Age and Sex in the Development of Hepatic Encephalopathy: Role of Alcohol.

Hepatic encephalopathy (HE) is a neurological condition linked to liver failure. Acute HE (Type A) occurs with acute liver failure, while chronic HE (Type C) is tied to cirrhosis and portal hypertension. HE treatments lag due to gaps in understanding its development by gender and age. We studied how sex and age impact HE and its severity with combined liver toxins. Our findings indicate that drug-induced (thioacetamide, TAA) brain edema was more severe in aged males than in young males or young/aged female rats. However, adding alcohol (ethanol, EtOH) worsens TAA's brain edema in both young and aged females, with females experiencing a more severe effect than males. These patterns also apply to Type A HE induced by azoxymethane (AZO) in mice. Similarly, TAA-induced behavioral deficits in Type C HE were milder in young and aged females than in males. Conversely, EtOH and TAA in young/aged males led to severe brain edema and fatality without noticeable behavioral changes. TAA metabolism was slower in aged males than in young or middle-aged rats. When TAA-treated aged male rats received EtOH, there was a slow and sustained plasma level of thioacetamide sulfoxide (TASO). This suggests that with EtOH, TAA-induced HE is more severe in aged males. TAA metabolism was similar in young, middle-aged, and aged female rats. However, with EtOH, young and aged females experience more severe drug-induced HE as compared to middle-aged adult rats. These findings strongly suggest that gender and age play a role in the severity of HE development and that the presence of one or more liver toxins may aggravate the severity of the disease progression.

The Orexin/Hypocretin System, the Peptidergic Regulator of Vigilance, Orchestrates Adaptation to Stress.

The orexin/hypocretin neuropeptide family has emerged as a focal point of neuroscientific research following the discovery that this family plays a crucial role in a variety of physiological and behavioral processes. These neuropeptides serve as powerful neuromodulators, intricately shaping autonomic, endocrine, and behavioral responses across species. Notably, they serve as master regulators of vigilance and stress responses; however, their roles in food intake, metabolism, and thermoregulation appear complementary and warrant further investigation. This narrative review provides a journey through the evolution of our understanding of the orexin system, from its initial discovery to the promising progress made in developing orexin derivatives. It goes beyond conventional boundaries, striving to synthesize the multifaceted activities of orexins. Special emphasis is placed on domains such as stress response, fear, anxiety, and learning, in which the authors have contributed to the literature with original publications. This paper also overviews the advancement of orexin pharmacology, which has already yielded some promising successes, particularly in the treatment of sleep disorders.

The effects of the urocortins on the hypothalamic-pituitary-adrenal axis - similarities and discordancies between rats and mice.

The urocortins (Ucn I, Ucn II and Ucn III) are structural analogues of corticotropin-releasing factor (CRF). The aim of our present experiments was to compare the effects of the urocortins on the hypothalamic-pituitary-adrenal (HPA) axis in rats and mice, including the hypothalamic adrenocorticotropic hormone (ACTH) secretagogues, such as CRF and arginine vasopressin (AVP). Therefore, male CFLP mice and male Wistar rats were injected intracerebroventricularly (icv) with 0.5, 1, 2 and 5 µg/2 µl of Ucn I, Ucn II or Ucn III. After 30 min the animals were decapitated, and then, hypothalamic CRF and AVP concentrations and plasma ACTH and corticosterone (CORT) levels were measured. All measurements were performed by enzyme-linked immunosorbent assays (ELISA), except that of the plasma CORT level, which was determined by chemofluorescent assay. Ucn I increased significantly the hypothalamic CRF and AVP concentrations in both rats and mice. Ucn II and Ucn III influenced significantly only the hypothalamic CRF concentration in rats, without affecting the hypothalamic AVP concentration. In contrast, Ucn II and Ucn III increased significantly only the hypothalamic AVP concentration in mice, without affecting the hypothalamic CRF concentration. The hypothalamic changes were reflected more or less accurately by changes of the plasma ACTH and CORT levels. The present experiments demonstrate that the urocortins regulate the HPA axis centrally via modulation of the hypothalamic ACTH secretagogues and that there are some similarities and discordancies between rats and mice regarding this regulation.

The effects of CRF and urocortins on the sociability of mice.

The aim of our study was to determine the role of corticotropin-releasing factor (CRF), the urocortins (Ucn1, Ucn2 and Ucn3) and their receptors (CRF1 and CRF2) in the sociability of mice. Male CFLP mice were administered intracerebroventricularly (icv) with CRF and urocortins alone or in combination with antalarmin (specific CRF1 antagonist) and astressin2B (specific CRF2 antagonist) and then investigated in a Crawley social interaction test arena, that consists of three chambers. An unknown male in a cage was put in the first chamber and an empty cage was put in the opposite chamber. The tested male was habituated with the middle chamber for 5min and then allowed to explore the remaining chambers for 5min, during which the number of entries and the time of interaction were measured. Intracerebroventricular administration of CRF decreased significantly the number of entries and the time of interaction with the unknown male and these effects were blocked by antalarmin, but not astressin2B. In contrast, central administration of Ucn1 increased significantly the number of entries into the chamber of the unknown male, without changing the time of interaction and this effect was blocked by astressin2B, but not antalarmin. Central administration of Ucn2 and Ucn3 didn't influence remarkably the number of entries, but it reduced the time of interaction between the male mice. Our study suggests that CRF and Ucn1 may play important, but different roles in sociability, and that Ucn2 and Ucn3, playing similar roles, must be also involved in social interactions.

The effects of CRF and urocortins on the preference for social novelty of mice.

The aim of the present study was to determine the role of corticotropin-releasing factor (CRF), the urocortins (UCN 1, UCN 2 and UCN 3) and their receptors (CRF1 and CRF2) in the preference for social novelty of mice. Male CFLP mice were administered intracerebroventricularly (ICV) with CRF, UCN 1, UCN 2 or UCN 3 and/or antalarmin or astressin 2B, selective antagonists of CRF1 receptor and CRF2 receptor, respectively. The mice were investigated in a Crawley social interaction test arena consisting of three chambers: an unknown female was set in the first chamber and a known female, with which the male was familiarized previously for 24h, was set in the third chamber. First the tested male was habituated with the middle chamber for 5min and then allowed to explore the remaining chambers for 5min, during which the number of entries and the time of interaction were measured. CRF decreased significantly the number of entries and the time of interaction with the unknown female, but not the known female. UCN 1 decreased significantly the number of entries into the chamber of the unknown female, but not the known female, without changing the time of interaction. All decreasing effects were reversed by antalarmin, but not astressin 2B. UCN 2 and UCN 3 didn't influence significantly any of the parameters. The present study suggests that CRF and UCN 1 decrease the preference for social novelty by activating CRF1 receptor, while UCN 2 and UCN 3, activating selectively CRF2 receptor, do not participate to male-female interaction.

Synthesis and structure-activity studies on novel analogs of human growth hormone releasing hormone (GHRH) with enhanced inhibitory activities on tumor growth.

The syntheses and biological evaluations of new GHRH analogs of Miami (MIA) series with greatly increased anticancer activity are described. In the design and synthesis of these analogs, the following previous substitutions were conserved: D-Arg2, Har9, Abu15, and Nle27. Most new analogs had Ala at position 8. Since replacements of both Lys12 and Lys21 with Orn increased resistance against enzymatic degradation, these modifications were kept. The substitutions of Arg at both positions 11 and 20 by His were also conserved. We kept D-Arg28, Har29 -NH2 at the C-terminus or inserted Agm or 12-amino dodecanoic acid amide at position 30. We incorporated pentafluoro-Phe (Fpa5), instead of Cpa, at position 6 and Tyr(Me) at position 10 and ω-amino acids at N-terminus of some analogs. These GHRH analogs were prepared by solid-phase methodology and purified by HPLC. The evaluation of the activity of the analogs on GH release was carried out in vitro on rat pituitaries and in vivo in male rats. Receptor binding affinities were measured in vitro by the competitive binding analysis. The inhibitory activity of the analogs on tumor proliferation in vitro was tested in several human cancer cell lines such as HEC-1A endometrial adenocarcinoma, HCT-15 colorectal adenocarcinoma, and LNCaP prostatic carcinoma. For in vivo tests, various cell lines including PC-3 prostate cancer, HEC-1A endometrial adenocarcinoma, HT diffuse mixed ß cell lymphoma, and ACHN renal cell carcinoma cell lines were xenografted into nude mice and treated subcutaneously with GHRH antagonists at doses of 1-5µg/day. Analogs MIA-602, MIA-604, MIA-610, and MIA-640 showed the highest binding affinities, 30, 58, 48, and 73 times higher respectively, than GHRH (1-29) NH2. Treatment of LNCaP and HCT-15 cells with 5µM MIA-602 or MIA-690 decreased proliferation by 40%-80%. In accord with previous tests in various human cancer lines, analog MIA-602 showed high inhibitory activity in vivo on growth of PC-3 prostate cancer, HT-mixed ß cell lymphoma, HEC-1A endometrial adenocarcinoma and ACHN renal cell carcinoma. Thus, GHRH analogs of the Miami series powerfully suppress tumor growth, but have only a weak endocrine GH inhibitory activity. The suppression of tumor growth could be induced in part by the downregulation of GHRH receptors levels.

Selective CRF2 receptor agonists ameliorate the anxiety- and depression-like state developed during chronic nicotine treatment and consequent acute withdrawal in mice.

The aim of the present study was to investigate the effects of the selective agonists of the corticotropin-releasing factor (CRF) 2 receptor, urocortin 2 (UCN 2) and urocortin 3 (UCN 3), on the anxiety- and depression-like signs induced by acute nicotine withdrawal in mice. In order to do so, male CFLP mice were exposed for 7 days to repeated intraperitoneal (IP) injection with nicotine or saline solution and 1day of acute withdrawal and then a single intracerebroventricular (ICV) injection with UCN 2, UCN 3 or saline solution. After 30min the mice were observed in an elevated plus-maze test or a forced swim test, for anxiety- and depression-like behavior. After 5min of testing, the plasma corticosterone concentration reflecting the activity of the hypothalamic-pituitary-adrenal (HPA) axis was also determined by a chemo-fluorescent method. Half of the animals were treated ICV and evaluated on the 8th day, the other half on the 9th day. On the 8th day, nicotine-treated mice presented signs of anxiolysis and depression, but no significant elevation of the plasma corticosterone concentration. On the 9th day, nicotine-treated mice exhibited signs of anxiety and depression and a significant increase of the plasma corticosterone levels. Central administration of UCN 2 or UCN 3 ameliorated the anxiety- and depression-like state including the hyperactivity of the HPA axis, developed during acute withdrawal following chronic nicotine treatment. The present study suggests that selective CRF2 receptor agonists could be used as a therapy in nicotine addiction.

The action of neuropeptide AF on passive avoidance learning. Involvement of neurotransmitters.

Neuropeptide AF (NPAF) is an amidated octadecapeptide, which is member of the RFamide peptide family. NPAF is encoded by the farp-1 gene and acts through the G protein coupled NPFF-1 and NPFF-2 receptors. NPAF is involved in several physiological functions of the central nervous system, however we have little evidence about the involvement of NPAF in learning and memory. Therefore, the aim of the present study was to investigate the action of NPAF on consolidation of memory in a passive avoidance learning paradigm in mice. We have also investigated the underlying neurotransmissions and the action of NPAF on ß-amyloid-induced memory impairment. Accordingly, mice were pretreated with a nonselective muscarinic acetylcholine receptor antagonist, atropine, a non-selective 5-HT2 serotonergic receptor antagonist, cyproheptadine, a mixed 5-HT1/5-HT2 serotonergic receptor antagonist, methysergide, a D2, D3, D4 dopamine receptor antagonist, haloperidol, a non-selective opioid receptor antagonist, naloxone, a nitric oxide synthase inhibitor, nitro-l-arginine, a α1/α2ß-adrenergic receptor antagonist, prazosin, a nonselective ß-adrenergic receptor antagonist, propranolol or ß-amyloid 25-35 in combination with NPAF administration. Our results demonstrate for the first time that NPAF improves the consolidation of passive avoidance learning. This effect is mediated through muscarinic cholinergic, 5HT1- and 5HT2-serotoninergic, dopaminergic, nitrergic and α- and ß-adrenergic neurotransmissions, but not by opioid transmission, since atropine, cyproheptadine, methysergide, haloperidol, nitro-l-arginine, prazosin and propranolol reversed the action of NPAF, whereas naloxone was ineffective. The present study also shows that NPAF reverses the ß-amyloid 25-35-induced memory impairment.

The effects of CRF and urocortins on the hippocampal glutamate release.

Corticotropin-releasing factor (CRF) is a hypothalamic neurohormone and an extrahypothalamic neurotransmitter that regulates the hypothalamic-pituitary-adrenal (HPA) axis. The urocortins (UCN I, UCN II and UCN III) are CRF-related peptides, which may also regulate the HPA axis directly or indirectly, by modulation of extrahypothalamic neurotransmitters, such as amygdalar GABA and hippocampal glutamate. Our previous in vitro superfusion studies have already demonstrated that CRF and UCN I stimulate the amygdalar GABA release in rats. The aim of the present study was to investigate the effects of CRF, UCN I, UCN II and UCN III on the glutamate release elicited electrically from rat hippocampal slices in similar in vitro conditions. In order to investigate the participation of CRF receptors (CRFR1 and CRFR2) in this process, hippocampal slices were pretreated with antalarmin, a selective antagonist of CRFR1 or astressin 2B, a selective antagonist of CRFR2. CRF and UCN I at 100 nM decreased significantly the hippocampal glutamate release evoked by electrical stimulation. In contrast, 100 nM of UCN II and UCN III did not affect significantly the hippocampal glutamate release enhanced by electrical stimulation. The decreasing effects of CRF and UCN I were reversed by antalarmin, but not by astressin 2B, both being administered in equimolar doses. Our results demonstrate that CRF and UCN I inhibit the glutamate release in the hippocampus via CRFR1 and that CRFR2 does not participate to this process. Based on the previous and the present results we conclude that CRFR1 agonists can activate the HPA axis not only directly, but also indirectly by increasing the amygdalar GABA release and decreasing the hippocampal glutamate release.

New therapeutic approach to heart failure due to myocardial infarction based on targeting growth hormone-releasing hormone receptor.

BACKGROUND: We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair. METHODS AND RESULTS: H9c2 cells were cultured in serum-free medium, mimicking nutritional deprivation. GHRH agonists decreased calcium influx and significantly improved cell survival. Rats with cardiac infarction were treated with GHRH agonists or placebo for four weeks. MI size was reduced by selected GHRH agonists (JI-38, MR-356, MR-409); this accompanied an increased number of cardiac c-kit+ cells, cellular mitotic divisions, and vascular density. One week post-MI, MR-409 significantly reduced plasma levels of IL-2, IL-6, IL-10 and TNF-α compared to placebo. Gene expression studies revealed favorable outcomes of MR-409 treatment partially result from inhibitory activity on pro-apoptotic molecules and pro-fibrotic systems, and by elevation of bone morphogenetic proteins. CONCLUSIONS: Treatment with GHRH agonists appears to reduce the inflammatory responses post-MI and may consequently improve mechanisms of healing and cardiac remodeling by regulating pathways involved in fibrosis, apoptosis and cardiac repair. Patients with cardiac dysfunction could benefit from treatment with novel GHRH agonists.

Neuropeptide AF induces anxiety-like and antidepressant-like behavior in mice.

Little is known about the action of neuropeptide AF (NPAF) on anxiety and depression. Only our previous study provides evidence that NPAF induces anxiety-like behavior in rats. Therefore, the aim of the present study was to investigate the action of NPAF on depression-like behavior and the underlying neurotransmissions in mice. In order to determine whether there are species differences between rats and mice, we have investigated the action of NPAF on anxiety-like behavior in mice as well. A modified forced swimming test (mFST) and an elevated plus maze test (EPMT) were used to investigate the depression and anxiety-related behaviors, respectively. Mice were treated with NPAF 30min prior to the tests. In the mFST, the animals were pretreated with a non-selective muscarinic acetylcholine receptor antagonist, atropine, a non-selective 5-HT2 serotonergic receptor antagonist, cyproheptadine, a mixed 5-HT1/5-HT2 serotonergic receptor antagonist, methysergide, a D2/D3/D4 dopamine receptor antagonist, haloperidol, a α1/α2ß-adrenergic receptor antagonist, prazosin or a non-selective ß-adrenergic receptor antagonist, propranolol 30min before the NPAF administration. In the mFST, NPAF decreased the immobility time and increased the climbing and swimming times. This action was reversed completely by methysergide and partially by atropine, whereas cyproheptadine, haloperidol, prazosin and propranolol were ineffective. In the EPMT, NPAF decreased the time spent in the arms (open/open+closed). Our results demonstrate that NPAF induces anti-depressant-like behavior in mice, which is mediated, at least in part, through 5HT2-serotonergic and muscarinic cholinergic neurotransmissions. In addition, the NPAF-induced anxiety is species-independent, since it develops also in mice.

The action of orexin B on passive avoidance learning. Involvement of neurotransmitters.

The extensive projection of orexigenic neurons and the diffuse expression of orexin receptors suggest that endogenous orexins are involved in several physiological functions of the central nervous system, including learning and memory. Our previous study demonstrated that orexin A improves learning, consolidation and retrieval processes, which involves α- and ß-adrenergic, cholinergic, dopaminergic, GABA-A-ergic, opiate and nitrergic neurotransmissions. However, we have little evidence about the action of orexin B on memory processes and the underlying neuromodulation. Therefore, the aim of the present study was to investigate the action of orexin B on passive avoidance learning and the involvement of neurotransmitters in this action in rats. Accordingly, rats were pretreated with the selective orexin 2 receptor (OX2R) antagonist, EMPA; the γ-aminobutyric acid subunit A (GABA-A) receptor antagonist, the bicuculline; a D2, D3, D4 dopamine receptor antagonist, haloperidol; the nonselective opioid receptor antagonist, naloxone; the non-specific nitric oxide synthase (NOS) inhibitor, nitro-l-arginine; the nonselective α-adrenergic receptor antagonist, phenoxybenzamine and the ß-adrenergic receptor antagonist, propranolol. Our results demonstrate that orexin B can improve learning, consolidation of memory and retrieval. EMPA reversed completely the action of orexin B on memory consolidation. Bicuculline blocked fully; naloxone, nitro-l-arginine, phenoxybenzamine and propranolol attenuated the orexin B-induced memory consolidation, whereas haloperidol was ineffective. These data suggest that orexin B improves memory functions through OX2R and GABA-ergic, opiate, nitrergic, α- and ß-adrenergic neurotransmissions are also involved in this action.

Orexin A-induced anxiety-like behavior is mediated through GABA-ergic, α- and ß-adrenergic neurotransmissions in mice.

Orexins are hypothalamic neuropeptides, which are involved in several physiological functions of the central nervous system, including anxiety and stress. Several studies provide biochemical and behavioral evidence about the anxiogenic action of orexin A. However, we have little evidence about the underlying neuromodulation. Therefore, the aim of the present study was to investigate the involvement of neurotransmitters in the orexin A-induced anxiety-like behavior in elevated plus maze (EPM) test in mice. Accordingly, mice were pretreated with a non-selective muscarinic cholinergic antagonist, atropine; a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist, bicuculline; a D2, D3, D4 dopamine receptor antagonist, haloperidol; a non-specific nitric oxide synthase (NOS) inhibitor, nitro-l-arginine; a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a ß-adrenergic receptor antagonist, propranolol 30min prior to the intracerebroventricular administration of orexin A. The EPM test started 30min after the i.c.v. injection of the neuropeptide. Our results show that orexin A decreases significantly the time spent in the arms (open/open+closed) and this action is reversed by bicuculline, phenoxybenzamine and propranolol, but not by atropine, haloperidol or nitro-l-arginine. Our results provide evidence for the first time that the orexin A-induced anxiety-like behavior is mediated through GABA-A-ergic, α- and ß-adrenergic neurotransmissions, whereas muscarinic cholinergic, dopaminergic and nitrergic neurotransmissions may not be implicated.

Transmitter mediation of the anxiolytic action of apelin-13 in male mice.

The widespread distribution of apelin-13 and apelin receptors in the brain and periphery suggests an important function of this neuropeptide in regulatory processes in the organism. In previous work we found that apelin-13 facilitates the consolidation of passive avoidance learning in rats. In the present work we demonstrate that apelin-13 exerts anxiolytic action in an elevated plus maze in mice. In order to assess the possible involvement of transmitters in this action, the animals were pretreated with the following receptor blockers in doses which themselves did not influence the behavioral paradigm: atropine (a nonselective muscarinic acetylcholine receptor antagonist), haloperidol (a D2, D3, D4 dopamine receptor antagonist), phenoxybenzamine (a nonselective α1-adrenergic receptor antagonist), methysergide (a nonselective 5-HT2 serotonergic receptor antagonist), propranolol (a ß-adrenergic receptor antagonist), naloxone (a nonselective opioid receptor antagonist) and bicuculline (a γ-aminobutyric acid subunit A receptor antagonist. Phenoxybenzamine, haloperidol, propranolol and methysergide prevented the action of apelin-13, whereas atropine, naloxone and bicuculline were ineffective. The data suggest that apelin-13 elicits its anxiolytic action via α-adrenergic, dopaminergic, ß-adrenergic and 5-HT2 serotonergic mediation.

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists.

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFß. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.

Interleukin-1ß (187-207)-induced hyperthermia is inhibited by interleukin-1ß (193-195) in rats.

Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine, which plays an important role in the immune response and signal transduction both in the periphery and the central nervous system (CNS). Various diseases of the CNS, including neurodegenerative disorders, vascular lesions, meningo-encephalitis or status epilepticus are accompanied by elevated levels of IL-1ß. Different domains within the IL-lß protein are responsible for distinct functions. The IL-lß domain in position 208-240 has pyrogenic properties, while the domain in position 193-195 exerts anti-inflammatory effects. Previous studies provide little evidence about the effect of the domain in position 187-207 on the body temperature. Therefore, the aim of the present study was to investigate the action of IL-1ß (187-207) and its interaction with IL-1ß (193-195) on the body temperature. IL fragments were administered intracerebroventricularly and the body temperature was measured rectally in male Wistar rats. IL-1ß (187-207) induced hyperthermia, while IL-1ß (193-195) did not influence the core temperature considerably. In co-administration, IL-1ß (193-195) completely abolished the IL-1ß (187-207)-induced hyperthermia. The non-steroid anti-inflammatory drug metamizole also reversed completely the action of IL-1ß (187-207). Our results provide evidence that the IL-lß domain in position 187-207 has hyperthermic effect. This effect is mediated through prostaglandin E2 stimulation and other mechanisms may also be involved in the action of IL-1ß (187-207). It also suggests that IL-lß domain in position 187-207 and IL-1ß (193-195) fragment may serve as novel target for treatment of disorders accompanied with hyperthermia.

The effect of urocortin I on the hypothalamic ACTH secretagogues and its impact on the hypothalamic-pituitary-adrenal axis.

Urocortin I (UCN I) is a structural analogue of corticotropin-releasing factor (CRF), which, together with arginine-vasopressin (AVP), are the principle adrenocorticotropic hormone (ACTH) secretagogues in mammals. The aim of the present study was to investigate the effects of UCN I on the hypothalamic CRF and AVP concentration and its impact on the hypothalamic-pituitary-adrenal (HPA) axis. First, male Wistar rats were injected intracerebroventricularly (ICV) with 0.5, 1, 2 and 5 µg of UCN I. After 30 min hypothalamic CRF and AVP concentrations were determined by immunoassays. In parallel, the trunk blood was collected and plasma ACTH and corticosterone concentration was determined by ELISA and chemofluorescent assay, respectively. Second, rats were pretreated ICV with selective antagonists of receptors being implicated in the regulation of the HPA axis (0.1 µg antalarmin for CRFR1, 1 µg astressin 2B for CRFR2 or 0.1 µg deamino-Pen1,Tyr2,Arg8-vasopressin for AVPR3) and treated ICV with the most effective dose of UCN I (5 µg). After 30 min plasma corticosterone concentration was determined by chemofluorescent assay. UCN I induced dose-dependent augmentation of the hypothalamic CRF and AVP concentration, associated with dose-dependent elevation of the plasma ACTH and corticosterone concentration. The most significant effect of UCN I on the plasma corticosterone concentration was inhibited by antalarmin, but was not influenced by astressin 2B or deamino-Pen1,Tyr2,Arg8-vasopressin. The present study demonstrates that UCN I modulates the concentration of the hypothalamic ACTH secretagogues in parallel with the concentration of the plasma ACTH and corticosterone. Our results suggest that UCN I may activate the HPA axis by stimulation of the hypothalamic CRF production, and this process is mediated by CRFR1, and not by CRFR2. UCN I may stimulate the AVP production, as well, but, based on the results with AVPR3 antagonist, this effect is not involved in the regulation of the HPA axis.

The actions of neuropeptide SF on the hypothalamic-pituitary-adrenal axis and behavior in rats.

Present experiments focused on measuring the effect of neuropeptide SF (NPSF) on the hypothalamus-pituitary-adrenal (HPA) axis and behavior. The peptide was administered in different doses (0.25, 0.5, 1, 2 µg) intracerebroventricularly to rats, and the behavior of which was then observed by telemetry and open-field test. Effect of NPSF on core temperature was also measured via telemetry. Plasma ACTH and corticosterone concentrations were measured to assess the influence of NPSF on the HPA activation. In addition, the changes in corticotrophin-releasing hormone (CRH) level in the hypothalamic paraventricular nucleus were continuously monitored by means of intracerebral microdialysis. Our results showed that NPSF augmented paraventricular CRH release and increased ACTH and corticosterone levels in the plasma. The release of corticosterone was successfully blocked by the pre-treatment of the CRH antagonist α-helical CRH9-41. Spontaneous and exploratory locomotor activity was also stimulated according to the telemetric and open-field studies. However, NPSF only tended to alter stereotyped behavior in the open-field experiments. These results demonstrate that NPSF may play a physiologic role in the regulation of such circadian functions as the activity of motor centers and the HPA axis, through the release of CRH.

Synthesis of new potent agonistic analogs of growth hormone-releasing hormone (GHRH) and evaluation of their endocrine and cardiac activities.

In view of the recent findings of stimulatory effects of GHRH analogs, JI-34, JI-36 and JI-38, on cardiomyocytes, pancreatic islets and wound healing, three series of new analogs of GHRH(1-29) have been synthesized and evaluated biologically in an endeavor to produce more potent compounds. "Agmatine analogs", MR-356 (N-Me-Tyr(1)-JI-38), MR-361(N-Me-Tyr(1), D-Ala(2)-JI-38) and MR-367(N-Me-Tyr(1), D-Ala(2), Asn(8)-JI-38), in which Dat in JI-38 is replaced by N-Me-Tyr(1), showed improved relative potencies on GH release upon subcutaneous administration in vivo and binding in vitro. Modification with N-Me-Tyr(1) and Arg(29)-NHCH3 as in MR-403 (N-Me-Tyr(1), D-Ala(2), Arg(29)-NHCH3-JI-38), MR-406 (N-Me-Tyr(1), Arg(29)-NHCH3-JI-38) and MR-409 (N-Me-Tyr(1), D-Ala(2), Asn(8), Arg(29)-NHCH3-JI-38), and MR-410 (N-Me-Tyr(1), D-Ala(2), Thr(8), Arg(29)-NHCH3-JI-38) resulted in dramatically increased endocrine activities. These appear to be the most potent GHRH agonistic analogs so far developed. Analogs with Apa(30)-NH2 such as MR-326 (N-Me-Tyr(1), D-Ala(2), Arg(29), Apa(30)-NH2-JI-38), and with Gab(30)-NH2, as MR-502 (D-Ala(2), 5F-Phe(6), Ser(28), Arg(29),Gab(30)-NH2-JI-38) also exhibited much higher potency than JI-38 upon i.v. administration. The relationship between the GH-releasing potency and the analog structure is discussed. Fourteen GHRH agonists with the highest endocrine potencies were subjected to cardiologic tests. MR-409 and MR-356 exhibited higher potency than JI-38 in activating myocardial repair in rats with induced myocardial infarction. As the previous class of analogs, exemplified by JI-38, had shown promising results in multiple fields including cardiology, diabetes and wound healing, our new, more potent, GHRH agonists should manifest additional efficacy for possible medical applications.

The interaction of Urocortin II and Urocortin III with amygdalar and hypothalamic cotricotropin-releasing factor (CRF)--reflections on the regulation of the hypothalamic-pituitary-adrenal (HPA) axis.

Urocortin II (Ucn II) and Urocortin III (Ucn III) are selective agonists of the CRF receptor type 2 (CRFR2). The aim of the present experiments was to investigate the effects of Ucn II and Ucn III on the central CRF and peripheral glucocorticoids in rats. Increasing doses (0.5-1-2-5 µg/2 µl) of Ucn II or Ucn III were administered intracerebroventricularly, then CRF concentration was determined by immunoassays in two different brain regions, the amygdala and the hypothalamus, and in two different time paradigms, 5 and 30 min after the administration of peptides. In parallel with the second determination, plasma corticosterone concentration was measured by chemofluorescent assay. The amygdalar CRF amount was increased significantly by 0.5 and 5 µg of UCN II and 2 and 5 µg of UCN III in the 5 min experiments and by 5 µg of UCN II and 0.5 and 5 µg of UCN III in the 30 min experiments. The hypothalamic CRF content was not affected considerably in the 5 min paradigm, but it was influenced significantly in the 30 min paradigm, with 0.5 and 1 µg of UCN II and 0.5-2 µg of UCN III decreasing, and 2 and 5 µg of UCN II and 5 µg of UCN III increasing the hormone concentration, respectively. The plasma corticosterone concentration was decreased by 1 and 2 µg of UCN II and UCN III and increased by 0.5 and 5 µg of UCN III. The present results demonstrate that central administration of Ucn II and Ucn III modulate time-dependently and dose-dependently the amygdalar and the hypothalamic CRF concentration, and, directly or indirectly, the plasma corticosterone concentration. The present experiments suggest that the role of CRFR2 in the regulation of the HPA axis can be inhibitory or stimulatory, depending on the actual concentration of their agonists.