The effect of the estrous cycle and estrogen on the release of immunoreactive alpha-melanocyte-stimulating hormone

Circulating levels and tissue content of alpha-MSH were measured on the morning of various days of the estrous cycle, and on the afternoon of proestrus in freely moving conscious rats. No surges of alpha-MSH were detected by RIA in the morning of various days of the cycle. The neurointermediate lobe content of alpha-MSH was slightly elevated on diestrus 1 as compared to the levels on diestrus 11 and proestrus but not to estrous levels. No changes in alpha-MSH content were detected in the anterior pituitary, the median eminence, mediobasal hypothalamus and the preoptic area at various stages of the estrous cycle. Plasma alpha-MSH levels were slightly elevated at 1500 hr of proestrus which was followed three hours later by a decline. This profile of plasma alpha-MSH on the afternoon of proestrus was reproduced by the SC administration of estradiol benzoate to long-term ovariectomized rats. These data suggest that, contrary to the results obtained by bioassay of alpha-MSH no surges of alpha-MSH occur on any day of the cycle, although a slight elevation on the afternoon of proestrus was detected. The altered pattern of release of this peptide on the afternoon of proestrus may be induced by estrogen.     

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock

Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5 μg/(kg_bodyweight h) CRH (or placebo) were intravenously administered for 24 h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16 h after starting the CRH infusion; 8 h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

Single injection of naked plasmid encoding alpha-melanocyte-stimulating hormone protects against thioacetamide-induced acute liver failure in mice

Oxidative stress has been implicated in the propagation of acute liver injury. The aim of our study was to investigate whether gene transfer of alpha-melanocyte-stimulating hormone (alpha-MSH), a potent anti-inflammatory peptide, could prevent fulminant hepatic failure in mice. Acute liver damage was induced by intraperitoneal administration of thioacetamide. Hydrodynamics-based gene transfection with alpha-MSH expression plasmid via rapid tail vein injection was initiated 1 day prior to intoxication. The mortality in the alpha-MSH-treated mice was significantly lower compared to the vehicle group 3 days after injury. Liver histology significantly improved and TUNEL-positive hepatocytes decreased in the treated mice. The degradation of IkappaBalpha, endogenous inhibitor of nuclear factor kappaB, and upregulation of inducible nitric oxide synthase and tumor necrosis factor-alpha mRNA levels were prevented in the alpha-MSH-treated group, indicating decreased oxidative stress and inflammation. These results suggest alpha-MSH gene therapy might protect against acute hepatic necroinflammatory damage with further potential applications.     

Glucocorticoid hormone receptors in rat pancreas

Although glucocorticoiods influence pancreatic function, it has not been established whether they act directly at the level of the pancreas, or indirectly by causing metabolic changes in other target tissues. As a step in elucidating the actions of glucocorticoids on the pancreas, a search was conducted for glucocorticoid hormone receptors in this tissue. Uptake and binding studies indicated that there were glucocorticoid hormone receptors in the high-speed cytosolic extract of rat pancreas. These receptors appear to be similar to other rat glucocorticoid receptors: they bind glucocorticoids rapidly in a reversible manner at 0°C, competitive binding analysis studies show that they have a preference for glucocorticoids and, like receptors, bind the synthetic steroids triamcinolone acetonide and dexamethasone with a higher affinity than corticosterone. Scatchard analysis demonstrated that there are 1.37 · 10^?13 mol glucocorticoid-binding sites/mg cytosolic protein. This demonstration of a glucocorticoid hormone receptor in pancreatic cytosol suggests that some of the effects glucocorticoids exert on pancreatic function are a consequence of their direct actions on this target tissue.     

The distribution of alpha-melanocyte stimulating hormone (alpha-MSH) in the central nervous system of the rat: an immunohistochemical study. II. Lower brain stem

The distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHI) in the rat lower brain stem was examined by indirect immunofluorescence or peroxidase- anti-peroxidase immunohistochemical method using an antiserum against synthetic alpha-MSH. The results confirmed the presence of alpha-MSHI fibers in the midbrain central gray matter and parabrachial area, and demonstrated a much more extensive distribution of these fibers in various parts of the lower brain stem areas previously thought not contain alpha-MSHI fibers. In addition, the commissural nucleus was identified as a new alpha-MSHI neurons-containing site. No alpha-MSHI neurons were seen in other regions of the rat lower brain stem.     

长短日照下绍鸭性成熟时间,下丘脑GnRH-I、POMC和NPY mRNA表达的差异

利用长日照和短日照环境调控绍鸭的性成熟时间,并采用反转录多聚酶链式反应(RT-PCR)方法, 检测两种日照组(n=6)鸭性成熟过程中下丘脑促性腺激素释放激素-Ⅰ(GnRH-Ⅰ)、阿片促黑激素皮质素 原(POMC)和神经肽Y(NPY)mRNA表达的发育性变化及日龄间差异。结果表明,长日照组性成熟时间较短 日照组提前30d。RT-PCR结果显示,两组鸭下丘脑GnRH-Ⅰ mRNA的发育性表达模式相似,从出雏至性成熟 过程中,其表达水平呈逐渐增加趋势;长日照组下丘脑GnRH-Ⅰ mRNA表达丰度均高于同日龄短日照组,并于 120日龄时其差异达显著水平。两组POMC mRNA的表达丰度呈现先增加后降低趋势,于性成熟前后显著下降; 日照长度对其基因表达具有显著影响。两组NPY mRNA表达水平分别于60和90日龄时显著升高,于性成熟时 显著下降,但光照对其基因表达无显著影响。下丘脑GnRH-Ⅰ基因表达的显著上调可能是禽类性成熟启动的关 键因素,也是长日照条件下绍鸭性早熟的主要原因;GnRH-Ⅰ表达的上调与下丘脑POMC和NPY mRNA表达变 化有关。     

Subcellular localization of alpha-melanocyte-stimulating hormone in the rat hypothalamus.

Homogenates of male rat hypothalami were fractionated by means of differential centrifugation, and α-melanocyte-stimulating hormone (α-MSH) in the various fractions was quantified by radioimmunoassay. Of the total quantity of α-MSH in the homogenate, 36% was recovered in the 11,500 g pellet and 31% sedimented between 11,500 and 105,000 g. α-MSH was not detected in the 105,000 g supernatant fluid. When the 900 g supernatant fluid was fractionated on continuous sucrose density gradients at non-equilibrium conditions, two populations of particles containing α-MSH were observed. When fractionated at equilibrium conditions, the two populations were recovered in a single band. These sedimentation characteristics indicate that the particles that contain α-MSH differ in size but are similar in density. After hypo-osmotic shock, the large particles containing α-MSH were not demonstrable, whereas the small particles appeared to be resistant to such treatment. In their sedimentation, the particles containing α-MSH were indistinguishable from particles containing thyrotropin releasing hormone (TRH) but were separable from those that contained luteinizing hormone releasing hormone (LHRH). It is suggested that the large particles containing α-MSH are synaptosomes.     

Immunohistochemical localization of porcine diazepam-binding inhibitor (DBI) to rat endocrine pancreas

Summary The occurrence of diazepam-binding inhibitor (DBI), isolated and characterized from porcine upper intestine, was examined in the pancreas of Sprague-Dawley albino rats using indirect immunofluorescence. The polypeptide was found in the endocrine Langerhans islets and, utilizing double-labelling controls, it was shown to be present within the peripherally located glucagon-containing cells. Regulation of islet hormone production may therefore be under DBI control.     

The source of an aggression-promoting olfactory cue,released by α-Melanocyte stimulating hormone,in the male mouse

Earlier experiments demonstrated that the injection of α-melanocyte stimulating hormone into one mouse of a male pair results, 15 minutes later, in the release of an olfactory cue which increases the aggressive behavior of its partner. Because the preputial gland, whose activity is dependent upon testosterone and α-MSH, produces an odor which stimulates aggressive behavior, its relationship with the MSH response was investigated. Preputialectomy failed to prevent the release of the odor by an MSH-treated subordinate mouse of a pair, although preputialectomy of one mouse of a naive pair resulted in it later displaying higher levels of aggression than its intact partner, which then became subordinate. Swabs taken from the perineal region of α-MSH-injected subordinates, contained more aggression-promoting factor than swabs taken from saline injected subordinates. It is concluded that the short-term effects of α-MSH upon aggression are probably via a product of glandular or urinary origin other than the preputial gland and that the latter is more involved in long-term aggression-promoting cues.     

A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase

As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and evaluated for their inhibitory potential in vitro. These compounds were designed and synthesized based on the structural attributes of a β-phenyl-α,β-unsaturated carbonyl scaffold template. Among these compounds, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (1e, MHY773) exhibited the greatest tyrosinase inhibition (IC₅₀ = 2.87 μM and 8.06 μM for monophenolase and diphenolase), and outperformed the positive control, kojic acid (IC₅₀ = 15.59 and 31.61 μM). The kinetic and docking studies demonstrated that MHY773 interacted with active site of tyrosinase. Moreover, a melanin quantification assay demonstrated that MHY773 attenuates α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine (IBMX)-induced melanin contents in B16F10 melanoma cells. Taken together, these data suggest that MHY773 suppressed the melanin production via the inhibition of tyrosinase activity. MHY773 is a promising for the development of effective pharmacological and cosmetic agents for skin-whitening.     

Vascular pattern in the pancreas of the cat

Summary The pathways of microcirculation in the pancreas of the cat were investigated by scanning electron microscopy of Mercox preparations of the vascular bed. A portal system from islet to exocrine vessels as well as a direct arterial flow to the exocrine pancreas have been observed. Sphincters appear to exist in portal capillaries at the border between the endocrine and exocrine portions of the pancreas. Islets possess an independent venous drainage.A portion of these results was presented on the occasion of the 6th European Anatomical Congress, Hamburg 1981 (see Syed Ali 1981, for abstract).     

Gastric protection by alpha-melanocyte-stimulating hormone against ethanol in rats: involvement of somatostatin

The proopiomelanocortin-derived tridecapeptide alpha-melanocyte-stimulating hormone (alpha-MSH) is a neuropeptide that exerts broad anti-inflammatory actions in mammals. This study aimed to investigate the effect of alpha-MSH on ethanol-induced gastric ulcer in rats and to evaluate the involvement of endogenous somatostatin in the actions of the peptide. The rats received 1 mL 75% ethanol or saline orally. alpha-MSH was given (25 micro g/rat; i.p.) alone or following the somatostatin antagonist cyclo-(7-aminoheptanoyl-PH-E-d-Trp-Lys-THR) (10 microM/kg; i.p.) administration. Gastric lesions were scored macroscopically and microscopically following decapitation at 30 min after ethanol challenge. Gastric malondialdehyde (MDA) level, myeloperoxidase (MPO) activity and mast cell counts were assessed. Ethanol-induced gastric hemorrhagic lesions were characterized by increased gastric MDA level, MPO activity and mast cell counts. alpha-MSH treatment decreased the extent of tissue injury and reversed tissue MDA level, MPO activity and mast cell counts. The effect of the peptide on the severity of gastric lesions, MDA level and MPO activity was reversed by the somatostatin antagonist. In conclusion, alpha-MSH is beneficial in a rat model of gastric ulcer via mechanisms which partly involve the endogenous somatostatin.     

Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas

Summary It was previously demonstrated that the two chemically related peptides calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) both occur in the pancreas. We have now examined the cellular localization of CGRP and IAPP in the rat and the mouse pancreas. We found, in both the rat and the mouse pancreas, CGRP-immunoreactive nerve fibers throughout the parenchyma, including the islets, with particular association with blood vessels. CGRP-immunoreactive nerve fibers were regularly seen within the islets. In contrast, no IAPP-immunoreactive nerve fibers were demonstrated in this location. Furthermore, in rat islets, CGRP immunoreactivity was demonstrated in peripherally located cells, constituting a major subpopulation of the somatostatin cells. Such cells were lacking in the mouse islets. IAPP-like immunoreactivity was demonstrated in rat and mouse islet insulin cells, and, in the rat, also in a few non-insulin cells in the islet periphery. These cells seemed to be identical with somatostatin/CGRP-immunoreactive elements. In summary, the study shows (1) that CGRP, but not IAPP, is a pancreati neuropeptide both in the mouse and the rat; (2) that a subpopulation of rat somatostatin cells contain CGRP; (3) that mouse islet endocrine cells do not contain CGRP; (4) that insulin cells in both the rat and the mouse contain IAPP; and (5) that in the rat, a non-insulin cell population apparently composed of somatostatin cells stores immunoreactive IAPP. We conclude that CGRP is a pancreatic neuropeptide and IAPP is an islet endocrine peptide in both the rat and the mouse, whereas CGRP is an islet endocrine peptide in the rat.     

Alpha-1-antitrypsin immunoreactivity in islet cells of adult human pancreas

alpha-1-antitrypsin immunoreactivity was demonstrated by immunofluorescence technique in the peripheral islet cells of all ten normal adult human pancreata examined; normal adult human liver was negative. The specificity of the reactions was confirmed by applying various control tests including absorption of the specific antisera with purified alpha-1-antitrypsin, inhibition and blocking tests and by ensuring the monospecificity of the antisera used. The findings suggest that the pancreatic islet may be an additional source of alpha-1-antitrypsin.     

Immunocytochemical localization of muscarinic acetylcholine receptors in the rat endocrine pancreas

Summary Immunocytochemical application of the antimuscarinic acetylcholine receptor antibody M35 to pancreas tissue revealed the target areas for the parasympathetic nervous system. Immunoreactivity in the endocrine pancreas was much higher than that in the exocrine part. Moreover, the endocrine cells at the periphery of the islets of Langerhans displayed the highest level of immunoreactivity. Based on these findings in the mantle of the islets, two types of islets have been distinguished: type-I islets with intensely stained mantle cells, and type-II islets with a much lower concentration of these cells. On average, type-I islets were larger (244.8 m±6.1 SEM) than type-II islets (121.5 m±3.8 SEM). M35-immunoreactivity was present on the majority of D cells, which were characterized by their immunoreactivity to somatostatin [of 446 D cells 356 (79.8%) were M35-immunopositive]. However, only a small proportion of the intensely stained mantle cells belonged to the D cell population. Therefore, it is concluded that the majority of the intensely stained mantle cells represent glucagon-secreting A and/or pancreatic polypeptide-secreting F cells. The intensity of M35-immunoreactivity at the periphery and central core of the islets paralleled the density of cholinergic innervation, suggesting a positive correlation between the intensity of cholinergic transmission and the number of muscarinic acetylcholine receptors at the target structures. The present study further revealed some striking parallels for the muscarinic acetylcholine receptor characteristics between the (endocrine) pancreas and the central nervous system.     

Quantification of the permeability of the blood-CSF barrier to alpha-MSH in the rat

The ability of alpha-MSH to cross the blood-CSF barrier of the rat was assessed by measurement of the rate of appearance of immunoreactive alpha-MSH in a cerebrospinal fluid (CSF) perfusate following intravenous injection of peptide. Comparisons were made with the rate of appearance of a simultaneously administered dose of 14C-inulin which is poorly permeable at the blood-CSF barrier. Concentrations of drugs measured in plasma were fitted to two-compartment pharmacokinetic models, and those measured in the CSF perfusate to one-compartment open systems receiving an input from the plasma compartment. The rate constant for entry of alpha-MSH into CSF was 0.00087 min-1, which was not significantly different from that for inulin of 0.00055 min-1. As alpha-MSH penetrated into CSF at a rate comparable to inulin, it was concluded that the limited entry of peptide was by aqueous diffusion along with other water-soluble macromolecules.     

Synthesis,biological evaluation,and metabolic stability of chlorogenic acid derivatives possessing thiazole as potent inhibitors of α-MSH-stimulated melanogenesis

A series of catechol and dioxolane analogs containing thiazole CGA derivatives have been synthesized and evaluated for their inhibitory activity against α-MSH. The inhibitory activity was improved by replacing an α,β-unsaturated carbonyl of previously reported caffeamides with thiazole motif. Surprisingly, compound 7d, one of the derivatives of dioxolane analogs, displayed the most potent inhibitory activity with an IC₅₀ of 0.90 μM. Further studies on metabolic stability and bioactivation potential were also accomplished.