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D G Lambert

Publications and source records attributed to D G Lambert.

At least 55 records · Page 3Linked to original sources
PubMed2000

Effects of intravenous anesthetic agents on glutamate release: a role for GABAA receptor-mediated inhibition.

BACKGROUND: Many anesthetic agents are known to enhance the alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) chloride current; however, they also depress excitatory neurotransmission. The authors evaluated two hypotheses: intravenous anesthetic agents inhibit glutamate release and any observed inhibition may be secondary to GABAA receptor activation. METHODS: Cerebrocortical slices were prepared from Wistar rats. After perfusion in oxygenated Krebs buffer for 60 min at 37 degrees C, samples for glutamate assay were obtained at 2-nmin intervals. After 6 min, a 2-min pulse of 46 mM K+ was applied to the slices (S1); this was repeated after 30 min (S2). Bicuculline (1-100 microM) was applied when the S1 response returned to basal level, and 10 min later, thiopental (1-300 micro/M), propofol (10 microM), or ketamine (30 microM) were also applied until the end of S2. Perfusate glutamate concentrations were measured fluorometrically, and the area under the glutamate release curves was expressed as a ratio (S2/S1). RESULTS: Potassium (46 mM) evoked a monophasic release of glutamate during S1 and S2, with a mean control S2/S1 ratio of 1.07 +/- 0.33 (mean +/- SD, n = 96). Ketamine and thiopental produced a concentration-dependent inhibition of K+-evoked glutamate release with half-maximum inhibition of release values of 18.2 and 10.9 /microM, respectively. Release was also inhibited by propofol. Bicuculline produced a concentration dependent reversal of thiopental inhibition of glutamate release with a half-maximum reversal of the agonist effect of 10.3 microM. Bicuculline also reversed the effects of propofol but not those of ketamine. CONCLUSIONS: The authors' data indicate that thiopental, propofol, and ketamine inhibit K+-evoked glutamate release from rat cerebrocortical slices. The inhibition produced by thiopental and propofol is mediated by activation of GABAA receptors, revealing a subtle interplay between GABA-releasing (GABAergic) and glutamatergic transmission in anesthetic action.

Anesthetics, Intravenous↗
PubMed1999

The effect of C-terminal truncation of the recombinant delta-opioid receptor on Ca2+i signaling.

We have previously shown a stimulatory coupling of the recombinant delta-opioid receptor to phospholipase C leading to production of inositol (1,4,5) triphosphate [Ins(1,4,5)P3] that is affected by truncation of the C-terminus of the receptor. Using a C-terminal mutant of the delta-opioid receptor lacking the final 37 amino acids (CHOdelta37), we examined its coupling to intracellular calcium ion concentration ([Ca2+]i) compared to the full length wild type receptor (CHOdeltaWT) in transfected Chinese hamster ovary (CHO) cells. D-[Pen2,5]enkephalin (DPDPE) mediated increases in [Ca2+]i were measured fluorimetrically in fura-2 loaded whole cell suspensions. DPDPE produced time- and concentration-dependent increases in [Ca2+]i in CHOdeltaWT and CHOdelta37. In both cell types the DPDPE simulated increase in [Ca2+]i was naloxone reversible and pertussis toxin and thapsigargin sensitive. Removal of the C-terminus resulted in a rightward shift of the Ca2+ release concentration-response curve [pEC50 = 8.43 +/- 0.13 and 6.08 +/- 0.25 for CHOdeltaWT and CHOdelta37, respectively]. These data indicate that the C-terminus of the recombinant delta-opioid receptor is important in [Ca2+]i coupling and may be attributed to the effect of C-terminus truncation on phospholipase C coupling reported previously.

Amino Acid Sequence↗
PubMed1999

Interaction of ketamine with mu2 opioid receptors in SH-SY5Y human neuroblastoma cells.

PURPOSE: Ketamine is known to interact with opioid receptors. However, because this agent does not produce opioid-like respiratory depression, it might not interact with mu(2) opioid receptors. Therefore, we have studied the interaction of ketamine with mu(2) opioid receptors expressed in SH-SY5Y cells. METHODS: SH-SY5Y cells (passage 70-80) were used to obtain ketamine dose-response curves for inhibition of 0.4 nM [(3)H][D-Ala(2),MePhe(4),Gly(ol)(5)] enkephalin (DAMGO) binding to mu(2) opioid receptors and of forskolin (1 microM)-stimulated cyclic AMP (cAMP) formation. RESULTS: Ketamine displaced [(3)H]DAMGO binding in SH-SY5Y cells with a K(i) of 12.1 microM. However, this concentrations did not inhibit forskolin-stimulated cAMP formation, although at supraclinical concentrations, significant inhibition was observed with an estimated IC(50) of 700 microM. CONCLUSION: The present study indicates that a clinically relevant concentration of ketamine interacts with mu(2) opioid receptors. However, no agonist activity was observed.

Journal Article↗
PubMed1999

Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(CH2-NH)Gly2]Nociceptin(1-13)NH2 ([F/G]NC(1-13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1-13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHO(NCR)) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHO(NCR) cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1-13)NH2 was as potent as NC(1-13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHO(NCR) cells was NCNH2> or =NC=NC(1-13)NH2>NC(1-12)NH2> >NC(1-11)NH2. [F/G]NC(1-13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1-13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5+/-4.9% and 7.39, 58.9+/-6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1-13)NH2, displayed a small (instrinsic activity alpha = 0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1-13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1-13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

Animals↗
PubMed1999

The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells.

1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the mu-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors. 2 E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively. E-2 displaced [3H]-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively. E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM. 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9. 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively. 4 E-1/E-2 (1 microM) increased [Ca2+]i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively. In single adherent cells E-1/E-2 (1 microM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells. 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor.

Analgesics, Opioid↗
PubMed1999

Neither nociceptin nor its receptor are present in human synovial fluid or tissue.

Our aim was to identify the nociceptin receptor and its endogenous ligand, nociceptin, in human peripheral tissue. Synovial tissue was obtained from 11 patients (ASA I-III, 66-84 yr) undergoing elective total knee replacement. Synovial fluid was obtained from another 10 patients (ASA I-III, 57-81 yr). Fluid was mixed with trifluoroacetic acid and the tissue with isopentone before freezing at -70 degrees C. Nociceptin receptor identification was performed using a [3H]nociceptin binding assay and nociceptin detection by radioimmunoassay. There was no specific [3H]nociceptin binding to knee synovial tissue and radioimmunoassay did not detect nociceptin. Neither the nociceptin receptor nor nociceptin was found in human synovial tissue or fluid.

Aged↗
PubMed1999

Stereoselective interaction of ketamine with recombinant mu, kappa, and delta opioid receptors expressed in Chinese hamster ovary cells.

BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.

Animals↗
PubMed1999

Analysis of vasoconstrictor responses to histamine in the hindlimb vascular bed of the rabbit.

Hemodynamic responses to histamine were investigated in the anesthetized rabbit. Intravenous injections of histamine induced dose-dependent decreases in systemic arterial pressure that were blocked by the H(1)-receptor antagonist pyrilamine but not the H(2) antagonist cimetidine. Injections of histamine and the H(1) agonist 6-[2-(4-imidazolyl)ethylamine]-N-(4-trifuormethylphenyl)-heptan ecardo xamide dimaleate (HTMT) into the hindlimb perfusion circuit increased hindlimb perfusion pressure, whereas the H(2) agonist dimaprit decreased perfusion pressure and the H(3)-receptor agonist R-(-)-alpha-methylhistamine did not alter perfusion pressure. Pyrilamine reduced hindlimb vasoconstrictor responses to histamine and HTMT but did not alter vasodilator responses to dimaprit. Cimetidine reduced the response to dimaprit but did not alter vasoconstrictor responses to histamine or HTMT. The H(3)-receptor antagonist thioperamide was without effect on responses to the histamine agonists. These data suggest the presence of H(1) and H(2) receptors and that histamine for the most part acts by stimulating H(1) receptors to produce vasoconstriction in the hindlimb vascular bed of the rabbit. Responses to histamine, HTMT, and norepinephrine were significantly enhanced by a nitric oxide synthase inhibitor at a time when vasodilator responses to dimaprit were unaltered and responses to acetylcholine were significantly reduced. Responses to histamine and the H(1) and H(2) agonists were not affected by the cyclooxygenase inhibitor meclofenamate or by ATP-sensitive K(+) channel, alpha-adrenergic, or angiotensin AT(1) receptor antagonists. The present data suggest that H(1) receptors mediate both systemic vasodepressor and hindlimb vasoconstrictor responses to histamine.

Adamantane↗
PubMed1999

The influence of candesartan and PD123319 on responses to angiotensin II in the hindquarters vascular bed of the rat.

The effects of the AT1 and AT2 receptor blockers candesartan and PD123319 on hemodynamic responses to angiotensin II (AngII) were investigated in the anesthetized rat. Injections of AngII caused dose-related increases in systemic arterial and in hindquarters perfusion pressure that were reduced by candesartan. The inhibitory effects of candesartan were insurmountable, and a vasodepressor or vasodilator response to AngII was not unmasked. The AT2 receptor antagonist PD 123319 had no effect on increases in systemic arterial and hindquarters perfusion pressure in response to AngII. The present results suggest that pressor responses to AngII are mediated by the activation of AT1 receptors, and that AT2 receptors do not appear to modulate hemodynamic responses to AngII in the anesthetized rat.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1999

Effects of candesartan and PD123319 on responses to angiotensin II in the anesthetized mouse.

The effects of candesartan (30 microg/kg i.v.) and PD123319 (10 mg/kg i.v.) on changes in systemic arterial pressure in response to angiotensin II (AngII) were investigated in the anesthetized mouse. Intravenous injections of AngII caused dose-related increases in systemic arterial pressure. Pressor responses to AngII were attenuated by candesartan but were not altered by PD123319. Neither candesartan nor PD123319 had a significant effect on baseline systemic arterial pressure or on the increase in arterial pressure in response to norepinephrine. The present results suggest that increases in systemic arterial pressure in response to AngII in the anesthetized mouse are mediated by AT1 receptors and that AT2 receptors do not modulate the pressor response to AngII.

Angiotensin II↗
PubMed1999

Analysis of the effects of candesartan on responses to angiotensin II in the hindquarters vascular bed of the cat.

The effects of the nonpeptide angiotensin II (AngII) AT1 receptor blocker candesartan on responses to AngII were investigated in the hindquarters vascular bed of the cat. Under constant-flow conditions, injections of AngII into the hindquarters perfusion circuit elicited dose-dependent increases in perfusion pressure. Candesartan in a dose of 3 microg/kg intravenously (i.v.) decreased vasoconstrictor responses to AngII in a surmountable manner. At doses of 30 and 300 microg/kg i.v., candesartan shifted the dose-response curve to AngII to the right in an insurmountable manner, indicating an insurmountable blockade of AT1 receptors. The inhibitory effects of the larger doses of candesartan on responses to AngII were long in duration, and the AT1 receptor blocker had little effect on baseline pressures. Candesartan was without effect on vasoconstrictor responses to norepinephrine, U46619, PGF2alpha, vasopressin, BAY K8644; biphasic responses to endothelin-1; or on vasodilator responses to acetylcholine, albuterol, or levcromakalim. These results indicate that candesartan is a potent and selective angiotensin AT1 receptor blocker that can induce both surmountable and insurmountable AT1 receptor blockade and provide support for the hypothesis that there are "spare" AT1 receptors in the hindquarters vascular bed of the cat.

Angiotensin II↗
PubMed1998

Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices.

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n = 7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7 +/- 8.3% (P < 0.05, n = 6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.

Animals↗
PubMed1998

Rat central ORL-1 receptor uncouples from adenylyl cyclase during membrane preparation.

Nociceptin/orphanin FQ is the endogenous agonist of the orphan receptor ORL-1. In this study, we sought to examine any possible regional differences of nociceptin binding using [125I]Tyr14-nociceptin, and of agonist induced inhibition of cAMP formation in membranes prepared from cerebrocortex, cerebellum and brainstem. The binding of [125I]Tyr14-nociceptin was concentration-dependent and saturable, with Bmax and pKd (pM) values of 179.7+/-15.3 fmol/mg protein and 10.26+/-0.09 (60.0), 12.4+/-1.8 fmol/mg protein and 10.44+/-0.07 (37.0), 52.3+/-0.8 fmol/mg protein and 10.16+/-0.08 (74.0) in cerebrocortical, cerebella and brainstem membranes, respectively. In all preparations, nociceptin up to 1 microM failed to inhibit basal and forskolin stimulated cAMP formation. In all tissues forskolin stimulated and nabilone (acting at the central cannabinoid receptor) inhibited cAMP formation. Collectively these data report regional differences in ORL-1 receptor expression and that these receptors uncouple during membrane preparation.

Animals↗
PubMed1998

Endomorphin 1 and 2, the endogenous mu-opioid agonists, produce biphasic changes in systemic arterial pressure in the cat.

The endogenous peptides endomorphin 1 and 2 are newly isolated, potent, selective mu-opioid receptor agonists. In the present study, responses to the endomorphin peptides were investigated in the systemic vascular bed of the cat. Endomorphin 1 and 2 induced dose-related biphasic changes in systemic arterial pressure when injected in doses of 1-30 nmol/kg i.v. The biphasic responses to endomorphin 1 and 2 were characterized by an initial increase followed by a decrease in systemic arterial pressure. In terms of relative vasodepressor activity, endomorphin 1 and 2 were similar in potency and approximately 10-fold less potent than the ORL1 ligand nociceptin (orphanin FQ) in decreasing systemic arterial pressure. The biphasic arterial pressure changes in response to endomorphin 1 and 2 were inhibited by the opioid receptor antagonist naloxone in a dose of 2 mg/kg i.v. These results demonstrate that endomorphin 1 and 2 produce significant, naloxone-sensitive changes in systemic arterial pressure that are characterized by an initial increase followed by a secondary decrease in arterial pressure in the cat.

Analgesics, Opioid↗
PubMed1998

Interaction of neuromuscular blocking drugs with recombinant human m1-m5 muscarinic receptors expressed in Chinese hamster ovary cells.

1. Neuromuscular blocking drugs (NMBD's) are known to produce cardiovascular side effects manifesting as brady/tachycardias. In this study we have examined the interaction of a range of steroidal NMBD's with recombinant human m1-m5 muscarinic receptors expressed in Chinese hamster ovary cells. Our main hypothesis is that NMBD's may interact with m2 (cardiac) muscarinic receptors. 2. All binding studies were performed with cell membranes prepared from CHO m1-m5 cells in 1 ml volumes of 20 mM HEPES, 1 mM MgCl2 at pH 7.4 for 1 h. Muscarinic receptors were labelled with [3H]-NMS and displacement studies were performed with pancuronium, vecuronium, pipecuronium, rocuronium and gallamine. In addition a range of muscarinic receptor subtype selective reference compounds were included. In order to determine the nature of any interaction the effects of pancuronium, rocuronium and vecuronium on methacholine inhibition of forskolin stimulated cyclic AMP formation in CHO m2 cells was examined. Cyclic AMP formation was assessed in whole cells using a radioreceptor assay. All data are mean +/- s.e.mean (n > or = 5). 3. The binding of [3H]-NMS was dose-dependent and saturable in all cells tested. Bmax and Kd values in m1-m5 cells were 2242+/-75, 165+/-13, 1877+/-33, 458+/-30, 127+/-2 fmol mg(-1) protein and 0.11+/-0.02, 0.15+/-0.01, 0.12+/-0.01, 0.12+/-0.01, 0.22+/-0.01 nM respectively. 4. The binding of [3H]-NMS was displaced dose dependently (pK50) by pirenzepine in CHO m1 membranes (7.97+/-0.04), methoctramine in CHO m2 membranes (8.55+/-0.1), 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) in CHO m3 membranes (9.38+/-0.03), tropicamide in CHO m4 membranes (6.98+/-0.01). 4-DAMP, pirenzepine, tropicamide and methoctramine displaced [3H]NMS in CHO m5 membranes with pK50 values of 9.20+/-0.14, 6.59+/-0.04, 6.89+/-0.05 and 7.22+/-0.01 respectively. These data confirm homogenous subtype expression in CHO m1-m5 cells. 5. [3H]NMS binding was displaced dose-dependently (pK50) by pancuronium (m1, 6.43+/-0.12; m2, 7.68+/-0.02; m3, 6.53+/-0.06; m4, 6.56+/-0.03; m5, 5.79+/-0.10), vecuronium (m1, 6.14+/-0.04; m2, 6.90+/-0.05; m3, 6.17+/-0.04; m4, 7.31+/-0.02; m5, 6.20+/-0.07), pipecuronium (m1, 6.34+/-0.11; m2, 6.58+/-0.03; m3, 5.94+/-0.01; m4, 6.60+/-0.06; m5, 4.80+/-0.03), rocuronium (m1, 5.42+/-0.01; m2, 5.40+/-0.02; m3, 4.34+/-0.02; m4, 5.02+/-0.04; m5, 5.10+/-0.03) and gallamine (m1, 6.83+/-0.05; m2, 7.67+/-0.04; m3, 6.06+/-0.06; m4, 6.20+/-0.03; m5, 5.34+/-0.03). 6. Cyclic AMP formation was inhibited dose dependently by methacholine in CHO m2 cells pEC50 for control and pancuronium (300 nM) treated cells were 6.18+/-0.34 and 3.57+/-0.36 respectively. Methacholine dose-response curves in the absence and presence of rocuronium (1 microM) and vecuronium (1 microM) did not differ significantly. Pancuronium, vecuronium and rocuronium did not inhibit cyclic AMP formation alone indicating no agonist activity. 7. With the exception of rocuronium there was a significant interaction with m2 muscarinic receptors with all NMBD's at clinically achievable concentrations suggesting that the brady/tachycardias associated with these agents may result from an interaction with cardiac muscarinic receptors. Furthermore pancuronium at clinically achievable concentrations antagonised methacholine inhibition of cyclic AMP formation in CHO m2 cells further suggesting that the tachycardia produced by this agent results from muscarinic antagonism. The mechanism of the bradycardia produced by vecuronium is unclear.

Analysis of Variance↗
PubMed1998

Effects of C-terminal truncation of the recombinant delta-opioid receptor on phospholipase C and adenylyl cyclase coupling.

Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. We have generated two C-terminal truncation mutants of the delta-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. D-[Pen(2,5)]-enkephalin (DPDPE) mediated Ins(1,4,5)P3 formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P3 mass formation in Chinese hamster ovary (CHO) cells expressing the delta(wt), delta15, and delta37 receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHO delta(wt), 60 s in CHO delta15, and 120 s in CHO delta37) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P3 levels. Whereas the dose-response curves to Ins(1,4,5)P3 formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC50 values, with cyclic AMP inhibition being the more potent, perhaps indicating G(i alpha) coupling to adenylyl cyclase and G(i beta/gamma) coupling to PLC. Collectively, these data indicate that the C terminus of the delta-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal "strong" coupling of receptor to Gi and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.

Adenylyl Cyclase Inhibitors↗