Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions

Abstract

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([ 125I-Tyr 11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu 8, d-Trp 22, 125I-Tyr 25] SS-28, referred to as LTT∗ SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10 −7–10 −3 M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the B max of the SS receptors, the binding affinity remaining unaltered. GTP (10 −4 M) decreased the B max of LTT∗ SS-28 binding sites to a greater extent than ATP (145 ± 10 and 228 ± 16 respectively, compared to control value of 320 ± 20 pmol mg −1). Under identical conditions GTP was less effective than ATP in reducing the number of T∗ SS-14 binding sites ( B max = 227 ± 15, respectively, compared to 340 ± 15 pmol mg −1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li + and Na + inhibited the binding of T∗ SS-14 to a greater extent than K +. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg 2+, Ba 2+, Mn 2+, Ca 2+ and Co 2+ augmented the binding of both T∗ SS-14 and LTT∗ SS-28, while higher than 4 mM Co 2+ inhibited binding of both ligands. LTT∗ SS-28 binding was reduced in the presence of high concentrations of Ba 2+ and Mn 2+ also. Interestingly Ca 2+ at higher than 10 mM preferentially inhibited LTT∗ SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT∗ SS-28 binding sites. These results provide direct evidence for the differential modulation by nucleotides and ions of the binding of SS-14 and SS-28 receptors in rat brain, but do not provide direct evidence for the presence of separate binding sites for these SS peptides.