Expression and Processing of the [Pro²,Met¹³]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.

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“…FMRFamide and α‐MSH were from Sigma (St. Louis, MO). The antiserum against PSS2 54‐66 was generated in rabbit by using synthetic PSS2 54‐66 conjugated to bovine thyroglobulin (Tostivint et al,2002). The somatostatin‐14 antiserum was from Biomeda (Foster City, CA).…”

“…The mRNAs encoding PSS1 and PSS2 are differentially distributed in frog tissues. Specifically, the PSS1 gene is widely expressed in the CNS and in peripheral organs including the pancreas and gastrointestinal tract whereas PSS2 is expressed only in the brain (Tostivint et al,1996) and in the intermediate lobe of the pituitary (Tostivint et al,2002). In the brain of all species investigated so far, the mRNA encoding PSS1 is more widely expressed than that encoding PSS2/prepro‐cortistatin (PCST) (Tostivint et al,1996; Lin et al,1999; Trabucchi et al,1999,2002,2003; Devos et al,2002).…”

“…However, at the peptide level, studies aimed at determining the specific distributions of SS1 and SS2/CST are hampered by the fact that antibodies directed against SS1 and SS2/CST may cross‐react to a certain extent with the other neuropeptide. To solve this problem, we have recently raised polyclonal antibodies against the N‐flanking region of SS2 (PSS2 54‐66 ) that specifically label PSS2‐expressing neurons but do not stain PSS1‐expressing cells (Tostivint et al,2002).…”

Somatostatin and its receptors from fish to mammals

“…As mentioned earlier, the processes of local and segmented duplication that occurred in the first stages of vertebrate evolution originated a large number of SST‐related peptides encoded by up to four different genes 1 . Because SST14 is present in all vertebrate phyla, 37–40 except for holocephalan fish, 41 and its nucleotide‐amino acidic sequence has been highly conserved across species, its coding gene, SS1, is considered the ancestral SST gene 2 . However, comparative genome organization studies suggest that SS1 gene shares with urotensin (UII) gene a common ancestor present in early vertebrates.…”

Effects of Pituitary Adenylate Cyclase‐Activating Polypeptide, Vasoactive Intestinal Polypeptide, and Somatostatin on the Release of Thyrotropin from the Bullfrog Pituitary

“…It thus appears that the two somatostatin isoforms may control the activity of both somatotrope and thyrotrope cells in amphibians. Consistent with this notion, it has been recently reported that the gene encoding the SS2 precursor is actively expressed in the pars intermedia of the frog pituitary, 24 suggesting that SS2, released by melanotrope cells, can diffuse toward the distal lobe to regulate the secretion of various adenohypophysial hormones.…”