[Tyr1]-Somatostatin

The rate of degradation of 125I-labelled [Tyr11]somatostatin by isolated rat hepatocytes was similar to that of unlabelled somatostatin. Reaction was dependent upon cell concentration and temperature, being rapid at 37 degrees C and negligible at 0 degrees C. The apparent Km for the overall degradative process was approximately the same for degradation by hepatocytes and by partially-purified liver plasma membranes. Extracellular breakdown of somatostatin, by proteases released from cells into the incubation medium, represented less than 10% of the cell-associated degradation. Homogenization of hepatocytes resulted in a 10--20-fold increase in the degrading ability of the cells. After incubation of 125I-labelled [Tyr11]somatostatin and 125I-labelled [Tyr1]somatostatin with hepatocytes, 125I-labelled tyrosine was the major radioactive product identified in the incubation medium. The rate of release of 125I-labelled tyrosine from the labelled [Tyr1]analogue was approximately 11 times greater than from the labelled [Tyr11] analogue. 125I-labelled [Tyr11]somatostatin bound to the cells in a non-saturable manner and approx. 70% of the cell-associated radioactivity could be dissociated by dilute acid. The rate of degradation of somatostatin was unchanged by reagents that inhibit the internalisation and lysosomal degradation of polypeptides by cell suspensions but was reduced by reagents that inhibit sulphydryl-dependent proteases. It is proposed that plasma-membrane associated proteolysis, involving both endo- and exopeptidases may represent the predominant degradative pathway of somatostatin in vivo.

GH4C1 cells are a clonal strain of rat pituitary tumor cells which synthesize and secrete prolactin and growth hormone. Somatostatin, a hypothalamic tetradecapeptide, inhibits the release of growth hormone and, under certain circumstances, also prolactin from normal pituitary cells. We have prepared [125I-Tyr1]somatostatin (approximately 2200 C1/mmol) and have shown that this ligand binds to a limited number of high affinity sites on GH4C1 cells. Half-maximal binding of somatostatin occurred at a concentration of 6 x 10(-10) M. A maximum of 0.11 pmol of [125I-Tyr1]somatostatin was bound per mg of cell protein, equivalent to 13,000 receptor sites per cell. The rate constant for binding (kon) was 8 x 10(7) M(-1) min(-1). The rate constant for dissociation (koff) was determined by direct measurement to be 0.02 min(-1) both in the presence and absence of excess nonradioactive somatostatin. Binding of [125I-Tyr1]somatostatin was not inhibited by 10(-7) M thyrotropin-releasing hormones. Substance P, neurotensin, luteinizing hormone-releasing hormone, calcitonin, adrenocorticotropin, or insulin. Of seven nonpituitary cell lines tested, none had specific receptors for somatostatin. Somatostatin was shown to inhibit prolactin and growth hormone production by CH4C1 cells. The dose-response characteristics for binding and the biological actions of somatostatin were essentially coincident. Furthermore, among several clonal pituitary cell strains tested, only those which had receptors for somatostatin showed a biological response to the hormone. We conclude that the characterized somatostatin receptor is necessary for the biological actions of somatostatin on GH4C1 cells.

AtT20/D16v is a clonal strain of mouse pituitary tumor cells which synthesizes and secretes ACTH. Somatostatin, a hypothalamic tetradecapeptide, has been shown to inhibit the release of PRL, GH, and TSH from the pituitary gland. We have characterized specific binding sites for somatostatin on AtT20/D16v cells and demonstrate that somatostatin inhibits stimulated ACTH release by these cells. Equilibrium binding studies with [125I]Tyr1]somatostatin showed the presence of a single class of noninteracting binding sites on AtT20/D16v cells. Half-maximal binding of somatostatin occurred at 1.7 X 10(-9) M, and there were 26,300 binding sites/cell. The binding of [125I]Tyr1]somatostatin was not significantly inhibited by the hypothalamic peptides TRH, LHRH, and substance P. Somatostatin had no consistent effect on basal ACTH secretion by AtT20/D16v cells, but it inhibited ACTH secretion stimulated with either 50 mM KCl or a hypothalamic extract. Half-maximal inhibition occurred with 4 X 10(-10) M somatostatin. TRH, LHRH, and substance P at concentrations of 10(-7) M were without effect. Somatostatin had no effect on either basal or stimulated hormone secretion by GH12C1 or F4C1 cells, two cell strains which lack specific somatostatin-binding sites. A critical concentration of extracellular calcium was required for the stimulation of ACTH secretion in AtT20/D16v cells. No response to 50 mM KCl occurred in the presence of EGTA or cobalt. Increased extracellular calcium overcame the inhibition of stimulated hormone secretion by EGTA, cobalt, and somatostatin. Therefore, we conclude that the inhibition of stimulated ACTH secretion by somatostatin involves the interaction of the peptide with specific binding sites on AtT20/D16v cells and the inhibition of stimulus-elicited calcium influx.