K. Raynor

Differential distribution of somatostatin receptor subtypes in rat brain revealed by newly developed somatostatin analogs

Somatostatin receptor subtypes were labeled with the somatostatin analogs [125I]CGP 23996 and [125I]MK 678 and the distribution of these receptors in rat brain was investigated using quantitative autoradiographic techniques. [125I]CGP 23996 and [125I]MK 678 specifically label different populations of somatostatin receptors in rat brain. In a number of brain regions striking differences in the distribution of the somatostatin receptor subtypes labeled by each peptide were observed. High levels of binding sites for both [125I]CGP 23996 and [125I]MK 678 were present in the cerebral cortex, CA1 region and subiculum of the hippocampus. In contrast, high levels of [125I]MK 678 binding were found in the dentate gyrus of the hippocampus while few [125I]CGP 23996 binding sites were observed in this brain region. [125I]CGP 23996 binding was detected in the central region of the interpeduncular nucleus whereas the dorsal and lateral subnuclei of this brain area expressed mainly somatostatin receptors with high affinity for MK 678. The locus coeruleus and regions of the superior colliculus and hypothalamus selectively express [125I]MK 678-sensitive somatostatin receptors. Furthermore, limbic structures such as the lateral septum, the nucleus accumbens and ventromedial striatum had much higher levels of [125I]MK 678 binding sites than [125I]CGP 23996 binding sites. Differences in the expression of the somatostatin receptor subtypes were also detected in the substantia nigra. [125I]CGP 23996 binding was present in the pars reticulata but not the pars compacta whereas the reverse distribution for [125I]MK 678 binding sites was observed. The differential distribution of [125I]CGP 23996 and [125I]MK 678 binding sites in rat brain supports the hypothesis that these peptides selectively label different somatostatin receptor subtypes in the central nervous system.

Analogues of Somatostatin Bind Selectively to Brain Somatostatin Receptor Subtypes

Abstract: Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane‐bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK‐678, such that SRIF, receptors are sensitive to MK‐678 and SRIF2 receptors are insensitive to MK‐678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I‐Tyr11 SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK‐678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF, receptor. The binding of 125I‐MK‐678 to SRIF, receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS‐201–995), and the hexapeptides (such as MK‐678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP‐23996‐like analogues did not inhibit 125I‐MK‐678 binding to SRIF, receptors. The binding of 125I‐CGP‐23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of < 1, indicating that 128I‐CGP‐23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP‐23996‐like compounds produced only partial inhibitions of 125I‐CGP‐23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I‐CGP‐23996. 125I‐Tyr11‐SRIF binding and 125I‐CGP‐23996 binding to SRIF receptors were likewise only partially affected by 100 μM guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS), a concentration that completely abolishes specific 125I‐MK‐678 binding to SRIF, receptors. The component of 125I‐CGP‐23996 labeling that was sensitive to GTPγS was also MK‐678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF, receptor is sensitive to cyclic hexapeptides such as MK‐678 and to GTPγS but insensitive to smaller CGP‐23996‐like compounds. The SRIF2 receptor is sensitive to the CGP‐23996‐like compounds and can be selectively labeled by 125I‐CGP‐23996 in the presence of high concentrations of the hexapeptides or GTPγS because, unlike the SRIF, receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype‐selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.

Synthesis of novel, highly potent cyclic-hexapeptide analogs of somatostatin. Potential application of orthogonal protection for affinity chromatography

Abstract The syntheses of new cyclic hexapeptides are described. These peptides potently displace [ 125 I] CGP-23996 (des-Ala 1 , Gly 2 -desamino-Cys 3 [Tyr 11 ]-dicarba 3,14 SRIF) from SRIF receptors on AtT-20 cells. Two of these compounds are of potential value for the isolation of SRIF receptors by affinity chromatography. A key feature in the design was the use of orthogonal protection to provide selective covalent bonding to the solid support.

Biochemical properties of somatostatin receptors

Somatostatin (SRIF) induces its biological actions by binding to and stimulating membrane-associated receptors. To investigate the molecular mechanisms by which SRIF induces its biological effects, we have characterized the biochemical properties of SRIF receptors. SRIF receptors can be solubilized in an active form with the detergent CHAPS and can be detected with the high-affinity SRIF analog [125I]MK 678. The pharmacological characteristics of solubilized SRIF receptors from brain are similar to the receptors in membranes, suggesting that the solubilized receptors retain their biological activity. Solubilized SRIF receptors appear to be tightly associated with GTP-binding proteins, since analogs of GTP can greatly reduce agonist labeling of the solubilized SRIF receptor. The solubilized SRIF receptor migrates as a mass of approximately 400 kd and is a glycoprotein since it can specifically interact with lectin columns. The solubilization of the SRIF receptor has allowed for its purification by affinity chromatography. The purified SRIF receptor migrates as a mass of 60 kd in denaturing gels. Using affinity chromatography, the receptor can be purified to near homogeneity. Present studies are directed toward sequencing and cloning cDNA encoding the SRIF receptor in order to further characterize its physical properties and expression.

Differential agonist modulation of the cloned opioid receptors reveals distinct cellular mechanisms of receptor regulation

Abstract Tolerance is a limitation to the clinical use of opioids. A cellular basis of tolerance may involve receptor downregulation/desensitization after exposure to agonists. We have investigated the effects of agonist pretreatments of cells expressing the cloned rat mu, and mouse delta and kappa receptors on both subsequent radioligand binding and coupling to adenylyl cyclase. Agonist pretreatment of cells expressing kappa and delta receptors diminished subsequent labelling of thoroughly washed membranes with agonist. Antagonist binding to the delta receptor was also diminished, but antagonist binding to the kappa receptor was unaffected. Neither agonist nor antagonist binding to the mu receptor was affected by agonist pretreatment. The loss of agonist binding to the delta and kappa receptors was paralleled by a loss of the ability of agonists to inhibit forskolin-stimulated cAMP accumulation. The mu receptor, in contrast, did not functionally desensitize. Desensitization of the kappa, but not delta, receptor could be blocked by cotransfection of the receptors with a dominant negative mutant of β-adrenergic receptor kinase. The mechanisms underlying the development of tolerance to opioid agents are likely multiple and divergent for the different receptor types.

Molecular biology of kappa and delta opioid receptors

The endogenous opioids, enkephalin and dynorphin induce their biological actions by interacting with delta and kappa receptors. To investigate the molecular mechanisms by which these peptides induce their physiological effects, we have cloned the kappa and delta opioid receptors from a mouse brain cDNA library. The kappa and delta receptors are 380 and 372 amino acids, respectively, and are 61% identical and 71% similar (1). They have relatively little similarity in amino acid sequence with any other receptors except somatostatin receptors. We have analyzed the pharmacological specificities of these receptors following their transient expression in COS cells or in PC12 and CHO cells stably expressing the individual receptors