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)

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5'-[gamma-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8-1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxinsensitive manner, but both the kinetics and the concentration-response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G1-dependent manner, and show that only a very small proportion of the cellular G1 population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.
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PMID:Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse delta opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation. 894 92

Neuroblastoma x glioma NG108-15 hybrid cells have been examined for the expression of opioid receptor-like receptor (ORL1). [3H]Nociceptin/orphanin FQ (OFQ) bound to the cell membrane specifically (Kd = 3.6 +/- 0.6 nM) and inhibited forskolin-stimulated cAMP accumulation (EC50 = 0.72 +/- 0.3 nM). The responsiveness of NG108-15 cells to nociceptin/OFQ was blocked by pertussis toxin but not by naltrindole. The inhibitory activity of nociceptin/OFQ was significantly reduced after a prechallenge with the same peptide but was not influenced by DPDPE pretreatment, indicating acute and homologous desensitization of ORL1 receptors. Naltrindole caused the overshoot of cAMP in DPDPE-pretreated cells but not in nociceptin/OFQ-pretreated cells. The results indicate that ORL1 is functionally expressed and does not cross-interact with specific ligands of the delta opioid receptor in NG108-15 cells.
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PMID:Functional expression, activation and desensitization of opioid receptor-like receptor ORL1 in neuroblastoma x glioma NG108-15 hybrid cells. 903 67

Morphine exerts direct effects on cultured cardiac myocytes from neonatal rats. These effects are mediated via the delta and the kappa opioid receptors, as mu opioid receptors are not present in neonatal cardiomyocyte cultures. Binding parameters to the delta and kappa opioid receptors were determined in membrane preparations from these cultures by heterologous competition to [3H]diprenorphine binding, with [D-Pen2, D-Pen5]-enkephalin (DPDPE) and trans-(dl)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate (U-50,488H) as specific displacers respectively. To define the components of morphine effects mediated via activation of either the delta or the kappa opioid receptor alone, cardiac myocytes were exposed to morphine in the presence of specific antagonists to the kappa or delta opioid receptor respectively. Activation of the kappa opioid receptors by morphine caused a transient increase in Ca2+ influx, leading to increase in amplitudes of [Ca2+]i transients and contraction, with no change in the intracellular pH. Activation of the delta opioid receptors alone by morphine caused a decrease in the amplitude of contraction. This decrease was mediated by a decrease in the intracellular pH leading to reduced responsiveness of the myofilaments to Ca2+. There was no change in Ca2+ influx and in the amplitude of [Ca2+]i transients. The effects mediated through the delta opioid but not through the kappa opioid receptors were pertussis toxin sensitive, indicating coupling of the delta opioid receptors to pertussis toxin sensitive GTP-binding proteins. The overall effects of morphine on the neonatal cardiac myocytes were the sum of the effects exerted by morphine when it activated each of the opioid receptors alone.
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PMID:Distinct components of morphine effects on cardiac myocytes are mediated by the kappa and delta opioid receptors. 914 Aug 28

G proteins couple delta opioid receptors to multiple cellular effector systems and are critical components of the delta opioid signal transduction cascade. To investigate the physical association of delta opioid receptors with G proteins, the cloned mouse delta opioid receptor was solubilized, and the G proteins associated with the receptor were identified through coimmunoprecipitation of the receptor/G protein complexes with antisera directed against different G(alpha) and G(beta) subunits. The delta receptor associates with G(i alpha1), G(i alpha3), G(o alpha), G(beta1) and G(beta2) subtypes. On agonist binding to the receptor, a greater proportion of the receptor is associated with G(i alpha) than with G(o alpha), G(i alpha1) dissociates from the receptor and G(i alpha2) associates with the receptor, whereas G(i alpha3) and the G(beta) subunits remain coupled to the delta receptor. These findings reveal dynamic changes in the G proteins associated with the receptor after agonist binding that may be linked to the activation of the delta receptor. In addition to pertussis toxin-sensitive G proteins, the delta receptor physically interacts with the pertussis toxin-insensitive G proteins G(q alpha) and G(z alpha). These interactions may be critical in linking delta receptors to phospholipase C. The diversity of G proteins associated with the delta opioid receptor may form the basis for the selective coupling of these receptors to multiple cellular effector systems.
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PMID:Changes in the association of G protein subunits with the cloned mouse delta opioid receptor on agonist stimulation. 919 Aug 86

The regulation of mitogenic signalling pathways by G-protein-coupled receptors has been studied in Rat-1 fibroblasts stably transfected with the murine delta opioid receptor. We showed recently that stimulation of this receptor led to the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase [Burt, Carr, Mullaney, Anderson and Milligan (1996) Biochem. J. 320, 227-235]. The present study has examined the role of the ribosomal S6 kinase p70(s6k) in mitogenic signalling by the delta opioid receptor. Treatment of Rat-1 fibroblasts expressing this receptor with the synthetic enkephalin [d-Ala,d-Leu]-enkephalin (DADLE) led to a dose-dependent increase in p70(s6k) enzyme activity. Activation of p70(s6k) was dependent on the level of delta opioid receptor expressed and was sustained above basal levels for several hours. Immunoblotting revealed that p70(s6k) was subject to increased phosphorylation, the extent of which coincided temporally with enzyme activation. Activation of p70(s6k) by DADLE, but not by platelet-derived growth factor, was blocked by pretreatment of cells with pertussis toxin. Activation of p70(s6k) was also partly blocked by wortmannin, indicating that phosphoinositide 3-OH kinase is required for full activation of p70(s6k) by opioid receptor agonists. Activation of the delta opioid receptor in transfected cells led to increased DNA synthesis. This increase was prevented by rapamycin, which also completely blocked activation of p70(s6k) by DADLE. In addition, prevention of the activation of p42 and p44 MAP kinases also blocked the induction of DNA synthesis by DADLE. These results suggest that the activation of both MAP kinases and p70(s6k) might be crucial to the induction of mitogenic responses by Gi-linked receptors such as the delta opioid receptor.
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PMID:Mitogenic signalling by delta opioid receptors expressed in rat-1 fibroblasts involves activation of the p70s6k/p85s6k S6 kinase. 922 49

A C6 glioma cell line stably transfected with the rat delta opioid receptor (C6delta) was used to characterize receptor binding and G protein activation by both peptide and nonpeptide delta opioid ligands. The ligand binding affinities for [3H]naltrindole and [3H]pCl-[D-Pen2,D-Pen5]enkephalin (DPDPE) were similar to those observed in monkey brain membranes. The nonpeptide agonists, BW373U86 and SNC80, as well as peptide agonist [D-Ser2, L-Leu5]enkephalyl-Thr maximally stimulated [35S]GTPgammaS binding by 640, 654 and 576%, respectively, over basal. The peptide agonists, DPDPE and deltorphin II, both stimulated [35S]GTPgammaS binding by 375%. Etorphine, diprenorphine, oxymorphindole and 7-spiroindanyloxymorphone were also partial agonists in this assay, although they were less efficacious than deltorphin II. Stimulation of [35S]GTPgammaS binding by agonists was blocked completely by pertussis toxin pretreatment. Both delta-1 and delta-2 selective antagonists 7-benzylidenenaltrexone and a benzofuran analog of naltrindole displayed high affinity for the cloned receptor (0.04 and 0.08 nM) and antagonized the stimulation of [35S]GTPgammaS binding by BW373U86 and DPDPE with similar potencies. Other evidence suggesting the lack of receptor subtypes includes the finding that stimulation of [35S]GTPgammaS binding by receptor subtype selective ligands DPDPE and deltorphin II was not additive. BW373U86, SNC80 and DPDPE maximally inhibited forskolin-stimulated adenylyl cyclase. These cells highly express a homogeneous population of delta opioid receptor that couple to inhibitory Go/Gi proteins. Ligand affinity for the delta opioid receptor correlates with ligand EC50 values for stimulation of [35S]GTPgammaS binding.
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PMID:Opioid efficacy in a C6 glioma cell line stably expressing the delta opioid receptor. 935 63

Maximally effective concentrations of the opioid agonist D-ala2-D-leu5-enkephalin resulted in some 2-3-fold enhancement of tyrosine phosphorylation of the p52 Shc adapter protein in a clone of Rat-1 fibroblasts transfected to express stably the murine delta opioid receptor. More limited modifications of the tyrosine phosphorylation status of the p46 and p66 forms of Shc were observed in parallel. Epidermal growth factor caused some 10-12-fold enhancement of tyrosine phosphorylation of p52 Shc and marked increases in the p46 and p66 forms. The effect of D-ala2-D-leu5-enkephalin was prevented by pretreatment of the cells with pertussis toxin and was not observed in non-transfected parental fibroblasts whereas the effect of epidermal growth factor was still manifest in both these situations. Half-maximal effects of D-ala2-D-leu5-enkephalin on p52 Shc tyrosine phosphorylation were produced with sub-nanomolar concentrations, in agreement with previous results on the tyrosine phosphorylation of p44MAPK (Burt et al., 1996). p52 Shc became tyrosine phosphorylated more rapidly than p44MAPK in response to D-ala2-D-leu5-enkephalin and its enhanced tyrosine phosphorylation was maintained for at least 10 min.
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PMID:Agonist-mediated tyrosine phosphorylation of isoforms of the shc adapter protein by the delta opioid receptor. 937 23

Gi/Go proteins are uncoupled from receptors by ADP-ribosylation with pertussis toxin (PTX). However, PTX treatment of delta opioid receptor-containing NG108-15 cells reduces, but does not eliminate, opioid inhibition of adenylyl cyclase. The present study explored potential mechanisms of this residual inhibition. Overnight treatment of NG108-15 cells with 100 ng/ml PTX eliminated both PTX-catalyzed [adenylyl-32P]NAD+-labeling of G proteins and agonist stimulation of low Km GTPase in membranes. Although PTX-treatment decreased the maximal opioid inhibition of adenylyl cyclase by 50-65%, the inhibition that remained was concentration-dependent and antagonist-reversible. This inhibition persisted in the absence of GTP (even though opioid inhibition of adenylyl cyclase in untreated membranes was GTP-dependent), but was eliminated by hydrolysis-resistant guanine nucleotide analogs, indicating that G-proteins were still involved in the coupling mechanism. However, assays of agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP indicated that PTX pretreatment eliminated stimulation of guanine nucleotide exchange by opioid agonists. These results suggest that in membranes from PTX-treated NG108-15 cells, a subpopulation of G proteins may transduce an inhibitory signal from agonist-bound opioid receptors without involvement of guanine nucleotide exchange.
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PMID:Opioid inhibition of adenylyl cyclase in membranes from pertussis toxin-treated NG108-15 cells. 949 66

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.
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PMID:Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors. 960 25

beta-Endorphin (beta-EP) and delta opioid receptor (DOR) agonists affect immune functions such as lymphocyte chemotaxis, proliferation, and cytokine production. Recent studies indicate that both neuronal DOR and novel G-protein-coupled receptors with high affinity for beta-EP and DOR agonists are expressed by mononuclear cells. In addition, proenkephalin A mRNA and enkephalin-related peptides are expressed by lymphocytes. These investigations were conducted to identify signal transduction pathways that mediate the effects of beta-EP and DOR agonists on T cells. Calcium mobilization was studied because it is central to T-cell activation initiated by antigen presentation to the T-cell receptor (TCR). Using the calcium-sensitive dye Fluo-3 and flow cytofluorometry to determine the concentration of free intracellular calcium, physiological concentrations of beta-EP were shown to enhance concanvalin. A (con A)-stimulated calcium mobilization by murine splenic T cells (p < 0.01). The DOR antagonist, naltrindole, inhibited this, whereas CTAP, a selective mu OR antagonist, was ineffective. In addition, N-Ac-beta-EP and the mu OR agonist DAMGO, failed to mimic the effects of beta-EP. Although it was less potent than beta-EP, DADLE, a DOR agonist, also enhanced Con-A-induced calcium mobilization (p < 0.01). A DOR-transfected human T-cell line (DOR-Jul.1) was developed to study signal transduction. Both DADLE and the selective DOR agonist, deltorphin, rapidly increased intracellular free calcium concentrations; ED50s were 10(-9) M. Pertussis toxin prevented the response, and EGTA significantly reduced it. In addition, DADLE inhibited forskolin-stimulated cAMP production (ED50: 10(-11) M). These findings with normal splenic T cells and DOR-transfected T-cell line indicate that beta-EP and DOR agonists affect calcium mobilization. This is likely to modulate downstream pathways that regulate T-cell activation and function.
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PMID:Signaling through delta opioid receptors on murine splenic T cells and stably transfected Jurkat cells. 962 68


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