A fully functional proopiomelanocortin/melanocortin-1 receptor system regulates the differentiation of human scalp hair follicle melanocytes

The proopiomelanocortin (POMC)-derived peptides, ACTH and alpha-MSH, are the principal mediators of human skin pigmentation via their action at the melanocortin-1 receptor (MC-1R). Recent data have demonstrated the existence of a functionally active beta-endorphin/mu-opiate receptor system in both epidermal and hair follicle melanocytes, whereby beta-endorphin can regulate melanogenesis, dendricity, and proliferation in these cells. However, a role for ACTH and alpha-MSH in the regulation of the human follicular pigmentary unit has not been determined. This study was designed to examine the involvement of ACTH and the alpha-MSH/MC-1R system in human follicular melanocyte biology. To address this question we employed RT-PCR and immunohisto/cytochemistry, and a functional role for these POMC peptides was assessed in follicular melanocyte cultures. Human scalp hair follicle melanocytes synthesized and processed POMC. ACTH and alpha-MSH in association with their processing enzymes and MC-1R are expressed in human follicular melanocytes at the message level in vitro and at the protein level both in situ and in vitro. The expression of the POMC/MC-1R receptor system was confined only to subpopulations of poorly and moderately differentiated melanocytes. In addition, functional studies revealed that ACTH and alpha-MSH are able to promote follicular melanocyte differentiation by up-regulating melanogenesis, dendricity, and proliferation in less differentiated melanocyte subpopulations. Thus, these findings suggest a role for these POMC peptides in regulating human hair follicle melanocyte differentiation.     

Molecular cloning and sequencing of a guinea-pig pro-opiomelanocortin cDNA.

The guinea-pig has high levels of circulating cortisol. Though adrenocorticotropin (ACTH) levels are similar to those in other mammals, guinea-pig ACTH has been reported to have a single amino-acid substitution which results in increased bioactivity of the peptide. Pro-opiomelanocortin (POMC) is the precursor for ACTH, gamma-melanocyte-stimulating hormone (gamma-MSH) and the endogenous opioid peptide beta-endorphin. Both to confirm this substitution in guinea-pig ACTH and to establish whether other non-conservative substitutions occur elsewhere in the precursor we cloned guinea-pig POMC. The guinea-pig alanine for proline substitution at position 24 of ACTH was confirmed. Potentially significant mutations were also identified in gamma-MSH and beta-endorphin. A similar pattern of POMC mRNA expression was obtained for guinea-pig and rat as determined by Northern analysis and in situ hybridization. Southern blot analysis indicated that guinea-pig POMC is a single-copy gene. Cloning and sequencing of guinea-pig POMC thus further demonstrate the divergence of the New World hystricomorph peptides from those in New World primates, and underscore the differences observed in other endocrine axes in the guinea-pig.     

A newly characterized melanotropin in proopiomelanocortin in pituitaries of an elasmobranch, Squalus acanthias.

Proopiomelanocortin (POMC) is a precursor for corticotropin (ACTH), three or fewer molecular types of melanotropin (MSH), and beta-endorphin. This protein is thought to have evolved by duplication of MSH genomic segments. Here we report that the POMC in the dogfish, an elasmobranch, contains a fourth type of MSH in addition to classical alpha-, beta-, and gamma-MSH. POMC cDNA was amplified by PCR from double-strand cDNA prepared from dogfish pituitary and ligated into lambdaZAP II. The POMC cDNA is composed of 1315 bp without a poly(A) tail. Northern blot analysis detected a 1.4-kb signal of dogfish POMC mRNA. An open reading frame of the POMC cDNA encodes 320 amino acids, including a signal peptide of 26 amino acids. The dogfish POMC includes gamma-MSH, ACTH, alpha-MSH, beta-MSH, and beta-endorphin at positions 50-61, 115-153, 115-127, 239-256, and 259-294, respectively. In addition to these classical peptides, a newly discovered MSH, which we have termed delta-MSH, is present in dogfish POMC at position (184-195). The four dogfish MSHs can be separated into two groups based on their sequence identities: one pair consists of alpha-MSH and gamma-MSH, and the other consists of beta-MSH and delta-MSH, suggesting that gamma-MSH and delta-MSH may have been duplicated evolutionarily from alpha-MSH and beta-MSH, respectively. gamma-MSH might first have appeared in early gnathostomes because it is absent in the most primitive vertebrate group, the agnathans. delta-MSH, which at this time is found only in chondrichthians, might have appeared after the divergence of chondrichthians from a lineage leading to osteichthyans and tetrapods.     

Atrial secretion of B-type natriuretic peptide.

In the normal heart, the endocrine capacity resides in the atria. Atrial myocytes express and secrete natriuretic hormones that regulate fluid homeostasis and blood pressure. But in ventricular disease, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression is also activated in ventricular myocytes. Plasma concentrations of natriuretic peptides and their biosynthetic precursors are accordingly increased in patients with marked ventricular dysfunction. In contrast, atrial peptide secretion in ventricular disease has received less attention, and our present understanding of the endocrine atria during ventricular dysfunction is still scarce. Although ventricular disease and increased circulating concentrations are associated, it does not entail that the ventricle is the sole or even the main source in all types of heart disease. Clearly, the endocrine atria are also active in heart failure. Plasma measurement of cardiac natriuretic peptides and their molecular precursors can perhaps help us to discriminate when, where and how.     

心肌营养素-1诱导心肌细胞肥大及辛伐他汀的干预作用

目的观察细胞因子心肌营养素1(CT-1)诱导大鼠心肌细胞的肥大效应,探讨辛伐他汀(Sim)对这一过程的干预作用及其信号转导机制。方法以培养的新生Sprague-Dawley(SD)大鼠心肌细胞为实验模型,分为对照组,CT-1诱导组,CT-1 Sim组,CT-1 Sim 甲羟戊酸(MVA)组,CT-1 AG490(JAK2拮抗剂)组。应用图像分析系统测定心肌细胞表面积,3H-亮氨酸掺入法测定心肌细胞蛋白合成速率,逆转录聚合酶链式反应(RT-PCR)检测心钠素(ANP)mRNA表达,RT-PCR检测细胞因子信号通路JAK/STAT的mRNA表达水平。结果(1)与对照组相比,CT-1组心肌细胞表面积,3H-亮氨酸掺入率,ANPmRNA表达水平均明显增高(P<0.01),JAK2拮抗剂AG490可显著的抑制CT-1的作用(P<0.01)。(2)与CT-1组相比,辛伐他汀共培养后心肌细胞表面积,3H-亮氨酸掺入率,ANPmRNA表达水平明显减小(P<0.01),外源性甲羟戊酸可显著的抑制辛伐他汀的作用(P<0.01);(3)CT-1可使心肌细胞JAK-STAT的mRNA表达水平显著增强,辛伐他汀显著抑制CT-1诱导心肌细胞JAK-STATmR-NA表达水平增高(P<0.01),而MVA和AG490可部分阻断辛伐他汀的效应(P<0.01)。结论心肌营养素1能够诱导心肌细胞肥大,辛伐他汀可抑制其这一过程并可能与细胞因子信号通路JAK-STAT活化有关。     

Cloning of a proopiomelanocortin cDNA from the pituitary gland of the sea bass (Dicentrarchus labrax) and assessment of mRNA expression in different tissues by means of real-time PCR

Proopiomelanocortin (POMC) cDNA was cloned from sea bass (Dicentrarchus labrax) pituitary gland. A 743 nucleotide sequence was obtained coding for the following sequences flanked by sets of proteolytic cleavage sites: ACTH (Ser(88)-Met(127)), alpha-MSH (Ser(88)-Gly(102)), CLIP (Pro(106)-Met(127)), beta-LPH (Glu(131)-Gln(208)), gamma-LPH (Glu(131)-Ser(175)), beta-MSH (Asp(159)-Ser(175)), and beta-endorphin (Tyr(178)-Gln(208)). No region homologous to gamma-MSH/joining peptide (a tetrapod POMC feature) was found. Amino acid sequence identity was high with other teleostean species considered (tilapia: 73%) and lower with elasmobranchs (dogfish: 42%). However, the presumed biologically active peptides were highly conserved within all species considered: alpha-MSH (93-100%), ACTH (80-95%) and beta-endorphin (54-90%). Real-time PCR allowed us to quantify the expression of the POMC in different tIssues of the sea bass: pituitary gland, liver, gonad and head kidney. No significant POMC expression was found in the integument. In pituitary gland, gonads, head kidney and liver, POMC expression was respectively, 1.26x10(10), 2.67x10(5), 2.06x10(4) and 1.67x10(4) copies/ micro g mRNA.     

Expression of Atrial and Brain Natriuretic Peptides and Their Genes in Hearts of Patients With Cardiac Amyloidosis

Objectives. We investigated the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and their genes in the hearts of patients with cardiac amyloidosis and those with isolated atrial amyloidosis.Background. The expression of ANP and BNP is augmented in the ventricles of failing or hypertrophied hearts, or both. The expression of ANP and BNP in the ventricles of hearts with cardiac amyloidosis, which is hemodynamically similar to restrictive cardiomyopathy, is not yet known. ANP is the precursor protein of isolated atrial amyloid.Methods. We analyzed the immunohistocytochemical localizations of ANP and BNP as well as the expression of their mRNAs by in situ hybridization in the myocardium and measured the plasma levels of ANP and BNP in patients with cardiac amyloidosis.Results. Four of the five right and all six left ventricular endomyocardial biopsy specimens obtained from six patients with cardiac amyloidosis were immunohistochemically positive for both ANP and BNP; none of the biopsy specimens from eight normal subjects were positive for ANP or BNP. All four of the right atria obtained at operation showed positive immunoreactions for both peptides. Electron microscopy identified specific secretory granules in ventricular myoctyes of the patients with cardiac amyloidosis, but not in ventricular myocytes from the normal control subjects. Double immunocytochemical analysis revealed the co-localization of ANP and BNP in the same granules and that isolated atrial amyloid fibrils were immunoreactive for ANP and BNP, whereas ventricular amyloid fibrils were negative for both peptides. Both ANP mRNA and BNP mRNA were expressed in the ventricles of the patients with cardiac amyloidosis but not in the normal ventricles. The autopsy study of four patients with cardiac amyloidosis revealed an almost transmural distribution of ANP and BNP, with predominance in the endocardial side. Plasma BNP levels in the patients were markedly elevated ([mean ± SD] 1,165.1 ± 561.2 pg/ml) compared with those in the control subjects (8.9 ± 6.0 pg/ml, p < 0.05).Conclusions. Expression of ANP and BNP and their genes was augmented in the ventricular myocytes of the patients with cardiac amyloidosis. Both regional mechanical stress by amyloid deposits and hemodynamic stress by diastolic dysfunction may be responsible for the expression of the peptides in patients with cardiac amyloidosis.     

Atrial natriuretic peptide and brain natriuretic peptide coexist in the secretory granules of human cardiac myocytes.

To elucidate the intracellular localization of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human cardiac myocytes, an immunocytochemical study was carried out by a double immunogold technique using antisera highly specific for ANP and BNP. Surgical and autoptic tissue specimens of human heart were studied. In the atrial myocytes, ANP was localized in almost all of the secretory granules, whereas BNP was colocalized with ANP in some of the granules. Although very few secretory granules were observed in ventricular myocytes, colocalization of ANP and BNP was basically the same as in atrial myocytes. No immunoreactive products were found in the control studies. These results suggest that secretion of BNP is under a regulatory mechanism similar to that of ANP.     

Cytosolic Ca2+ during atrial natriuretic peptide secretion from cultured neonatal cardiomyocytes

Mechanisms of atrial natriuretic peptide (ANP) release were studied in neonatal rat heart atrial and ventricular myocytes cultured on Cytodex 3 microcarriers. For simultaneous observations of cytosolic free calcium concentration ([Ca2+]f) and ANP secretion, the culture was packed in a chromatography column, inserted into the cell holder of a spectrofluorometer was perifused with a buffer solution. [Ca2+]f was measured by the fluorescent calcium indicator Fura-2 and ANP in the effluent perfusate by radioimmunoassay. No cell damage was observed and the basal ANP secretion rate and [Ca2+]f were comparable with values obtained by other methods. K(+)-induced depolarization raised [Ca2+]f by 50%, but it rapidly declined again to a steady level 10-20% above the baseline. The calcium channel agonist Bay k8644 elicited a similar temporal pattern of [Ca2+]f changes and 1 microM ionomycin induced a 100-fold increase in [Ca2+]f with a slow re-establishment of the original baseline. None of these stimuli increased the ANP secretion rate of the atrial or ventricular myocytes. Protein kinase C activation by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated ANP secretion from the atrial myocytes, while the ventricular myocytes were unresponsive to TPA. It is concluded that Ca2+ is not the main mediator in the regulation of ANP release in cultured neonatal heart cells.     

Left ventricular assist device support reverses altered cardiac expression and function of natriuretic peptides and receptors in end-stage heart failure

OBJECTIVE: Atrial (ANP) and B-type natriuretics peptides (BNP) via their guanylyl cyclase-A (GC-A) receptor not only regulate arterial blood pressure and volume but also exert local antihypertrophic, antifibrotic and lusitropic effects in the heart. To elucidate whether cardiac hypertrophy/insufficiency and reversal is associated with changes in the local responsiveness to NPs, we compared the mRNA expression of ANP, BNP and receptors and the responsiveness of GC-A to ANP in left ventricular tissue obtained from 10 patients with congestive heart failure (CHF) before and after hemodynamic unloading by left ventricular assist device (LVAD) support. METHODS AND RESULTS: Quantitative "real time" RT-PCR demonstrated that the mRNA expression levels of ANP, BNP and the NP-metabolizing NPR-C receptor were both markedly increased in human failing hearts. GC-A mRNA expression levels were not different from nonfailing hearts, but cGMP production by GC-A in response to ANP was nearly abolished. Reversal of cardiomyocyte hypertrophy during LVAD support was accompanied by normalization of ANP, BNP and NPR-C mRNA levels and a significant recovery of GC-A responsiveness to ANP. CONCLUSION: In CHF patients, increased local clearance by NPR-C receptors and diminished responsiveness of cardiac GC-A might impair the local antihypertrophic effects of natriuretic peptides and contribute to the progression of cardiac hypertrophy and insufficiency. Reverse remodeling during LVAD support reverses these changes and can thereby recuperate the local protective effects of ANP and BNP.     

The functional significance of glycosylation of pro-opiomelanocortin in melanotrophs of the mouse pituitary gland

Pro-opiomelanocortin (POMC) is a glycoprotein precursor for a number of neuropeptides and peptide hormones. The functional significance of the glycosylation of POMC has never been established. Using the antibiotic tunicamycin to block glycosylation of the prohormone in the mouse pars intermedia, we have compared processing of non-glycosylated prohormone with that of glycosylated prohormone in pulse-chase experiments. The peptides produced from non-glycosylated prohormone were shown to be correct cleavage products. Therefore it was concluded that, with the possible exception of peptides from the N-terminal region of the prohormone, the carbohydrate on POMC plays no role in directing cleavage or in protecting the prohormone from random proteolysis. Tunicamycin treatment retarded N-terminal acetylation of melanotrophin but had no apparent effect on acetylation of beta-endorphin. The mouse pars intermedia synthesizes two forms of POMC which differ in their degree of glycosylation. Our results indicated that, during secretion, the melanotrophs make no distinction between peptides derived from the two prohormones.     

心钠素及内皮素对心肌细胞肥大及心脏成纤维细胞增殖的影响

目的观察心钠素(ANP)、内皮素-1(ET-1)对心肌细胞肥大和心脏成纤维细胞增殖的调节作用。方法采用体外乳鼠心肌细胞和心脏成纤维细胞培养,一定浓度的ET-1、ANP单独或联合作用细胞24h,以3H-胸腺嘧啶脱氧核苷(3H-TdR)、3H-亮氨酸(3H-Leu)掺入法测定细胞DNA、蛋白合成,Brad-ford法测细胞总蛋白含量,RT-PCR法测心肌细胞心钠素ANPmRNA的表达,评价ET-1、ANP对心肌细胞和成纤维细胞的作用。结果对于心肌细胞,ET-1显著促进3H-TdR、3H-Leu掺入,总蛋白含量增加,呈剂量依赖性,ANPmRNA表达明显增加;对于心脏成纤维细胞,ET-1促进3H-TdR、3H-Leu掺入,总蛋白含量增加,以上作用可被ANP明显抑制。结论ET-1促进ANP分泌的同时也促进心肌细胞肥大和心脏成纤维细胞增殖,ANP反之抑制ET-1的促心肌肥厚作用。     

Estradiol regulation of proopiomelanocortin gene expression and peptide content in the hypothalamus.

Previous studies have shown that the hypothalamic concentrations of beta-endorphin (beta-EP) and other proopiomelanocortin (POMC)-derived peptides change in the female rat following castration and gonadal steroid replacement. In this study we have measured POMC mRNA by solution hybridization assay in the medial basal hypothalamus (MBH) of ovariectomized rats treated with a regimen of estradiol (E2) that we have previously shown alters brain beta-EP peptide content. In addition the effect of progesterone (P) was also studied. In the first experiment the concentration of beta-EP and alpha-melanocyte-stimulating hormone (alpha-MSH) in the MBH of castrated rats decreased significantly after 3 weeks of E2 treatment compared to castrated unreplaced rats: beta-EP decreased from 6.00 +/- 0.46 to 4.32 +/- 0.38 ng/mg protein and alpha-MSH decreased from 3.00 +/- 0.23 to 2.35 +/- 0.15 ng/mg protein (p less than 0.05). A similar decrease in peptide content was noted in the anterior hypothalamus/preoptic area. A parallel reduction in the concentration of POMC mRNA was measured in the MBH of the E2-replaced animals: 1.17 +/- 0.14 vs. 0.72 +/- 0.08 pg/microgram RNA (p less than 0.02). In a second study castrated rats were studied after 2 weeks of E2 or E2 plus P treatment. After 2 weeks, POMC peptide levels did not change significantly in the MBH of either the E2- or E2 plus P-treated rats. POMC mRNA, however, was significantly reduced from 1.10 +/- 0.10 pg/micrograms RNA in the unreplaced rats to 0.58 +/- 0.05 and 0.61 +/- 0.06 pg/microgram RNA after E2 or E2 plus P, respectively (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Light and electron microscopic localization of brain natriuretic peptide in relation to atrial natriuretic peptide in porcine atrium. Immunohistocytochemical study using specific monoclonal antibodies.

BACKGROUND. Although first isolated from the porcine brain, brain natriuretic peptide (BNP) is also found in the heart, particularly in the atria. METHODS AND RESULTS. We examined the immunocytochemical localization of BNP in relation to atrial natriuretic peptide (ANP) in five porcine atria using highly specific monoclonal antibodies. With the use of an indirect immunoperoxidase method, serial sections examined by light microscopy showed foci of multihormonal myocytes containing both ANP and BNP localized in the subendocardium. Monohormonal myocytes containing ANP only were observed in the subepicardium and part of the subendocardium. The staining intensity of ANP and BNP showed a transmural gradient from the subendocardium to the subepicardium. At the electron microscopic level, double immunocytochemistry using a two-face immunogold-staining method revealed two types of granules: Type 1 is a monohormonal granule containing ANP alone, and type 2 is a multihormonal granule containing both ANP and BNP. Although most atrial myocytes contained both, type 1 granules were frequently observed in the epicardial side, and type 2 granules were frequently observed in the endocardial side. We observed a few type 2 granules even in the light microscopically monohormonal myocytes. CONCLUSIONS. These data suggest that the atrial myocyte population comprises single, multipotential cells able to synthesize both natriuretic peptides in varying proportions. The atrial transmural gradient of BNP and ANP may be related to responses to wall stress.     

Expression of A-, B-, and C-type natriuretic peptide genes in failing and developing human ventricles. Correlation with expression of the Ca(2+)-ATPase gene.

Brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are novel natriuretic peptides, originally isolated from porcine brain. Similar to atrial natriuretic peptide (ANP), BNP is also synthesized in and secreted from cardiocytes, but CNP is not expressed at significant levels in normal adult myocardium. Previous studies have indicated that the serum level and ventricular expression of the ANP gene were augmented in patients with heart failure. Recently, the serum level of BNP was also reported to increase in human heart failure. To examine whether or not the expression of these natriuretic peptides is regulated in ventricular myocardium in a concordant manner, we performed Northern blot analysis using total cellular RNA isolated from the diseased left ventricles of 30 cardiac transplant recipients with end-stage heart failure, seven ventricles from organ donors (control group), and two ventricles of artificially aborted 17- and 19-week-old fetuses. The levels of mRNAs encoding both BNP and ANP increased significantly (p less than 0.01) in the left ventricular myocardium from the patients with end-stage heart failure as compared with the control group. The levels of BNP mRNA correlated positively with those of ANP mRNA (r = 0.73, p less than 0.01) and negatively with those of sarcoplasmic reticulum Ca(2+)-ATPase mRNA (r = -0.66, p less than 0.01) in the left ventricular myocardium from the patients with heart failure. There was also a negative correlation between the levels of ANP and the sarcoplasmic reticulum Ca(2+)-ATPase mRNAs (r = -0.65, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)