Characterization of somatostatin receptors which mediate inhibition of insulin secretion in RINm5F insulinoma cells

Somatostatin (SRIF) is a neuropeptide which inhibits secretion from a variety of target cells including pancreatic beta-cells. In this study we have used the RINm5F rat insulinoma cell line to characterize high affinity receptors for SRIF. The binding of 0.03 nM [125I-Tyr11]SRIF to RINm5F cells reached a plateau level within 4 h at 37 C at which time 80% of the total binding could be displaced by 100 nM unlabeled SRIF. In contrast, 100 nM concentrations of eight structurally unrelated peptides did not inhibit [125I-Tyr11]SRIF binding. Scatchard analysis indicated that RINm5F cells contained a single class of noninteracting binding sites (910 +/- 190 sites per cell) with high affinity for [125I-Tyr11]SRIF [equilibrium dissociation constant (Kd) = 0.04 +/- 0.01 nM]. Competition experiments with SRIF analogs showed that the binding affinity for [I-Tyr11]SRIF (Kd = 0.03 +/- 0.02 nM) was higher than that for either SRIF (0.24 +/- 0.04 nM) or [Tyr11]SRIF (0.27 +/- 0.04 nM) and that reduced SRIF analogs bound poorly (Kd greater than 50 nM). These results demonstrate that RINm5F cells possess specific, high affinity binding sites for SRIF. Insulin release stimulated by 20 mM leucine or 15 mM glyceraldehyde was inhibited as much as 80% by maximal concentrations (100 nM) of SRIF. The IC50 for SRIF inhibition of leucine-stimulated insulin secretion was 0.43 +/- 0.15 nM, in good agreement with the apparent Kd for binding. In fact, this close correlation between binding affinity and potency to inhibit insulin release was observed for six SRIF analogs, indicating that the characterized binding sites are the receptors which mediate the biological actions of SRIF in RINm5F cells.     

Somatostatin receptors: identification and characterization in rat brain membranes

We have identified and characterized specific receptors for tetradecapeptide somatostatin (SRIF; somatotropin release-inhibiting factor) in rat brain using [125I]Tyr11]SRIF as the radioligand. These receptors are present in membranes obtained from a subfraction of synaptosomes. Membranes derived from cerebral cortex bind SRIF with high affinity (Ka = 1.25 X 10(10) M-1) and have a maximum binding capacity (Bmax) of 0.155 X 10(-12) mol/mg. Neither opiates nor other neuropeptides appear to influence the binding of SRIF to brain membranes. Synthetic analogs with greater biological potency than SRIF--[D-Trp8]SRIF, [D-Cys14]SRIF, and [D-Trp8, D-Cys14]SRIF--bind to the receptors with greater avidity than SRIF, whereas inactive analogs [(2H)Ala3]SRIF and [Ala6]SRIF exhibit low binding. The ratio of receptor density to endogenous somatostatin is high in the cortex, thalamus, and striatum, low in the hypothalamus, and extremely low in the brain stem and cerebellum. Thus, SRIF receptors in the brain appear to be a distinct, new class of receptors with a regional distribution different from that of endogenous somatostatin.     

Characterization of pituitary membrane receptors for somatostatin in the rat

We have demonstrated the presence of specific receptors for somatostatin (SRIF) in normal rat pituitary membranes using ([125I]Tyr11]SRIF) as the radioligand. These receptors bind SRIF with high affinity (Ka approximately 0.47 X 10(10) M-1) and have a maximal binding capacity of 0.095 pmol/mg membrane protein. Two other radioiodinated SRIF analogs which contain N-terminally situated radiolabel, [125I-Tyr1]SRIF and [125I-N-Tyr] SRIF, were found unsuitable for receptor binding studies due to loss of the radiolabel from the ligand molecule under the experimental conditions employed. Binding of [125I]Tyr11]SRIF to these receptors was specific and was not influenced by a variety of other neuropeptides. The specificity of SRIF receptors was also examined using 10 synthetic SRIF analogs as well as catfish somatostatin I. Catfish somatostatin I was 8.3 times less potent than SRIF in binding to SRIF receptors, although it has been reported to be equipotent in terms of in vitro GH inhibition. Analogs which exhibit greater potency for GH inhibition in vitro bound to these receptors with greater affinities than SRIF, whereas biologically inactive analogs showed markedly reduced binding, suggesting that the in vitro GH inhibitory actions of SRIF analogs are related to their ability to interact with SRIF receptors.     

Somatostatin receptors in the rat adrenal cortex: characterization and comparison with brain and pituitary receptors

Specific receptors for tetradecapeptide somatostatin (S-14) in rat adrenal cortical membranes were quantitated by direct binding studies using [125I-Tyr11]S-14. Competitive inhibition of this radioligand by S-14 showed that these receptors constitute a single class of high affinity binding sites [dissociation constant (Kd) = 1.08 nM and maximum binding capacity (Bmax) = 0.35 pmol/mg membrane protein]. Structural analogs of S-14 with halogenated Trp8 moiety exhibited 4- to 46-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 and [des-Ala1]S-14 bound to these receptors with reduced affinity whereas [Phe4]S-14 exhibited 1.5-fold greater affinity than S-14. Somatostatin-28 (S-28) and S-14 were equipotent, whereas the N-terminal fragments of S-28 [S-28(1-14) and S-28(1-12)] were inactive. High affinity binding sites were also quantitated using a radioligand prepared from the tyrosinated S-28 analog, [Leu8, D-Trp22, Tyr25]S-28 (Kd = 1.2 nM; Bmax = 0.21 pmol/mg membrane protein). Both S-14 and S-28 exhibited comparable relative potencies for inhibiting the specific binding of this radioligand and [125I-Tyr11]S-14. Extracts of whole adrenal or the adrenal medulla and cortex contained very low levels of S-14-like immunoreactivity (2.4 pg/mg protein). These studies confirm the presence of specific receptors for S-14 in the adrenal cortex and suggest that 1) with respect to S-14 biological activity, Trp8-modified S-14 analogs should be more potent than S-14, S-28 equipotent with S-14, and N-terminal fragments of S-28 inactive in this tissue. 2) Direct binding studies using radioiodinated [Tyr11]S-14 and [Leu8,D-Trp22, Tyr25]S-28 appear to quantitate the same receptor sites in adrenocortical tissue. 3) The ligand specificity of the adrenocortical S-14 receptor differs from that previously reported for the pituitary and brain providing further evidence for the heterogeneity of the S-14 receptor. 4) In view of the very low concentrations of endogenous S-14-like immunoreactivity, the adrenal actions of S-14 and S-28 are probably mediated through an endocrine mechanism.     

Purification of a putative brain somatostatin receptor.

The brain somatostatin (somatotropin release-inhibiting factor; SRIF) receptor was purified by affinity chromatographic techniques. A protein of 60 kDa could be purified from rat brain. The protein was eluted from a [D-Trp8]SRIF affinity column with either sodium acetate (pH 5.5) or free [D-Trp8]SRIF. The binding of the protein to the affinity column was prevented by free [D-Trp8]SRIF or the stable SRIF analogue SMS 201-996 but not by the inactive somatostatin 28-(1-14). The purified receptor could be covalently labeled by the 125I-labeled SRIF analogue CGP 23996. Excess [D-Trp8]SRIF blocked the binding of 125I-labeled CGP 23996 to the purified receptor, but somatostatin 28-(1-14) did not affect the binding. A 60-kDa protein was also purified from the anterior pituitary cell line AtT-20, which has a high expression of SRIF receptors. In contrast, no 60-kDa protein could be purified from CHO cells, which have no detectable SRIF receptors. These findings present evidence for the purification of the SRIF receptor.     

Distribution of somatostatin receptors in RINm5F insulinoma cells

Previous studies with heterogeneous populations of pancreatic cells have provided evidence for the presence of somatostatin (SRIF) receptors in cytosol and secretion vesicles, as well as the plasma membrane. To examine the distribution of SRIF receptors between soluble and membrane fractions in a homogeneous pancreatic islet cell population, we have used the clonal RINm5F insulinoma cell line. These cells contain specific, high affinity binding sites for [125I-Try11]SRIF on the cell surface, and occupancy of these sites by SRIF and SRIF analogs correlates with inhibition of insulin secretion. Stable, steady state binding was achieved using both intact cells and membranes by performing binding incubations with [25I-Tyr11]SRIF at 22 C. Half-maximal inhibition of [125I-Tyr11]SRIF binding occurred with 0.21 +/- 0.11 nM SRIF in membranes and 0.35 +/- 0.30 nM SRIF in cells. In contrast, the binding of [125I-Tyr11]SRIF to cytosolic macromolecules was not reduced by concentrations of SRIF as high as 100 nM, demonstrating that this binding was of much lower affinity. RINm5F membranes were further purified using a Percoll gradient to prepare a microsomal fraction, which was enriched in adenylate cyclase activity, and a secretory granule fraction, which was enriched in insulin. [125I-Tyr11]SRIF binding to the microsomal fraction (3.8 +/- 0.3 fmol/mg) was 3 times higher than to secretion granules (1.2 +/- 0.2 fmol/mg). Thus, high affinity SRIF binding sites were most abundant in microsomal membranes and were low or undetectable in secretory granules and cytosol. To determine whether translocation of SRIF receptors to the plasma membrane accompanied insulin secretion, we examined the effects of various insulin secretagogues on [125I-Tyr11]SRIF binding to intact cells. Leucine (20 mM), glyceraldehyde (15 mM), forskolin (1 microM), and glucagon (1 microM) stimulated insulin release 1.5- to 4.0-fold in different experiments. However, these secretagogues did not increase [125I-Tyr11]SRIF binding. In summary, our results indicate that high affinity SRIF receptors in RINm5F cells are located primarily on the plasma membrane and that the concentration of SRIF receptors at the cell surface is independent of the secretory activity of the cells.     

Expression cloning of a rat brain somatostatin receptor cDNA.

We have used an expression-cloning strategy to isolate a cDNA encoding a somatostatin (somatotropin release-inhibiting factor, SRIF) receptor from rat cortex and hippocampus. A positive clone was identified by autoradiography after binding of radiolabeled SRIF to COS-1 cells previously transfected with pools of cDNA clones. The deduced amino acid sequence of the receptor displays sequence and structural homology to the family of G-protein-coupled receptors. The affinity of various SRIF analogs to the expressed receptor resembles their effects on growth hormone release from pituitary cells. In addition, the distribution of the mRNA in various tissues corresponds to that described for native SRIF receptors. Therefore, we conclude that we have isolated a rat brain SRIF receptor cDNA.     

Cyclic cholecystokinin analogues with high selectivity for central receptors.

Taking as a model the N-terminal folding of the cholecystokinin tyrosine-sulfated octapeptide [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] deduced from conformational studies, two cyclic cholecystokinin (CCK) analogues were synthesized by conventional peptide synthesis: Boc-D-Asp-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 [compound I (Ahx, 2-aminohexanoic acid)] and Boc-gamma-D-Glu-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 (compound II). The binding characteristics of these peptides were investigated on brain cortex membranes and pancreatic acini of guinea pig. Compounds I and II were competitive inhibitors of [3H]Boc[Ahx28,31]CCK-(27-33) binding to central CCK receptors and showed a high degree of selectivity for these binding sites (compound I: Ki for pancreas/Ki for brain, 179; compound II: Ki for pancreas/Ki for brain, 1979). This high selectivity was associated with a high affinity for central CCK receptors (compound I: Ki, 5.1 nM; compound II: Ki, 0.49 nM). Similar affinities and selectivities were found when 125I Bolton-Hunter-labeled CCK-8 was used as a ligand. Moreover, these compounds were only weakly active in the stimulation of amylase release from guinea pig pancreatic acini (EC50 greater than 10,000 nM) and were unable to induce contractions in the guinea pig ileum (to 10(-6) M). The two cyclic CCK analogues, therefore, appear to be synthetic ligands exhibiting both high affinity and high selectivity for central CCK binding sites. These compounds could help clarify the respective role of central and peripheral receptors for various CCK-8-induced pharmacological effects.     

The somatostatin receptor is directly coupled to adenylate cyclase in GH4C1 pituitary cell membranes

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. In this study we examined the interaction between the SRIF receptor and adenylate cyclase 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 two 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 microM) or the nonhydrolyzable GTP analog guanyl-5'-yl-imidodiphosphate (100 microM) 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 microM) increased the equilibrium dissociation constant for SRIF from 0.11 to 0.40 nM, whereas 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 (t 1/2 = 180 min). However, in the presence of a half-maximal concentration of guanyl-5'-yl-imidodiphosphate (0.3 microM), [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, whereas the remainder dissociated 15 times more rapidly (t 1/2 = 9.6 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Reciprocal interactions of somatostatin with thyrotropin-releasing hormone and vasoactive intestinal peptide on prolactin and growth hormone secretion in vitro

Reciprocal interactions of somatostatin (SRIF) and vasoactive intestinal peptide (VIP) or TRH on in vitro PRL and GH release from male rats hemipituitaries were investigated. SRIF did not modify basal PRL release, but TRH- or VIP-induced release was inhibited by SRIF in a dose-dependent manner [effective concentration-fifty (EC50) = 1.7 +/- 0.9 nM for SRIF inhibition of TRH stimulation and EC50 = 0.8 +/- 0.5 nM for SRIF inhibition of VIP stimulation]. VIP and TRH did not affect GH release by themselves, but reduced the inhibition of GH secretion elicited by SRIF (EC50 = 7.6 +/- 3.4 nM for TRH blockade of SRIF inhibition and EC50 = 4.6 +/- 3.1 nM for VIP blockade of SRIF inhibition). Secretin, a partial structural analog of VIP, also blocked SRIF-induced inhibition of GH and stimulated PRL release. Secretin stimulation of PRL release was also prevented by SRIF. [D-Trp8,D-Cys14]SRIF, a potent analog of SRIF, antagonized VIP stimulation of PRL secretion with the same apparent affinity as the native peptide. The maximal stimulation, but not the apparent affinity of VIP action on prolactin release was reduced by SRIF, suggesting that the interaction is of a noncompetitive nature. This conclusion as further substantiated by the observation that neither TRH nor VIP were able to displace specific 125I-labeled [Tyr1] SRIF high affinity binding to pituitary membranes. The three peptides tested thus appear to exhibit reciprocal interactions mediated by independent receptor sites on GH as well as on PRL-producing cells.     

Modulation of somatostatin binding to rat pituitary membranes by exogenously administered growth hormone

The effect of exogenously administered GH on somatostatin (SRIF) receptor regulation was studied in rat anterior pituitary membranes. A single class, high affinity specific receptor for SRIF was identified by binding studies with [125I-Tyr11]SRIF [binding capacity (mean +/- SD), 129.4 +/- 23.3 fmol/mg protein; binding affinity, 2.8 +/- 0.6 X 10(10) M-1]. A single injection of rat GH (150 micrograms) caused a significant reduction in capacity, but not in affinity, of SRIF receptors 2 and 6 h after injection (mean decrease, 23% and 24%, respectively) from that in controls. In contrast, mean SRIF binding capacity 24 h after a single injection of rat GH was increased 48% above control values, but affinity was unaffected. Measurement of membrane SRIF content indicated that these changes could not be explained by alterations in receptor occupancy. When rat GH was injected repeatedly for 3 days (150 micrograms/rat X day), the mean binding capacity, though not the affinity, of SRIF receptors was decreased 23% from that in controls 24 h after the last injection. The results can be explained by stimulation of SRIF release from the hypothalamus by GH and somatomedins, with subsequent internalization of the pituitary plasma membrane SRIF receptor. They suggest yet another level of neuroendocrine regulation of GH secretion.     

Nucleotide regulation of growth hormone-releasing factor binding to rat pituitary receptors

The specific binding of a GRF radioligand, [His1,125I-Tyr10,Nle27]hGRF-1-32NH2, to rat pituitary homogenates is reduced by the addition of GTP and its nonhydrolyzable analogs 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). GDP and cAMP had no effect while the nonhydrolyzable ATP analogs 5'-adenylylimidodiphosphate and adenosine 5'-O-(3-thiotriphosphate) did elicit a significant reduction in GRF binding. The effect of GppNHp was half-maximal at 0.2 microM, and the maximum inhibition achieved was 85%. The effect of 0.1 microM GppNHp on GRF competitive displacement experiments indicated a significant reduction in affinity for the ligand (Kd = 0.51 +/- 0.11 nM in the absence of GppNHp and 2.1 +/- 1.1 nM in its presence) without an effect on receptor number. The GRF radioligand dissociates slowly from its receptor (t1/2 = 250 +/- 50 min), but the addition of 0.1 microM GppNHp converts approximately half of the receptors present to a more rapidly dissociating form (t1/2 = 9 +/- 10 min). These results are consistent with existing models for receptor-G-protein interactions, and thus, we conclude that transduction of the GRF response across the cell membrane involves a guanine nucleotide-binding protein, presumably Gs.     

Somatostatin analogs. Dissociation of brain receptor binding affinities and pituitary actions in the rat

We have recently demonstrated the presence of specific receptors for somatostatin (SRIF) in rat brain synaptosomal membranes which appear to mediate its action. Using this system as a radioreceptor assay, we have examined the ability of a wide range of SRIF analogs to interact with these receptors. Although structural modifications in the Trp8 moiety of SRIF resulted in significant enhancement of affinity for binding to the brain SRIF receptors, the different relative specificities of des AA1,2,4,5,12,13 D-Trp8 SRIF (oligo D-Trp8 SRIF), D-Trp8 SRIF and D-5-Br-Trp8 SRIF in the pituitary and the central nervous system (CNS) suggest that basic differences exist between SRIF receptors present in the brain and the pituitary.     

Characterization and distribution of somatostatin binding sites in goldfish brain

Somatostatin (SRIF) binding sites were characterized in goldfish brain. Binding of (125)I-[Tyr(11)]-SRIF-14 to a brain membrane preparation was found to be saturable, reversible, and time-, temperature-, and pH-dependent. Binding was also displaceable by different forms of SRIF. Under optimal conditions (22 degrees C, pH 7.2), the equilibrium binding of (125)I-[Tyr(11)]-SRIF-14 to goldfish brain membranes was achieved after 60 min incubation. Analysis of saturable equilibrium binding revealed a one-site model fit with K(a) of 1.3 nM. SRIF-14, mammalian SRIF-28, and salmon SRIF-25 displaced (125)I-[Tyr(11)]-SRIF-14 binding with similar affinity, whereas other neuropeptides, e.g., substance P, were unable to displace (125)I-[Tyr(11)]-SRIF-14. Autoradiography studies demonstrated that (125)I-[Tyr(11)]-SRIF-14 binding sites are found throughout the goldfish brain. A high density of (125)I-[Tyr(11)]-SRIF-14 binding sites was found in the forebrain, including the nucleus preopticus, nucleus preopticus periventricularis, nucleus anterioris periventricularis, nucleus lateralis tuberis, nucleus dorsomedialis thalami, nucleus dorsolateralis thalami, nucleus ventromedialis thalami, and nucleus diffusus lobi inferioris. In midbrain, (125)I-[Tyr(11)]-SRIF-14 binding sites were found in the optic tectum. The facial and vagal lobes and the mesencephalic-cerebellar tract were found to have a high density of binding sites. This study provides the first characterization and distribution of specific binding sites for SRIF in a fish brain.     

Somatostatin octapeptides (lanreotide, octreotide, vapreotide, and their analogs) share the growth hormone-releasing peptide receptor in the human pituitary gland

The binding affinity of somatostatin-14 (SRIF), various SRIF derivatives, and some peptides belonging to the growth hormone-releasing peptide (GHRP) family to specific receptors for SRIF and GHRP in the human pituitary gland has been measured. GHRP receptors have been identified using [¹²⁵I]Tyr-Ala-hexarelin, a peptide that thas been demonstrated to be a potent growth hormone (GH) releaser in humans. Tyr-Ala-hexarelin binding was displaced in a dose-dependent manner by different GHRPs (hexarelin, GHRP-2, and EP-51216). Surprisingly, some SRIF octapeptide derivatives such as vapreotide, lanreotide, octreotide, and their analogs were also able to displace the GHRP ligand. By contrast, no inhibition of Tyr-Ala-hexarelin binding was observed in the presence of SRIF or SRIF derivatives (SRIF H-2186, H-2485, and H-3382) that are known to have a weak SRIF-like activity. When [¹²⁵I]Tyr¹-SRIF-14 was used as a ligand, we observed displacement with SRIF and the octapeptide SRIF analogs but not with GHRPs and other SRIF derivatives. The results point to a sharing of the GHRP receptor with the octapeptide SRIF analogs, but not SRIF. Our data are consistent with the hypothesis that the putative natural GH secretagogue ligand may be a growth hormone release inhibiting factor that is different from SRIF and that is antagonized by GHRP.     

Development of antibodies against the rat brain somatostatin receptor.

Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).     

Specific high affinity binding sites for somatostatin-28 on pancreatic beta-cells: differences with brain somatostatin receptors

Saturable and high affinity binding sites have been obtained for an iodinated somatostatin-28 (SS-28) analog, [Leu8,D-Trp22,125I-Tyr25] SS-28, in a membrane preparation from hamster insulinoma, mainly composed of pancreatic beta-cells. Specific binding is maximal after 1 hour incubation at 22C and represents 65% of the total binding. KD for [Leu8,D-Trp22,Tyr25] SS-28 is 0.25 nM with the number of sites corresponding to 68 fmol/mg protein. The KD for SS-28 (1nM) is more than 5 times lower than that for SS-14. SS-28 analogs, such as [D-Trp22] SS-28 and analogs which selectively inhibit insulin release in vivo (Des-Asn5[D-Trp8,D-Ser13] SS-14 and Des-Asn19[D-Trp22,D-Ser27] SS-28), are the most potent compounds in this assay. C-terminal replacement of L-cysteine by D-cysteine reduces the apparent affinity of SS analogs. Unrelated peptides and the inactive analog Des-Trp8-SS-14 have no affinity for insulinoma binding sites. There are differences between the insulinoma SS binding sites and those monitored under similar conditions from rat cerebral cortex. In cerebral cortex, SS-14 and SS-28 have similar affinity for the binding sites, and the insulin selective analogs are less potent than SS-14. It is concluded that pancreatic beta-cells, as well as brain, possess high affinity binding sites for SS, but that they differ in some of their pharmacological properties.     

A hypothalamic hormone-somatostatin--from endocrinology to neurophysiology]

The densest distribution of somatostatin (SRIF) neuron perikarya is localized in the hypothalamic periventricular nucleus (Pe) close to the third ventricle, from which many fibers are projected to the median eminence. The release of SRIF in the neurohemal organ into the anterior pituitary modulates GH secretion from pituitary somatotrophs. When SRIF input from the hypothalamus to rat anterior pituitary is reduced by either neurosurgery or SRIF antiserum iv injection, the responsiveness of the pituitaries to human GH releasing factor (hGRF) in an in vitro perifusion system is markedly attenuated. Moreover, SRIF pretreatment facilitates the GH release response of dispersed anterior pituitary cells to hGRF. The long lasting SRIF effect to sensitize somatotrophs appears to take place beyond cAMP formation or as an unknown distal effect. These findings indicate that SRIF neurons in the Pe play a role in maintaining the pituitary responsiveness to GRF in addition to the original action to inhibit GH secretion. Neuronal networks between Pe-SRIF neurons, and intra- and extrahypothalamic nuclei are identified by Pe stimulation test on GRF-GH secretion. In addition to the physiological role in maintaining pituitary responsiveness, Pe SRIF neurons have a wide influence on specific SRIF receptor binding in various brain regions as well as in the anterior pituitary. Shortly after lesioning the Pe neurons, there is a continuous increase in plasma GH level with a transient increase in specific binding of 125I-Tyr 11-SRIF-14 to the anterior pituitary. Furthermore, there is a similar but a little longer increase in binding of the radioligand to some brain areas such as the cerebral cortex, hippocampus, and amygdala nuclei. However, neuronal connections between the SRIF neurons and nuclei which are up-regulated by the lesioning have not been fully proven. When the labeled ligand is infused into the lateral ventricle, it is rapidly and widely distributed in many periventricular structures in the lateral and third ventricles. These findings suggest that SRIF produced in the Pe neurons is transported to other brain areas via cerebrospinal fluid in addition to neuronal connections for modulating the activity of neurons which have SRIF receptors. Thus, hypothalamic Pe SRIF neurons have dualistic roles for controlling anterior pituitary function and modulating CNS neuron activity.     

Coexpression of somatostatin receptor subtype 5 affects internalization and trafficking of somatostatin receptor subtype 2

The somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor subtypes sst(2A) and sst(5) are frequently coexpressed in SRIF-responsive cells, including endocrine pituitary cells. We previously demonstrated that sst(2A) and sst(5) exhibit different subcellular localizations and regulation of cell surface expression, although they have similar signaling properties. We investigated here whether sst(2A) and sst(5) functionally interact in cells coexpressing the two receptor subtypes. We stimulated both transfected cells stably expressing sst(2A) alone (CHO-sst(2A)) or together with sst(5) (CHO-sst(2A+5)) and the pituitary cell line AtT20, which endogenously expresses the two receptor subtypes, with either the nonselective agonist [D-Trp(8)]-SRIF-14 or the sst(2)-selective agonist L-779,976. In CHO-sst(2A) cells, stimulation with either ligand resulted in the loss of approximately 75% of cell surface SRIF binding sites and massive internalization of sst(2A) receptors. The cells were desensitized to subsequent stimulation with [D-Trp(8)]-SRIF-14, which failed to inhibit forskolin-evoked cAMP accumulation. Similarly, in CHO-sst(2A+5) and AtT20 cells, [D-Trp(8)]-SRIF-14 induced the loss of 60-70% of SRIF binding sites as well as massive sst(2A) endocytosis. By contrast, in cells expressing both sst(2A) and sst(5), selective stimulation of sst(2A) with L-779,976 resulted in only 20-40% loss of cell surface binding and markedly reduced sst(2A) internalization. Consequently, whereas CHO-sst(2A+5) and AtT20 cells stimulated with [D-Trp(8)]-SRIF-14 were desensitized to a second stimulation with the same agonist, cells prestimulated with L-779,976 were not desensitized to subsequent [D-Trp(8)]-SRIF-14 stimulation. These findings indicate that the presence of sst(5) in the same cells modulates trafficking and cell surface regulation of sst(2A) and cellular desensitization to the effects of SRIF.