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D G Lambert

Publications and source records attributed to D G Lambert.

At least 109 records · Page 6Linked to original sources
PubMed1995

Characterisation of a new human neuroblastoma cell line, NAL-GT.

This paper describes the establishment of a new polyclonal human neuroblastoma cell line NAL-GT. Pharmacological characterisation of a cell line comprising > 70% neurones using radioligand binding, Ins(1,4,5)trisphosphate (Ins(1,4,5)P3) mass formation, intracellular Ca2+ ([Ca2+]i) determinations and cyclic adenosine monophosphate (cAMP) formation has been performed. Carbachol (1 mM) and noradrenaline (10 microM) increased Ins(1,4,5)P3 formation 2-3-fold basal. Noradrenaline (10 microM) and morphine (10 microM) reduced forskolin stimulated cAMP formation by 19.7 and 30.5%, respectively. Carbachol (1 mM) and K+ (50 mM) increased [Ca2+]i. These data indicate that polyclonal (heterogeneous) NAL-GT cells express muscarinic, alpha 1 and alpha 2 adrenoceptors, opioid receptors and voltage-sensitive Ca2+ channels and may prove useful in the study of the cellular basis of drug action.

Calcium↗
PubMed1995

Glutamate uptake is not a major target site for anaesthetic agents.

We have examined the effects of thiopentone, propofol and ketamine 3-300 mumol litre-1, 3.6%, 2.4 rat MAC of isoflurane, 3.0%, 2.4 rat MAC of halothane and morphine 0.1-10 mumol litre-1 on uptake of [3H]glutamate into rat cerebrocortical and cerebellar synaptosomes. Corticol and cerebellar synaptosomes took up [3H]glutamate in a time-, concentration-, Na(+)-dependent and L-transpyrrolidine-2,4-dicarboxylate inhibitory manner. The Km and Vmax values for uptake were 8.6 mumol litre-1 and 1.7 nmol/min/mg protein and 2.2 mumol litre-1 and 0.7 nmol/min/mg protein in cortical and cerebellar preparations, respectively. At clinically relevant concentrations none of the agents tested influenced the uptake process. Our data suggest that the uptake of glutamate is not a major target site for anaesthetic or analgesic agents.

Anesthetics, Dissociative↗
PubMed1995

Do nitrous oxide and halothane influence opioid receptor binding in SH-SY5Y human neuroblastoma cells?

The site of interaction of opioids and inhalation anaesthetic agents is unknown, but may be at the level of the opioid receptor. In this study we have used SH-SY5Y human neuroblastoma cells, which express both mu and delta receptors, to examine the effects of halothane on the receptor binding profiles of [3H]diprenorphine (DPN), an opioid receptor antagonist, and [3H] [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO), a mu receptor selective agonist. Binding of [3H]DPN and [3H]DAMGO was performed at 37 degrees C for 60 min in the presence of air, nitrous oxide (75%) or air containing halothane (0.5-5.0% v/v). Compared with air controls, neither 75% nitrous oxide nor 0.5, 1.0, 2.0 and 5.0% halothane influenced DPN binding variables. Binding of [3H]DAMGO was unaffected by 1.0% halothane, but 5.0% halothane reduced the affinity, with a modest increase in Kd (1.15 (0.16) to 1.7 (0.2) nmol litre-1) without effect on Bmax. Our data suggest that the site of opioid and volatile anaesthetic interaction is not at the opioid receptor.

Analgesics↗
PubMed1995

Activation of phospholipase C in SH-SY5Y neuroblastoma cells by potassium-induced calcium entry.

1. We used SH-SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved. 2. Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura-2. 3. Depolarization with K+ caused a time- and dose-dependent increase in [Ca2+]i (peak at 27 s, EC50 of 50.0 +/- 9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4 +/- 1.1 mM). 4. Both the K(+)-induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose-dependently by the L-type voltage-sensitive Ca2+ channel closer, (R+)-BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively. 5. These data show a close temporal and dose-response relationship between Ca2+ entry via L-type voltage-sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH-SY5Y cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Desensitization of the mu-opioid activation of phospholipase C in SH-SY5Y cells: the role of protein kinases C and A and Ca(2+)-activated K+ currents.

1. In SH-SY5Y cells, mu-opioids cause a rapidly desensitizing activation of phospholipase C (PLC), that appears secondary to Ca2+ influx via L-type voltage-sensitive Ca2+ channels (VSCCs). The aim of the present study was to characterize the mechanisms of desensitization of the mu-opioid-induced inositol (1,4,5) triphosphate (Ins(1,4,5)P3) response, by use of a stereospecific radioreceptor mass assay. 2. (R+)-Bay K 8644 (1 nM-10 microM) dose-dependently inhibited fentanyl-induced Ins(1,4,5)P3 formation, with an IC50 of 28.5 nM, confirming our earlier observations that mu-opioids open L-type VSCCs, thus allowing Ca2+ influx to activate PLC. 3. Ro 31-8220 (0.1 nM-10 microM), a protein kinase C inhibitor, dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 20.0 nM), whilst acute phorbol 12,13-dibutrate (1 microM) abolished the response. 4. H-89 (1 nM-10 microM), a protein kinase A inhibitor, also dose-dependently enhanced fentanyl-induced Ins(1,4,5)P3 formation (EC50 = 93 nM), whilst dibutryl cyclic AMP (0.5 mM) abolished the response. 5. Blockade of Ca(2+)-activated K+ currents with 4-aminopyridine (2 mM) or iberiotoxin (10 nM) had no effect on fentanyl-induced Ins(1,4,5)P3 formation but further increased the Ro 31-8220-enhanced response. 6. All three mechanisms had additive, or even supra-additive, effects, but only at later (120-300 s) time points. In addition, fentanyl-induced Ins(1,4,5)P3 formation, even if enhanced by H-89, Ro 31-8220 and/or 4-aminopyridine, was inhibited by nifedipine (1 nM-10 microM). 7. In conclusion, desensitization of the mu-opioid-induced activation of PLC is multifactorial, involving protein kinases C and A and Ca(2+)-activated K+ efflux, but the L-type VSCC is of critical importance and may be a possible common site of action.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Characterisation of mu-opioid receptors on SH-SY5Y cells using naloxonazine and beta-funaltrexamine.

The irreversible opioid receptor antagonists naloxonazine and beta-funaltrexamine have been used to determine whether multiple mu-opioid receptors exist on undifferentiated SH-SY5Y human neuroblastoma cells. Naloxonazine binds irreversibly to the mu 1-opioid receptor subtype and reversibly to the mu 2-opioid receptor subtype. On SH-SY5Y cells naloxonazine afforded a Ki of 3.4 +/- 0.7 nM, and was fully reversible, indicating the mu-opioid receptor population on SH-SY5Y cells was solely of the mu 2-opioid receptor subtype. The alkylating agent beta-funaltrexamine was maximally able to alkylate only 60% of the mu-opioid receptor sites on SH-SY5Y cells, labelled with [3H]diprenorphine or [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO). The reversible binding of naloxonazine and the insensitivity of a percentage of the mu-opioid receptor sites to alkylation by beta-funaltrexamine suggests that differences do exist in the mu 2-opioid receptor population on undifferentiated SH-SY5Y cells. This may indicate further heterogeneity or the inability of beta-funaltrexamine to alkylate all relevant nucleophilic groups in a single population of receptors.

Amino Acid Sequence↗
PubMed1994

Halothane and isoflurane enhance basal and carbachol-stimulated inositol(1,4,5)triphosphate formation in SH-SY5Y human neuroblastoma cells.

The cellular mechanisms underlying the clinical effects of volatile anaesthetics remain unknown, although the plasma membrane and its associated proteins are likely targets. One such protein is the enzyme phospholipase C (PLC), which catalyses the formation of the second messenger inositol(1,4,5)triphosphate [Ins(1,4,5)P3]. Using SH-SY5Y human neuroblastoma cells we have demonstrated that halothane (0.50, 0.75 and 1.00%) enhances basal Ins(1,4,5)P3 mass formation approximately 1.8-fold. Halothane also caused a dose-dependent enhancement of carbachol-stimulated biphasic Ins(1,4,5)P3 formation at both the peak (half-maximal stimulation, EC50 = 0.76%) and plateau (EC50 = 0.74%) phases. At 1%, halothane did not alter the affinity for carbachol at either the peak (IC50: air = 9.4 +/- 1.5, halothane = 12.7 +/- 1.0 microM) or plateau (EC50: air = 11.7 +/- 1.2, halothane = 11.6 +/- 1.0 microM) phase, but did increase the maximum Ins(1,4,5)P3 response at both phases (air vs halothane: peak, 79.9 +/- 0.5 vs 124.8 +/- 2.5; plateau, 33.2 +/- 0.5 vs 47.9 +/- 0.6 pmol/mg protein). Isoflurane (2%) also enhanced basal and carbachol-stimulated Ins(1,4,5)P3 formation 2-fold and 1.5-fold, respectively. In summary, clinically relevant doses of the volatile anaesthetics halothane and isoflurane enhance basal and carbachol-stimulated Ins(1,4,5)P3 formation. Thus, activation of PLC, and subsequent potential Ins(1,4,5)P3-mediated rises in intracellular calcium, could play a part in the cellular mechanisms of volatile agent-induced anaesthesia.

Carbachol↗
PubMed1994

mu-Opioid receptor stimulation of inositol (1,4,5)trisphosphate formation via a pertussis toxin-sensitive G protein.

The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both mu- and delta-opioid receptors), we demonstrated that fentanyl, a mu-preferring opioid, caused a dose-dependent (EC50 = 16 nM) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1-2 min. This response was of similar magnitude (25.4 +/- 0.8 pmol/mg of protein for 0.1 microM fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 mM carbachol (18.3 +/- 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole- (a delta antagonist), reversible. Further studies using [D-Ala2, MePhe4, Gly(ol)5]enkephalin and [D-Pen2,5]enkephalin confirmed that the response was specific for the mu receptor. Incubation with Ni2+ (2.5 mM) or in Ca(2+)-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 microM fentanyl, 26.9 +/- 1.5 pmol/mg of protein; pertussis-treated plus 0.1 microM fentanyl, 5.1 +/- 1.3 pmol/mg of protein). In summary, we have demonstrated a mu-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca(2+)-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca2+, could play a part in the cellular mechanisms of opioid action.

Calcium↗
PubMed1994

Studies on the interaction of steroidal neuromuscular blocking drugs with cardiac muscarinic receptors.

Interaction of the steroidal neuromuscular blocking drugs pancuronium, pipecuronium, rocuronium and vecuronium with cardiac muscarinic receptors in rat hearts was investigated in vitro by a tritiated N-methyl hyoscine binding assay. We showed an interaction with cardiac muscarinic receptors with a rank order of potency pancuronium > vecuronium > pipecuronium > rocuronium and demonstrated complex binding characteristics for pancuronium, vecuronium and rocuronium, with "Hill coefficient" of less than unity. We conclude that the haemodynamic differences seen during the use of these neuromuscular blocking drugs may be a result of their interactions with cardiac M2 muscarinic receptors.

Androstanols↗
PubMed1994

Fentanyl inhibits the release of [3H]noradrenaline from SH-SY5Y human neuroblastoma cells.

We have examined the effect of fentanyl on [3H]noradrenaline release in a human neuroblastoma cell preparation, SH-SY5Y. Fentanyl produced a significant, concentration-dependent inhibition of [3H]noradrenaline release with IC50 values of 5.5 x 10(-6) mol litre-1 and 15.5 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively. The small difference in IC50 between the two evoking stimuli may be explained by the weak binding affinity of fentanyl to muscarinic receptors (Ki = 570 nmol litre-1). The minimum concentrations at which a significant effect was observed were 0.3 x 0.10(-6) mol litre-1 and 10.0 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively; these values are considerably in excess of the serum concentration of fentanyl required to produce analgesia. Naloxone failed to antagonize the fentanyl inhibition and, furthermore, morphine and an enkephalin had no effect on evoked release, implying a non-opioid receptor mediated effect.

Atropine↗