IDENTIFICATION AND PHARMACOLOGICAL CHARACTERIZATION OF SOMATOSTATIN RECEPTORS IN RAT LUNG

1 [I-125]-[LTT]SRIF-28 and [I-125]-SMS 201-995 were used to identify a nd characterize somatostatin (SRIF) receptors localized in rat lung ti ssue. In vitro autoradiography of rat lung tissue sections showed the existence of specific, high affinity binding sites for [I-125]-[LTT]SR IF-28 without any significant specific binding of the sst(2)/sst(5)-re ceptor selective ligand [I-125]-SMS 201-995. 2 In radioligand binding studies, specific binding of [I-125]-[LTT]SRIF-28 to membranes of rat lung was linearly related to the concentration of membrane protein use d with only a small portion of nonspecific binding. With [I-125]-SMS 2 01-995 no specific binding could be observed up to a membrane concentr ation of 0.1 mg of protein/assay tube. 3 [I-125]-[LTT]SRIF-28 bound ra pidly to rat lung membranes with an apparent association rate constant (k(app)) of 1.8 +/- 0.1 h(-1) (n = 3). The equilibrium of specific bi nding was reached after an incubation period of approximately 90 min a t room temperature and remained constant for the next 3 h. The associa tion rate constant (k(1)) was calculated to be 3.7 x 10(10) M-1 h(-1). The dissociation reaction followed first order kinetics with a dissoc iation rate constant (k(-1))=0.44+/-0.07 h(-1) corresponding to a half -time of 95+/-15 min (n=3). From these kinetic experiments an equilibr ium dissociation constant (K-D) for the binding of [I-125]-[LTT]SRIF-2 8 was calculated to be 11.9 pM. 4 Saturation binding of [I-125]-[LTT]S RIF-28 revealed an equilibrium dissociation constant (K-D) of 50.1 pM (pK(D)=10.3+/-0.1; n=3) and a receptor density (B-max) of 78+/-3 fmol mg(-1) protein. A Hill coefficient not significantly different from 1 indicated saturable binding to a single class of high affinity binding sites. 5 Specific binding of [I-125]-[LTT]SRIF-28 to rat lung membran es was inhibited by SRIF-14, SRIF-28 and different SRIF analogues. SRI F and different synthetic short chain SRIF analogues exhibited the fol lowing rank order of potency: SRIF-28 > SRIF-14 > CGP 23996 > > RC 160 > BIM 23014> SMS 201-995 > BIM 23056 > MK 678. 6 The binding affiniti es for SRIF and the various SRIF analogues determined using rat lung t issue were in close correlation to those obtained with Chinese hamster ovary (CHO) cells stably expressing sst(1) (r=0.92) and sst(4)(r=0.95 ) receptors, respectively. 7 Reverse transcriptase - polymerase chain reaction (RT-PCR) showed the predominant expression of mRNA specific f or sst(4) receptors as well as some weak sst(1) mRNA expression. 8 The findings suggest that sst(4) receptor expression is the predominant f orm of the somatostatin receptors identified in rat lung tissue. In th is study we demonstrated for the first time the existence of sst(4) re ceptors in mammalian tissue.