PDFlib PLOP: PDF Linearization, Optimization, Protection Page inserted by evaluation version www.pdflib.com –
[email protected]
Localization of Amidating Enzymes (PAM) in Frog (Rana temporaria) Endocrine Pancreasa JAVIER BUTRÓN, MARIA E. BODEGAS, ANA C. VILLARO, JUAN C. ETAYO, AND PILAR SESMA Department of Histology, Faculties of Medicine and Science, University of Navarra, 31080 Pamplona, Navarra, Spain
Carboxy-terminal α-amidation is an essential posttranslational process associated with the activation of many neuroendocrine peptides.1 The amidating precursor peptidylglycine α-amidating monooxygenase (PAM) includes two enzymatic domains, known as peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-αhydroxyglicine α-amidating lyase (PAL), responsible for the two steps that constitute the amidating reaction.2 The presence of PAM in endocrine pancreas has received little attention, which has been mainly focused in mammals.3–6 To our knowledge, studies in non-mammalian vertebrate pancreas have been carried out only in fishes (Lophius americanus).7–9
PAM IN RANA TEMPORARIA PANCREAS Avidin-biotin complex (ABC) technique was applied to paraffin and epon-embedded frog pancreas, using specific antisera against two PHM different sequences, PAL, insulin, glucagon, glucagon-like peptide (GLP-1), pancreatic polypeptide (PP), somatostatin, met-enkephalin, big-endothelin-1 and 7B2. Amidating enzymes were located in a part of the pancreatic endocrine population. As observed in serial sections, antibodies against PHM and PAL stained the same cells in the islets. PAL antiserum rendered stronger immunoreaction . The study of serial sections showed that all the PAM-positive cells were also immunoreactive for insulin, met-enkephalin, big-endothelin-1 and 7B2. In contrast, cells immunoreactive for glucagon, GLP-1, PP and somatostatin were negative for the PAM antibodies used.
CONCLUSIONS To our knowledge this is the first description of the presence of PAM immunoreactivity in amphibian pancreas. PAM immunoreactivity has been demonstrated in β-cells of Rana temporaria pancreas, since both PHM and PAL immunoreactivities have been colocalized with insulin, met-enkephalin, big-endothelin-1 and 7B2, present in frog β-cells. a This study was supported by the “Ministerio de Educación y Ciencia” (DGICYT, PB930711).
486
BUTRON et al.: AMIDATING ENZYMES
487
Neither PHM nor PAL immunoreactivity has been found in the rest of endocrine cell types: α-cells, immunoreactive for glucagon, GLP-1 and PP, or δ-cells immunoreactive for somatostatin.
ACKNOWLEDGMENTS We wish to thank B.A. Eipper, Johns Hopkins University, Baltimore, MD, USA; F. Cuttita, and A. Treston National Cancer Institute, Rockville, MD, USA; and J. Polak, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK for the generous gift of antisera. REFERENCES 1. E IPPER B.A., S TOFFELS D.A. & M AINS R.E. 1992. The biosynthesis of neuropeptides: Peptide α-amidation. Ann. Rev. Neurosci. 15: 57–85. 2. PERKINS S.N., H USTEN E.J. & E IPPER B.A. 1990. The 108-kDA peptidylglycine alpha-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation. Biochem. Biophys. Res. Commun. 171: 926–932. 3. M ARTÍNEZ A., M ONTUENGA L., S PRINGALL D., T RESTON A., C UTTITA F., & P OLAK J. 1993. Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas. J. Histochem. Cytochem. 41: 375–380. 4. SCHARFMANN R., L EDUQUE P., A RATAN -SPIRE S., D UBOIS P., BASMACIOGULLARI A. & C ZERNICHOW P. 1988. Persistence of peptidylglycine alpha-amidating monooxygenase activity and elevated thyrotropin-releasing hormone concentrations in fetal islets in culture. Endocrinology 123: 1329–1334. 5. M ALTESE J.Y., G IRAUD P., K OWALSKI C., O UAFIK L.H., SALERS P., P ELEN F. & O LIVER C. 1989. Ontogenic expression of peptidyl-glycine alpha-amidating monooxygenase mRNA in the rat pancreas. Biochem. Biophys. Res. Commun. 158: 244–250. 6. K APUSCINSKI M. & S HULKES A. 1995. Peptide amidating activity and gastrin processing in the developing sheep pancreas. J. Endocrinol. 145: 137–142. 7. M ACKI R.B., F LACKER J.M., M ACKIN J.A. & N OE B.D. 1987. Peptidylglycine alphaamidating monooxygenase is present in islet secretory granules of the anglerfish Lophius americanus. Gen. Comp. Endocrinol. 67: 263–269. 8. N OE B.D., K ATOPODIS A.G. & MAY S.W. 1991. Kinetic analyses of peptidylglycine alpha-amidating monooxygenase from pancreatic islets. Gen. Comp. Endocrinol. 83: 183–192. 9. M C D ONALD J.K., K LEIN K. & N OE B.D. 1995. Distribution of peptidylglycine alphaamidating monooxygenase immunoreactivity in the brain, pituitary and islet organ of the anglerfish (Lophius americanus). Cell Tissue Res. 280: 159–170.
