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D G Lambert

Publications and source records attributed to D G Lambert.

At least 91 records · Page 5Linked to original sources
PubMed1996

Characterisation of the rat cerebella CB1 receptor using SR141716A, a central cannabinoid receptor antagonist.

We describe the use of SR141716A, a central cannabinoid antagonist, in radioligand binding and adenylyl cyclase (AC) inhibition studies in rat cerebella membranes. The binding of [3H]SR141716A was dose-dependent and saturable, with Kd and Bmax of 0.61 +/- 0.12 nM and 1752 +/- 294 fmol/mg protein, respectively. Kinetic analysis of [3H]SR141716A binding afforded a Kd of 0.72 nM. In addition [3H]SR141716A was displaced dose-dependently by unlabelled SR141716A yielding a pKi of 8.37 +/- 0.07. Cannabinoid receptor agonists displaced [3H]SR141716A in a dose-dependent manner, (pKi) nabilone (8.29 +/- 0.08), WIN 55,212-2 (7.75 +/- 0.15), delta 9-tetrahydrocannabinol (7.29 +/- 0.21), delta 8-tetrahydrocannabinol (6.53 +/- 0.09) and anandamide (5.92 +/- 0.04). The affinity of anandamide was increased (6.26 +/- 0.13) by co-incubation with a serine protease inhibitor. A range of 13 commonly used non-cannabinoid ligands included at 100 microM were unable to displace [3H]SR141716A. WIN 55,212-2 inhibited basal cAMP formation dose-dependently with a pIC50 of 7.61 +/- 0.12 (24.3 nM) in an SR141716A (1 microM) reversible manner.

Animals↗
PubMed1996

mu- and kappa-opioids inhibit K+ evoked glutamate release from rat cerebrocortical slices.

We have examined the effects of a range of opioid receptor subtype selective agonists on K+ evoked glutamate release from perfused rat cerebrocortical slices. Dual application (S1 and S2) of K+ (46 mM) evoked dual monophasic glutamate release profiles. When areas under the release curves were calculated an S2/S1 ratio for control slices of 1.07 +/- 0.08 (n = 75) was obtained, this was reduced by 80% with EGTA (0.1 mM) treatment confirming the presence of a Ca2+ regulated release process, Morphine produced a dose-dependent inhibition of the S2/S1 ratio. At 1 microM this amounted to 78 +/- 12% (mean +/- SEM; n = 6). (D-Ala2,MePhe4,gly(ol)5)enkephalin (DAMGO; 60 +/- 12%, n = 6 at 1 microM), and spiradoline (53 +/- 14% at 1 and 71 +/- 11% at 100 microM, both n = 6) also inhibited glutamate release in a cyprodime (10 microM) and norbinaltorphimine (10 microM) reversible manner. (D-Pen2.5) enkephalin (DPDPE; 1 microM) was ineffective. All agents tested did not affect basal glutamate release. Collectively these data implicate a role for mu and kappa opioids in the control of evoked glutamate release and their potential for neuroprotective therapy.

Analgesics↗
PubMed1996

Effects of propofol and thiopentone on potassium- and carbachol-evoked [3H]noradrenaline release and increased [Ca2+]i from SH-SY5Y human neuroblastoma cells.

We have examined the effects of two intravenous anaesthetic induction agents, propofol and thiopentone, on K+ and carbachol evoked [3H]noradrenaline release from a human neuroblastoma cell line, SH-SY5Y. In this model, we have previously demonstrated that K+ evoked [3H]noradrenaline release was dependent on Ca2+ entry and carbachol evoked release was extracellular Ca(2+)- independent. Propofol inhibited K+ (100 mM)-evoked (IC50 of 42 +/- 11 microM), but not carbachol (1 mM)-evoked, [3H]noradrenaline release. Thiopentone inhibited both K+- and carbachol-evoked release with IC50 values of 116 +/- 15 microM and 169 +/- 39 microM, respectively. These inhibitory effects were not due to changes in the release dynamics, as assessed using perfused cells. Furthermore, thiopentone inhibition of carbachol-evoked release was not due to muscarinic receptor antagonism. Both propofol and thiopentone caused noncompetitive inhibition of K+-stimulated Ca2+ influx, with IC50 values of 127 +/- 7 microM and 121 +/- 10 microM, respectively. These effects were not due to interaction with GABAA receptors, but suggest that both compounds block voltage-sensitive Ca2+ channels. Thiopentone, but not propofol, inhibited carbachol-stimulated increased intracellular Ca2+ concentrations in the presence and absence of extracellular Ca2+. However, thiopentone had no effect on carbachol-stimulated inositol (1,4,5)-triphosphate formation, suggesting that thiopentone may directly inhibit Ca2+ release from intracellular stores.

Calcium↗
PubMed1996

Tyr-D-Arg2-Phe-sarcosine4 activates phospholipase C-coupled mu2-opioid receptors in SH-SY5Y cells.

The dermorphin analogue Tyr-D-Arg2-Phe-sarcosine4 acts as a mu1-opioid receptor agonist, but as a mu2-opioid receptor antagonist, in vivo, yet the biochemical effects of Tyr-D-Arg2-Phe-sarcosine4 are unknown. Therefore, we characterized the effects of Tyr-D-Arg2-Phe-sarcosine4 on the mu-opioid receptor-mediated stimulation of inositol(1,4,5)trisphosphate, and inhibition of cAMP, in SH-SY5Y cells. We report here for the first time that Tyr-D-Arg2-Phe-sarcosine4 has no effect on basal cAMP or inositol(1,4,5)trisphosphate formation, but reversed the effects of fentanyl on these second messengers, consistent with Tyr-D-Arg2-Phe-sarcosine4 acting as a mu2-opioid receptor antagonist, and confirming that the mu-opioid receptors in SH-SY5Y cells are of the mu2 subtype.

Analgesics↗
PubMed1996

[MPR14]-rADM(14-50), a novel analog of adrenomedullin, possesses potent vasodilator activity in the hindlimb vascular bed of the cat.

Responses to [Mpr14]-ADM(14-50), a novel analog of adrenomedullin, were investigated in the hindlimb vascular bed of the cat under conditions of controlled blood flow. Intraarterial injections of [Mpr14]-rADM(14-50) in doses of 0.003-1 nmol caused dose-related decreases in hindlimb perfusion pressure. In terms of relative vasodilator activity, [Mpr14]-rADM(14-50) was more potent than human synthetic adrenomedullin (hADM) in doses of 0.003-0.1 nmol. The recovery half-times (T 1/2) for the vasodilator response to [Mpr14]-rADM(14-50) were significantly greater than the recovery half-times for hADM in all doses studied. Decreases in hindlimb perfusion pressure in response to [Mpr14]-rADM(14-50) were not altered by the calcitonin gene-related peptide receptor antagonist rCGRP(8-37) at the same time vasodilator responses to calcitonin gene-related peptide were significantly reduced. The present data demonstrate that [Mpr14]-(14-50) has potent and long-lasting vasodilator activity when compared to hADM, and that vasodilator responses to [Mpr14]-rADM(14-50) are not dependent on the activation of CGRP receptors in the hindlimb vascular bed of the cat.

Adrenomedullin↗
PubMed1996

The stimulatory effects of opioids and their possible role in the development of tolerance.

Opioids have stimulatory as well as the traditional inhibitory effects on neurotransmission, but the underlying mechanisms are poorly understood. Here, Darren Smart and David Lambert review the stimulatory effects of opioids on second messengers, including inositol (1,4,5)-trisphosphate (IP3), protein kinase C (PKC), Ca2+, and cAMP, and propose that these coordinated changes at the cellular level underlie the facilitatory effects of opioids on neurotransmission. The evidence for a possible role for these stimulatory effects, particularly the activation of PKC by opioids, in the development of tolerance is also discussed.

Calcium↗
PubMed1996

delta-Opioids stimulate inositol 1,4,5-trisphosphate formation, and so mobilize Ca2+ from intracellular stores, in undifferentiated NG108-15 cells.

delta-Opioids mobilize Ca2+ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [D-Pen 2,5] enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [D-Pen 2,5] enkephalin caused a dose-dependent (EC50= 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7+/-1.2 pmol/mg of protein with 1 microM, n=9) at 30 s and returned to basal levels (10.6+/-0.9 pmol/mg of protein, n=9) within 4-5 min. This response was fully naloxone (1 microM) reversible and pertussis toxin (100ng/ml for 24 h) sensitive. Preincubation with Ni2+ (2.5 mM) or nifedipine (1 microM) had no effect on the [D-Pen 2,5] enkephalin (1 microM)-induced inositol 1,4,5-triphosphate response, and K+ (80mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca2+ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 microM) enhanced, whereas acute expo sure to phorbol 12,13-dibutyrate (1 microM) abolished, the [D-Pen 2,5] enkephalin (0.1 microM)-induced inositol 1,4,5-triphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [D-Pen 2,5] Enkephalin also dose-dependently (EC50 =2.8 nM) increased the intracellular [Ca2+], which was maximal (24 nM increase with 1 microM, n=5) at 30 s. This close temporal and dose-response relationship strongly suggests that delta-opioid receptor-mediated increases in intracellular [Ca2+] results from inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular stores, in undifferentiated NG108-15 cells.

Analgesics↗
PubMed1996

Interaction of i.v. anaesthetic agents with 5-HT3 receptors.

Using N1E-115 neuroblastoma cells as an experimental model, we have examined if four commonly used i.v. anaesthetic induction agents interact with 5-HT3 receptors. Specifically, we tested the hypothesis that the antiemetic effects of propofol may result from 5-HT3 receptor antagonism. Binding of tropisetron (a 5-HT3 selective reference compound), etomidate, ketamine, thiopentone and propofol to 5-HT3 receptors was assessed by measuring the displacement of [3H]BRL 43694 from whole N1E-115 cells. The rank order potency (Ki) was tropisetron (1.7 (SEM 0.2) nmol litre-1) >> etomidate (83.(4) mumol litre-1) > or = ketamine (97 (4) mumol litre-1) > thiopentone (177 (9) mumol litre-1) > propofol (819 (171) mumol litre-1). With the exception of thiopentone these effects were outside the clinical range and suggest that anaesthetic agents are unlikely to interact directly with 5-HT3 receptors, and that other mechanism(s) must underlie the antiemetic effects of propofol.

Anesthetics, Intravenous↗
PubMed1996

Effects of morphine on human nasal cilia beat frequency in vitro.

Using human nasal cytological brushings, we have investigated the effects of morphine on ciliary function by measurement of cilia beat frequency in vitro, and we have also determined opioid receptor binding in these specimens. We obtained ciliated samples from seven volunteers, and measured cilia beat frequency using the transmitted light technique during exposure to morphine 10 mumol litre-1 for 4 h. Mean cilia beat frequency of the samples exposed to morphine was 11.1 (95% confidence interval 10.9-11.5) Hz and that of the controls 11.3 (11.1-11.7) Hz. There was no significant effect of morphine on human cilia beat frequency in vitro (MANOVA for repeated measures and nested, F = 0.61, P = 0.66). In a separate study, we obtained nasal brushings from 20 patients and measured the binding of the opioid antagonist tritiated diprenorphine ([3H]DPN). Mean disintegrations per minute (dpm) for total and non-specific binding were 9036 (8105-9967) dpm and 9130 (8054-10206) dpm, respectively. These values did not differ significantly (paired t test, t = 0.22, P = 0.83). We conclude that morphine had no effect on cilia beat frequency in vitro and we were unable to demonstrate any significant numbers of opioid receptors on nasal ciliated epithelium.

Adult↗
PubMed1996

Fentanyl increases intracellular Ca2+ concentrations in SH-SY5Y cells.

Classically, opioids inhibit Ca2+ influx, but recent reports suggest opioids may also stimulate Ca2+ entry. Therefore, we have measured the effect of opioids on intracellular Ca2+ ([Ca2+]i), fluorimetrically, in Fura-2-loaded SH-SY5Y cells. Fentanyl 0.3 mumol litre-1 caused a mean increase in [Ca2+]i of 18.8 (SEM 2.1) nmol litre-1 in some (30.3%) batches of SH-SY5Y cells. In responding cells, the fentanyl-induced increase in [Ca2+]i was dose-dependent, with an EC50 of 0.73 mumol litre-1. This response was naloxone-reversible, and the delta opioid agonist [D-Pen2,5]enkephalin had no effect on [Ca2+]i, suggesting the fentanyl-induced Ca2+ response was entirely mediated by the mu opioid receptor. Fentanyl 0.3 mumol litre-1 increased [Ca2+]i without preactivation of phospholipase C by another agonist, and this was markedly reduced by Ni2+ 2.5 mmol litre-1. These data suggest that mu opioids directly increase [Ca2+]i by stimulating Ca2+ influx in SH-SY5Y cells.

Analgesics, Opioid↗
PubMed1996

I.v. anaesthetic agents inhibit dihydropyridine binding to L-type voltage-sensitive Ca2+ channels in rat cerebrocortical membranes.

Previous studies have implicated the neuronal L-type voltage-sensitive Ca2+ channel (VSCC) as a target site for i.v. anaesthetic agents. It is unclear if these agents interact with the L-channel alpha-subunit 1,4-dihydropyridine (DHP) binding site. In this study, we have examined the interaction of thiopentone, pentobarbitone, ketamine, etomidate, propofol and alphaxalone, and the non-anaesthetic barbiturate, barbituric acid, with the DHP binding site on rat cerebrocortical membranes. Binding assays were performed in 1-ml volumes of Tris-HCl 50 mmol litre-1, pH 7.4, for 90 min at room temperature containing 200 micrograms of membrane protein with [3H]PN200-110 as a radiolabelled DHP. Non-specific binding was defined in the presence of nifedipine 10(-5) mol litre-1. The interaction of i.v. anaesthetics was determined by displacement of [3H]PN200-110 0.2 nmol litre-1. All i.v. anaesthetics showed some interaction with the DHP binding site. The concentrations of anaesthetic producing 25% inhibition of specific binding (corrected for the competing mass of [3H]PN200-110), K25 were (mumol litre-1): thiopentone 48 (SEM 2), pentobarbitone 95 (7), propofol 40 (2), etomidate 25 (2), alphaxalone 17 (3) and ketamine 198 (16). Barbituric acid was ineffective. With the exception of ketamine, there was a significant correlation between K25 and peak serum concentration during anaesthesia (P = 0.033) and serum concentrations on wakening (P = 0.018), suggesting that the L-channel DHP binding site may be a target for i.v. anaesthetic agents.

Anesthetics, Intravenous↗
PubMed1996

I.v. anaesthetic agents do not interact with the verapamil binding site on L-type voltage-sensitive Ca2+ channels.

In this study we have examined if the i.v. anaesthetic agents thiopentone, pentobarbitone, ketamine, etomidate, propofol and alphaxalone interact with the verapamil binding site on L-type voltage-sensitive Ca2+ channels in rat cerebrocortical membranes. Binding assays were performed in 1 ml volumes of Tris HCl 50 mmol litre-1, pH 7.4, for 90 min at 20 degrees C, with cerebrocortical membranes (200 micrograms of protein), the verapamil binding sites of which were radiolabelled with [3H]verapamil. Non-specific binding was defined in the presence of verapamil 10(-6) mol litre-1. The interaction of i.v. anaesthetics was determined by displacement of [3H]verapamil 0.2 nmol litre-1. The mean concentrations of anaesthetic producing 25% inhibition of specific binding (corrected for the competing mass of [3H]verapamil), K25, were (mmol litre-1): thiopentone 0.68 (SEM 0.14); pentobarbitone 1.22 (0.13); propofol 0.66 (0.10); etomidate 0.24 (0.03); alphaxalone 0.19 (0.02); and ketamine 0.75 (0.04). These concentrations exceeded those seen during anaesthesia and suggest that the neuronal verapamil binding site may not be an important target for i.v. anaesthetic agents.

Anesthetics, Intravenous↗
PubMed1996

Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices.

Nociceptin, an endogenous ligand for the orphan receptor ORL1, has recently been described. In this study we have shown that nociception inhibits 46 mM K(+)-stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68 +/- 14% and was naloxone (10 microM)-insensitive excluding an activation of mu, delta and kappa opioid receptors. These data demonstrate the functional coupling of ORL1 in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission.

Amino Acid Sequence↗
PubMed1995

Studies on the neurotoxicity of 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) in SH-SY5Y cells.

We have studied the hypothesis that 6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) is neurotoxic. Salsolinol induced a significant time and dose related inhibition of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazoyl blue (MTT) reduction, and increased lactate dehydrogenase release (LDH) release from human SH-SY5Y neuroblastoma cells, at concentrations within the range of 1-methyl-4-phenylpyridinium (MPP+) cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of antioxidants, monoamine oxidase inhibitors or imipramine. In confluent monolayers, salsolinol stimulated catecholamine uptake with EC50 values of 17 muM and 11 muM, for noradrenaline and dopamine, respectively. Conversely, at concentrations above 100 muM, salsolinol inhibited the uptake of noradrenaline and dopamine, with IC50 values of 411 muM and 379 muM, respectively. The inhibition of catecholamine uptake corresponded to the increase displacement of [3H]nisoxetine from the uptake 1 site by salsolinol, as the Ki (353 muM) for displacement was similar to the IC50 (411 and 379 muM) for uptake. Salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, or elevation of cAMP, cGMP, or inhibition of protein kinase C. Salsolinol also inhibited both carbachol (1 mM) and K+ (100 mM, Na+ adjusted) evoked released of noradrenaline from SH-SY5Y cells, with IC50 values of 500 muM and 120 muM, respectively. In conclusion, salsolinol appears to be cytotoxic to SH-SY5Y cells, via a mechanism that does not require uptake 1, bioactivation by monoamine oxidase, or membrane based free radical damage. The effects of salsolinol on catecholamine uptake, and the mechanism of toxicity require further investigation.

Adenylyl Cyclases↗
PubMed1995

Adenylyl cyclase in SH-SY5Y human neuroblastoma cells is regulated by intra- and extracellular calcium.

Adenylyl cyclase exists as a family of closely related subtypes which differ in their tissue distribution and regulatory properties. Submicromolar rises in [Ca2+]i produced via activation of phospholipase C (PLC) or Ca2+ channel opening, provide a mechanism by which Ca2+/calmodulin (CaM) or protein kinase C (PKC)-sensitive isoforms of adenylyl cyclase can be regulated. In this study we have examined, in detail, the muscarinic (M3) regulation of adenylyl cyclase in SH-SY5Y cells and report a role for both [Ca2+]e and [Ca2+]i. Carbachol (1 mM) and potassium (100 mM) caused a time (T1/2 = 3 and 4 min, respectively) and dose (EC50 = 6.95 microM and 34.7 mM respectively) related increase in cAMP formation. This amounted to an approximate two-fold increase over basal levels. Carbachol and potassium also caused a biphasic increase in [Ca2+]i with basal, peak and plateau values of 118.4 nM, 697.6 nM, 253.0 nM and 104.0 nM, 351.6 nM, 181.5 nM, respectively. Calcium channel blockade with nickel (2.5 mM) abolished potassium-stimulated cAMP formation and rises in [Ca2+]i. However, carbachol-stimulated cAMP formation was significantly decreased only at the later time points, where rises in [Ca2+]i were also essentially abolished. Further evidence for a role for [Ca2+]e and [Ca2+]i is provided by the stimulation of cAMP formation by carbachol in the absence of added Ca2+, followed by a further increase on its re-addition. Carbachol- and potassium-stimulated cAMP formation were inhibited by the CaM antagonist trifluoperazine (100 microM). The mu-opiate agonists, morphine and fentanyl also inhibited carbachol-stimulated cAMP formation. In addition, cAMP formation in SH-SY5Y cell membranes was significantly increased in the presence of Ca2+ (1.46 microM), CaM (200 nM) and forskolin (1 microM). PKC inhibition with Ro 31 8220 did not affect carbachol-stimulated cAMP formation. Taken collectively, these data suggest that SH-SY5Y cells express type 1, and possibly type 8 isoforms of adenylyl cyclase, which can be regulated by intra- and extracellular Ca2+.

Adenylyl Cyclases↗
PubMed1995

Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.

We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated.

Calcium↗