Occurrence of members of the insulin superfamily in central nervous system and digestive tract of protochordates.

Antisera specific for mammalian insulin-like growth factor 1 (IGF-1) and mammalian insulin and the double immunofluorescence technique were used for this study. IGF-1-like-immunoreactivity was localized in entero-endocrine cells in the gastro-intestinal tract of the protochordates Ciona intestinalis and Branchiostoma lanceolatum. Some of the specimens also showed IGF-1-like-immunoreactive (-IR) perikarya and fibers in the central nervous system. Whilst in rat endocrine pancreas, IGF-1-IR and insulin-IR occurred in different cell populations, in Ciona and Branchiostoma the vast majority of entero-endocrine cells and central neurons were IGF-1-like- +insulin-IR. A minor portion exhibited IGF-1-like-IR alone. For further characterization of the IGF-1-like-IR material, in Ciona intestinalis, peptides related to IGF-1 were identified by radioimmunoassay and gel chromatography. In accordance with the immunohistochemical results, IGF-I-like-IR was detected both in cerebral ganglion and in gastro-intestinal tract. Using acid gel chromatography, in Ciona gastro-intestinal tract the IGF-1-like-IR was found to occur in two peaks, with apparent molecular weights of approximately 16 kDa and 3 kDa. Absorption studies with insulin- and IGF-related peptides, with crude extracts and the peak material obtained after gel chromatography, indicated that the IGF-1-like peptides in Ciona are different from mammalian insulin and IGF-1. The findings are in accordance with the presence of a common insulin/IGF precursor molecule in protochordates.

Immunocytochemistry of cardiac polypeptide hormones (cardiodilatin/atrial natriuretic polypeptide) in brain and hearts of Myxine glutinosa (Cyclostomata).

With the use of different region-specific antisera against partial sequences of porcine cardiodilatin (CDD)-126 and the peroxidase-antiperoxidase (PAP) technique, the central nervous system as well as the systemic and the portal vein heart of the cyclostomian species Myxine glutinosa were investigated for a possible existence of cardiac polypeptides. In contrast to mammals, CDD-immunoreactions were obtained only with antisera directed against the C-terminus of CDD (CDD 99-126) which is identical to human atrial natriuretic polypeptide (alpha hANP). CDD-immunoreactive myocardiocytes were found in high densities in the atrium of the systemic heart and in the portal vein heart. In the ventricle of the systemic heart, CDD-immunoreactive cells were extremely scarce. In agreement with the immunohistochemical results, myoendocrine cells analyzed by electronmicroscopy exhibited specific granules of an average diameter of 0.21 + 0.02 micron in equivalent localizations. Furthermore, with the use of the protein A-gold (PAG) technique, CDD-immunoreactivity was ultrastructurally localized within the specific granules of atrial myocardiocytes. In the central nervous system of Myxine glutinosa, CDD-immunoreactive perikarya and/or fibers were present on all levels from the telencephalon to the spinal cord. The results of the present study are compared with those obtained in mammals and their possible functional relevance and their meaning in phylogeny are discussed as well.

Electronmicroscopical, immunohistochemical, immunocytochemical and biological evidence for the occurrence of cardiac hormones (ANP/CDD) in chondrichthyes.

As representatives of the vertebrate class of chondrichthyes the plagostomian species Squalus acanthias, Scyliorhinus canicula and Raja clavata as well as the holocephalan species Chimaera monstrosa were investigated for the presence of cardiac hormones of the atrial natriuretic polypeptide/cardiodilatin- (ANP/CDD-) family. ANP/CDD-immunoreactive cells were detected in the atria and the ventricles of all species studied. While these cells failed to react with antisera raised against the N-terminus of CDD-126 (= gamma-ANP) they reacted with all antisera directed against sequences of the C-terminus of CDD-126 (CDD 99-126) which is identical to alpha-ANP. The ANP/CDD-immunoreactive cells were found in high numbers in all regions of the atria and in moderate density also in the ventricles. In correspondence, in the electron microscope, myoendocrine cells which were characterized by dense-cored secretory granules were identified in the atrial and ventricular myocardium. With the use of the protein A-gold technique, ANP/CDD-immunoreactivity was determined within the secretory granules. Furthermore, in the bioassay, prepurified extracts of the atria and the ventricles of Scyliorhinus and Chimaera exerted dose-dependent relaxations of the pre-contracted mammalian (rabbit) aorta. In both cases the atrial extracts proved to be more potent than the ventricular extracts. The present findings indicate that myoendocrine cells occur in the atria and ventricles of chondrichthyes and that these cells contain homologous cardiac hormones of the ANP/CDD-family in their secretory granules. The results are compared with those obtained earlier for the other vertebrate classes and their phylogenetic and functional significance is discussed.