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D G Lambert

Publications and source records attributed to D G Lambert.

At least 73 records · Page 4Linked to original sources
PubMed1998

Effects of i.v. anaesthetic agents and halothane on [3H]tetracaine binding to rat cerebrocortical membranes.

In an attempt to determine if there is any overlap in local and general anaesthetic binding sites, we have examined the effects of thiopental, phenobarbital, pentobarbital, propofol, ketamine (racemic and R(+)/S(-)), alfaxalone, etomidate and halothane on [3H]tetracaine binding to rat cerebrocortical membranes. Membranes were prepared in Tris HCI 50 mmol litre-1, pH 7.4, by homogenization and centrifugation. Binding assays were performed in 1-ml volumes of Tris HCI buffer at room temperature or 37 degrees C for halothane. Binding of [3H]tetracaine was displaced dose-dependently by unlabelled tetracaine with a mean pIC50 value of 6.91 (SEM 0.07) (123 nmol litre-1). With the exception of propofol (at high concentrations), all i.v. anaesthetic agents failed to displace the binding of [3H]tetracaine. In contrast, halothane produced a dose-dependent and statistically significant reduction in total [3H]tetracaine binding at clinically achievable concentrations (0.289, 0.885 and 1.484 mmol litre-1 equivalent to 1.0, 3.1 and 5.1 rat MAC) without markedly affecting the pIC50. Collectively these data may suggest some overlap in the binding sites for [3H]tetracaine and volatile but not i.v. general anaesthetic agents.

Anesthetics, Inhalation↗
PubMed1998

Inhibitory effects of candesartan on responses to angiotensin peptides in the hindquarters vascular bed of the cat.

The effects of the nonpeptide angiotensin II AT1 receptor antagonist candesartan on responses to angiotensin II were investigated in the hindquarters vascular bed of the cat. Under constant-flow conditions, injections of angiotensin II into the hindquarters perfusion circuit elicited dose-dependent increases in perfusion pressure. Candesartan in a dose of 3 micrograms/kg i.v. decreased vasoconstrictor responses to angiotensin II in a competitive manner. However, at doses of 10-1000 micrograms/kg i.v., candesartan shifted the dose-response curve to angiotensin II to the right in a nonparallel manner, suggesting a noncompetitive blockade. The inhibitory effects of candesartan on responses to angiotensin II were long in duration, and the AT1 receptor antagonist had little effect on baseline pressures. Candesartan was without effect on vasoconstrictor responses to norepinephrine, U46619, PGF2 alpha, and BAY K8644; on biphasic responses to endothelin-1; and on vasodilator responses to acetylcholine. Candesartan significantly attenuated hindquarters vasoconstrictor responses to angiotensin III and IV with a parallel shift at the 3 micrograms/kg iv dose and a nonparallel shift to the right at the high dose of the AT1 receptor antagonist. The results of the present study indicate that candesartan is a potent angiotensin AT1 receptor antagonist that can induce both competitive and noncompetitive blockade of responses to angiotensin II, III, and IV n the hindquarters vascular bed of the cat.

Angiotensin II↗
PubMed1997

Etomidate inhibits [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells.

We have examined the effects of the intravenous anaesthetic induction agent etomidate on K+ and carbachol evoked [3H]noradrenaline ([3H]NA) release and the associated increase in [Ca2+]i in SH-SY5Y human neuroblastoma cells in a attempt to study potential anaesthetic target site(s). Preincubation with etomidate produced a dose-dependent inhibition of both K+ and carbachol evoked [3H]NA release with estimated IC50 values of 88 and 69 microM, respectively. Only K+ stimulated increase in [Ca2+]i was inhibited by etomidate preincubation with an IC50 of 146 microM. Acute addition of etomidate after K+ challenge also inhibited the increase in [Ca2+]i with an IC50 of 99 microM. In addition etomidate displaced the binding of [3H]PN200-110 to L-type voltage sensitive Ca2+ channels with a Ki of 48 microM. As K+ but not carbachol evoked [3H]NA release is extracellular Ca2+ dependent and was inhibited by etomidate these data coupled with the PN200-110 displacement studies suggest that etomidate may interact with L-type voltage sensitive Ca2+ channels. The inhibition of carbachol evoked release without affecting the associated increase in [Ca2+]i suggests that etomidate may exert additional effects at either the muscarinic receptor or the secretory machinery in these cells.

Calcium↗
PubMed1997

Coupling of the cloned rat kappa-opioid receptor to adenylyl cyclase is dependent on receptor expression.

This study describes the coupling of the recombinant rat kappa-opioid receptor expressed in Chinese hamster ovary (CHO) cells to adenylyl cyclase and the effects of receptor density. The binding of [3H]diprenorphine ([3H]DPN) was dose dependent and saturable in membranes prepared from cells of early (p4-7) and late (p14-17) passage after transfection. As passage increased the receptor numbers (Bmax) declined from 231 +/- 24 (early) to 31 +/- 2 fmol/mg protein (late) but the equilibrium dissociation constant (Kd) did not change. Spiradoline dose dependently displaced [3H]DPN from membranes prepared from early and late cells revealing both high (Ki[H]) and low (Ki[L]) affinity binding sites. There were no significant differences in the proportion of these sites (approximately 50% Ki(L):50% Ki[H]), and whilst spiradoline was generally less potent in late cells the differences were small and failed to reach statistical significance. In contrast, spiradoline produced a dose dependent inhibition of forskolin stimulated cAMP formation in whole cells with pIC50 of 8.62 and 8.00 in early compared with late cells. In addition, the maximum inhibition was dramatically reduced from 47 to 22%. Etorphine, (+/-)bremazocine, ICI-204,448 and (+/-)trans-U-50488 methanesulfonate (1 microM), compounds with activity at kappa-receptors, produced a greater inhibition of cAMP formation in early (42.2, 45.8, 50.2 and 50.5%, respectively) than late (12.9, 11.8, 13.5 and 7.8%, respectively) cells, indicating that expression dependent inhibition of cAMP formation was not kappa-agonist specific. Collectively, these data suggest that in CHO cells, kappa-opioid receptor coupling to adenylyl cyclase is dependent on receptor expression levels.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1997

Comparison of responses to rat and human adrenomedullin in the hindlimb vascular bed of the cat.

Responses to rat (r) adrenomedullin (ADM) and human (h) ADM were compared in the hindlimb vascular bed of the cat under conditions of controlled blood flow. Intra-arterial injections of rADM and hADM in doses of 0.03-1 nmol caused dose-related decreases in hindlimb perfusion pressure. In terms of relative vasodilator activity, rADM was similar to hADM. The time course of the vasodilator response and the recovery half times (T1/2) for the vasodilator response to rADM and hADM were not significantly different. Decreases in hindlimb perfusion pressure in response to rADM and hADM 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 (CGRP) were significantly reduced. The T1/2 of the vasodilator response to rADM and hADM were significantly greater after administration of the cAMP-selective, type IV phosphodiesterase inhibitor, rolipram. These data demonstrate that decreases in hindlimb perfusion pressure in response to rADM and hADM are similar and that vasodilator responses to rADM are not dependent on the activation of CGRP receptors in the hindlimb vascular bed of the cat. These data further suggest that decreases in hindlimb perfusion pressure in response to rADM are mediated by smooth muscle increases in cAMP levels.

Adrenomedullin↗
PubMed1997

A comparative study of L-type voltage sensitive Ca2+ channels in rat brain regions and cultured neuronal cells.

Radioligand binding studies using the L-type voltage sensitive Ca2+ channel (VSCC) antagonist (+)-[3H]PN200-110 revealed the following rank order channel density in rat brain and cultured neuronal cell homogenates: striatum > or = cerebrocortex > > cerebellum = brainstem > SH-SY5Y cell line > NG108-15 cell line > 1321N1 cell line > PC12 cell line. There were no significant differences in the equilibrium dissociation constant, Kd for (+)-[3H]PN200-110 or pK50 for nifedipine. K+ depolarization in SH-SY5Y cells and NG108-15 cells evoked a biphasic and monophasic increase in [Ca2+]i. The L-type Ca2+ channel antagonist nifedipine (1 microM) produced a 66 and 87% inhibition of the K(+)-evoked rise in the peak and plateau phase [Ca2+]i in SH-SY5Y cells and abolished the monophasic response in NG108-15 cells. The L-channel activator S(-)Bay K 8644 (1 microM) enhanced the K(+)-evoked increase in [Ca2+]i in both cell lines. These data demonstrate a comparatively low density of L-VSCC in undifferentiated SH-SY5Y cells, NG108-15 cells, 1321N1 cells and PC12 cells that are functionally active in at least SH-SY5Y cells and NG108-15 cells.

Animals↗
PubMed1997

Adrenomedullin (16-31) has pressor activity in the rat but not the cat.

Responses to a newly synthesized human adrenomedullin (hADM) analog, hADM (16-31), were investigated in the rat and cat. Unlike the full-sequence peptide, which has potent hypotensive activity, hADM (16-31) had pressor activity in the rat but not in the cat. Injection of hADM (16-31) in doses of 10-300 nmol/kg i.v. induced dose-dependent increases in systemic arterial pressure in the rat, and the peptide was approximately 10-fold less potent than norepinephrine when doses are compared on a nanomole basis. In contrast, injection of hADM (16-31) in doses up to 1,000 nmol/kg i.v. had no significant effect on systemic arterial pressure in the cat. Increases in systemic arterial pressure in response to hADM (16-31) in the rat were significantly reduced after administration of phentolamine or reserpine. These data suggest that increases in systemic arterial pressure in response to hADM (16-31) are mediated by release of catecholamines and activation of alpha-adrenergic receptors in the rat. These data show that hADM (16-31) has significant pressor activity and that there are marked species differences in the response to hADM (16-31).

Adrenergic Uptake Inhibitors↗
PubMed1997

Tone-dependent vasodilator responses to proadrenomedullin NH2-terminal 20 peptide in the hindquarters vascular bed of the rat.

Responses to proadrenomedullin NH2-terminal 20 peptide (PAMP) were investigated in the systemic and hindquarters vascular bed of the rat. Intravenous injections of PAMP and adrenomedullin (ADM) produced dose-related decreases in systemic arterial and hindquarters perfusion pressure, which were not altered by alpha-receptor or adrenergic nerve terminal blocking agents. PAMP was 100-fold less potent than ADM, and hindquarters vasodilator responses to both peptides were similar in innervated and denervated preparations. When baseline tone was increased with phenylephrine and U46619 or decreased with sodium nitroprusside, vasodilator responses to PAMP and ADM were correlated with the basal level of tone, suggesting that responses to both peptides are dependent on the baseline level of vasoconstrictor tone in the hindquarters vascular bed of the rat.

Adrenomedullin↗
PubMed1997

The effects of recombinant rat mu-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase.

1. The rat mu-opioid receptor has recently been cloned yet its second messenger coupling remains unclear. The endogenous mu-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+]i and inhibits adenylyl cyclase (AC). We have examined the effects of mu-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+]i and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned mu-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+]i was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the Bmax varied between passages. Fentanyl (10 pM 1 microM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 +/- 0.1), and was best fit to a two site model, with pK1 values (% of sites) of 9.97 +/- 0.4 (27 +/- 4.8%) and 7.68 +/- 0.07 (73 +/- 4.8%). In the presence of GppNHp (100 microM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 +/- 0.1) with a pK1 value of 6.76 +/- 0.04. 4. Fentanyl (0.1 nM-1 microM) had no effect on basal, but dose-dependently inhibited forskolin (1 microM)-stimulated, cyclic AMP formation (pIC50 -7.42 +/- 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K1 = 1.7 nM). Morphine (1 microM) and [D-Ala2, MePhe4, gly(ol)5]-enkephalin (DAMGO, 1 microM) also inhibited forskolin (1 microM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 microM) did not. 5. Fentanyl (0.1 nM-10 microM) caused a naloxone (1 microM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 +/- 0.15 (n-5), PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2 (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 microM) and DAMGO (1 microM), but not DPDPE (1 microM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 microM) also caused an increase in [Ca2+]i (80 +/- 16.4 nM, n-6), reaching a maximum at 26.8 +/- 2.5 s. The increase in [Ca2+]i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 microM). Pre-incubation with naloxone (1 microM, 3 min) completely abolished fentanyl-induced increases in [Ca2+]i. 6. In conclusion, the cloned mu-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+]i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous mu-opioid receptor.

Adenylyl Cyclases↗
PubMed1997

Do local anaesthetics interact with dihydropyridine binding sites on neuronal L-type Ca2+ channels?

We have examined the interaction of procaine, prilocaine, lignocaine, bupivacaine, amylocaine and R(+) and S(-) ropivacaine with L-type voltage-sensitive Ca2+ channels in rat cerebrocortical membranes. Membranes were prepared in Tris HCl 50 mmol litre-1, pH 7.4, by homogenization and centrifugation. Binding assays were performed in 1-ml volumes of Tris HCl 50 mmol litre-1, pH 7.4, for 90 min at room temperature using approximately 200 micrograms of protein. Non-specific binding was defined in the presence of nifedipine 10(-5) mol litre-1, and bound and free radioactivity were separated by vacuum filtration. The effects of local anaesthetics were determined by displacement of [3H]PN200-110 (approximately 0.2 nmol litre-1), a radiolabelled 1,4- dihydropyridine (DHP) L-channel antagonist. The concentration of displacer producing 50% displacement was corrected for the competing mass of [3H]PN200-110 to yield the affinity constant, K50. All local anaesthetics displaced [3H]PN200-110 in a dose-dependent manner with a rank order potency of (K50, mmol litre-1) bupivacaine (0.48), amylocaine (0.74), lignocaine (1.09), prilocaine (2.06) and procaine (2.09). Ropivacaine enantiomers did not show stereo-selective displacement, with K50 values of 0.99 and 0.92 mmol litre-1 for R(+) and S(-) ropivacaine, respectively. There was a significant correlation between pK50 and p (octanol:buffer partition coefficient) (r2 = 0.872, P = 0.020), pK50 and p (local anaesthetic potency) (r2 = 0.816, P = 0.036), pK50 and p (relative conduction blocking potency) (r2 = 0.843, P = 0.028) and between pK50 and p (IC50 for inhibition of cardiac output) (r2 = 0.897, P = 0.015). These data suggest that DHP binding sites may be involved in both the mechanism of local anaesthesia and the cardiotoxicity of these agents.

Anesthetics, Local↗
PubMed1997

Effect of halothane on K+ and carbachol stimulated [3H]noradrenaline release and increased [Ca2+]i in SH-SY5Y human neuroblastoma cells.

We have examined the effects of the volatile general anaesthetic agent, halothane, on K+ and carbachol stimulated [3H]noradrenaline release and associated increases in intracellular Ca2+ in a cultured human neuroblastoma cell line, SH-SY5Y. K+ (but not carbachol) stimulated [3H]noradrenaline release, and the increase in intracellular Ca2+ concentration was entirely extracellular Ca2+ dependent. Halothane produced a dose-dependent reduction in K+ evoked release of [3H]noradrenaline with significant inhibition (17%) occurring from 1.26 atm%. Basal and carbachol evoked release were unaffected. Halothane also produced a dose-dependent reduction in K+ evoked increases (measured at the peak) in intracellular Ca2+ with significant inhibition (29%) occurring from 0.88 atm%. K+ plateau, basal and carbachol evoked increases in intracellular Ca2+ were unaffected. These data suggest that halothane reduced Ca2+ entry through voltage-sensitive Ca2+ channels and implicate this important class of ion channel in the mechanism of anaesthesia.

Anesthetics, Inhalation↗
PubMed1997

Studies on the binding of [3H]amethocaine to rat cerebrocortical membranes.

We have examined the binding of the local anaesthetic agent [3H]amethocaine to rat cerebrocortical membranes. All studies were performed in Tris buffer 50 mmol litre-1 at pH 7.4. Bound and free radioligand were separated by rapid vacuum filtration. [3H]Amethocaine binding at room temperature was dose-dependent and saturable, with mean Kd and Bmax values of 153 (SEM 18) nmol litre-1 and 9.4 (1.6) pmol/mg protein, respectively. [3H]Amethocaine binding was displaced in a dose-dependent manner (pIC50) by unlabelled amethocaine (6.89), procaine (5.20), lignocaine (3.46) and prilocaine (2.81). Ropivacaine and bupivacaine did not produce 50% displacement at the highest concentrations used (10(-4) and 10(-3) mol litre-1, respectively). We examined the nature of the binding site further with a range of ion channel antagonists (nifedipine, verapamil, diltiazem, omega-conotoxin, tetrodotoxin, tetraethylammonium and 4-aminopyridine) and ion channel coupled receptor ligands (L-glutamate, MK801, GABA, glycine and nicotine). With the exception of tetraethylammonium (pIC50 3.07) and 4-aminopyridine (pIC50 3.68), all non-anaesthetic agents failed to displace [3H]amethocaine. Collectively our data suggest that it is unlikely that there is a single target site for all local anaesthetic agents.

Anesthetics, Local↗
PubMed1997

Analysis of responses to human synthetic adrenomedullin and calcitonin gene-related peptides in the hindlimb vascular bed of the cat.

Vasodilator responses to human adrenomedullin (hADM), a newly discovered hypotensive peptide, human calcitonin gene-related peptide-alpha (hCGRP-alpha) and hCGRP-beta, which share structural homology with hADM, were compared in the hindlimb vascular bed of the cat under constant flow conditions. Injections of hADM (0.003-1 nmol), hCGRP-alpha, and hCGRP-beta (0.003-0.3 nmol) into the perfusion circuit caused dose-related decreases in hindlimb perfusion pressure. Vasodilator responses to hCGRP-alpha and hCGRP-beta were similar in potency and duration, and the doses of hCGRP-alpha and hCGRP-beta required to reduce hindlimb perfusion pressure 40 mm Hg (ED40 mm Hg) were significantly lower than the ED40 mm Hg for hADM. The duration of the hindlimb vasodilator responses to hCGRP-alpha and hCGRP-beta were significantly longer than the duration of the response to hADM. Amylin, a peptide that shares structural homology with ADM and with CGRP, had no significant effect on hindlimb perfusion pressure when injected in doses up to 1 nmol. Decreases in hindlimb perfusion pressure in response to hADM, hCGRP-alpha, and hCGRP-beta were not altered by L-N5-(1-iminoethyl)-ornithine (L-NIO) in a dose of the nitric oxide synthase inhibitor that decreased the vasodilator response to acetylcholine or by the cyclooxygenase inhibitor, meclofenamate, in a dose that decreased the vasodilator response to archidonic acid. The present data demonstrate that hADM, hCGRP-alpha, and hCGRP-beta have potent, but relatively short-lasting, vasodilator activity, and that vasodilator responses are not dependent on the release of nitric oxide or vasodilator prostaglandins in the hindlimb vascular bed of the cat.

Acetylcholine↗