Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0043167 (pertussis)
 19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using explant cultures of human fetal anterior pituitary glands (9-19 weeks fetal age) and an acute (3-h) test protocol, we investigated the role of two signal transduction pathways (Gs-adenylate cyclase-Gi, Ca2+ channels) in GH-releasing factor (GRF)/somatostatin (SRIF) regulation of GH secretion during the first half of gestation. Data have been analyzed for ontogenic changes using three age groups: 9-10, 12-13, and 15-19 weeks fetal age. The fetal somatotrope shows dose-related responses to forskolin (0.1-10 microM), dibutyryl cAMP (dBcAMP; 0.1-1 mM), and theophylline (0.01-1 mM), all factors that increase intracellular concentrations of cAMP; there is a significant age-related increase in the stimulatory effects of 1 mM dBcAMP and 1 mM theophylline. When any one of these three factors is added with 10 nM GRF, there are no significant additive or synergistic effects on GH secretion. Although 10 nM SRIF has no effect at 9-10 weeks, it is inhibitory in the 12-13 and 15-19 week groups, significantly suppressing the stimulatory effect of 1 microM forskolin and completely blocking the effects of 1 mM dBcAMP or theophylline. Pretreatment of cultures with pertussis toxin completely blocks SRIF inhibition of both basal and GRF-stimulated GH release. KCl (5-50 mM) and Bay K 8644 (0.1-10 microM), both activators of Ca2+ channels, have dose-related stimulatory effects on GH release; 50 mM KCl shows an age-related increase in stimulatory activity. If 10 nM GRF, 1 microM forskolin, 1 mM theophylline or 1 mM dBcAMP is added with either 50 mM KCl or 1 microM Bay K 8644, there is an additive response. SRIF (10 nM) completely blocks the stimulatory effects of 1 microM Bay K 8644 and markedly inhibits the effects of 50 mM KCl from as early as the ninth week of fetal age. The Ca2+ channel blocker nifedipine (1-10 microM) significantly inhibits basal as well as stimulated (GRF, forskolin, dBcAMP, theophylline, KCl, and Bay K) GH release from as early as 9 weeks fetal age; in contrast, the calmodulin blocker trifluoperazine (0.01-10 microM) has no effects on basal GH secretion and only slightly inhibits the stimulatory effects of KCl. Pretreatment with 10 nM GRF for 24 h significantly decreases a subsequent 3-h response to 10 nM GRF, but does not alter the subsequent response to 1 mM theophylline or 50 mM KCl.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vitro modulation of growth hormone (GH) secretion from early to midgestation human fetal pituitaries by GH-releasing factor and somatostatin: role of Gs-adenylate cyclase-Gi complex and Ca2+ channels. 809 16

To determine the effect of somatostatin (SRIF) on airway smooth muscle contractile and relaxant responses and its mechanism of action, we studied rabbit tracheal smooth muscle under isometric conditions in vitro. SRIF did not change the contractile responses to electrical field stimulation and acetylcholine, but it inhibited the relaxant responses to isoproterenol in a concentration-dependent manner, an effect that was reversed by treatment of tissues with pertussis toxin (PTX). In contrast, the forskolin- and verapamil-induced relaxations were not altered by SRIF. SRIF also attenuated the increase in intracellular cyclic AMP levels in response to isoproterenol. These results suggest that SRIF may decrease beta-adrenoceptor-mediated muscle relaxation by acting at the site proximal to cyclic AMP synthesis, especially at PTX-sensitive GTP-binding regulatory protein, Gi, coupled to adenylate cyclase.
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PMID:Effect of somatostatin on contractile and relaxant responses of tracheal smooth muscle in rabbits. 810 92

The recent molecular cloning of the genes and cDNAs encoding multiple somatostatin (SRIF) receptor subtypes has allowed for the individual expression of these receptors in mammalian cells and characterization of their respective pharmacological profiles. Previously, we fully described and compared the pharmacological properties of the first three SRIF receptor subtypes, SRIF receptor type (SSTR)1, SSTR2, and SSTR3. In the present study, we have investigated the properties of the newly cloned SRIF receptor subtypes SSTR4 and SSTR5 with regard to pharmacological profiles, the regulation of high affinity agonist binding to these receptors by stable GTP analogues, Na+, or prior exposure to agonists, and the inhibition of forskolin-stimulated cAMP accumulation mediated by these receptors. We labeled SSTR4 and SSTR5 expressed in Chinese hamster ovary (CHO-K1) and COS-1 cells, respectively, with the metabolically stable SRIF analogue 125I-CGP 23996. Radioligand binding competition 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)], and linear SRIF analogues. SSTR4 bound compounds in all structural classes with high to moderate affinities, and several compounds were identified that are > 100-fold selective for SSTR4, compared with the other cloned SRIF receptors, including the linear SRIF analogue BIM-23052 and the CGP 23996-like SRIF analogue L-362,855. In contrast, SSTR5 bound very few SRIF analogues with high affinity. Both receptors could be regulated by prior exposure to agonist. In addition, agonist binding to SSTR4 was reduced by stable GTP analogues, Na+, and pertussis toxin, but agonist binding to SSTR5 was not affected by these treatments. SSTR4 is efficiently coupled to the inhibition of adenylyl cyclase activity, whereas SSTR5 appears not to couple to this cellular effector system. Such differences between the cloned SRIF receptors provide useful strategies for identifying regions of these receptor subtypes that may be involved in ligand-binding specificities and G protein and cellular effector system coupling. The identification of subtype-selective SRIF analogues may lead to more specific therapeutic interventions.
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PMID:Characterization of cloned somatostatin receptors SSTR4 and SSTR5. 810 85

Somatostatin regulates diverse cellular effectors, including adenylyl cyclase, ion channels, and ion exchangers. We expressed two somatostatin receptor subtypes, SSTR1 and SSTR2, stably in mouse fibroblast Ltk- cells and transiently in human embryonic kidney HEK293 cells to investigate subtype-specific pharmacological and functional properties. The effects of GTP gamma S and pertussis toxin on [125I-Tyr11]somatostatin-14 binding indicated that SSTR2 may couple exclusively to pertussis toxin-sensitive G proteins, whereas SSTR1 may couple to both pertussis-sensitive and -insensitive G proteins. When expressed either stably or transiently, both receptor subtypes mediated somatostatin inhibition of cAMP accumulation by a pertussis toxin-sensitive mechanism. In contrast, only SSTR1 mediated somatostatin inhibition of Na(+)-H+ exchange activity, and this action was insensitive to pertussis toxin. We generated two chimeric receptors by replacing sequential residues of SSTR2 with cognate sequences of SSTR1 to identify molecular determinants unique to SSTR1 that may confer coupling to the exchanger. SSTCR4 included a SSTR1 segment encompassing determinants within the fifth and sixth hydrophobic domains and the entire third cytoplasmic loop, while SSTCR5 contained a SSTR1 segment spanning the second through sixth hydrophobic domains, including both second and third cytoplasmic loops. Although both chimeric receptors mediated somatostatin inhibition of cAMP accumulation, only SSTCR5 mediated the inhibition of Na(+)-H+ exchange activity, and this effect was pertussis-insensitive. These findings demonstrate both pharmacological and functional differences between SSTR1 and SSTR2. The ability of SSTR1 to selectively attenuate Na(+)-H+ exchange activity requires determinants outside the third cytoplasmic domain.
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PMID:Subtype-specific signaling mechanisms of somatostatin receptors SSTR1 and SSTR2. 814 17

The pharmacology, signal transduction, and coupling to G proteins of the rat somatostatin (SRIF) receptor (SSTR)1 have been characterized in transfected Chinese hamster ovary (CHO) (K1 strain) cells. The expressed receptor exhibited saturable, high affinity binding of several radioiodinated SRIF analogues. Three different radioligands were used to determine the pharmacological properties of this SSTR subtype. [125I-Tyr11]SRIF-14 (125I-S-14), [Leu8,D-Trp22,125I-Tyr25]SRIF-28 (125I-S-28), and cyclo(D-Trp-Lys-Abu-Phe-MeAla-125I-Tyr) (125I-peptide C) displayed the following rank order of affinity (Kd) for the SSTR1 subtype: 125I-S-14 > or = 125I-S-28 > 125I-peptide C. Competition of 125I-S-14 with S-14, S-28, or peptide C displayed the same rank order of potency. Chemical cross-linking of specifically bound 125I-S-28 to membranes from CHO cells expressing the receptor indicated that the molecular weight of the SSTR1 expressed in CHO cells is approximately 70,000, suggesting that it is heavily glycosylated. Previous reports have suggested that the human SSTR1 [Mol. Pharmacol. 42:28-34 (1992)] couples poorly to G proteins. The coupling of the rat SSTR1 to G proteins was demonstrated by three independent methods. (a) Binding of 125I-S-14 to the SSTR1 subtype was inhibited in a dose-dependent fashion by incubation of membranes with guanosine-5'-O-(3-thio)triphosphate. (b) Treatment of cells with pertussis toxin decreased binding by 80%. (c) Immunoprecipitation of 125I-S-14 binding was observed with antiserum specific for Gi alpha 1,2, but not with antiserum specific for Gs alpha, in membranes from transfected cells. In CHO cells transfected with the SSTR1 cDNA, SRIF inhibited forskolin-stimulated cAMP accumulation by up to 50%, in a dose-dependent fashion (ED50 = 1.1 nM). Pertussis toxin treatment decreased both the efficacy and the potency of the SRIF-mediated inhibition of cAMP accumulation (from 50% to 22%), compared with control untreated cells. These data suggest that the rat SSTR1 inhibits cAMP accumulation by coupling to pertussis toxin-sensitive G proteins.
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PMID:Rat somatostatin receptor type 1 couples to G proteins and inhibition of cyclic AMP accumulation. 814 28

Somatostatin (SRIF) receptors of GH4C1 cells occupied with biotinyl-NH-[Leu8,D-Trp22,Tyr25] somatostatin28 (bio-S28) have been affinity purified over streptavidin affinity columns (Eppler, C. M., Zysk, J. R., Corbett, M., and Shieh, H.-M. (1992) J. Biol. Chem. 267, 15603-15612). This procedure results in the copurification of a single subtype of SRIF receptor (SSTR2) and associated guanine nucleotide-binding proteins (G proteins) that are coupled to these receptors. For accurate quantification it was necessary to: (i) use homogenous recombinant standards; (ii) accurately assess the purity of standards; (iii) determine recovery of G proteins during sample preparation and Western blotting; and (iv) account for cross-reactivity among antisera. Four pertussis toxin-sensitive G proteins were quantified with previously characterized polyclonal antisera. Gi alpha 1 also was measured with a novel, more sensitive monoclonal antibody (7H7). Go alpha and Gi alpha 2 but not Gi alpha 1 and Gi alpha 3 were detected in membrane extracts prepared from GH4C1 cells. In contrast, the G proteins copurified with SSTR2 receptors were predominantly Gi alpha 2 (50% of total G protein) and Gi alpha 3 (36% of total G protein), whereas Go alpha and Gi alpha 1 were negligible. G beta subunits also were detected. Silver staining confirmed the absence of a 39-kDa protein, corresponding to the M(r) of Go alpha associated with purified SRIF receptor-G protein complexes. These data suggest that SRIF receptors selectively couple to two G proteins, one of which is sparsely expressed in GH4C1 cells; the data conform to the notion that SRIF receptors discriminate between similar pertussis toxin-sensitive G proteins.
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PMID:Identification and quantification of Gi-type GTP-binding proteins that copurify with a pituitary somatostatin receptor. 844 59

The inhibitory neuropeptide somatostatin (SRIF) initiates many of its physiological effects by binding to membrane receptors which are coupled to pertussis toxin-sensitive G-protein(s). We have solubilized such a SRIF receptor-G-protein complex and purified it using a biotinylated SRIF analog and guanine nucleotide-dependent affinity chromatography. Following [125I-Tyr11]SRIF binding to membranes from AR4-2J pancreatic acinar cells, only two detergents, dodecyl-beta-D-maltoside (D beta M) and sucrose monolaureate, extracted greater than 70% of the prebound peptide in association with receptor. The D beta M-solubilized ligand-receptor complex was extremely stable: the half-time (t1/2) for dissociation was 11 days at 4 degrees C. However, guanosine 5'-3-O-(thio)triphosphate (10 microM) elicited rapid dissociation of the [125I-Tyr11]SRIF-receptor complex (t1/2 < 30 s), and this effect was concentration-dependent (ED50 = 4.0 + 0.3 nM). [125I-Tyr11]SRIF dissociation was also stimulated by GDP (ED50 = 4.1 +/- 0.3 microM), and the potency of GDP was increased 4-fold by 30 microM AlF4-. Thus, the solubilized receptor was functionally associated with G-proteins. Cross-linking of the soluble [125I-Tyr11]SRIF-receptor complex resulted in the covalent labeling of a 70-90-kDa band, the same band that was specifically labeled in membranes. Affinity purification of the SRIF receptor-G-protein complex was accomplished by prebinding a biotinylated SRIF analog, [N-biotinyl-Leu8,D-Trp22,Tyr25]SRIF28, to membranes followed by solubilization of the ligand-receptor-G-protein complex, adsorption to streptavidin-agarose, and specific elution with 100 microM GDP, 30 microM AlF4-. G-proteins were identified in the eluate by immunoblotting with specific antipeptide antisera. Using this protocol, the G-protein subunits alpha i, alpha i-3, and beta 36 were shown to be specifically associated with the AR4-2J cell SRIF receptor. Thus, we have developed a new, generally applicable, procedure for the efficient solubilization and affinity purification of a stable SRIF receptor-G-protein complex and have characterized the specific G-protein subunits associated with pancreatic SRIF receptors.
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PMID:Affinity purification of a somatostatin receptor-G-protein complex demonstrates specificity in receptor-G-protein coupling. 845 39

Modulation of the Ca- and voltage-dependent K channel--KCa--by receptors coupled to the G proteins G(i)/G(o) and Gs has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the KCa channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous alpha subunits of either G(i)- or G(o)-type G proteins also inhibited the KCa channel (IC50 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses KCa channel activity via a membrane-delimited pathway that involves the alpha subunits of PTX-sensitive G proteins G(i) and/or G(o). In outside-out patches, activation of Gs either by beta-agonists or with cholera toxin (CTX) increased KCa channel activity, consistent with a membrane-delimited stimulatory pathway linking the beta-adrenergic receptor to the KCa channel via Gs. In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of beta-agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous alpha i or alpha o. Taken together these data suggest that KCa channel activity is enhanced by activation of Gs and blocked by activated G(i) and/or G(o). Further, KCa channel stimulation by activated Gs may be "direct," while inhibition by G(i)/G alpha may involve deactivation of Gs. In inside-out patches KCa channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the KCa channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous alpha i or alpha o. These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G(i)/G(o)-induced deactivation of Gs.
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PMID:Characterization of the G protein coupling of SRIF and beta-adrenergic receptors to the maxi KCa channel in insulin-secreting cells. 860 61

The effect of somatostatin on Bombesin-induced contraction of isolated rabbit colonic smooth muscle cells was examined. Preincubation of muscle cells with somatostatin 10(-6) M inhibited bombesin-induced contraction. To characterize somatostatin receptors, muscle cells (10(5) cells/tube) were incubated at 24 degrees C with 125I-Tyr0-SS-28. Binding reached a plateau at 60 sec and was reversible by addition of excess synthetic SS-28. Scatchard analysis revealed high and low affinity bindings sites (Ka = 0.48 +/- 0.01 and 40 +/- 13 (nM +/- S.E.), 1830 +/- 433 and 65820 +/- 13183 receptors/cell +/- S.E.). Inhibition of 125I-Tyr0-SS-28 binding was possible with biologically active analogs of somatostatin, indicating the specificity of the receptors to somatostatin. Binding of 125I-Tyr0-SS-28 was inhibited by GTP gamma s, a nonhydrolysable analog of guanosine 5'-triphosphate, whereas adenosine 5'-triphosphate at a high concentration (100 microM) slightly inhibited the binding. Further, pretreatment of muscle cells with pertussis toxin at 37 degrees C abolished binding of 125I-Tyr0-SS-28, although pretreatment of cells with cholera toxin had no effect. Inasmuch as Gi protein is postulated as a signal protein, muscle cells were labeled with 3H-methionine, before stimulation with Bombesin (10(-6) M), in the presence and absence of somatostatin (10(-6) M). The cells were then lysed and Gi was precipitated by a Gi specific antibody. Gi synthesis was stimulated by bombesin at 60 sec and somatostatin inhibited it (6114 +/- 986 vs. 2998 +/- 841 cpm +/- S.E., P < .05). These data suggest that colonic smooth muscle cells contain specific receptor for somatostatin-28 and that somatostatin reverses bombesin-induced contraction regulated by Gi-type G protein.
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PMID:Somatostatin inhibits bombesin-stimulated Gi-protein via its own receptor in rabbit colonic smooth muscle cells. 863 41

1. The human recombinant somatostatin (SRIF) receptors, sst1 and sst2, have been stably expressed in mouse fibroblast (Ltk-) cells. Two stable clones, LSSR 1/20 and LSSR 11/13, expressing sst1 and sst2 receptors, respectively, have been used to characterize these receptor types using radioligand binding assays as well as measurements of changes in extracellular acidification rates using microphysiometry. 2. [125I]-[Tyr11]-SRIF bound to sst1 and sst2 receptors expressed in Ltk- cells with high affinity, Kd values being 1.52 nM, and 0.23 nM respectively. 3. In Ltk- cells expressing sst1 receptors, SRIF, SRIF-28, [D-Trp8]-SRIF and CGP 23996 all displaced [125I]-[Tyr11]-SRIF binding with high potency (IC50 values of 0.43 - 1.27 nM) whilst seglitide, BIM-23027, BIM-23056 and L-362855 were either weak inhibitors of binding or were ineffective. 4. In contrast MK-678 (seglitide) and BIM-23027 were the most potent inhibitors of [125I]-[Tyr11]-SRIF binding in Ltk- cells expressing sst2 receptors with IC50 values of 0.014 and 0.035 nM, respectively. 5. SRIF and a number of SRIF agonists, including seglitide and BIM-23027, caused concentration-dependent increases in extracellular acidification rates in Ltk- cells expressing sst2 receptors but not in Ltk- cells expressing sst1 receptors. The maximum increase in acidification rate produced by SRIF was 11.3 +/- 0.7% above baseline (0.1-0.28 pH unit min-1). The relative potencies of the SRIF agonists examined in causing increases in extracellular acidification rates in Ltk- cells expressing sst2 receptors correlated well with their relative potencies in inhibiting [125I]-[Tyr11] -SRIF binding (r = 0.94). 6. The increase in extracellular acidification produced by SRIF was markedly inhibited by pretreatment of cells with pertussis toxin (100 ng ml-1) indicating the involvement of pertussis toxin-sensitive G proteins. 7. SRIF (1 microM) had no effect on basal cyclic AMP levels in Ltk- cells expressing sst1 or sst2 receptors nor did it inhibit forskolin stimulated increases in cyclic AMP levels in either cell type. 8. The results from the present study describe the operational characteristics of human sst2 receptors expressed in Ltk- cells where receptor activation causes increases in extracellular acidification rates. This receptor is coupled to a pertussis toxin-sensitive G protein. In contrast, activation of sst1 receptors, at a similar transfection density, did not cause increases in extracellular acidification rates.
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PMID:Differences in the operational characteristics of the human recombinant somatostatin receptor types, sst1 and sst2, in mouse fibroblast (Ltk-) cells. 864 8


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