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A Martinez, R Pio, J Lopez, and F Cuttitta

Adrenomedullin (AM) is a ubiquitous peptide hormone which, among other functional roles, reduces insulin secretion in the pancreas. Recently we have described the interaction between AM and the complement regulator protein factor H, which results in mutual modulation of their respective functions. Here we identify the expression of factor H in the beta cells of the rat pancreatic islets by immunohistochemistry and multiple immunofluorescence followed by confocal microscopy. In addition, double immunogold staining under the electron microscope showed coexistence of insulin and factor H immunoreactivities within the same secretory granules; interestingly, factor H staining was found in the electron-lucent haloes whereas the insulin antibody labeled preferentially the dense cores. The existence of factor H mRNA in the pancreas was confirmed by RT-PCR and in situ hybridization. The function of factor H in the pancreas was investigated with an insulin secretion assay. Addition of factor H to freshly isolated islets in the presence of AM resulted in a further reduction in insulin secretion with a concomitant elevation of cAMP, suggesting that factor H increases AM function in glucose homeostasis. The expression of factor H in the pancreas may play other important roles such as protection against complement-mediated cell lysis.

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S Kapas, A Martinez, F Cuttitta, and JP Hinson

This study was designed to investigate the synthesis and action of adrenomedullin in the rat adrenal gland. The results obtained from in situ hybridization and immunocytochemical studies suggest that adrenomedullin is synthesized not only in the medulla, but also within the zona glomerulosa of the rat adrenal cortex. Findings from in situ hybridization and binding studies also suggested that specific adrenomedullin receptors are expressed in the zona glomerulosa, and that low levels are present in the inner zones of the cortex. The Kd of the zona glomerulosa adrenomedullin receptor (5.5 nmol/l) suggests that it may respond to locally produced adrenomedullin rather than circulating concentrations of the peptide, which are in a lower range. It was found that adrenomedullin acted on zona glomerulosa cells in vitro to stimulate aldosterone release and cAMP formation, but in this tissue did not stimulate inositol phosphate turnover. The effect of adrenomedullin on aldosterone secretion was significantly attenuated by a protein kinase A inhibitor, suggesting that cAMP mediates the effects of adrenomedullin on aldosterone secretion. Adrenomedullin did not significantly affect the response of zona glomerulosa cells to stimulation by either ACTH or angiotensin II. Adrenomedullin did not affect the release of catecholamines, either adrenaline or noradrenaline, by intact adrenal capsular tissue. These data suggest that both adrenomedullin and its specific receptor are expressed in the rat adrenal zona glomerulosa, leading to the hypothesis that adrenomedullin may have an autocrine/paracrine role in the regulation of the rat adrenal zona glomerulosa.

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A Martinez, L Saldise, MJ Ramirez, S Belzunegui, E Zudaire, MR Luquin, and F Cuttitta

Adrenomedullin (AM) immunoreactivity has been found in granules of the glomus (type I) cells of the carotid bodies in rats. The identity of these cells was ascertained by colocalization of immunoreactivities for AM and tyrosine hydroxylase in their cytoplasm. Exposure of freshly isolated carotid bodies to synthetic AM resulted in a concentration- and time-dependent degranulation of glomus cells as measured by dopamine (DA) release. DA release reached a zenith 30 min after exposure to AM (94.2% over untreated controls). At this time-point, the response to AM was similar to the one elicited by 5 min of exposure to 100 mM K+. Nevertheless, injection of 1 micro l 60 nM AM/g body weight into the tail vein of the rats did not induce statistical differences in DA release from the carotid bodies. Exposure of the oxygen-sensitive cell line PC-12 to hypoxia elicited an increase in AM mRNA expression and peptide secretion into serum-free conditioned medium. Previous data have shown that elevation of AM expression under hypoxia is mediated through hypoxia-inducible factor-1, and that exposure of chromaffin cells to AM results in degranulation. All these data suggest that AM is an important autocrine regulator of carotid body function.