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D G Lambert

Publications and source records attributed to D G Lambert.

At least 19 recordsLinked to original sources
PubMed2005

Effects of pre-eclampsia on maternal plasma, cerebrospinal fluid, and umbilical cord urotensin II concentrations: a pilot study.

BACKGROUND: Urotensin II (UII) is the most potent endogenous vasoconstrictor identified to date. Pre-eclampsia is associated with arteriolar vasospasm but the precise underlying mechanism is uncertain and we hypothesized that UII concentrations might also be elevated. In this study we measured UII concentrations in maternal plasma and cerebrospinal fluid (CSF), and umbilical vein plasma from pre-eclamptic (PET) and normotensive patients undergoing elective Caesarean section under spinal or combined spinal-epidural anaesthesia. METHODS: With LREC approval and informed consent we recruited two groups of 10 patients; control [mean (range) age, 29 (22-43) yr; BMI, 25 (20-32); gestation, 273 (267-281) days; mean arterial pressure (MAP) on day of delivery, 81 (75-96) mm Hg] and PET [age, 34 (22-40) yr; BMI, 25 (21-46); gestation, 253 (203-289) days; MAP on day of delivery, 106 (88-128) mm Hg]. Maternal blood and CSF samples and umbilical vein blood samples were taken. UII was extracted and concentrations measured using a radioimmunoassay. RESULTS: Two plasma and two CSF samples in the control and two CSF samples in the PET group were below the assay detection limits. There were no differences in maternal plasma or CSF or umbilical vein UII concentrations between the groups. However, there was a small ( approximately 40%) but significant increase in cord UII concentrations when compared with paired plasma in the PET group. There was a weak but significant negative correlation (r=-0.4, P=0.049) between cord UII concentrations and gestation in the PET group. In addition, we observed a significant positive correlation between plasma and CSF (r(2)=+0.57, P=0.0009, n=16), plasma and cord (r(2)=+0.43, P=0.0031, n=18) and CSF and cord (r(2)=+0.32, P=0.022, n=16) UII concentrations for the whole data set. CONCLUSIONS: Collectively the data indicate that UII concentrations do not increase in PET compared with controls but, in PET patients, cord UII concentrations are elevated relative to paired plasma samples. Elevated umbilical vein UII concentrations may simply indicate reduced placental viability and possibly UII metabolism as a result of reduced blood flow or possibly that the placenta is producing UII.

Adult↗
PubMed2005

Characterization and comparison of recombinant human and rat TRPV1 receptors: effects of exo- and endocannabinoids.

BACKGROUND: TRPV1 is a ligand-gated ion channel whose activation by capsaicin increases intracellular Ca(2+) ([Ca(2+)](i)). TRPV1 and cannabinoid CB(1) receptor activation are capable of eliciting analgesia. In this study, using recombinant human (h) and rat (r) TRPV1 receptors expressed in HEK293 cells, we have performed a comparison of both TRPV1 species at 22 and 37 degrees C and compared endo- and exocannabinoid activity at both receptors. METHODS: [Ca(2+)](i) was measured in Fura-2-loaded HEK293(hTRPV1) and HEK293(rTRPV1) cells. To assess native CB(1) receptor activity, [(35)S]GTPgammaS binding to membranes prepared from rat cerebellum was measured. RESULTS: Both capsaicin (pEC(50) rat approximately 6.9 and pEC(50) human approximately 6.8 at 37 degrees C) and anandamide (pEC(50) rat approximately 5.3 and pEC(50) human approximately 5.8 at 37 degrees C) produced a concentration-dependent increase in [Ca(2+)](i) in rat and human systems and at 22 and 37 degrees C. In HEK293(rTRPV1) cells, anandamide appeared to be a partial agonist. Capsazepine demonstrated competitive antagonism at both human and rat TRPV1 receptors and at both temperatures studied. Capsazepine effects were not temperature dependent: pK(B) at rTRPV1 was 5.98 at 22 degrees C and 6.02 at 37 degrees C, and pK(B) at hTRPV1 was 6.76 at 22 degrees C and 6.75 at 37 degrees C. However, there was a consistent 6-fold increase in capsazepine potency for hTRPV1 relative to rTRPV1. The exocannabinoid Delta(9)-tetrahydrocannabinol failed to increase [Ca(2+)](i), although its solvent ethanol was an effective TRPV1 activator. In the [(35)S]GTPgammaS binding assay using rat cerebellar membranes, anandamide (pEC(50) approximately 5.8) and Delta(9)-tetrahydrocannabinol (pEC(50) approximately 7.1), but not capsaicin, stimulated binding. Delta(9)-tetrahydrocannabinol was a partial agonist. pEC(50) values for anandamide at rTRPV1 and rCB(1) were similar. CONCLUSIONS: There were small differences in the pharmacology of rat and human TRPV1 receptors. Whilst capsaicin activated TRPV1 and Delta(9)-tetrahydrocannabinol activated CB(1), anandamide is an endogenous agonist for both receptor systems.

Animals↗
PubMed2003

Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys10,14]N/OFQ(1-14)NH2 and c[Nphe1,Cys10,14]N/OFQ(1-14)NH2.

In this study we describe the activity of two cyclic nociceptin/orphanin FQ (N/OFQ) peptides; c[Cys(10,14)]N/OFQ(1-14)NH(2) (c[Cys(10,14)]) and its [Nphe(1)] derivative c[Nphe(1),Cys(10,14)]N/OFQ(1-14)NH(2) (c[Nphe(1),Cys(10,14)]) in native rat and mouse and recombinant human N/OFQ receptors (NOP). Cyclisation may protect the peptide from metabolic degradation. In competition binding studies of rat, mouse and human NOP the following rank order pK(i) was obtained: N/OFQ(1-13)NH(2)(reference agonist)>N/OFQ=c[Cys(10,14)]>>c[Nphe(1)Cys(10,14)]. In GTPgamma(35)S studies of Chinese hamster ovary cells expressing human NOP (CHO(hNOP)) c[Cys(10,14)] (pEC(50) 8.29) and N/OFQ(1-13)NH(2) (pEC(50) 8.57) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. Following 30 min pre-incubation c[Nphe(1)Cys(10,14)] competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pA(2) and slope factor of 6.92 and 1.01 respectively. In cAMP assays c[Cys(10,14)] (pEC(50) 9.29, E(max) 102% inhibition of the forskolin stimulated response), N/OFQ(1-13)NH(2) (pEC(50) 10.16, E(max) 103% inhibition) and c[Nphe(1)Cys(10,14)] (~80% inhibition at 10 microM) displayed agonist activity. In the mouse vas deferens c[Cys(10,14)] (pEC(50) 6.82, E(max) 89% inhibition of electrically evoked contractions) and N/OFQ(1-13)NH(2) (pEC(50) 7.47, E(max) 93% inhibition) were full agonists whilst c[Nphe(1)Cys(10,14)] alone was inactive. c[Nphe(1)Cys(10,14)] (10 microM) competitively antagonised the effects of N/OFQ(1-13)NH(2) with a pK(B) of 5.66. In a crude attempt to assess metabolic stability, c[Cys(10,14)] was incubated with rat brain membranes and then the supernatant assayed for remaining peptide. Following 60 min incubation 64% of the 1 nM added peptide was metabolised (compared with 54% for N/OFQ-NH(2)). In summary, we report that c[Cys(10,14)] is a full agonist with a small reduction in potency but no improvement in stability whilst c[Nphe(1)Cys(10,14)] displays tissue (antagonist in the vas deferens) and assay (antagonist in the GTPgamma(35)S assay and agonist in cAMP assay) dependent activity.

Animals↗
PubMed2003

Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone-inducible mammalian expression system.

(1) Partial agonism is primarily dependent upon receptor density and coupling efficiency. As these parameters are tissue/model dependent, intrinsic activity in different tissues can vary. We have utilised the ecdysone-inducible expression system containing the human nociceptin/orphanin FQ (N/OFQ) peptide receptor (hNOP) expressed in Chinese hamster ovary cells (CHOINDhNOP) to examine the activity of a range of partial agonists in receptor binding, GTPgamma35S binding and inhibition of adenylyl cyclase studies. (2) Incubation of CHOINDhNOP cells with ponasterone A (PON) induced hNOP expression ([leucyl-3H]N/OFQ binding) of 24, 68, 191 and 1101 fmol mg-1 protein at 1, 2, 5 and 10 microm PON, respectively. At 191 fmol mg-1, protein hNOP pharmacology was identical to that reported for other traditional expression systems. (3) pEC50 values for GTPgamma35S binding ranged from 7.23 to 7.72 (2-10 microm PON) for the partial agonist [Phe1psi(CH2-NH)Gly2]N/OFQ(1-13)-NH2 ([F/G]N/OFQ(1-13)-NH2) and 8.12-8.60 (1-10 microm PON) for N/OFQ(1-13)-NH2 and Emax values (stimulation factor relative to basal) ranged from 1.51 to 3.21 (2-10 microm PON) for [F/G]N/OFQ(1-13)-NH2 and 1.28-6.95 (1-10 microm) for N/OFQ(1-13)-NH2. Intrinsic activity of [F/G]N/OFQ(1-13)-NH2 relative to N/OFQ(1-13)-NH2 was 0.3-0.5. [F/G]N/OFQ(1-13)-NH2 did not stimulate GTPgamma35S binding at 1 microm PON, but competitively antagonised the effects of N/OFQ(1-13)-NH2 with a pKB=7.62. (4) pEC50 values for cAMP inhibition ranged from 8.26 to 8.32 (2-10 microm PON) for [F/G]N/OFQ(1-13)-NH2 and 9.42-10.35 for N/OFQ(1-13)-NH2 and Emax values (% inhibition) ranged from 19.6 to 83.2 for [F/G]N/OFQ(1-13)-NH2 and 40.9-86.0 for N/OFQ(1-13)-NH2. The intrinsic activity of [F/G]N/OFQ(1-13)-NH2 relative to N/OFQ(1-13)-NH2 was 0.48-0.97. (5) In the same cellular environment with receptor density as the only variable, we show that the profile of [F/G]N/OFQ(1-13)-NH2 can be manipulated to encompass full and partial agonism along with antagonism.

Animals↗
PubMed2003

Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the nociceptin receptor (NOP). In an attempt to identify high potency NOP agonists for use in the brain we have compared the activity of a novel N/OFQ analogue [Phe(1)Psi(CH(2)-O)Gly(2)]N/OFQ(1-13)NH(2) ([F/G-O]) with the existing [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]). Both peptides are modified between the first two N-terminal amino acids and are further compared with the agonist template N/OFQ(1-13)NH(2) in [(3)H]N/OFQ binding, GTPgamma[(35)S] binding and cAMP inhibition studies using Chinese hamster ovary cells expressing the recombinant human NOP. All peptides displaced [(3)H]N/OFQ, stimulated GTPgamma[(35)S] binding and inhibited cAMP formation. In [(3)H]N/OFQ binding and GTPgamma[(35)S] binding the rank order affinity and potency was N/OFQ(1-13)NH(2)>[F/G-O]>[F/G]. In GTPgamma[(35)S] binding [F/G] was a clear partial agonist with intrinsic activity (E(max) stimulation factor, mean+/-SEM, n=4) of 7.75+/-1.02 compared with N/OFQ(1-13)NH(2) of 11.13+/-1.76. The efficacy of [F/G-O] (10.17+/-1.88) approached that of the full agonist N/OFQ(1-13)NH(2). Downstream, at the level of cAMP formation, all peptides were full agonists with the following rank order potency: N/OFQ(1-13)NH(2)>[F/G-O]=[F/G]. The enhanced potency and intrinsic activity of the novel [F/G-O] modification makes this an interesting peptide for further in vivo analysis.

Animals↗
PubMed2003

UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPgamma(35)S binding studies.

Studies of the pharmacology of nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) have been hampered by the lack of a range of high potency antagonists. In this study we have examined the effects of a novel N/OFQ analogue [Nphe(1),Arg(14),Lys(15)]N/OFQ NH(2) hereafter referred to as UFP-101. [(3)H]N/OFQ competition binding and GTPgamma(35)S binding assays were performed using CHO cells expressing the human NOP receptor (CHO(hNOP)). UFP-101 (pK(i) of 10.14+/-0.09) and a range of NOP selective agonists displaced [(3)H]N/OFQ binding with the following rank order of affinity: [Arg(14),Lys(15)]N/OFQ>[( pF)Phe(4)]N/OFQ(1-13)NH(2)>N/OFQ(1-13)NH(2)>UFP-101>N/OFQ>Ro64-6198>[Nphe(1)]N/OFQ(1-13)NH(2). N/OFQ, N/OFQ(1-13)NH(2), [( pF)Phe(4)]N/OFQ(1-13)NH(2), [Arg(14),Lys(15)]N/OFQ and Ro64-6198 also produced a concentration dependent (pEC(50) values of 8.75+/-0.11, 9.28+/-0.15, 9.69+/-0.04, 9.12+/-0.11 and 8.09+/-0.07 respectively) and saturable stimulation of GTPgamma(35)S binding and all were full agonists. UFP-101 did not stimulate GTPgamma(35)S binding per se, but produced a concentration dependent and parallel rightward shift in the concentration response curves to all agonists. UFP-101 yielded pA(2) values in the range 8.4-9.0. For comparison a pA(2) for [Nphe(1)]N/OFQ(1-13)NH(2) (the template for UFP-101) against N/OFQ of 7.33+/-0.08 was obtained. Slope factors for the Schild regression lines were approximately 1 indicating competitivity. When UFP-101 is compared with its template molecule [Nphe(1)]N/OFQ(1-13)NH(2), Arg(14),Lys(15) substitution produced approximately 1 log greater potency. We suggest that due to its high potency UFP-101 should prove a further useful tool in the evaluation of the N/OFQ-NOP receptor system.

Animals↗
PubMed2003

Orexinergic neurons and barbiturate anesthesia.

Orexins (OXs) regulate sleep with possible interactions with brain noradrenergic neurons. In addition, noradrenergic activity affects barbiturate anesthesia. As we have also recently reported that OXs selectively evoke norepinephrine release from rat cerebrocortical slices we hypothesized that barbiturate anesthesia may result from of an interaction with central orexinergic systems. To test this hypothesis, we performed a series of in vivo and in vitro studies in rats. In vivo, the effects of i.c.v. OX A, B and SB-334867-A (OX1 receptor antagonist) on pentobarbital, thiopental or phenobarbital-induced anesthesia times (loss of righting reflex) was assessed. In vitro effects of barbiturates and SB-334867-A on OX-evoked norepinephrine release from cerebrocortical slice was examined. In Chinese hamster ovary cells expressing human OX1/OX2 receptors OX A- and B-evoked increases in intracellular Ca2+ were measured with and without barbiturates. OX A and B significantly decreased pentobarbital, thiopental and phenobarbital anesthesia times by 15-40%. SB-334867-A increased thiopental-induced anesthesia time by approximately by 40%, and reversed the decrease produced by OX A. In vitro, all anesthetic barbiturates inhibited OX-evoked norepinephrine release with clinically relevant IC50 values. A GABAA antagonist, bicuculline, did not modify the inhibitory effects of thiopental and the GABAA agonist, muscimol, did not inhibit norepinephrine release. In addition there was no interaction of barbiturates with either OX1 or OX2 receptors. Collectively our data suggest that orexinergic neurons may be an important target for barbiturates, and GABAA, OX1 and OX2 receptors may not be involved in this interaction.

Animals↗
PubMed2002

Comparison of the binding of [(3)H]nociceptin/orphaninFQ(1-13)NH(2), [(3)H]nociceptin/orphaninFQ(1-17)OH and [(125)I]Tyr(14)nociceptin/orphaninFQ(1-17)OH to recombinant human and native rat cerebrocortical nociceptin/orphanin FQ receptors.

Nociceptin/orphanin FQ (N/OFQ) is a 17-amino acid endogenous neuropeptide ligand for the nociceptin receptor (NOP). We have prepared a [(3)H]-labelled truncated N/OFQ peptide, [(3)H]N/OFQ(1-13)NH(2) and compared its binding characteristics with [(3)H]N/OFQ(1-17)OH and [(125)I]Y(14)N/OFQ(1-17)OH in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHO(hNOP)) and the rat cerebrocortex. [(3)H]N/OFQ(1-13)NH(2), [(3)H]N/OFQ(1-17)OH and [(125)I]Y(14)N/OFQ(1-17)OH binding to CHO(hNOP) was concentration dependent and saturable with receptor density (B(max)) and radioligand equilibrium dissociation constant (pK(d)) values (mean +/- SEM) of 1043 +/- 58 fmol/mg protein and 10.35 +/- 0.03, 1348 +/- 44 fmol/mg protein and 10.06 +/- 0.04, and 1169 +/- 76 fmol/mg protein and 10.45 +/- 0.06, respectively. In the rat, B(max) and pK(d) values for [(3)H]N/OFQ(1-13)NH(2) and [(3)H]N/OFQ(1-17)OH were 130 +/- 1 fmol/mg protein and 10.70 +/- 0.03, and 157 +/- 4 fmol/mg protein and 10.34 +/- 0.02, respectively. The binding of all radioligands was displaced by a range of peptide and non-peptide ligands. There was a strong correlation (r(2) = 0.92, P = 0.002) between pK(i) values estimated with [(3)H]N/OFQ(1-13)NH(2) and [(3)H]N/OFQ(1-17)OH. No such correlation was observed in comparison with the [(125)I]-labelled peptide (poor agreement with low affinity N/OFQ(1-9)NH(2), Dynorphin-A and Naloxone benzoylhydrazone). We suggest that [(3)H]N/OFQ(1-13)NH(2) may be a useful alternative to [(3)H]N/OFQ(1-17)OH.

Animals↗
PubMed2002

Nociceptin/orphanin FQ inhibits glutamate release from rat cerebellar and brain stem slices.

We have previously demonstrated that nociceptin/orphanin FQ (N/OFQ), inhibits K(+) depolarisation-evoked glutamate release from rat cerebrocortical slices. In this study we have examined the effects of N/OFQ on glutamate release from rat cerebellar and brain stem slices as there are regional differences in nociceptin/orphanin FQ receptor (NOP) expression. Slices were depolarised with two pulses of 46 mM K(+) (S(1) and S(2)) with N/OFQ added after S(1). Glutamate (non-radioactive) was measured using a fluorescence-based assay. N/OFQ effects were assessed by measuring area under S(1) and S(2) release curves and calculation of S(2)/S(1) ratios. In cerebellar slices K(+) evoked S(2)/S(1) ratio was 1.17+/-0.10 (n=28). This was reduced in a concentration dependent (EC(50) 22 nM; E(max) 46%) and naloxone (10 microM) insensitive manner by N/OFQ. In the brain stem K(+) evoked glutamate release was considerably reduced compared to cerebellum. In several preparations K(+) failed to evoke a significant release. In those that did K(+) evoked S(2)/S(1) ratio was 1.03+/-0.07 (n=13). A total of 100 nM N/OFQ reduced this by 38+/-12% and this response was naloxone insensitive. Due to this small response and its variability we could not construct a full concentration response curve. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate.

Animals↗
PubMed2002

Characterisation of the non-peptide nociceptin receptor agonist, Ro64-6198 in Chinese hamster ovary cells expressing recombinant human nociceptin receptors.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like receptor or nociceptin receptor (NOP). We have compared a novel non-peptide NOP agonist Ro64-6198 with N/OFQ in a series of GTPgamma35S binding and inhibition of forskolin stimulated cAMP formation assays. GTPgamma35S binding assays were performed in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP). cAMP inhibition studies were performed in whole CHOhNOP cells. Both Ro64-6198 and N/OFQ stimulated GTPgamma35S binding with pEC50 values(95%CL) of 7.61(0.18) and 8.58(0.21) respectively. Both Ro64-6198 and N/OFQ inhibited cAMP formation with pEC50 values of 8.45(0.9) and 9.28(028) respectively. In each assay Ro64-6198 and N/OFQ were full agonists. Ro64-6198 stimulation of GTPgamma35S binding and inhibition of cAMP formation was competitively antagonised by the NOP antagonists [Nphe1]NC(1 - 13)NH2 (10microM), J-113397 (100nM) and III-BTD (1microM) with pKB values of 7.04(0.34) and 6.29(0.10), 8.65(0.34) and 7.90(0.30) and 7.59(0.22) and 7.60(0.22) respectively. Despite the slightly reduced potency of Ro64-6198 compared with N/OFQ, by virtue of high selectivity and relative metabolic stability this molecule will be of considerable use in studies of the actions of the NOP.

Animals↗
PubMed2002

Effects of naloxone benzoylhydrazone on native and recombinant nociceptin/orphanin FQ receptors.

We have studied the effects of naloxone benzoylhydrazone (NalBzoH) at recombinant human OP4 receptors expressed in Chinese hamster ovary (CHO) cells (CHOhOP4) and native OP4 sites in isolated tissues from various species. In CHOhOP4 membranes, nociceptin (NC) and NalBzoH displaced [125I]Tyr14-NC with pKi values of 10.1 and 7.3. In the presence of 100 microM GDP, NC stimulated GTPgamma35S binding (pEC50 = 8.5). NalBzoH was ineffective but antagonized the effects of NC (pA2 = 6.9). At 5 microM GDP, there was an increase in potency (pEC50 = 9.3) and efficacy (4.3-fold) of NC. NalBzOH was a partial agonist (pEC50 = 7.0, Emax = 13% relative to NC). In CHOhOP4 cells, NC and NalBzoH inhibited cAMP formation with pEC50 and Emax values of 9.8 and 100% and 6.0 and 44%, respectively. In the rat vas deferens, NalBzoH (10 microM) did not modify electrically induced twitches but competitively antagonized the inhibitory action of NC (pA2 = 6.2). In the mouse vas deferens (mVD) and guinea pig ileum (gpI), NalBzoH inhibited twitches with pEC50 and Emax values of 7.6 and 78% and 8.5 and 77%, respectively. The effect of 3 microM NalBzoH was fully inhibited by 3 microM naloxone in mVD and 30 microM in gpI. Under these conditions, NalBzoH antagonized the actions of NC in both preparations with pA2 values of 6.3 and 6.8, respectively. Collectively, these data demonstrate that NalBzoH is a nonselective OP4 ligand with system-dependent behaviour.

Animals↗
PubMed2001

Structure-activity studies of the Phe(4) residue of nociceptin(1-13)-NH(2): identification of highly potent agonists of the nociceptin/orphanin FQ receptor.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.

Animals↗
PubMed2001

Temperature-dependent activation of recombinant rat vanilloid VR1 receptors expressed in HEK293 cells by capsaicin and anandamide.

Capsaicin activates vanilloid (VR1) receptors found on sensory neurons. These ligand-gated ion channels are also sensitive to low pH, elevated temperature and the endocannabinoid, anandamide. In this study, we have measured capsaicin- and anandamide-induced elevations in intracellular calcium concentrations ([Ca(2+)](i)) in fura-2 loaded HEK293 cells stably expressing the rat VR1 receptor at 22, 37 and 50 degrees C. Both capsaicin and anandamide produced a concentration-dependent elevation in [Ca(2+)](i) at all temperatures. pEC(50) values were 7.74 and 5.69 at 22 degrees C and 6.90 and 5.15 at 37 degrees C for capsaicin and anandamide, respectively. At 50 degrees C, the pEC(50) value for capsaicin was 6.36 but the response to anandamide did not saturate. Responses to both agonists were sensitive to ruthenium red and capsazepine at all temperatures. This temperature-dependent reduction in potency may result from desensitization.

Animals↗
PubMed2001

Interaction of local anaesthetic agents with the endogenous norepinephrine transporter in SH-SY5Y human neuroblastoma cells.

Use of intravenous guanethidine for the treatment of complex regional pain syndrome type I is of variable efficacy. Guanethidine injection is painful, so local anaesthetic is co-administered. We hypothesize that local anaesthetic inhibits uptake of guanethidine and hence reduces its efficacy. In this study we have examined the effects of a range of local anaesthetic agents on the uptake of [3H]norepinephrine ([3H]NE) (as a surrogate for guanethidine) and the binding of [3H]nisoxetine to the NE transporter in cultured SH-SY5Y human neuroblastoma cells. All local anaesthetic agents inhibited NE uptake with a rank order cocaine>tetracaine>procaine(esters), dibucaine > bupivacaine > prilocaine > lidocaine (amides). In addition all anaesthetic agents displaced [3H]nisoxetine with a rank order cocaine > tetracaine > dibucaine > procaine > prilocaine > bupivacaine > lidocaine. There was a positive correlation between [3H]NE uptake and [3H]nisoxetine binding. Our data suggest that when local anaesthetic and guanethidine are co-administered the former may reduce uptake of the latter and hence reduce the clinical efficacy of guanethidine.

Anesthetics, Local↗
PubMed2001

Effects of nociceptinNH2 and [Nphe1]nociceptin(1-13)NH2 on rat brain noradrenaline release in vivo and in vitro.

We have investigated the effects of nociceptin/orphanin FQ (NC) receptor agonist NCNH2 and a competitive NC receptor antagonist, [Nphe1]NC(1-13)NH2 on noradrenaline (NA) release in vivo using microdialysis in freely moving animals and in vitro from cerebrocortical slices. One nmol of NCNH2 injected into rat locus coeruleus inhibited NA release from the prefrontal cortex (E(max) 27.4+/-5.7% 30 min after injection) which was partially (33%) reversed by 100 nmol of [Nphe1]NC(1-13)NH2. In cerebrocortical slices NCNH2 inhibited NA release in a concentration-dependent manner (EC50 12 nM, E(max) 29.4%) that was reversed by [Nphe1]NC(1-13)NH2. In both preparations, [Nphe1]NC(1-13)NH2 per se was inactive. These data demonstrate an inhibition of NA release by NCNH2 in a [Nphe1]NC(1-13)NH2 sensitive manner in both in vivo brain microdialysis and in vitro cerebrocortical slices studies in rats.

Animals↗
PubMed2001

Characterisation and comparison of novel ligands for the nociceptin/orphanin FQ receptor.

Studies of nociceptin/orphanin FQ (NC) have been hampered by the paucity of available ligands with activity at the nociceptin receptor (NCR). In this study we have compared the agonist profile of NC and a novel NCR agonist, Ro65-6570, in a series of radioligand binding studies and effects on forskolin-stimulated cAMP formation in Chinese hamster ovary (CHO) cells expressing the recombinant human NCR (CHOhNCR). In addition, we report the effects of three antagonists, [Nphe1]NC(1-13)NH2, J-113397 and III-BTD, on these responses. In radioligand binding studies Ro65-6570, [Nphe1]NC(1-13)NH2, J-113397 and III-BTD displaced [3H]NC with similar pKi values (8.4-8.8). This compares with a pK(D) of 10.2 for NC in a direct saturation experiment. [Nphe1]NC(1-13)NH2 and J-113397 showed at least 100-fold selectivity over classical opioid receptors. Both NC and Ro65-6570 produced a concentration-dependent inhibition of cAMP formation with pEC50 values of 9.56+/-0.06 and 8.68+/-0.04, respectively. Maximum inhibition achieved was 100%. [Nphe1]NC(1-13)NH2, J-113397 and III-BTD produced a parallel rightward shift in the concentration-response curves to both NC and Ro65-6570 with pK(B) values of approximately 6.5, approximately 7.5 and approximately 7.7, respectively. Importantly, all three antagonists were devoid of residual agonist activity. Collectively, these data indicate the value of Ro65-6570, [Nphe1]NC(1-13)NH2, J-113397 and III-BTD in studies of the physiological role played by NC. However, due to the relatively poor selectivity of Ro65-6570 and III-BTD caution should be exercised when using tissues that co-express micro-opioid receptors.

Animals↗