Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

An epitope-tagged form of an inwardly rectifying and G protein-coupled K+ channel (GIRK1-cp) was expressed at high levels in transfected mammalian cells. Immunoblot analysis of transfected human embryonic kidney cells (HEK293) and mouse insulinoma cells (beta TC3) revealed several GIRK1-cp polypeptides, including the major 59-kDa band, corresponding to the predicted mass of the GIRK1 polypeptide plus the epitope tag. Immunohistochemical staining using two anti-tag antibodies showed abundant immunoreactive material, which was predominantly concentrated in the perinuclear area in both transfected cell types. While functional GIRK1-cp message was present in poly(A)+ RNA prepared from HEK293 cells expressing GIRK1-cp protein, appropriate K+ currents could not be detected. In contrast, whole cell recordings made directly from transfected beta TC3 cells expressing GIRK1-cp revealed inwardly rectifying, pertussis toxin-sensitive currents activated by norepinephrine and galanin. Single channel recordings in excised patches of beta TC3 cells expressing GIRK1-cp showed rectifying K+ currents when activated by 50 microM guanosine 5'-O-(thiotriphosphate), with a slope conductance of 39.1 +/- 1.0 picosiemens. This is the first report of stable heterologous expression of a functional G protein-coupled K+ channel in mammalian cells. The activity of an epitope-tagged channel in insulinoma cells demonstrates the utility of this system for further biochemical and biophysical analyses of G protein-K+ channel interactions.
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PMID:Functional expression of an epitope-tagged G protein-coupled K+ channel (GIRK1). 754 Jan 74

Using a combination of polymerase chain reaction and genomic library screening we have cloned a human gene for a subtype of the somatostatin (SST) receptor (SSTR) termed human SSTR5 (hSSTR5), which is located on chromosome 16. The predicted amino acid sequence of hSSTR5 displays 75% sequence identity with a recently identified rat SSTR [Mol. Pharmacol. 42:939-946 (1992)], suggesting that it is the human homologue of this receptor. hSSTR5 consists of a 363-residue polypeptide exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. The receptor displays considerable sequence identity to hSSTR1 (42%), hSSTR2 (48%), hSSTR3 (47%), and hSSTR4 (46%). Membranes prepared from COS-7 cells transiently expressing the hSSTR5 gene bound 125I-Leu8,D-Trp22,Tyr25-SST-28 (125I-LTT-SST-28) with high affinity and in a saturable manner. SST-14, SST-28, and various synthetic SST peptide agonists produced dose-dependent inhibition of radioligand binding with the following rank order of potency: LTT-SST-28 > SST-28 > D-Trp8-SST-14 > SST-14 approximately RC-160 approximately BIM 23014 > MK-678 > SMS 201-995. hSSTR5 bound SST-28 with a 12.6-fold greater affinity (Ki = 0.19 nM), compared with SST-14 (Ki = 2.24 nM), indicating that the receptor is SST-28 selective. Addition of GTP, guanosine-5'-O-(3-thio)triphosphate, Na+ ions, or pertusis toxin greatly reduced 125I-LTT-SST-28 binding, thereby indicating that hSSTR5 is coupled to pertussis toxin-sensitive G proteins. Both SST-14 and SST-28 displayed dose-dependent inhibition of forskolin-stimulated cAMP accumulation, consistent with functional coupling of the receptor to adenylyl cyclase inhibition. Northern blot analysis of SSTR5 mRNA revealed a 2.4-kilobase transcript in normal rat pituitary and GH3 rat pituitary tumor cells and a 4.0-kilobase transcript in normal human pituitary. Reverse transcriptase polymerase chain reaction revealed expression of the hSSTR gene in fetal human pituitary and hypothalamus but not in human cerebral cortex. In situ hybridization of the rat pituitary showed that SSTR5 mRNA is selectively localized in the anterior lobe. SSTR5 mRNA was not expressed in four human pituitary tumors (somatotroph adenoma, prolactinoma, and chromophobe adenomas) or in a human insulinoma. Although hSSTR5 displays approximately 75% sequence identity with rat SSTR5, the two receptors display significantly different pharmacological profiles, especially with respect to their binding affinities for the SST analogue SMS 201-995.
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PMID:Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28. 790 5

Experiments have been carried out to examine the effects of GTP on the opening of K+ channels in insulin-secreting cells by diazoxide (0.2 mM) and cromakalim (0.5 mM). Using rat pancreatic beta-cells and RINm5F insulinoma cells, patch-clamp recordings of unitary ATP-sensitive potassium (K+ATP) channel currents were made in either the open cell or outside-out patch recording configurations. Adding diazoxide or cromakalim to either the inside or the outside face of the membrane was found regularly to cause the activation of K+ATP channels in the presence of 0.5 mM ATP. We now demonstrate that in the absence of ATP but in the presence of GTP (0.5-1 mM), both diazoxide and cromakalim activate channels. Effects are rapid in onset, sustained and readily reversible. Both the diazoxide- and cromakalim-induced activation of K+ATP channels were mediated by increases in channel open-state probability, and were not associated with any significant change in either channel amplitude or by an increase in the number of channels in the patch. The actions of both diazoxide and cromakalim were not affected by overnight pretreatment of cells with pertussis toxin, suggesting that PTX-sensitive GTP-binding proteins are not involved in mediating the actions of either compound. These data indicate that diazoxide and cromakalim open K+ATP channels in a manner not solely dependent upon intracellular ATP, but by mechanisms involving other cytosolic nucleotides, including GTP.
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PMID:Properties of diazoxide and cromakalim-induced activation of potassium channels in cultured rat and RINm5F insulin-secreting cells; effects of GTP. 844 11

To elucidate the regulatory pathway through which pancreastatin inhibits insulin secretion, RINm5F insulinoma cells were challenged with physiological and pharmacological probes known to stimulate insulin release through different mechanisms. Utilizing the electrophysiological technique of capacitance measurements as a correlate to exocytosis, pancreastatin was found to significantly diminish maximum capacitance changes evoked by glyceraldehyde, an effect which was attenuated in pertussis toxin-treated cells. In static incubations of this cell line, pancreastatin significantly inhibited insulin secretion stimulated by glyceraldehyde, carbachol and A23187, secretagogues known to directly elevate beta-cell cytosolic Ca2+. This peptide also inhibited insulin secretion stimulated by phorbol myristate acetate (PMA), but only at incubation times < or = 15 min. It was without effect on insulin secretion stimulated by mastoparan and longer incubations (30 min) with PMA, where the secretory mechanisms are not necessarily Ca(2+)-dependent. Additionally, pancreastatin had no effect on carbachol-generated inositol phosphate accumulation but inhibited simultaneously stimulated insulin secretion. All inhibitory effects of pancreastatin were pertussis toxin sensitive. These results suggest that pancreastatin inhibits insulin secretion in RINm5F cells through a G-protein regulated mechanism at a control point involved in the Ca(2+)-directed exocytotic machinery, a feature shared by other physiologic inhibitors of insulin secretion.
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PMID:Pancreastatin inhibits insulin secretion in RINm5F cells through obstruction of G-protein mediated, calcium-directed exocytosis. 868 70

Galanin is a ubiquitous neuropeptide that regulates a wide array of physiological processes via interaction with specific G protein-coupled receptors. A rat galanin receptor cDNA was cloned from the Rin14B insulinoma cell line. The isolated cDNA encodes a 346 amino acid G protein-coupled receptor that is 92% identical to the recently reported human GALR1 galanin receptor. [125I]Galanin binds with high affinity to two receptor states in COS1 cell membranes containing the rat GALR1 receptor, consistent with coupling of the receptor to a G protein in these membranes. N-terminal galanin fragments and the putative galanin receptor antagonists galantide, C7, M35 and M40 bind with high affinity to the rat GALR1 receptor. In contrast, C-terminal galanin fragments do not bind to this receptor. Galanin inhibits basal and forskolin-stimulated cAMP formation in CHO cells expressing the rat GALR1 receptor via a pertussis toxin-sensitive G protein. The GALR1 receptor is expressed in rat spinal cord, small intestine, Rin14B insulinoma cells and several brain regions, particularly ventral hippocampus, amygdala, supraoptic nucleus, hypothalamus, thalamus, lateral parabrachial nucleus and locus coeruleus. Cloning of the rat GALR1 galanin receptor cDNA will permit many new experimental strategies to be applied to studies of the structure and function of galanin receptors.
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PMID:Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells. 875 Aug 21

Somatostatin significantly suppressed cell growth of the mouse insulinoma-derived cell line MIN6. MIN6 cells exhibited high-affinity binding of somatostatin with 50% inhibitory concentration value of 0.9 nM. RNA blot analysis revealed that MIN6 cells expressed only SSTR3 among the five somatostatin receptors so far identified. Treatment of MIN6 cells with somatostatin significantly reduced the serum-induced c-fos expression levels. On the other hand, somatostatin (100 nM) treatment of MIN6 cells cultured in medium containing 10% serum transiently increased c-fos expression levels to 282 +/- 4.7% and then significantly decreased them to 27 +/- 7.6% of the levels before treatment. Mitogen-activated protein (MAP) kinase activity transiently increased to 656 +/- 91.2% and decreased thereafter to 39 +/- 13.3% of the activity before the addition of somatostatin (100 nM) into the medium. In addition, the stimulatory effect of somatostatin on c-fos expression and MAP kinase activity (early effect) was not altered by pertussis toxin (PTX), whereas the suppressive effect of somatostatin on c-fos expression and MAP kinase activity (late effect) was mitigated by PTX. These findings suggest that an inhibition of c-fos expression mediated by cross talk between PTX-sensitive G protein signaling and receptor tyrosine kinase signaling is one of the mechanisms by which somatostatin inhibits cell growth in MIN6 cells.
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PMID:Involvement of MAP kinase and c-fos signaling in the inhibition of cell growth by somatostatin. 917 74

It is well known that alpha 2-adrenergic agonism inhibits insulin secretion and stimulates glucagon secretion in both animal and human studies. Recently, alpha 2-adrenergic blockers (DG-5128, MK-912, and SL 84.0418) have been studied as antihyperglycemic agents in human subjects. To clarify the action mechanism(s) of these agents, we investigated the effects of alpha 2 agonists and antagonists (10(-10) to 10(-4) mol/L) and pretreatment by pertussis toxin (PTX) on glucose-stimulated insulin secretion using the hamster insulinoma cell line HIT-T15. The imidazoline-derivative alpha 2-adrenoceptor agonists clonidine and oxymetazoline at concentrations as low as 10(-8) mol/L significantly inhibited glucose-stimulated insulin secretion by 63% and 65%, respectively (P < .01 for both). These inhibitory effects were abolished by 20-hour preincubation of these cells with PTX 100 ng/mL. The imidazoline-derivative alpha 2-adrenoceptor antagonist DG-5128 at a concentration of 10(-4) mol/L doubled insulin secretion with or without pretreatment by PTX (P < .01 for both). Furthermore, both clonidine and oxymetazoline at a high concentration of 10(-4) mol/L stimulated insulin secretion with pretreatment of the cells by PTX (P < .05 for both). These results indicate that glucose-stimulated insulin secretion is inhibited by alpha 2-adrenoceptor agonists through PTX-sensitive G-protein in HIT-T15 cells. It is also suggested that imidazoline compounds at high concentrations directly stimulate insulin secretion.
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PMID:Effects of alpha 2-adrenergic agonism, imidazolines, and G-protein on insulin secretion in beta cells. 932 97

The lysosphingolipids sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) reportedly increase free cytosolic Ca2+ concentration ([Ca2+]i) in a variety of cell types, apparently by activating G protein-coupled plasma membrane receptors. We investigated whether and how sphingolipids modulate Ca2+ homeostasis in the insulinoma cell line RINm5F. The addition of SPPC and glucopsychosine (GPS) did not affect basal [Ca2+]i but inhibited the KCl (30 mM)-induced increase in [Ca2+]i in a pertussis toxin-insensitive and concentration-dependent manner (EC50 approximately 5 micro M). Similar inhibitory effects were observed with dihydro-SPPC and psychosine, whereas SPP and various N-acylated sphingolipids (at 10 micro M each) had little or no effect on the KCl-induced [Ca2+]i increase. Because in RINm5F cells the primary pathway for depolarization-induced [Ca2+]i increase are L-type Ca2+ channels, we studied whether sphingolipids reduce L-type Ca2+ current (ICa.L). When added to the bath, GPS and SPPC, but not SPP (10 micro M each), rapidly reduced maximal ICa.L by approximately 35%, similar to the alpha2-adrenoceptor agonist clonidine (30 micro M). However, when applied internally, GPS had no effect on ICa. L. When the electrode solution contained the stable GDP analog guanosine-5'-O-(2-thio)diphosphate (1 and 10 mM), the inhibitory effect of GPS was abolished. In conclusion, a novel cellular action of lysosphingolipids is observed in RINm5F cells (i.e., a guanine nucleotide-sensitive inhibition of L-type Ca2+ currents). The pharmacological profile of this inhibition is unique and unlike any known lysosphingolipid receptor-mediated action.
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PMID:Guanine nucleotide-sensitive inhibition of L-type Ca2+ current by lysosphingolipids in RINm5F insulinoma cells. 958 12

Neuropeptide Y (NPY) has been shown to inhibit insulin secretion from the islets of Langerhans. We show that insulin secretion in the insulinoma cell line RIN 5AH is inhibited by NPY. 125I-Peptide YY (PYY) saturation and competition-binding studies using NPY fragments and analogues on membranes prepared from this cell line show the presence of a single class of NPY receptor with a Y1 receptor subtype-like profile. Inhibition of insulin secretion in this cell line by NPY fragments and analogues also shows a Y1 receptor-like profile. Both receptor binding and inhibition of insulin secretion showed the same orders of potency with NPY > [Pro34]-NPY > NPY 3-36 >> NPY 13-36. The Y1 receptor antagonist, BIBP 3226, blocks NPY inhibition of insulin secretion from, and inhibits 125I-PYY binding to, RIN 5AH cells. Northern blot analysis using a Y1-receptor specific probe shows that NPY Y1 receptors are expressed by RIN 5AH cells. Y5 receptors are not expressed in this cell line. Neuropeptide Y inhibition of insulin secretion is blocked by incubation with pertussis toxin, implying that the effect is via a G-protein (Gi or Go) coupled receptor. Neuropeptide Y inhibits the activation of adenylyl cyclase by isoprenaline in RIN 5AH cell lysates, and the stimulation of cAMP by glucagon-like peptide-1 (7-36) amide (GLP-1). It also blocks insulin secretion stimulated by GLP-1, but not by dibutyryl cyclic AMP. Hence, we suggest that NPY inhibits insulin secretion from RIN 5AH cells via a Y1 receptor linked through Gi to the inhibition of adenylyl cyclase.
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PMID:Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells. 986 16

We compared internalization of three radioiodinated octreotide (OCT) somatostatin (SS) analogs-[125I-Tyr3]OCT, [DTPA degrees, 125I-Tyr3]OCT, and [DOTA degrees,125I-Tyr3]OCT-by somatostatin receptor (SSR)-positive mouse AtT20 pituitary tumor cells and human insulinoma cells. The three SS analogs were internalized in a specific, time-dependent manner. Internalization was significantly inhibited by pertussis toxin (100 microg/l) by 38%, 43%, and 31%, and by an inhibitor of receptor-mediated endocytosis (phenyl arsine oxide; 10 microM) by 98%, 94%, and 92%, respectively. Binding affinities of the three radioligands were comparable (0.2, 0.2, and 0.3 nM, respectively). However, [DOTA degrees,125I-Tyr3]OCT was internalized in a five-fold higher amount in comparison with the two other radioligands. A comparably high uptake of [DOTA degrees, 125I-Tyr3]OCT was found in SSR-positive organs (pituitary, pancreas, and adrenals) in vivo in rats (a ten-fold, five-fold, and eight-fold higher uptake 4 hr post injection, respectively, compared with the two other radioligands). This resulted in very high target-background ratios for [DOTA degrees,125I-Tyr3]OCT 4 hr post injection amounting to 274, 566, and 623 in the pituitary, adrenals, and pancreas, respectively. Both in vivo and in vitro there was a rapid dissociation of radioactivity from the SSR-positive cells. Main conclusions are that: 1) coupling of chelating groups like DTPA or DOTA to the SS analog [Tyr3]OCT does not prevent the internalization of OCT after binding to SSRs; 2) [DOTA degrees, 125I-Tyr3]OCT is internalized in a significantly higher amount by AtT20 and human insulinoma cells and in vivo in rats in SSR-positive organs, in comparison with [DTPA degrees,125I-Tyr3]OCT and [125I-Tyr3]OCT; and 3) the very high target-background ratios in vivo make radioiodinated [DOTA degrees,Tyr3]OCT a very suitable ligand for SSR-targeted radioguided surgery of SSR-positive human neuroendocrine tumors.
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PMID:Internalization of [DOTA degrees,125I-Tyr3]Octreotide by somatostatin receptor-positive cells in vitro and in vivo: implications for somatostatin receptor-targeted radio-guided surgery. 989 58


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