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)

In ND8-47 cells, a neuroblastoma x dorsal root ganglion hybrid cell line, activation of delta-opioid receptors induced an increase in the intracellular free calcium concentration ([Ca2+]i) through dihydropyridine-sensitive calcium channels. This effect was mediated by pertussis toxin-sensitive G proteins. The G protein alpha subunits alpha i2, alpha i3, alpha q, and alpha s were detected using Western blots, whereas alpha o and alpha i1 were not found in ND8-47 cell membranes. To identify the specific G protein alpha subunit(s) responsible for the increase in [Ca2+]i, we treated ND8-47 cells with antisense oligodeoxynucleotides (AS) complementary to the mRNA for each G protein alpha subunit (alpha i2, alpha i3, or alpha s), at a concentration of 10 microM, for up to 6 days and examined their effects on opioid-induced increases in [Ca2+]i and on the levels of G protein alpha subunits. [Ca2+]i was measured in adherent cells using the fluorescent dye fura-2. Treatment of cells with alpha i2-AS (10 microM, for 6 days) resulted in a 73% inhibition of the [D-Ser2,Leu5]-enkephalin-Thr-induced increase in [Ca2+]i. In contrast, pretreatment of cells with alpha i3-AS (10 microM, for 6 days) or alpha s-AS (10 microM, for 6 days) had no effect on the [D-Ser2,Leu5]-enkephalin-Thr-induced responses. Western blots indicated that the levels of alpha i2 were decreased when cells were exposed to alpha i2-AS (10 microM) for 6 days, whereas the levels of alpha i3, alpha s, and alpha q were not affected by this treatment. Treatment of the cells with alpha i3-AS or alpha s-AS for 6 days significantly reduced alpha i3 or alpha s levels, respectively. These results indicate that the opioid-induced increase in [Ca2+]i in ND8-47 cells is mediated by G alpha i2.
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PMID:Antisense oligodeoxynucleotide to the Gi2 protein alpha subunit sequence inhibits an opioid-induced increase in the intracellular free calcium concentration in ND8-47 neuroblastoma x dorsal root ganglion hybrid cells. 765 50

A desensitizing protocol to i.c.v. substance P (SP) (from 0.1-10 nmol x 2 at 25-min interval) diminished the supraspinal mu-mediated antinociceptive activity of morphine, D-Ala2-N-MePhe4-Gly-ol5-enkephalin (DAMGO), beta-endorphin-(1-31), D-Ala2-D-Leu5-enkephalin and of the alpha-2 agonist clonidine, whereas the activity of the highly selective delta ligands [D-Pen2,5]-enkephalin and [D-Ala2]-Deltorphin II remained unchanged. This effect was noncompetitive as the slopes for the antinociceptive dose-response curves diminished after SP pretreatment. The antagonism was evident within a few hours after SP and lasted longer than 15 days. The N-acetyl derivative of beta-endorphin-(1-31) (1 pmol) increased the antinociceptive response of DAMGO, D-Ala2-D-Leu5-enkephalin and clonidine, but not of morphine, in SP-pretreated mice. ED80 values of opioid agonists or naltrexone did not prevent SP from reducing the antinociceptive activity of opioids and clonidine. The effect of N-acetyl beta-endorphin-(1-31) was transitory and disappeared within 48 hr, after this period the long-lasting antagonism of SP was revealed. Clonidine (150 nmol) also enhanced opioid antinociception in SP-treated mice. This effect was reversed by the alpha-2 antagonist yohimbine (50 nmol) when given 10 min before clonidine. In mice undergoing treatment with pertussis toxin (0.5 micrograms i.c.v.), an agent that impairs the function of GTP-binding regulatory proteins (Gi/Go), the SP desensitizing protocol did not reduce further the antinociception of DAMGO or morphine. These results suggest a modulatory role for the SP system and the neuropeptide N-acetyl beta-endorphin-(1-31) upon mu and alpha-2 but not delta-mediated supraspinal antinociception in mice.
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PMID:N-acetyl beta-endorphin-(1-31) and substance P regulate the supraspinal antinociception mediated by mu opioid and alpha-2 adrenoceptors but not by delta opioid receptors in the mouse. 768 46

Neuroblastoma NS20Y cells possess a high density of stereoselective delta opioid receptors as determined by competition binding with 3H-diprenorphine and various opioid ligands. Scatchard analysis of [3H]diprenorphine saturation binding data revealed a Kd = 0.79 +/- 0.17 nM and Bmax = 370 +/- 50 fmol/mg protein. These opioid binding sites have highest affinity for delta opioid receptor selective agonists and lowest affinity for mu opioid receptor selective agonists. Agonist binding was sensitive to the presence of the monovalent cation, Na+. Activation of receptor with D-Ala2, D-Leu5-enkephalin (DADLE) resulted in dose-dependent inhibition of forskolin-stimulated intracellular [3H]cAMP accumulation, which was antagonized by (-)-naloxone but not (+)-naloxone. Relative potencies of various opioid agonists to inhibit intracellular cAMP production paralleled those observed in neuroblastoma x glioma NG108-15 hybrid cells. Pretreatment of NS20Y cells with pertussis toxin (PTX) eliminated opioid agonist inhibition of adenylyl cyclase activity. Chronic DADLE treatment resulted in desensitization and down-regulation of opioid receptor. An increase in intracellular [3H]cAMP level above the control was observed in the presence of naloxone after chronic DADLE treatment. Therefore, opioid binding sites in neuroblastoma NS20Y cells possess properties of the classical delta opioid receptor type. After neuroblastoma NS20Y was growth arrested by culturing the cells in serum-free medium for 72 hr, proliferation was reinitiated by addition of fetal calf serum (FCS), 0.01% to 12%, and was monitored by either [3H]thymidine incorporation or by dye viability assay. It was demonstrated that naloxone and naltriben but not Met5-enkephalin could attenuate FCS-induced proliferation in a dose-dependent manner. Naltriben was 54-fold more potent than naloxone to attenuate NS20Y proliferation. The maximal level of viable cells per well was reduced (35.2 +/- 1.9%) with no alteration in FCS concentration-dependent stimulation of growth. Similar inhibition by naloxone (37.3 +/- 2.7%) was observed with [3H]thymidine incorporation studies. This naloxone effect was serum concentration-dependent and could be blocked by culturing NS20Y cells in the presence of both naloxone and Met5-enkephalin. Although pretreatment of NS20Y cells with pertussis toxin could attenuate FCS-stimulated proliferation, naloxone effect on growth was not affected by pertussis toxin pretreatment. Furthermore, the naloxone effect was not NS20Y specific. A similar naloxone effect was observed with neuroblastoma N1E115, although not with neuroblastoma x glioma NG108-15, nor human neuroblastoma SHSY5Y, cell lines that have been reported to contain delta opioid receptors. Therefore, activation of delta opioid receptor could modulate FCS-induced growth in some but not all neuroblastoma cell lines.
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PMID:Properties of delta opioid receptor in neuroblastoma NS20Y: receptor activation and neuroblastoma proliferation. 781 47

The question of whether short- and long-term opioid agonist activities could be affected by receptor density is being addressed with Chinese hamster ovary (CHO) cell lines stably expressing delta-opioid receptors. CHO cells expressing different levels of delta-opioid binding sites were isolated and characterized. The opioid binding sites in these cell lines have stereoselective high affinity for 3H-diprenorphine (0.18 to 0.91 nM), with agonist binding sensitive to both Na+ and GTP gamma S, and are of the delta-2-opioid receptor subtype. This is conclude from observations that the delta-2-opioid receptor-selective agonist naltriben (NTB) has higher affinity than the delta-1-opioid receptor-selective agonist 7-benzylidenenaltrexone (BNTX). It also could be demonstrated that delta-opioid agonists inhibited forskolin-stimulated intracellular 3H-cAMP production and that agonist inhibition could be blocked by pretreating cells with pertussis toxin. Again, NTB is more potent than BNTX or naloxone in reversing DPDPE inhibition of intracellular 3H-cAMP production. When the ability of [D-Ala2,D-Leu5]enkephalin (DADLE) to regulate adenylyl cyclase activity was examined in three separate CHO cell lines: CHODORX1-15, CHODORX1-8 and CHODORX1-4, which express 1.42 +/- 0.08, 0.74 +/- 0.07 and 0.27 +/- 0.04 pmol/mg-protein of receptor, respectively, maximal inhibitory levels in clones X1-15 and X1-8 were similar, whereas maximal inhibitory level for clone X1-4 was 66% of the other two clones. However, when maximal inhibitory levels of other opioid receptor-selective agonists were examined, different levels of inhibition were observed among these three CHO clones.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Analysis of delta-opioid receptor activities stably expressed in CHO cell lines: function of receptor density? 799 85

To determine whether the previously demonstrated ability of delta-opioid receptors to interact simultaneously with multiple G-proteins was a function of high receptor levels, this interaction was investigated in Chinese hamster ovary cells stably expressing 10 different levels of cloned delta-opioid receptors, ranging from 18,000 to 1.6 x 10(6) receptors/cell. The opioid agonist D-Ala2,D-Leu5-enkephalin (DADLE) inhibited forskolin-stimulated adenylyl cyclase activity in all 10 clones with variable maximal inhibitory levels. Furthermore, opioid agonists altered incorporation of [alpha-32P]azidoanilido-GTP into at least four G-protein alpha-subunits in all 10 clones, three of which were determined to be Gi3 alpha, Gi2 alpha and Go2 alpha. This effect was concentration-dependent, naloxone-reversible, and delta-opioid agonist-specific and was blocked by pretreatment with pertussis toxin. Although DADLE induced an increase in the incorporation of [alpha-32P]azidoanilido-GTP into three of the four G alpha proteins that was independent of receptor density, the magnitude of this response was greater as receptor density increased. In addition, concentrations of DADLE required to promote 50% maximal labeling were similar for all four G alpha proteins within each clone and did not appear to be affected by receptor density. Therefore, the ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density and there is also no apparent correlation between the amount of G-protein activated and the maximal effect of an agonist.
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PMID:Ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density. 806 54

Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.
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PMID:Opioids mobilize calcium from inositol 1,4,5-trisphosphate-sensitive stores in NG108-15 cells. 815 47

In rat olfactory bulb, muscarinic and opioid receptor agonists stimulate basal adenylyl cyclase activity in a GTP-dependent and pertussis toxin-sensitive manner. However, in the present study, we show that in the same brain area activation of these receptors causes inhibition of adenylyl cyclase activity stimulated by Ca2+ and calmodulin (CaM) and by forskolin (FSK), two direct activators of the catalytic unit of the enzyme. The opioid and muscarinic inhibitions consist of a decrease of the maximal stimulation elicited by either CaM or FSK, without a change in the potency of these agents. [Leu5]-Enkephalin and selective delta- and mu-, but not kappa-, opioid receptor agonists inhibit the FSK stimulation of adenylyl cyclase activity with the same potencies displayed in stimulating basal enzyme activity. Similarly, the muscarinic inhibition of FSK-stimulated adenylyl cyclase activity shows agonist and antagonist sensitivities similar to those characterizing the muscarinic stimulation of basal enzyme activity. Fluoride stimulation of adenylyl cyclase is not affected by either carbachol or [Leu5]enkephalin. In vivo treatment of olfactory bulb with pertussis toxin prevents both opioid and muscarinic inhibition of Ca2+/CaM- and FSK-stimulated enzyme activities. These results indicate that in rat olfactory bulb delta- and mu-opioid receptors and muscarinic receptors, likely of the M4 subtype, can exert a dual effect on cyclic AMP formation by interacting with pertussis toxin-sensitive GTP-binding protein(s) and possibly by affecting different molecular forms of adenylyl cyclase.
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PMID:Activation of opioid and muscarinic receptors stimulates basal adenylyl cyclase but inhibits Ca2+/calmodulin- and forskolin-stimulated enzyme activities in rat olfactory bulb. 820 25

Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, gi and Go, in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2,D-Leu5]enkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the alpha subunit of Gs (Gs alpha) but had no effect on the alpha subunit of Gi/o (Gi/o alpha), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/o alpha selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with Gs as well as Gi. Cholera toxin treatment attenuated opioid-stimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to Gs and thereby mediate stimulation of adenylate cyclase. This Gs-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.
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PMID:Direct coupling of opioid receptors to both stimulatory and inhibitory guanine nucleotide-binding proteins in F-11 neuroblastoma-sensory neuron hybrid cells. 838 55

In neuroblastoma x glioma NG108-15 hybrid cells, opioid agonists inhibited both basal and prostaglandin E1-stimulated adenylate cyclase activities assayed in the presence of the phosphodiesterase (PDE) inhibitors isobutylmethylxanthine and ZK62711 (rolipram). However, when intracellular [3H]cAMP was measured in the absence of the PDE inhibitors the maximal inhibitory level was increased, using the opioid agonist D-Ala2,D-Leu5-enkephalin. This increase in opioid activity was due to agonist stimulation of cAMP degradation, because when the degradation rate of [3H] cAMP was measured in intact hybrid cells it was observed to increase from the control value of 0.495 +/- 0.003 min-1 to 0.760 +/- 0.003 min-1 in the presence of 1 microM D-Ala2,D-Leu5-enkephalin; this was reversed by naloxone. Dose-dependent studies with various opioid agonists, partial agonists, and antagonists revealed that there was a direct correlation between the abilities of these opioid ligands to inhibit adenylate cyclase activity and to stimulate PDE activity, with enkephalin and its analogs being the most potent agonists. Chronic agonist treatment also resulted in a reduction of the opioid agonist stimulation of cAMP degradation, with an apparent decrease in the PDE activity upon addition of naloxone after chronic treatment. However, treatment of the hybrid cells with pertussis toxin, which attenuated the agonist inhibition of adenylate cyclase activity, did not abolish this opioid response. When selective inhibitors for various types of PDE were used, the type I PDE inhibitor W-7 attenuated the opioid effect, whereas the type II PDE inhibitor trequinsin (HL725), the type III PDE inhibitor indolidan, and the type IV PDE inhibitor rolipram had no effect on opioid-stimulated cAMP degradation. The stimulation of type I PDE activity by delta-opioid receptors was independent of extracellular Ca2+ and was not observed with membrane preparations. Therefore, in NG108-15 cells delta-opioid receptors regulate intracellular cAMP levels by coupling to a pertussis toxin-insensitive guanine nucleotide-binding protein, resulting in an increase in intracellular Ca2+ and in Ca2+/calmodulin-dependent PDE activity.
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PMID:delta-Opioid receptor activates cAMP phosphodiesterase activities in neuroblastoma x glioma NG108-15 hybrid cells. 838 86

It is currently accepted that occupancy of opioid receptors by agonists, but not antagonists, promotes the association of the receptors to guanine nucleotide binding proteins (G-proteins) and stimulates a high affinity GTPase as part of the mechanism that links the receptor-ligand complex to adenylate cyclase inhibition. In this work we report that in rat brain membranes selective delta-opioid antagonists, the peptides N,N-Diallyl-Tyr-D-Leu-Gly-Tyr-Leu-OH (Diallyl-G) and N-N-Diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174,864), inhibit the low Km GTPase activity in a concentration dependent way. On the other hand the delta-opioid agonists D-Ala2-D-Leu5-enkephalin (DADLE) and D-Ser2-Leu5-Thr6-enkephalin stimulate dose-dependently the low Km GTPase activity in rat brain membranes. This stimulation was blocked in the presence of Diallyl-G, and reciprocally the inhibition induced by Diallyl-G was reversed by DADLE. The inhibitory effect of Diallyl-G as well as the stimulation induced by DADLE were abolished when membranes were exposed to low concentrations of N-ethylmaleimide or by ADP ribosylation with pertussis toxin which interferes with the ability of the receptor to couple to G-proteins. These observations indicate that the inhibitory effect of Diallyl-G on GTPase requires a functional G-protein and suggest that certain delta-opioid antagonists exhibit negative intrinsic activity and may have the ability to inhibit the receptor-mediated activation of G-proteins.
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PMID:Effect of delta-opioid antagonists on the functional coupling between opioid receptors and G-proteins in rat brain membranes. 839 41


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