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.     

Brain and Spinal Cord Distribution of Biphalin: Correlation with Opioid Receptor Density and Mechanism of CNS Entry

Abstract: Biphalin [(Tyr-d -Ala-Gly-Phe-NH)₂] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [¹²⁵I-Tyr¹]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [¹²⁵I-Tyr¹]-biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ- and μ-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [¹²⁵I-Tyr¹]biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [¹²⁵I-Tyr¹]biphalin was decreased with naloxone, d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ- and μ-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [¹²⁵I-Tyr¹]biphalin, which could be described by Michaelis-Menten kinetics with a K_m of 2.6 ± 4.8 µM, V_max of 14.6 ± 2.89 pmol^?1·min^?1·g^?1, and K_d of 0.568 ± 0.157 µl·min^?1·g^?1. Brain entry of [¹²⁵I-Tyr¹]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and l -phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.     

Preferential suppression of insulin-stimulated proliferation of cultured hepatocytes by somatostatin: Evidence for receptor-mediated growth regulation

The role of somatostatin (SS-14) in the regulation of rat liver regeneration was examined by using thymidine incorporation into hepatocyte DNA labeled with tritiated thymidine, a nuclear-labeling index, and the binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes isolated at various times after partial hepatectomy. The data demonstrated no suppressive effect of SS-14 on insulin and glucagon-stimulated thymidine incorporation into hepatocyte DNA as early as 2 h after partial hepatectomy. These data were substantiated by a nuclear labeling index studies. At 2 h, ¹²⁵I-tyr¹¹-SS-14 binding to its specific sites on isolated hepatocytes was undetectable. There was a time-dependent increase in binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes obtained at various times after partial hepatectomy. There was a significant decrease in the number of binding sites after partial hepatectomy as determined by Scatchard analysis. The data were supported by autoradiography analysis of affinity labeled ¹²⁵I-tyr¹¹-SS-14-binding protein complex followed by SDS-PAGE. SS-14 also inhibited intracellular cAMP in hepatocytes obtained at 18 h after hepatectomy. The data are consistent with the hypothesis that SS-14 participates via its own receptor in the regulation of the liver regeneration. © 1995 Wiley-Liss, Inc.     

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.     

High affinity binding sites for a somatostatin-28 analog in rat brain

Using an iodinated analog of a large (28 residues) and biologically active form of somatostatin, ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵]SS-28, it was possible to demonstrate saturable and high affinity binding sites (dissociation constant = 0.46 ± 0.04 nM) in rat cortical membranes. Somatostatin, somatostatin-28, as well as two potent analogs, [D-Trp⁸] somatostatin and [D-Trp²²] somatostatin-28, could completely displace the radiogland in the nanomolar range whereas the inactive analog Des-Trp⁸-somatostatin and the unrelated peptide GnRH showed no affinity for these binding sites; octa- and nona-peptide analogs of somatostatin were inactive. High binding was found in hippocampus, amygdala, tuberculum olfactorium, caudate-putamen and cortex; moderate binding in midbrain and hypothalamus, and no binding in the cerebellum. These results suggest that specific somatostatin receptors can be measured within the brain with ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵] SS-28 as radioligand.     

Autoradiographic Localization of [125I-Tyr0]Bradykinin Binding Sites in Brains of Wistar-Kyoto and Spontaneously Hypertensive Rats

1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [¹²⁵I-Tyr⁰]bradykinin ([¹²⁵I-Tyr⁰]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [¹²⁵I Tyr⁰]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with K _D values in the range of 100 pM and a B _max of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg⁹-BK. The B₂ receptor antagonist icatibant inhibited [¹²⁵I-Tyr⁰]BK binding with a K _i value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while K _i values for the B> ₁ receptor agonist DesArg⁹-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [¹²⁵I-Tyr⁰]BK B₂ binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B₂ receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.     

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.     

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)     

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [³H]Boc[Nle^28,31]CCK_27^?33, designated [³H]Boc[diNle]CCK₇, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK₇ displaces [¹²⁵I]CCK₈ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK₈. The order of potencies to displace [³H]Boc[diNle]CCK₇ from its binding sites was the same on mouse brain and rat pancreas: $\text{[}{}^3\text{HBoc[diNle]CCK}{}_7\text{>}\text{CCK}{}_8\text{,}\text{Boc-CCK}{}_7\text{>}\text{non-sulfated CCK}{}_8$, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [³H]Boc[diNle]CCK₇ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.     

Evidence for an intracellular somatostatin receptor in pancreas: a comparative study with reference to gastric mucosa

Specific binding sites for ¹²⁵I-Tyr₁somatostatin-14 were comparatively demonstrated in isolated rat pancreatic and gastric parietal cells. In both materials, the sites occurred mostly in cytosol, with apparent affinities of 1×10^?10M and 3×10^?11M, respectively, in pancreatic and gastric cells. Somatostatin-14 stimulated cytosolic phosphoprotein phosphatases (PPPases) in pancreas as well as in gastric mucosa with concentrations for half maximal effect consistent with binding affinities. Somatostatin 28 mimicked somatostatin 14 stimulation with a higher efficacy but an equivalent potency. Secretin and cholecystokinin C terminal octapeptide were ineffective. Furthermore, in intact isolated cells, somatostatinic stimulation of PPPases was blocked by 5×10^?4M dinitrophenol. We therefore suggest that in pancreas as in gastric mucosa, somatostatin's inhibitory effect on secretory functions could involve protein dephosphorylation mediated by an intracellular receptor.     

New specific radioligand for one subpopulation of brain somatostatin receptors

Cyclic octapeptide analogues of somatostatin (SS) like SMS 201-995 [H-(D) Phe-Cys-Phe-(D) Trp-Lys-Thr-Cys-Thr(ol)] or its Tyr3-derivative 204-090, displaced [125I-Tyr11]-SS 100% from pancreatic membranes but only 62-75% from brain membranes; the remaining sites were displaced by SS. These data indicate that some mini-somatostatins bind to a subpopulation of SS receptors in rat brain. The iodinated Tyr3-derivative (125I-204-090) can be considered a selective radioligand for one rat brain SS receptor subpopulation: It shows saturable and high affinity binding (KD = 0.29 nM; Bmax = 350 fmoles/mg protein) to rat cortex. The pharmacological properties of 125I-204-090 binding sites are similar to those of [125I-Tyr11]-SS sites. Distribution of these sites correspond to SS receptor-rich areas such as cortex, hippocampus, striatum, pituitary, pancreatic beta-cell. SS as well as SMS 201-995 bind to these sites with high affinity. The stability and high specific vs non-specific binding ratio makes 204-090 a radioligand of choice to measure this SS receptor subpopulation in CNS but also the SS receptors in pituitary and pancreas.     

High affinity GnRH binding to testicular membrane homogenates

The binding of GnRH to membrane homogenates from collagenase-dissociated normal rat interstitial cells is shown to be of high affinity and specific. The dissociation constant for the radioligand used, [¹²⁵I-Tyr⁵, D-Ala⁶, N^αMeLeu⁷, Pro⁹-NEt]-GnRH is 0.26 ± 0.02 nM and the number of binding sites is 17 fmoles/mg membrane homogenate. There is one GnRH antagonist whose affinity is greater for the pituitary than for the testis. This antagonist has the same affinity for testis homogenates as for ovarian homogenates.     

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.     

Atrial and brain natriuretic peptides share binding sites on cultured cells from the rat trachea

Summary We examined the distribution of binding sites for alpha-atrial natriuretic peptide (¹²⁵I-ANP_1–28) and the recently discovered porcine brain natriuretic peptide (¹²⁵I-pBNP) on immunocytochemically identified cells in dissociated culture preparations of the rat trachea. Specific binding sites for both ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP were evenly distributed over distinet subpopulations of smooth muscle myosin-like immunoreactive muscle cells, fibronectin-like immunoreactive fibroblasts and S-100-like immunoreactive glial cells. Neither keratin-like immunoreactive epithelial cells nor protein gene product 9.5-like immunoreactive paratracheal neurones expressed natriuretic peptide binding sites, although autoradiographically labelled glial cells were seen in close association with both neuronal cell bodies and neurites. The binding of each radiolabelled peptide was abolished by the inclusion of either excess (1 M) unlabelled rat ANP or excess unlabelled porcine BNP, suggesting that ANP and BNP share binding sites in the trachea. Furthermore, the ring-deleted analogue, Des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANF_4–23-NH₂, strongly competed for specific ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP binding sites in the tracheal cultures; this suggests that virtually all binding sites were of the clearance (ANP-C or ANF-R₂) receptor subtype.     

[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.     

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown

Analogs of luteinizing hormone- releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct ³H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe⁶,desGly¹⁰]-LHRH ethylamide and [D-Ser(Bu^t)⁶,desGly¹⁰]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al₂O₃ catalyst to high specific radioactives. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe⁶,desGly¹⁰]- and [D-Ser(Bu^t)⁶, desGly¹⁰]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of tha analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.     

The two subtype 1 somatostatin receptors of rainbow trout, Tsst1A and Tsst1B, possess both distinct and overlapping ligand binding and agonist-induced regulation features

In the present study, two isoforms of somatostatin receptor subtype one, previously obtained from the brain of rainbow trout, Tsst1A and Tsst1B, were stably transfected in the Chinese hamster ovary cell line (CHO-K1) and their binding properties were characterized. High affinity binding of somatostatin by expressed receptors was saturable and ligand selective. Both Tsst1A and Tsst1B preferentially bound peptides derived from preprosomatostatin I (PPSS I; e.g., SS-14-I) over those derived from PPSS II (containing Tyr7, Gly10-SS-14-I at their C-terminus; e.g., SS-25-II). The rank order of ligand affinities for Tsst1A was SS-28-I>SS-14-I>SS-26-I?SS-28-II>SS-14-II>SS-25-II. The rank order for Tsst1B was SS-14-I>SS-28-I>SS-26-1?SS-28-II>SS-25-II>SS-14-II. Agonist-induced regulation of Tsst1A and Tsst1B was also investigated. After 30 min of SS-14-I exposure, both Tsst1A and Tsst1B underwent rapid internalization; ca. 60% of membrane Tsst1A was internalized and only about 40% of membrane Tsst1B was internalized. Prolonged agonist exposure (up to 48 h) induced up-regulation of membrane-expressed Tsst1A, but had no effect on Tsst1B. These results indicate that Tsst1s display both distinct and overlapping ligand binding and agonist-induced regulation features. Such features may form the basis of ligand-selection and have important consequences on target organ responsiveness.     

Two Saturable Recognition Sites for (-)[125I]Iodo-N6-(4-Hydroxyphenyl-Isopropyl)-Adenosine Binding on Purified Cardiac Sarcolemma

Abstract

Analysis of (-)[¹²⁵]iodo-N⁶-(4-hydroxyphenylisopropyl)-adenosine ([¹²⁵I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 μmol/1) was used as non-specific binding marker. In the presence of 1 mmol/1 theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [¹²⁵I]HPIA binding, with a high affinity potency rank order typical of interaction with A₁-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/1. In brain membranes, specific binding of [¹²⁵I]HPIA as well as of [³H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [¹²⁵I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.     

Binding of luteinizing hormone-releasing hormone analogues to dispersed rat pituitary cells

The binding of luteinizing hormone-releasing hormone (LHRH) to dispersed rat pituitary cells was studied by using ¹²⁵I-labeled analogues of the neurohormone: a superactive agonist [D Ser (Bu^t)⁶]LHRH(1–9) ethylamide and an antagonist DpGlu¹, DPhe², DTrp^3,6-LHRH. Although these cells were exposed to proteolytic enzymes, their ability to respond to LHRH stimulation by gonadotropin release, is preserved. The time course of binding of the two analogues at different temperatures has demonstrated that highest specific binding is evident at 4°C and that equilibrium is reached after 90 min of incubation at this temperature. Incubation of pituitary cells with the labeled analogues together with increasing concentrations of LHRH or unlabeled analogues exhibited parallel competition curves, suggesting binding to the same receptor sites but with different affinities. Biologically inactive analogues of LHRH or unrelated peptides such as TRH did not compete for binding sites. K_a values for the agonist, LHRH and the antagonist were 2.1 × 10⁹M^?1, 0.92 × 10⁸M^?1 and 0.76 × 10⁹M^?1, respectively, and the binding capacity was 116 fmoles/10⁶ pituitary cells.     

Subcellular distribution of somatostatin-14, somatostatin-28 and somatostatin-28 (1-12) in rat brain cortex and comparisons of their respective binding sites in brain and pituitary

Subcellular distribution and binding characteristics of the three endogenous peptides somatostatin-14 (SRIF-14), somatostatin-28 (SRIF-28) and somatostatin-28(1-12) (SRIF-28(1-12] derived from preprosomatostatin were investigated in the rat brain cortex. The three peptides are predominantly recovered from a crude mitochondrial pellet (P2), containing the pinched off nerve endings. Specific high affinity binding sites for 125I-N-Tyr-SRIF-14 and 125I-N-Tyr-SRIF-28 are present on pituitary and brain membranes. Under the same conditions, 125I-N-Tyr-SRIF-28(1-12) binding is undetectable. Moreover, SRIF-28(1-12) does not displace 125I-N-Tyr-SRIF-14 or 125I-N-Tyr-SRIF-28 binding. SRIF-28 is more potent than SRIF-14 to displace 125I-N-Tyr-SRIF-28 binding to brain and pituitary membranes, while both peptides are equipotent to displace 125I-N-Tyr-SRIF-14 binding. Finally, the regional distribution of 125I-N-Tyr-SRIF-14 and 125I-N-Tyr-SRIF-28 binding sites in the brain is identical. In conclusion, the present results are consistent with a neurotransmitter and neurohormonal role for SRIF-14 and SRIF-28. The function of SRIF-28(1-12) in brain remains to be elucidated. Additionally, a differential role for SRIF-14 and SRIF-28 both in adenohypophysis and brain cannot be ascertained at the present time.