unique features not present in the adult. 4. Barriers to protein at blood-brain and blood-cerebrospinal fluid (CSF) interfaces (tight junctions) are present from very early in development; immunocytochemical and permeability data show that proteins are largely excluded from extracellular space in developing brain. 5. Cerebrospinal fluid in developing brain contains high concentrations of proteins largely derived from plasma. This protein is transferred from blood by an intracellular mechanism across the epithelial cells of the immature choroid plexus. Only a small proportion of choroid plexus cells is involved. The route is an intracellular system of tubulo-endoplasmic reticulum continuously connected across the epithelial cells only early in brain development. 6. High concentrations of proteins in CSF in developing brain are largely excluded from the brain's extracellular space by barriers at the internal and external CSF-brain interfaces. These consist of membrane specializations between surfaces of cells forming these interfaces (neuroependyma on the inner surface; radial glial end feet on the outer surface). In contrast with tight junctions present at the blood-brain and blood-CSF barriers, at the CSF-brain barriers of the immature brain, other junctional types are involved: strap junctions in the neuroependyma and a mixture of junctions at the outer CSF-brain barrier (plate junctions, strap junctions and wafer junctions). These barriers are not present in the adult. 7. Permeability to small lipid-insoluble molecules is greater in developing brain; more specific mechanisms, such as those involved in transfer of ions and amino acids, develop sequentially as the brain grows.