Biomedical subjects
Publications and source records attributed to T Reisine.
Opiate receptors mediate the physiological actions of opioid peptides and the clinical effects of the synthetic opioid agonists and antagonists. Site-directed mutagenesis studies have revealed regions of opiate receptors that are essential for ligand recognition, and this could aid the design of more selective opioid ligands.
Explore the source record for details and available documents.
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.
Explore the source record for details and available documents.
Several lines of evidence link the opioid binding cell adhesion molecule (OBCAM) to opioid function. When delta-opioid receptor cDNA (DOR-1) was expressed in CHO cells, OBCAM immunoreactivity (OBCAM-ir) was detected. Transfected cell lines which displayed high opioid binding also expressed high cell surface OBCAM-ir, while untransfected CHO and vector control cells did not. The positive control, neural cell adhesion molecule (NCAM), a protein with structural homology to OBCAM, displayed the same levels of immunofluorescence in transfected and nontransfected cell lines. Membranes from CHO cells transfected with and expressing a variety of muscarinic and dopamine receptors were tested for immunoreactivity. No significant OBCAM-ir was detected in any of these cell membranes. When anti-OBCAM peptide antibodies were used for immunoblots of CHO cells, untransfected, non-binding transfected, and high binding transfected cells revealed the same banding patterns with approximately equal intensity. These observations suggest that in untransfected cells OBCAM is either not present on the extracellular side of the CHO cell membrane or that it exists in an altered conformation which changes upon transfection with opioid receptors to allow recognition of the non-denatured protein by anti-OBCAM antibodies.
Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.
Somatostatin induces its multiple biological actions by interacting with a family of receptors, referred to as sstr1-sstr5. To determine the molecular mechanisms of action of somatostatin, we have investigated the interaction of the different cloned receptors with G proteins and cellular effector systems. sstr2, sstr3 and sstr5 associate with pertussis toxin-sensitive G proteins and are able to mediate the inhibition of adenylyl cyclase activity by somatostatin. Two forms of sstr2, sstr2A and sstr2B, are generated by alternative splicing and differ in their C-terminal amino acid sequence. sstr2B couples to adenylyl cyclase whereas sstr2A does not. To investigate the basis for the differential coupling to adenylyl cyclase, we truncated sstr2B to the point of amino acid sequence divergence from sstr2A. The truncated sstr2B mediated the inhibition of cAMP formation by somatostatin, indicating that the C-terminus is not needed for coupling sstr2 to adenylyl cyclase. It is likely that the C-terminus of sstr2A hinders coupling to adenylyl cyclase. sstr2A associates with Gi alpha 3 and G(o) alpha but does not effectively interact with Gi alpha 1, a G protein that is necessary for coupling somatostatin receptors to adenylyl cyclase. The differential association of the splice variants with Gi alpha 1 may explain their contrasting effects on adenylyl cyclase activity. sstr3 also couples to adenylyl cyclase. Gi alpha 1 links sstr3 to adenylyl cyclase and mutagenesis studies have shown that the C-terminus of Gi alpha 1 is necessary for this coupling. The C-terminus of the Gi alpha proteins differ by only a few amino acid residues and only Gi alpha 1 couples sstr3 to adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)
The diverse physiological effects of somatostatin are mediated by a family of cell surface receptors that bind somatostatin selectively and with high affinity. The somatostatin receptors are members of the seven transmembrane segment receptor superfamily and molecular cloning studies have identified five types, designated sstr1-5. The human somatostatin receptors vary in size from 364 (sstr5) to 418 (sstr3) amino acids with 46-61% amino acid identity between receptors, and 105 amino acids are invariant. The sequences of the seven putative alpha-helical membrane-spanning domains are more highly conserved than those of the extracellular N- and intracellular C-terminal domains. Two forms of sstr2 have been identified in the mouse, sstr2A and sstr2B, which differ in size and sequence of the intracellular C-terminal domain. These two forms of sstr2 are products of a common gene and are generated by alternative splicing with sstr2A and sstr2B being the products of the unspliced and spliced forms, respectively, of sstr2 mRNA. Thus, functional diversity within the somatostatin receptor family may result from the expression of multiple types as well as from alternative splicing. The five somatostatin receptors have distinct patterns of expression in the central nervous system and peripheral tissues. They have also been expressed in vitro and shown to have different pharmacological properties. Somatostatin analogues selective for sstr2, sstr3 and sstr5 have been identified which will facilitate in vivo studies of the functions of these somatostatin receptors. Such studies to date suggest that sstr2 mediates inhibition of growth hormone secretion and sstr5 mediates inhibition of insulin secretion. The molecular cloning and functional characterization of the somatostatin receptor family is a first step in elucidating the diverse effects of somatostatin on cellular functions.
1. Somatostatin (SRIF) exerts diverse physiological actions in the body including regulation of hormone and neurotransmitter release and neuronal firing activity. Analogs of SRIF are used clinically to treat tumors and cancers and to block the hypersecretion of growth hormone in acromegaly. 2. The recent cloning of five SRIF receptor subtypes has allowed for the identification of the molecular basis of the cellular actions of SRIF. The ligand binding domains and regions involved in coupling to G proteins and cellular effector systems are being identified and the processes by which SRIF inhibits cell growth and proliferation are being established. Furthermore, subtype selective agonists have been generated which are being used to investigate the specific biological roles of each SRIF receptor subtypes. 3. Such information will be useful in developing a new generation of SRIF drugs that could be employed to treat metabolic diseases, disorders of the gut, cancer and abnormalities in the central nervous system such as epilepsy and Alzheimer's disease.
The neuropeptide somatostatin is widely distributed in the central nervous system and in peripheral tissues and may be involved in the regulation of a number of physiological functions including movement and cognition. Somatostatin may also have a role in the development of the central nervous system, in particular, the cerebellum and spinal cord. Somatostatin induces its actions by interacting with a family of membrane associated receptors. Recently, five somatostatin receptors have been cloned and referred to as SSTR1-SSTR5. The distribution of the expression of the mRNAs for these receptors are distinct but overlapping. Preliminary pharmacological analysis of these receptors may lead to the development of selective ligands at these receptors. These compounds may be useful in identifying the selective functions of these receptor subtypes. Some somatostatin analogues have antiproliferative actions and are used presently to treat carcinoids. Development of subtype selective somatostatin analogues could be helpful in further identifying somatostatin receptor-expressing tumors and in the treatment of cancer. The cloning of these receptors has now opened up the possibility of more clearly investigating the functions of somatostatin in the brain and peripheral tissues and will facilitate the generation of new somatostatin drugs that may be employed for the treatment of a number of diseases.
Somatostatin (SRIF) induces its multiple biological actions by interacting with a family of receptors, referred to as SSTR1-SSTR5. These receptors are capable of associating with particular guanine nucleotide binding proteins to couple the receptors to distinct cellular effector systems. Therefore, G proteins have an important role in directing SRIF signalling and may provide the molecular basis for the diverse cellular actions of SRIF.
Somatostatin induces its biological actions by activating a family of receptor subtypes. The recent cloning of five somatostatin receptor subtypes has led to the development of subtype-selective agonists. These compounds are revealing distinct functions of the individual receptor subtypes. Mutagenesis studies have revealed domains of several of the receptors involved in specific recognition of somatostatin analogues. Molecular modeling of both of these ligand-binding domains and the constrained somatostatin analogues that they interact with may lead to the development of nonpeptide somatostatin drugs that could be useful in the treatment of tumors and various metabolic, gastrointestinal, and central nervous system disorders.
Explore the source record for details and available documents.
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)
Opium and its derivatives are potent analgesics that can also induce severe side effects, including respiratory depression and addiction. Opioids exert their diverse physiological effects through specific membrane-bound receptors. Three major types of opioid receptors have been described, termed delta, kappa, and mu. The recent molecular cloning of these receptor types opens up the possibility to identify the ligand-binding domains of these receptors. To identify the ligand-binding domains of the kappa and delta receptors, we have expressed in COS-7 cells the cloned mouse delta and kappa receptors and chimeric delta/kappa and kappa/delta receptors in which the NH2 termini have been exchanged. The opioid antagonist naloxone binds potently to wild-type kappa receptor but not to wild-type delta receptor. The kappa/delta chimera bound [3H]naloxone with high affinity. In contrast, the kappa-specific agonist [3H]U-69,593 did not bind to the kappa/delta chimera. These findings indicate that selective agonists and antagonists interact with different recognition sites in the kappa receptor and localize the antagonist-binding domain to the NH2 terminus. Consistent with the results of radioligand-binding studies, the kappa/delta chimera did not mediate kappa-agonist inhibition of cAMP formation. In contrast, the delta/kappa chimera did mediate kappa-agonist inhibition of cAMP formation, but this effect was not blocked by naloxone. Furthermore, a truncated kappa receptor lacking its NH2 terminus was able to mediate agonist inhibition of cAMP accumulation in a naloxone-insensitive manner. This result further indicates that the NH2 terminus of the kappa receptor contains the selective antagonist-binding domain. The ability to dissociate agonist- and antagonist-binding sites will facilitate the development of more specific kappa agonists, which could have analgesic properties devoid of side effects.
The mu (mu) opioid receptors, which mediate the effects of morphine, are widely distributed in brain. We have examined the distribution of mRNA encoding a mu opioid receptor in rat brain with in situ hybridization histochemistry at the single-cell level to obtain information about the cell types synthesizing this receptor. Only neurons, not glia, were labeled in discrete brain regions. High levels of labeling were detected in the thalamus, striosomes of the caudate-putamen, globus pallidus, and brain regions involved in nociception, arousal, respiratory control, and, possibly, addiction. The general distribution of the receptor mRNA paralleled that of mu opioid binding sites with some notable exceptions. These include the cerebral cortex, which contains binding sites, but very few labeled neurons. No labeling was observed in the cerebellum, a region devoid of mu binding sites. Three main findings emerged from these experiments: 1) the mRNA was present in regions mediating both the therapeutic (analgesia) and the unwanted (respiratory depression, addiction) effects of morphine, 2) the mRNA was very densely expressed by neurons known to receive dense enkephalin-containing inputs, and 3) the dissociation between the presence of binding sites and absence of mRNA in some brain regions supports a presynaptic localization of mu opioid receptors in these areas. Alternatively, other subtypes of mu opioid receptors may be encoded by a different mRNA. These results provide new insights into the receptor types and neuronal circuits involved in the effects of endogenous opioids and morphine.
The distribution of the kappa opioid receptor mRNA in adult mouse brain has been determined using the technique of in situ hybridization histochemistry. The mRNA for the kappa opioid receptor was expressed in distinct areas throughout the brain. The telencephalon showed high levels of expression in the deeper layers of the parietal and temporal cortex, olfactory tubercle, nucleus accumbens, claustrum, endopiriform nucleus, nucleus of the vertical and horizontal limb of the diagonal band, and medial and central nuclei of the amygdala. In the diencephalon, kappa opioid receptor mRNA was present in multiple medial thalamic nuclei including the centromedial, paraventricular, parafasicular, central, and peritenial nuclei, as well as in most hypothalamic nuclei including the ventromedial, periventricular, supraoptic, arcuate, and dorsomedial nuclei. The mesencephalon showed highest levels of kappa receptor mRNA in the substantia nigra pars compacta, ventral tegmental area, zona incerta, interpeduncular nucleus, superior colliculus, inferior colliculus, central grey, and the raphe nucleus. In the metencephalon, kappa opioid receptor mRNA was expressed in the parabrachial nuclei, locus coeruleus, dorsal and ventral tegmental nuclei, and the raphe pontine nuclei. The distribution of the kappa receptor mRNA closely coincides with the localization of binding sites in rat brain for [3H]U-69,593, a specific kappa 1 opioid receptor ligand. The mRNA distribution also correlates with neuroanatomical sites of actions of kappa agonists and distribution of the endogenous kappa receptor ligand dynorphin.