Expression and Processing of the [Pro 2 ,Met 13 ]Somatostatin-14 Precursor in the Intermediate Lobe of the Frog Pituitary
Abstract
The biosynthesis of various hypothalamic neuropeptides has been previously reported in anterior pituitary cells but not in intermediate lobe cells. We have recently demonstrated the occurrence of two somatostatin isoforms in the frog brain, namely somatostatin-14 (SS1) and [Pro(2),Met(13)]somatostatin-14 (SS2). In the present study, we demonstrate that the gene encoding the SS2 precursor (PSS2) is actively expressed in the intermediate lobe of the frog pituitary. High concentrations of PSS2 mRNA have been detected by Northern blot analysis and in situ hybridization in the frog pars intermedia but not in the pars distalis or pars nervosa. The distribution of PSS1- and PSS2-derived peptides has been investigated by immunohistochemistry using two antisera directed against SS1 and the sequence 54-66 of PSS2 (PSS2(54-66)), respectively. The SS1 antiserum stained only a network of fibers in the neural lobe and a few nerve processes in the intermediate lobe. In contrast, the PSS2(54-66) antiserum produced intense labeling of melanotrope cells in the pars intermedia. Biochemical characterization of the immunoreactive materials present in pituitary extracts was performed by combining high-performance liquid chromatography analysis and RIA detection. The SS1 RIA revealed the existence of two major immunoreactive peaks that exhibited the same retention times as synthetic SS1 and SS2. The PSS2(54-66) RIA detected a single peak that likely corresponds to the N-flanking peptide of SS2 (PSS2(1-66)). The present study reveals that melanotrope cells of the frog pituitary selectively express the PSS2 gene and fully process PSS2 to generate the mature somatostatin variant SS2. Taken together, these data provide the first evidence that the gene encoding a hypophysiotropic neuropeptide is intensely expressed in the intermediate lobe of the pituitary.