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Opioid drugs exert a wide spectrum of physiological and behavioral effects, including effects on pain perception, mood, motor control and autonomic functions. The effects of opioids are mediated via a family of membrane-bound receptors, of which the most extensively characterized are the mu, delta and kappa receptors. We have now cloned the human homolog of the mu opioid receptor and, in the present study, we have examined its pharmacological profile. The human mu receptor has high affinities for several alkaloids of high abuse potential as well as a variety of peptide and nonpeptide drugs characterized previously as mu-selective, but not delta- or kappa-selective. Most importantly, the human mu receptor has higher affinity for morphine and methadone than does the rat mu receptor, despite the fact that these receptors are 95% identical at the amino acid level. The labeling of the receptor by agonist was decreased by nonhydrolyzable GTP analogs and by pertussis toxin treatment of cells expressing the human mu receptor, consistent with the coupling of the receptor to guanine nucleotide binding proteins. The human mu receptor functionally couples to the inhibition of adenylyl cyclase in a stereospecific and naloxone-reversible manner. We have also investigated the distribution of mRNAs encoding the mu receptor in human brain by Northern analysis, which demonstrates the existence of multiple transcripts of 13.5, 11, 4.3 and 2.8 kb, which were highly expressed in the hypothalamus, thalamus and subthalamic nucleus, more moderately expressed in the amygdala and caudate nucleus and which demonstrated lowest levels of expression in the hippocampus, substantia nigra and corpus callosum.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin (SRIF) induces its biological actions by interacting with a family of five recently cloned receptors. SRIF receptor subtype, SSTR1, has high affinity for SRIF, but no ligand has been available that selectively binds to this receptor. Desamino acid(1,2,5) [DTryptophan8, N-p-isopropl-4-aminomethyl-l-phenylalanine9]SRIF(des-AA1,2,5 [DT rp8, IAmp9]SRIF inhibits the binding of [125ITyr11]SRIF to the cloned human SSTR1 with an affinity of 1.8+0.7nM, but does not bind to the other cloned SRIF receptors. des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF bound selectively, potently and saturably to SSTR1 with a Kd of 0.5 + 0.1 nM and a maximal binding density of 226 +/- 56 fmol/mg of protein. The binding of des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF to SSTR1 was potently inhibited by SRIF, [DTrp8]SRIF, des-AA1,2,5[DTrp8,IAmp9,DSer13]SRIF and SRIF 28 with K, values of 0.7+0.3, 0.2+0.2, 4.3+0.7 and 0.6+0.1 nM, respectively. SRIF analogs that selectively bind to SSTR2 and SSTR5 were impotent in displacing des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF from human SSTR1. des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF binding to SSTR1 expressed in COS-7 cells was reduced by GTPgS, and this effect was prevented by pertussis toxin treatment. In contrast, the binding of[125ITyr11]SRIF to SSTR1 was not affected by these treatments. These findings indicate that des-AA1,5[125ITyr2,DTrp8,IAmp9]SRIF may bind to SSTR1 in a defferent manner than SRIF. des-AA1,2,5[DTrp8,IAmp9]SRIF and its tyrosine analog are the first ligands that selectively bind to SSTR1 with high affinity and should be useful in localizing and determining the functional properties of this receptor.

The characteristics of somatostatin (SRIF) inhibition of calcium influx stimulated by corticotropin releasing factor (CRF), an activator of adenylate cyclase, and K+, a membrane depolarizing agent, in AtT-20 cells were assessed. Changes in cytosolic calcium levels were measured using the fluorescence probe Quin 2. Both CRF and K+ raise cytosolic calcium levels by stimulating calcium influx. SRIF induced an immediate inhibition of CRF and K+-stimulated calcium influx. This effect was concentration-dependent with IC50 values for SRIF's blockade of CRF and K+ stimulation of 64 +/- 13 pM and 100 +/- 15 pM, respectively. The SRIF analogs, SRIF 28, Trp8-SRIF and Tyr11-SRIF had the same rank order of potency to block CRF and K+-induced calcium influx. The inhibitory effects of SRIF on AtT-20 cells were abolished by pertussis toxin pretreatment. SRIF inhibition of both CRF and K+-induced calcium influx desensitized. The desensitization was rapid (T1/2 approximately 2.5 min), dependent on the concentration of SRIF and not due to the degradation of the peptide. The ability of SRIF to block CRF (cyclic AMP-dependent) and K+ (cyclic AMP-independent)-stimulated calcium influx into AtT-20 cells cannot be separated.

To investigate the interaction of guanine nucleotide-binding regulatory proteins (G proteins) with the agonist-bound brain somatostatin (SRIF) receptor, rat brain SRIF receptor/G protein complexes were solubilized and immunoprecipitated with peptide-directed antisera selective for the different subtypes of G protein alpha subunits (G alpha). In the absence of agonist, solubilized SRIF receptor/G proteins complexes could be immunoprecipitated by antiserum 8730, which is directed against the carboxyl-terminal region of Gi alpha and recognizes all Gi alpha subtypes, and by antiserum 3646, which selectively interacts with internal regions of Gi alpha 1. In contrast, antiserum 1521, which is directed against an internal region of Gi alpha 2, and antiserum 9072, which is directed against the carboxyl-terminal region of Go alpha, did not immunoprecipitate the SRIF receptor. After the binding of agonist to solubilized SRIF receptors, antisera 9072 and 1521, as well as antisera 8730 and 3646, were able to immunoprecipitate the agonist-bound SRIF receptor/G protein complexes, indicating that agonist interaction with SRIF receptors maintained receptor association with Gi alpha 1 and promoted receptor association with Go alpha and, to a lesser extent, Gi alpha 2. Antiserum 1518, which is directed against Gi alpha 3, uncoupled SRIF receptors from Gi alpha and did not immunoprecipitate the agonist-bound or agonist-free brain SRIF receptor. These findings indicate that differences exist in the interaction of the agonist-free and agonist-bound SRIF receptors with G proteins. The binding of agonists to SRIF receptors promotes the association of the receptor with Go alpha and, to a lesser extent, Gi alpha 2, indicating that these G proteins, along with Gi alpha 1 and Gi alpha 3, may be involved in coupling SRIF receptors to cellular effector systems.

Previous studies have shown that at least two subtypes of somatostatin (SRIF) receptors (SRIF1 and SRIF2) are expressed in mammalian cells. SRIF1 receptors have high affinity for MK 678, whereas SRIF2 receptors have no affinity for MK 678 but selectively bind peptides with structures similar to that of CGP 23996. Recently, two SRIF receptor genes have been cloned from human and mouse genomic libraries. In the present study, the pharmacological properties of these two cloned SRIF receptors, expressed in Chinese hamster ovary (CHO) cells, were investigated, to determine whether they have any similarity to the previously described SRIF1 and SRIF2 receptor subtypes. Both cloned receptors could be labeled with 125I-Tyr11-SRIF and exhibited high affinity for SRIF. The SSTR1 receptor could also bind CGP 23996-like compounds but not MK 678. In contrast, the SSTR2 receptor was insensitive to CGP 23996-like compounds but bound MK 678 with high affinity. These findings indicate that the peptide specificities of the cloned SSTR1 and SSTR2 receptors differ from each other. Pretreatment of CHO cells expressing the two cloned SRIF receptors with SRIF abolished high affinity agonist binding to the cloned SSTR2 receptor but not the cloned SSTR1 receptor. Agonist binding to SSTR1 receptors was not significantly affected by guanosine-5'-)-(3-thiotriphosphate) or pertussis toxin pretreatment, whereas agonist binding to SSTR2 receptors was inhibited by both treatments. These findings suggest that SSTR2 receptors can be regulated and they associate with pertussis toxin-sensitive guanine nucleotide-binding proteins, whereas SSTR1 receptors do not. SRIF is a potent inhibitor of adenylyl cyclase activity in mammalian cells. However, neither the cloned SSTR2 nor SSTR1 receptor mediated SRIF inhibition of adenylyl cyclase activity in stably transformed CHO cells or COS-1 cells transiently expressing the cloned receptors, suggesting that neither cloned receptor couples to adenylyl cyclase. The results of these studies indicate that the two cloned SRIF receptors have different pharmacological properties. The characteristics of the cloned SSTR2 receptor are similar to those of the previously described SRIF1 receptor, and the characteristics of the cloned SSTR1 receptor are similar to those of the previously described SRIF2 receptor.

The recent molecular cloning of the genes encoding three somatostatin (SRIF) receptor subtypes has allowed for the individual expression of these receptors in mammalian cells and characterization of their respective pharmacological profiles. In the present study, we have investigated the affinities of a battery of SRIF analogues to bind to SRIF receptor subtypes SSTR1 (cloned somatostatin complex), SSTR2, and SSTR3, as well as their abilities to inhibit the release of growth hormone from anterior pituitary cells in vitro. We labeled SSTR1 and SSTR3 receptors expressed in Chinese hamster ovary and COS-1 cells, respectively, with the metabolically stable SRIF analogue 125I-CGP 23996. SSTR2 receptors expressed in Chinese hamster ovary cells were labeled with the SSTR2-specific radioligand 125I-MK-678. Inhibition studies were performed using SRIF analogues of differing structures, including hexapeptide analogues similar to MK-678, octapeptide analogues similar to SMS 201-995, pentapeptide analogues similar to c[Ahep-Phe-D-Trp-Lys-Thr(Bzl)] (SA), and linear SRIF analogues. SSTR1 bound SRIF and SRIF-28 with high affinity and the peptide SA and its structural analogues with low affinity. The hexapeptides did not interact with SSTR1 at concentrations as high as 1 microM, and only a few of the octapeptides or linear peptides bound, with very low affinities. In contrast, 125I-MK-678 binding to SSTR2 was potently inhibited by the hexapeptides, octapeptides, and some of the linear compounds, whereas SA and its analogues did not bind to SSTR2. The potencies of the various SRIF agonists to inhibit growth hormone release in vitro was highly correlated with their potencies to inhibit radioligand binding to SSTR2, but not to SSTR1 or SSTR3. SSTR3 bound analogues of each class but with moderate to low affinities, with the exception of several linear peptides and one of the octapeptides. We report for the first time the binding affinities of linear analogues of SRIF, some of which display subnanomolar affinities and are highly selective for SRIF receptor subtypes. Most importantly, these studies identify several peptide analogues that are highly potent, specific, and selective for individual subtypes of SRIF receptors. Such information, coupled with the knowledge of the distribution of these receptor subtypes in normal and pathological tissues, will be critical for more specific experimental and therapeutic interventions.

Addition of somatostatin (SRIF) inhibits corticotropin-releasing factor- and forskolin-stimulated cyclic AMP accumulation and adrenocorticotropin hormone secretion from mouse anterior pituitary tumor cells (AtT-20/D16-16). However, prior exposure of these cells to SRIF reduced the potency of SRIF to inhibit both corticotropin-releasing factor- and forskolin-stimulated cyclic AMP accumulation and adrenocorticotropin hormone release. This SRIF desensitization is time- and concentration-dependent and reversible. Cross-desensitization to SRIF analogs also occurred whereas SRIF pretreatment did not affect the inhibition by SRIF of 8-bromo-cyclic AMP-stimulated adrenocorticotropin hormone release or did it affect basal cyclic AMP levels, protein content or phosphodiesterase activity. These data indicate that SRIF can regulate the sensitivity of its own receptor and that SRIF desensitization may involve either a down-regulation of SRIF receptors or an uncoupling of these inhibitory receptors from adenylate cyclase.