High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

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

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-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] 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 Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax 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 (Bmax = 227 +/- 8 and 182 +/- 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) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ 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.(ABSTRACT TRUNCATED AT 400 WORDS)     

To the problem of biologically active conformation of enkephalin

Summary A semi-rigid structural analog of [Leu⁵] enkephalin, possessing the azo-bridge between Tyr¹ and Phe⁴ residues, was synthesized, along with two other linear enkephalin analogs: [4′-amino Phe⁴] enkephalin and [4′hydroxyphenyl/-azo Phe⁴] enkephalin. The results of the determination of the analgesic activity of the synthesized compounds suggest that the biologically active conformation of the enkephalin molecule should be such that both aromatic rings, Tyr¹ and Phe⁴, are situated in close proximity.     

Metabolism of somatostatin and its analogues by the liver

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°C and negligible at 0°C. The apparent K_m for the overall degradation 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.     

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Abstract: Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13–day-old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [¹²⁵I-Tyr⁰,D-Trp⁸]SRIF-14 ([¹²⁵I-Tyr⁰,d -Trp⁸]S14) and ^I25I-SMS 204–090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13–day-old rat cerebellum showed that the A'_D values for [¹²⁵I-Tyr⁰,D-Trp⁸]S14 and ¹²⁵I-SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding B_max values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SSI). Competition studies showed that different D-Trp-sub-stituted analogs displaced [¹²⁵I-Tyr⁰,d -Trp⁸]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr⁰,d -Trp⁸]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day-old cerebellum and adult cerebral cortex. The higher-affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower-affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn²⁺ was the most efficient cation for promoting binding of [¹²⁵I-Tyr⁰,d -Trp⁸]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF-Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same K_D and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF-binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF-Rs. The extremely high density of SRIF-Rs found in the external granule cell layer in 13–day-old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.     

Impaired negative cooperativity of the semisynthetic analogues human [LeuB24]- and [LeuB25]-insulins

Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [Leu^B24]- and [Leu^B25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature $\text{28}$:121–125). [Leu^B24]- and [Leu^B25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [Leu^B24]-insulin to induce negative cooperativity was clearly impaired, that of [Leu^B25]-insulin was almost abolished. [Leu^B25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.     

Binding capacity of luteinizing hormone-releasing hormone and its analogues for pituitary receptor sites.

Competition for luteinizing hormone-releasing hormone (LH-RH) receptor sites by the inhibitory analog [D-Phe², D-Trp³, D-Phe⁶]-LH-RH and by the superactive stimulatory analog [D-Trp⁶]-LH-RH was observed in adenohypophysial homogenates incubated at 4°C. Competition for LH-RH binding sites was less evident with adenohypophysial plasma membranes. The binding affinities of these analogues to LH-RH pituitary receptors can explain at least in part their respective action in blocking ovulation and in inducing a greater release of luteinizing hormone and follicle stimulating hormone than the parent hormone.     

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.     

Bombesin acts in preoptic area to produce hypothermia in rats

To determine a neuroanatomic location where bombesin produces hypothermia, bombesin or an inactive bombesin analog, [D-Trp⁸]-bombesin, were microinjected in nanogram quantities into various regions of the brain of unanesthetized, unrestrained, cold-exposed rats. Bombesin, but not [D-Trp⁸]-bombesin, produced hypothermia when injected into or near the preoptic area, whereas injections into other forebrain and midbrain areas failed to lower body temperature. These results support a possible involvement of a bombesin-like peptide in thermoregulation.     

Chemical cross-linking of somatostatin receptors in rat adrenal cortex

Adrenocortical somatostatin receptors have been shown to interact with somatostatin-14 (S-14) and somatostatin-28 (S-28). To determine whether these peptides interact with the same or different receptor proteins, we chemically cross-linked these receptors using disuccinimidyl suberate to radioligands prepared from tyrosinated S-14 and S-28 analogs. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and subsequent autoradiography of [125I-Tyr11] S-14 and [Leu8, D-Trp22, 125I-Tyr25] S-28 cross-linked to their binding sites following solubilization in the presence of 50 mM DTT revealed the presence of a single labelled protein of Mr = 200,000. When the cross-linked material was treated under non-reducing conditions, this band was not observed. Furthermore, addition of excess S-14 and S-28 at the time of binding inhibited the incorporation of both radioligands into the receptor protein. These results demonstrate that adrenocortical membrane receptors for somatostatin contain a single receptor protein sub-unit or sub-units of identical size which interact with both S-14 and S-28.     

Synthesis and Structure-Activity Relationship of [Nle10]Neurokinin A (4–10) Analogs with Constraint in the Backbone and at Position Six

Steric requirements of binding [Nle¹⁰]NKA(4–10) to NK-2 receptor were studied by introducing conformationally constrained amino acid analogs into its sequence. Two series of [Nle¹⁰]NKA(4–10) analogs were synthesized to investigate (i) the significance of a putative β-turn in the receptor-ligand interaction by insertion of either (S)- or (R)-Gly⁸{ANC-2}Leu⁹ γ-lactams to mimic a β-turn constraint, and (ii) the effect of hindered rotation in the Φ, χ¹ and χ² dihedral angle space of the crucially important Phe⁶ which was replaced systematically with d-Phe, d- and l-Tyr, as well as with their conformationally constrained analogs, Tic, HOTic and β-MePhe. Competition binding experiments with [³H]NKA were performed using cloned human NK-2 receptors expressed in CHO cells. The analog possessing only an (R)-Gly⁸{ANC-2}Leu⁹ constraint, had the same binding affinity as that of the parent peptide. The rank order of potency of the other analogs showed a cumulative effect of different structural modifications in decreasing the binding affinity, i.e., when changing the configuration of the lactam ring to S, replacing Phe⁶ with constrained analogs, Tic or β-MePhe, changing the configuration of the amino acid at position six to d, and introducing a hydroxyl group on the aromatic ring. Ferenc ?tv?s and Dmitry S. Gembitsky - Made an equal contribution. Abbreviations used for amino acids and peptides follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature, Eur. J. Biochem. (1984) 138, 9–37     

Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R₁ and MCH‐R₂, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R₁ was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R₁ and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R₁ (SLC‐1), the radioligand was [¹²⁵I]–[Tyr¹³]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [¹²⁵I]–[D‐Phe¹³, Tyr¹⁹]‐MCH served as radioligand. The bioassay used for MCH‐R₁ was intracellular Ca²⁺ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R₁ and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe¹³ in [Phe¹³, Tyr¹⁹]‐MCH was well tolerated by both MCH‐R₁ and B16 MCH‐R, change of configuration to D‐Phe¹³ in [D‐Phe¹³, Tyr¹⁹]‐MCH or [D‐Phe¹³]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R₁. By contrast, the D‐Phe¹³ residue increased the affinity of [D‐Phe¹³, Tyr¹⁹]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe¹³, Tyr¹⁹]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R₁ in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R₁.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

[Lys(DOTA)4]BVD15, a novel and potent neuropeptide Y analog designed for Y1 receptor-targeted breast tumor imaging

We substituted a truncated neuropeptide Y (NPY) analog, [Pro³⁰, Tyr³², Leu³⁴]NPY(28-36)NH₂ also called BVD15, at various positions with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7-10-tetraacetic acid) and evaluated the effect of the coupling position with the binding affinity for NPY Y₁ receptors (NPY1R). Our data suggest that [Lys(DOTA)⁴]BVD15 (K_i = 63 ± 25 nM vs. K_i = 39 ± 34 nM for BVD15) is a potent NPY analog suitable for radiolabeling with metallo positron emitters for PET imaging of breast cancer.     

Somatostatin inhibits insulin-like growth factor-I receptor expression in the gill of a teleost fish (Oncorhynchus mykiss)

Rainbow trout gill tissue was used to examine the role of somatostatin (SS) on insulin-like growth factor-I (IGF-I) receptor expression. In vivo implantation of fish with somatostatin-14 (SS-14) reduced expression of IGF-I receptor mRNAs as well as [(125)I]-IGF-I binding. In vitro incubation of gill filaments with SS-14 or various SS isoforms, including SS-28 and [Tyr(7), Gly(10)]-SS-14-containing peptides, directly inhibited IGF-I receptor mRNA expression. SS-14 also inhibited [(125)I]-IGF-I binding in vitro. These data indicate that SSs inhibit the mRNA and functional expression of IGF-I receptors in gill, and suggest that SSs regulate growth in an extrapituitary manner by reducing sensitivity to IGF-I.     

Characterization, Regional Distribution, and Subcellular Distribution of 125I-Tyr1-Somatostatin Binding Sites in Rat Brain

Abstract: ¹²⁵I-Tyr₁-somatostatin binds reversibly, in a saturable manner, and with high affinity to membranes from rat brain. Kinetic and saturation data measured at equilibrium lead to K_Dvalues of 0.4 nM for cortical membranes. The binding is not affected significantly by seven neuropeptides and drugs unrelated structurally to somatostatin (SRIF) while native SRIF, Tyr₁-SRIF, and D-Trp⁸-D-Cys¹⁴-SRIF displace ¹²⁵I-Tyr₁-SRIF in a dose-dependent manner, with K_i of 0.23 nM, 0.90 nM, and 0.11 nM, respectively. Binding sites for ¹²⁵I-Tyr₁-SRIF were found in 9 out of 11 central structures; there was a significant correlation between binding capacity and endogenous SRIF levels measured by radioimmunoassay. In each of the two structures containing the most binding sites, the cortex and the preoptic area, Scatchard analysis suggests a single population of sites with apparent affinities of 0.8 nM and 1.4 nM, respectively. Subcellular fractionation of these two regions reveals that more than 60% of ¹²⁵I-Tyr₁-SRIF specific binding of the homogenate is found in the crude mitochondrial pellet (P₂), which contains synaptosomes. When P₂ is further fractionated on a discontinuous sucrose gradient, most of the initial P₂ binding is recovered from membrane fractions. Each of nine SRIF analogs, with a single alanine substitution, displaces ¹²⁵I-Tyr₁-SRIF binding on cortical membranes in the same order of potency as on adenohypophyseal membranes (r= 0.84). The data demonstrate the presence of SRIF binding sites in the rat brain, with kinetic characteristics comparable to those found in the adenohypophysis, and they provide a biochemical basis for the multiple functions of SRIF in brain.     

Verification of both the sequence and conformational specificity of neurotensin in binding to mast cells

Neurotensin (NT) analogs, modified at Arg⁸ and Arg⁹, were used to assess the role of Arg in NT binding to mast cells. [D-Arg⁸]- and [D-Arg⁹]-NT bound 4–5 times better than NT, whereas [D-Arg^8,9]-NT had the same binding affinity as NT. Binding of [Ala⁸]-NT was not parallel to NT and exhibited a dissociation constant 38-fold lower than NT while [Ala⁹]-NT had 32% binding. C-terminal peptides, NT_8–13 and NT_9–13, had about 65% binding. These data suggest that Arg⁸ plays a greater role than Arg⁹ in the binding to mast cell NT receptors. Reduction of the disulfide bond in [Cys^2,13]-NT produced an analog 4-times more potent than NT, while the cyclized form had only 3% binding. Thus, a linear peptide with a free C-terminus appears to be required for binding.     

Somatostatin-28 regulates GLP-1 secretion via somatostatin receptor subtype 5 in rat intestinal cultures

Five somatostatin receptors (SSTRs) bind somatostatin-14 (S-14) and somatostatin-28 (S-28), but SSTR5 has the highest affinity for S-28. To determine whether S-28 acting through SSTR5 mediates inhibition of glucagon-like peptide-1 (GLP-1), fetal rat intestinal cell cultures were treated with somatostatin analogs with relatively high specificity for SSTRs 2-5. S-28 dose-dependently inhibited GLP-1 secretion stimulated by gastrin-releasing peptide more potently than S-14 (EC(50) 0.01 vs. 5.8 nM). GLP-1 secretion was inhibited by an SSTR5 analog, BIM-23268, more potently than S-14 and nearly as effectively as S-28. The SSTR5 analog L-372,588 also suppressed GLP-1 secretion equivalent to S-28, but a structurally similar peptide, L-362,855 (Tyr to Phe at position 7), was ineffective. An SSTR2-selective analog was less effective than S-28, and an SSTR3 analog was inactive. Separate treatment with GLP-1-(7-36)-NH(2) increased S-28 and S-14 secretion by three- and fivefold; BIM-23268 abolished S-28 without altering S-14, whereas the SSTR2 analog was inactive. The results indicate that somatostatin regulation of GLP-1 secretion occurs via S-28 through activation of SSTR5. GLP-1-stimulated S-28 secretion is also autoregulated by SSTR5 activation, suggesting a feedback loop between GLP-1 and S-28 modulated by SSTR5.     

Specificity of receptors to vasoactive intestinal peptide in guinea pig brain.

The specific binding of VIP to guinea pig brain membranes was tested by 1/ the ability of eight VIP and secretin analogs and fragments to inhibit the binding of ¹²⁵I-VIP and 2/ the capacity of the same peptides to influence basal and VIP-stimulated adenylate cyclase activities. Among all peptides tested, only VIP, secretin, [Val⁵] secretin, and [Gln⁹, Asn¹⁵] secretin (5–27) were able to inhibit ¹²⁵I-VIP binding. The adenylate cyclase activity was stimulated by VIP, secretin and [Val⁵] secretin. [Gln⁹, Asn¹⁵] secretin (5–27) although inactive per se was able to inhibit the VIP-stimulated adenylate cyclase activity competitively.