The Somatostatin Receptor Is Directly Coupled to Adenylate Cyclase in GH4Ci Pituitary Cell Membranes*

Abstract

Somatostatin (SRIF) inhibits vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation in the GH4C1 strain of rat pituitary tumor cells, and this effect is responsible for SRIF inhibition of VIP-stimulated hormone release. The interaction between the SRIF receptor and adenylate cyclase was examined in GH4C1 cell membranes. Maximal concentrations of VIP (50 nM) increased membrane adenylate cyclase activity 4.2-fold; half-maximal stimulation was observed with 0.75 nM VIP, SRIF noncompetitively inhibited the stimulatory effect of VIP, but it did not alter basal adenylate cyclase activity. The relative potencies of SRIF and 2 SRIF analogs as inhibitors of VIP-stimulated adenylate cyclase activity in membranes and of VIP-stimulated cAMP accumulation in intact cells were similar. Furthermore, the concentration of SRIF that caused half-maximal inhibition of adenylate cyclase activity (ED50 = 2.3 nM) was close to the equilibrium dissociation constant for SRIF (Kd = 0.40 nM) measured in membrane preparations in the presence of GTP. Therefore, SRIF inhibition of adenylate cyclase appears to be receptor mediated. As with receptors known to regulate adenylate cyclase by interaction with a guanine nucleotide regulatory subunit, SRIF receptor binding was decreased in the presence of guanine nucleotides. Addition of GTP (150 .mu.M) or the nonhydrolyzable GTP analog guanyl-5''-yl-imidodiphosphate (100 .mu.M) decreased the specific binding of [125I-Tyr1]SRIF to 31 and 13% of the control value, respectively. This decrease in specific binding was due entirely to decreased receptor affinity for SRIF. GTP (150 .mu.M) increased the equilibrium dissociation constant for SRIF from 0.11 to 0.40 nM; the number of binding sites was unaffected by the nucleotide (Bmax = 0.2 pmol/mg protein). Analysis of dissociation kinetics demonstrated that in the absence of guanyl nucleotides, the rate of [125I-Tyr1]SRIF dissociation was first order (t1/2 = 180 min). However, in the presence of a half-maximal concentration of guanyl-5''-yl-imidodiphosphate (0.3 .mu.M), [125I-Tyr1]SRIF dissociation occurred with biphasic kinetics. Fifty percent of the specifically bound peptide dissociated at the same rate as that observed in the absence of nucleotide; the remainder dissociated 15 times more rapidly (t1/2 = 9.6 min). The SRIF receptor can exist in 2 interconvertible forms and that guanyl nucleotides regulate the equilibrium between low and high affinity states of the receptor. Apparently, SRIF receptor occupancy is coupled to inhibition of adenylate cyclase activity by a guanine nucleotide-binding regulatory protein.