Dopamine D2 receptor inhibition of adenylyl cyclase is abolished by acute ethanol but restored after chronic ethanol exposure (tolerance).

Dopamine D2 (D2) receptors seem to mediate reinforcing responses to addicting drugs. A stably transfected NG108-15 cell line expressing the long form of the rat brain D2 receptor (D2L) was used to determine how ethanol modifies D2 receptor coupling to adenylyl cyclase. Activation of D2L receptors by the D2 receptor-specific agonist R-(-)-2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) inhibits both basal and receptor-stimulated cAMP production in these cells. Ethanol added acutely prevents D2L receptor inhibition of cAMP production. After chronic exposure to ethanol, however, D2L receptor coupling to adenylyl cyclase becomes tolerant to rechallenge with ethanol, i.e., ethanol no longer inhibits D2L receptor coupling and NPA inhibition of cAMP production is restored. Acute ethanol does not change NPA binding to D2 receptor in cell membranes but abolishes guanosine-5'-O-(3-thio)triphosphate induction of a lower-affinity state; chronic ethanol is without effect. The protein kinase A (PKA) inhibitor adenosine 3',5' cyclic monophosphorothioate, Rp-isomer, prevents acute ethanol inhibition of D2L receptor coupling. In contrast, the PKA activator adenosine 3',5' cyclic monophosphorothioate, Sp-isomer, reverses chronic ethanol-induced tolerance of D2L receptor coupling, restoring coupling to an ethanol-sensitive state. These results suggest that D2L receptor coupling to adenylyl cyclase via G(i) develops tolerance to ethanol inhibition, which appears to be influenced by PKA activity.     

Measurement of the proportion of D2 receptors configured in state of high affinity for agonists in vivo: a positron emission tomography study using [11C]N-propyl-norapomorphine and [11C]raclopride in baboons

Dopamine D2 receptors are configured in interconvertible states of high (D(2 high)) or low (D(2 low)) affinity for agonists. The in vivo proportion of sites in high-affinity state remains poorly documented. Previous studies have established the D2 agonist [11C]N-propyl-norapomorphine (NPA) as a suitable positron emission tomography radiotracer for imaging D(2 high) in the living brain. To elucidate the proportion of D2 receptors configured in D(2 high) states in vivo, imaging studies were conducted in three baboons with both [11C]NPA and the D2 receptor antagonist [11C]raclopride. These studies were performed under noncarrier- and carrier-added conditions, to compare the Bmax of [11C]NPA and [11C]raclopride in the same animals. [11C]raclopride in vivo KD and Bmax were 1.59 +/- 0.28 nM (n = 3) and 27.3 +/- 3.9 nM (n = 3), respectively. The in vivo KD of [11C]NPA was 0.16 +/- 0.01 nM (n = 3), consistent with its affinity for D(2 high) reported in vitro. The maximal density of sites for [11C]NPA was 21.6 +/- 2.8 nM (n = 3), i.e., 79% of the [11C]raclopride Bmax. This result suggested that 79% of D2 receptors are configured as D(2 high) in vivo. This large proportion of D(2 high) sites might explain the vulnerability of D2 radiotracers to competition by endogenous dopamine, and is consistent with a previous report that the in vivo binding of agonist radiotracer [11C]NPA is more vulnerable to competition by endogenous dopamine than that of antagonist radiotracer [11C]raclopride.     

G-protein sensitivity of ligand binding to human dopamine D(2) and D(3) receptors expressed in Escherichia coli: clues for a constrained D(3) receptor structure

Human dopamine D(2) and D(3) receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with G(i/o)-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [(125)I]iodosulpride and [(3)H]7-OH-DPAT, were similar for hD(3) receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD(2) receptors in E. coli versus CHO cell membranes (measured with [(3)H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([(3)H]7-OH-DPAT) binding at hD(2) receptors but not at hD(3) receptors. Also, [(3)H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD(2) but clearly measurable at hD(3) receptors in E. coli membranes. Addition of a G(i/o)-protein mix to E. coli membranes increased high affinity [(3)H]7-OH-DPAT binding in a concentration-dependent manner at hD(2) and hD(3) receptors; this effect was reversed by addition of GppNHp. The potency of the G(i/o)-protein mix to reconstitute high affinity binding was similar for hD(2) and hD(3) receptors. Thus, agonist binding to D(3) receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D(3) receptors (versus D(2) receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.     

Comparison of the ligand binding and signaling properties of human dopamine D(2) and D(3) receptors in Chinese hamster ovary cells.

Human dopamine D(2) (hD(2)) and D(3) (hD(3)) receptors were expressed at similar, high expression levels in Chinese hamster ovary (CHO) cells, and their coupling to G proteins and further signal transduction pathways were compared. In competition radioligand-binding experiments, guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) treatment of hD(2S)- or hD(3)-CHO cell membranes induced a rightward shift and steeping of the dopamine inhibition curve. This effect was pronounced for hD(2) receptors and small for hD(3) receptors. Activation of G proteins was investigated in [(35)S]GTPgammaS-binding assays. Dopamine stimulated [(35)S]GTPgammaS binding 330 and 70% over basal levels on hD(2)-CHO and hD(3)-CHO cell membranes, respectively. (+)-7-(Dipropylamino)-5, 6,7,8-tetrahydro-2-naphthalenol and PD128907 were partial agonists for both receptors. Haloperidol, risperidone, raclopride, and nemonapride inhibited dopamine-stimulated [(35)S]GTPgammaS binding with potencies comparable to their binding affinities for hD(2) and hD(3) receptors in CHO cell membranes; inverse agonism could not be detected with this assay. Receptor stimulation by dopamine inhibited forskolin-induced cyclic AMP formation in hD(2)-CHO and hD(3)-CHO cells by 70%. Furthermore, the extracellular acidification rate increased when hD(2)-CHO and hD(3)-CHO cells were stimulated by dopamine; this effect was abolished by pertussis toxin pretreatment. In this study, we could demonstrate clear functional effects at different levels of the signaling cascade of hD(2) and hD(3) receptors in CHO cells when expressed at high levels. High-affinity agonist binding to hD(2) and hD(3) receptors was still present, but effects of receptor-G protein uncoupling at hD(3) receptors were small, indicating that hD(3) receptors maintain relatively high-affinity agonist binding in the absence of G proteins.     

Pharmacological differences between the high-affinity muscarinic agonist binding states of the rat heart and cerebral cortex labeled with (+)-[3H]cismethyldioxolane

The high-affinity agonist binding state of muscarinic receptors in the rat heart and cerebral cortex has been pharmacologically characterized in parallel studies. Muscarinic sites were labeled and studied with the aid of a highly specific, rapid filtration binding assay using the potent muscarinic agonist (+)-[3H]CD. Homogenates of both tissues were found to contain a saturable high-affinity (Kd = 1-2 nM), low capacity (6-17% of (-)-[3H]QNB sites) (+)-[3H]CD binding state which demonstrated stereoselectivity and drug specificity typical of a muscarinic site. However, comparative studies of drug potency profiles in competition for myocardial and cerebral cortical (+)-[3H]CD-labeled membranes revealed several major pharmacological differences between muscarinic sites in these tissues. Whereas the muscarinic agonists pilocarpine and McN-A-343, the nonclassical antagonist pirenzepine, and the acetylcholinesterase inhibitor physostigmine reduced (+)-[3H]CD binding in both tissues, their inhibitory effects were more potent (4- to 77-fold) in cerebral cortical membranes. Conversely, gallamine, a nicotinic cholinergic antagonist, demonstrated a 36-fold greater potency at the high-affinity (+)-[3H]CD binding state in myocardial membranes. By comparison, other classical muscarinic agonists and antagonists were nearly equipotent as inhibitors of high-affinity (+)-[3H]CD binding in these two tissues. Thus, these studies for the first time demonstrate that muscarinic receptors in the heart and cerebral cortex can be distinguished pharmacologically by certain drugs which interfere with the high-affinity agonist binding state of the muscarinic recognition site and provide support for the subclassification of these receptors.     

SK&F 89124, A POTENT AND SELECTIVE AGONIST AT PREJUNCTIONAL DOPAMINE RECEPTORS

SK&F 89124 (4-[2-(N,N-di-n-propylamino)ethyl]-7-hydroxy-2(3H) indolone) can be considered as a derivative of N,N-di-n-propyldopamine (DPDA) in which the meta-hydroxyl is replaced by a cyclic amide function. SK&F 89124 is at least one order of magnitude more potent than DPDA as an agonist at peripheral inhibitory prejunctional dopamine receptors (DA2 receptors) in the isolated perfused rabbit ear artery. A potent agonist action of SK&F 89124 at the DA2 receptor can also be demonstrated by inhibition of radioactive overflow from prelabelled canine coronary artery or saphenous vein, and in the anesthetized dog as an inhibition of the tachycardia induced by cardioaccelerator nerve stimulation or the increase in hind-limb perfusion pressure induced by stimulation of the lumbar sympathetic chain. SK&F 89124 is a potent inhibitor of the binding of [3H]spiroperidol to D2 receptors in bovine pituitary homogenates. High concentrations of SK&F 89124 do not activate the adenylate cyclase D1 receptor in rat caudate homogenates, nor produce activation of alpha 2-adrenoceptors or H2-histamine receptors in the guinea pig atrium. Although some alpha 1-adrenoceptor mediated vasoconstriction is produced in the rabbit ear artery and rabbit aorta, the concentrations required are several orders of magnitude higher than those active at the DA2 receptor. From these data it is evident that this structural modification can increase both the potency and selectivity of DPDA as a DA2 receptor agonist. The potency and selectivity of SK&F 89124 make this agent a useful tool for determination of the functional role of the DA2 receptor.     

Cocaine-induced increases in vesicular dopamine uptake: role of dopamine receptors

The vesicular monoamine transporter-2 is the sole transporter responsible for sequestration of monoamines, including dopamine (DA), into synaptic vesicles. Previous studies demonstrate that agents that inhibit DA transporter function, such as cocaine, increase vesicular [(3)H]DA uptake and binding of the ligand [(3)H]dihydrotetrabenazine ([(3)H]DHTBZ), as assessed in vesicles prepared from treated rats. The present studies examine the role of DA receptors in these cocaine-induced effects. Results demonstrate that administration of the D(2) DA receptor antagonist, eticlopride, but not the D(1) DA receptor antagonist, SCH23390, inhibited these cocaine-induced increases. Similar to the effects of cocaine, treatment with the D(2) agonist, quinpirole, increased both vesicular [(3)H]DA uptake and [(3)H]DHTBZ binding. In contrast, administration of the D(1) agonist, SKF81297, was without effect on vesicular [(3)H]DA uptake or [(3)H]DHTBZ binding. Finally, coadministration of quinpirole and cocaine did not further increase vesicular [(3)H]DA uptake or [(3)H]DHTBZ binding when compared with treatment with either agent alone. These data suggest that cocaine-induced increases in vesicular DA uptake and DHTBZ binding are mediated by a D(2) receptor-mediated pathway. Furthermore, results indicate that D(2) receptor activation, per se, is sufficient to increase vesicular DA uptake.     

S32504, a novel naphtoxazine agonist at dopamine D3/D2 receptors: I. Cellular, electrophysiological, and neurochemical profile in comparison with ropinirole

S32504 [(+)-trans-3,4,4a,5,6,10b-hexahydro-9-carbamoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine] displayed marked affinity for cloned, human (h)D(3) receptors (pK(i), 8.1) at which, in total G-protein ([(35)S]GTPgammaS binding, guanosine-5'-O-(3-[(35)S]thio)-triphosphate), Galpha(i3) (antibody capture/scintillation proximity), and mitogen-activated protein kinase (immunoblot) activation procedures, it behaved as an agonist: pEC(50) values, 8.7, 8.6, and 8.5, respectively. These actions were blocked by haloperidol and the selective D(3) receptor antagonist S33084 [(3aR,9bS)-N-[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl]-(4-phenyl) benzamide)]. S32504 showed lower potency at hD(2S) and hD(2L) receptors in [(35)S]GTPgammaS binding (pEC(50) values, 6.4 and 6.7) and antibody capture/scintillation proximity (hD(2L), pEC(50), 6.6) procedures. However, reflecting signal amplification, it potently stimulated hD(2L) receptor-coupled mitogen-activated protein kinase (pEC(50), 8.6). These actions were blocked by haloperidol and the selective D(2) receptor antagonist L741,626 [4-(4-chlorophenyl)-1-(1H-indol-3-ylmethyl)piperidin-4-ol]. The affinity of S32504 for hD(4) receptors was low (5.3) and negligible for hD(1) and hD(5) receptors (pK(i), <5.0). S32504 showed weak agonist properties at serotonin(1A) ([(35)S]GTPgammaS binding, pEC(50), 5.0) and serotonin(2A) (G(q), pEC(50), 5.2) receptors and low affinity for other (>50) sites. In anesthetized rats, S32504 (0.0025-0.01 mg/kg, i.v.) suppressed electrical activity of ventrotegmental dopaminergic neurons. Correspondingly, S32504 (0.0025-0.63 mg/kg, s.c.) potently reduced dialysis levels (and synthesis) of dopamine in striatum, nucleus accumbens, and frontal cortex of freely moving rats, actions blocked by haloperidol and L741,626 but not by S33084. In contrast, S32504 only weakly inhibited serotonergic transmission and failed to affect noradrenergic transmission. Actions of S32504 were expressed stereospecifically versus its less active enantiomer S32601 [(-)-trans-3,4,4a,5,6,10b-hexahydro-9-carbomoyl-4-propyl-2H-naphth[1,2-b]-1,4-oxazine]. Although the D(3)/D(2) agonist and antiparkinsonian agent ropinirole mimicked the profile of S32504, it was less potent. In conclusion, S32504 is a potent and selective agonist at dopamine D(3) and D(2) receptors.     

Functional selectivity of dopamine receptor agonists. II. Actions of dihydrexidine in D2L receptor-transfected MN9D cells and pituitary lactotrophs

D(2)-like dopamine receptors mediate functional changes via activation of inhibitory G proteins, including those that affect adenylate cyclase activity, and potassium and calcium channels. Although it is assumed that the binding of a drug to a single isoform of a D(2)-like receptor will cause similar changes in all receptor-mediated functions, it has been demonstrated in brain that the dopamine agonists dihydrexidine (DHX) and N-n-propyl-DHX are "functionally selective". The current study explores the underlying mechanism using transfected MN9D cells and D(2)-producing anterior pituitary lactotrophs. Both dopamine and DHX inhibited adenylate cyclase activity in a concentration-dependent manner in both systems, effects blocked by D(2), but not D(1), antagonists. In the MN9D cells, quinpirole and R-(-)-N-propylnorapomorphine (NPA) also inhibited the K(+)-stimulated release of [(3)H]dopamine in a concentration-responsive, antagonist-reversible manner. Conversely, neither DHX, nor its analogs, inhibited K(+)-stimulated [(3)H]dopamine release, although they antagonized the effects of quinpirole. S-(+)-NPA actually had the reverse functional selectivity profile from DHX (i.e., it was a full agonist at D(2L) receptors coupled to inhibition of dopamine release, but a weak partial agonist at D(2L) receptor-mediated inhibition of adenylate cyclase). In lactotrophs, DHX had little intrinsic activity at D(2) receptors coupled to G protein-coupled inwardly rectifying potassium channels, and actually antagonized the effects of dopamine at these D(2) receptors. Together, these findings provide compelling evidence for agonist-induced functional selectivity with the D(2L) receptor. Although the underlying molecular mechanism is controversial (e.g., "conformational induction" versus "drug-active state selection"), such data are irreconcilable with the widely held view that drugs have "intrinsic efficacy".     

Desensitization of adenosine and dopamine receptors in rat brain after treatment with adenosine analogs

Maximally tolerated doses of N6-[(R)-1-methyl-2-phenylethyl] adenosine (0.50 nmol/hr/2 wk), 5'-N-ethylcarboxamide adenosine (NECA, 0.04 nmol/hr/2 wk) or deoxycoformycin (5 nmol/hr/1 wk) were administered i.c.v. to rats using mini-osmotic pumps. Adenosine receptor function was subsequently assayed using both ligand binding and adenylate cyclase assays. Binding to A1 receptors was quantitated using [3H]N6-[(R)-1-methyl-2-phenylethyl]adenosine, a selective agonist ligand at A1 receptors. Differences in the binding of this ligand and that of [3H]NECA, which binds to A1 and A2 receptors with similar affinities, were used to quantitate A2 receptors. None of the treatments affected A1 receptor function as assessed by both ligand binding and adenylate cyclase assays. A2 receptor binding and A2 receptor-mediated stimulation of adenylate cyclase were blunted in striatal membranes from NECA- and deoxycoformycin-treated rats but unaffected in striatal membranes from N6-[(R]-1-methyl-2-phenylethyl]adenosine-treated rats. All three pretreatments attenuated D1 dopamine receptor-mediated stimulation of adenylate cyclase in striatal membranes. These results suggest that 1) the A2 adenosine receptor system is susceptible to desensitization and 2) different mechanisms are involved in the NECA- and deoxycoformycin-induced desensitization of A2 adenosine receptor and D1 dopamine receptor systems. It is suggested that the D1 dopamine receptor desensitization is, in fact, due to the tonic stimulation of adenosine A1 receptors.     

N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds: novel allosteric enhancers of gamma-aminobutyric acidB receptor function

N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds are described as novel allosteric enhancers of GABA(B) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes from a GABA(B)(1b/2)-expressing Chinese hamster ovary cell line at low micromolar concentrations, but do not stimulate [35S]GTPgammaS binding by themselves. Similar effects of GS39783 are seen on native GABA(B) receptors in rat brain membranes. Concentration-response curves with GABA in the presence of different fixed concentrations of GS39783 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B)(1b/2) heterodimer. In radioligand binding experiments, GS39783 reduces the kinetic rate constants of the association and dissociation of [3H]3-aminopropylphosphinic acid, resulting in a net increase in affinity for the agonist radioligand. In equilibrium binding experiments (displacement of the antagonist ligand [3H]CGP62349), GS39783 increases agonist affinities. Agonist displacement curves are biphasic, probably reflecting the G protein-coupled and uncoupled states of the receptor. The proportion of the high-affinity component is increased by GS39783, suggesting that the G protein coupling of the receptor is also promoted by the positive modulator. We also show that GS39783 has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus oocytes and Ca2+ signaling in human embryonic kidney 293 cells. In a more physiological context, GS39783 is shown to suppress paired pulse inhibition in rat hippocampal slices. This effect is reversed by the competitive GABA(B) receptor antagonist CGP55845A and is produced most likely by enhancing the effect of synaptically released GABA at presynaptic GABA(B) receptors.     

Cannabinoid agonist signal transduction in rat brain: comparison of cannabinoid agonists in receptor binding, G-protein activation, and adenylyl cyclase inhibition

To investigate differences in agonist affinity, potency, and efficacy across rat brain regions, five representative cannabinoid compounds were investigated in membranes from three different rat brain regions for their ability to maximally stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding and bind to cannabinoid receptors (measured by inhibition of [(3)H]antagonist binding) under identical assay conditions. In all three brain regions, the rank order of potency for the stimulation of [(35)S]GTPgammaS binding and the inhibition of [(3)H]SR141716A binding for these compounds were identical, with CP55940 approximately levonantradol > WIN55212-2 >/= Delta(9)-tetrahydrocannabinol (Delta(9)-THC) > methanandamide. The rank order of efficacy was not related to potency, and relative maximal agonist effects varied across regions. Receptor binding fit to a three-site model for most agonists, stimulation of [(35)S]GTPgammaS binding fit to a two-site model for all agonists, and high-affinity receptor binding did not appear to produce any stimulation of [(35)S]GTPgammaS binding. WIN55212-2, methanandamide, and Delta(9)-THC also were assayed for the inhibition of adenylyl cyclase in cerebellar membranes. The rank orders of potency and efficacy were similar to those for [(35)S]GTPgammaS binding, but the efficacies and potencies of methanandamide and Delta(9)-THC compared with WIN55212-2 were higher for adenylyl cyclase inhibition, implying receptor/G-protein reserve.     

Effect of chronic ethanol and withdrawal on the mu-opioid receptor- and 5-Hydroxytryptamine(1A) receptor-stimulated binding of [(35)S]Guanosine-5'-O-(3-thio)triphosphate in the fawn-hooded rat brain: A quantitative autoradiography study

Previous studies have shown that chronic ethanol influences the density of central mu-opioid receptors and serotonin(1A) (5-hydroxytryptamine(1A)) receptors. To determine whether the functional coupling of these two receptors to G proteins in the rat brain, particularly in mesocorticolimbic regions, is affected by ethanol, receptor-mediated [(35)S]guanosine-5'-O-(3-thio)-triphosphate ([(35)S]GTPgammaS) binding stimulated by [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO) or L694,247 was used. By quantitative autoradiography, receptor-mediated [(35)S]GTPgammaS binding activated by the two agonists was mapped throughout brain sections at the level of the nucleus accumbens and hippocampus from groups of alcohol-preferring Fawn-Hooded (FH) rats after different ethanol consumption paradigms. Significant DAMGO (mu-opioid receptor agonist)-stimulated binding of [(35)S]GTPgammaS was obtained in the striatum, nucleus accumbens, and lateral septum, whereas L694,247 (5-hydroxytryptamine(1A/1B/1D) receptor agonist)-stimulated binding of [(35)S]GTPgammaS was observed in the lateral septum, amygdala, and cingulate cortex. Chronic ethanol self-administration significantly reduced DAMGO-stimulated [(35)S]GTPgammaS binding in the nucleus accumbens (-19%), lateral septum (-15%), and striatum (-23%), which recovered toward control levels after ethanol withdrawal. However, chronic ethanol, as well as ethanol withdrawal, failed to produce any significant alteration in L694,247-stimulated [(35)S]GTPgammaS binding in all tested brain regions. The region-specific and receptor-specific alteration of agonist-stimulated [(35)S]GTPgammaS binding suggests that the change of functional coupling of mu-opioid receptors to G proteins induced by chronic ethanol drinking may have a pathophysiological role in the consequences of ethanol consumption.     

Influence of Mg++ on the effect of diltiazem to increase dihydropyridine binding to receptors on Ca++-channels in chick cardiac and skeletal muscle membranes

The influence of Mg++ on the effect of diltiazem to increase ligand binding to a high-affinity state of dihydropyridine receptors on voltage-dependent Ca-channels has been studied in chick cardiac and skeletal muscle membranes at 25 degrees C. The high-affinity binding of the Ca-channel inhibitors (+)-[3H]PN 200-110 and [3H]nitrendipine to cardiac membranes was depressed markedly by EDTA and restored fully by the addition of free Mg++ (Ptasienski et al., Biochem. Biophys. Res. Commun. 129: 910-917, 1985). Similar results have now been obtained with skeletal muscle membranes. In the presence of EDTA alone, diltiazem, which binds to another receptor on the Ca-channel, increased the high-affinity binding of both ligands to cardiac and skeletal muscle membranes. However, in the presence of added Mg++, diltiazem had smaller or no effects on the binding of these dihydropyridines. Analyses of the data indicated that both Mg++ and diltiazem could increase the maximum binding (Bmax) for these ligands, but the effect of diltiazem was smaller than, and not additive to, that of Mg++. Specific binding of the Ca-channel activator [3H]Bay k 8644 was only observed in assays containing Mg++ in excess of EDTA. The Bmax for [3H]Bay k 8644 in skeletal muscle membranes was less than that for [3H]PN 200-110 and [3H]nitrendipine, whereas with cardiac membranes equal Bmax values were obtained for all ligands. Diltiazem increased the Bmax for [3H]Bay k 8644 in skeletal muscle, but not in cardiac membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]domperidone binding to the kidney inner medullary collecting duct dopamine-2K (DA2K) receptor.

Previous studies by our laboratory have indicated that inner medullary collecting ducts (IMCDs) express a novel dopamine (DA) receptor, designated DA2K, that is linked to stimulation of prostaglandin E2 production. This receptor has a distinct pharmacological profile and is similar in size, but not homologous to, the brain D2 receptor. Because the DA2-selective antagonist domperidone blocks DA-mediated stimulation of prostaglandin E2 production in IMCD cells, we utilized [3H]domperidone to study the binding characteristics of the DA2K receptor in IMCD cells. [3H]Domperidone binding was saturable and best fit to a single high density site (KD, 57.6 +/- 10.5 nM; Bmax, 14.9 +/- 2.7 pmol/mg protein). The specificity of [3H]domperidone binding in IMCD cells was verified by competition analysis with a variety of dopaminergic and nondopaminergic agents. Dopaminergic drugs were less potent competitors for [3H]domperidone binding in IMCD cells than previously reported for brain DA receptors, but the rank order was consistent with the labeling of a DA receptor [antagonists: domperidone greater than spiperone greater than haloperidol greater than Sch 23390 much greater than (-)-sulpiride; agonists: norapomorphine greater than fenoldopam much greater than dopamine = quinpirole], and was better correlated with the pharmacological profile for the brain D2 receptor than the brain D3 receptor. In addition, quinpirole, the most D3-selective ligand currently available, did not compete for [3H]domperidone binding in IMCD cells. These results add further support to the existence of a novel high density DA receptor, DA2K, expressed in IMCD cells.     

Reciprocal modulation of agonist and antagonist binding to A1 adenosine receptors by guanine nucleotides is mediated via a pertussis toxin-sensitive G protein

In the present study, we have characterized the effects of guanine nucleotides on agonist and antagonist binding to A1 adenosine receptors, which mediate inhibition of adenylate cyclase via the inhibitory G protein (Gi) in adipocytes. Our data indicate that guanosine-triphosphate (GTP) and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) enhance the binding of 8-(4-[(([(2-amino-ethyl)amino]carbonyl) methyl)oxyl]phenyl)-1,3-dipropylxanthine ([3H]XAC) to adipocyte membranes in a dose-dependent manner, with EC50 values being 1.8 and 2.2 microM, respectively. The stimulatory effect of GTP was abolished in pertussis toxin-intoxicated membranes, implying a role of a pertussis toxin-sensitive G protein in mediating this effect. Furthermore, the ranked order of efficacy for a series of guanine nucleotides to enhance [3H]XAC binding was GTP = Gpp(NH)p greater than GDP greater than GDP beta S = cGMP, which paralleled their ability to inhibit forskolin-stimulated adenylate cyclase activity. Saturation isotherms performed in the absence and presence of GTP and Gpp(NH)p indicate that the guanine nucleotide decreased the equilibrium dissociation constant (KD) but had no effect on the maximal binding (Bmax) of [3H]XAC. In contrast, Gpp(NH)p decreased agonist binding as manifested by a decrease in the percentage of A1 adenosine receptors in the agonist high affinity state (from 81% to 27%) without changing the high (KH) and low (KL) affinity constants. Kinetic experiments conducted to assess the effect of guanine nucleotide on [3H]XAC binding parameters demonstrate that Gpp(NH)p enhanced the observed rate of association (Kobs) of the radioligand with the receptor by 2-fold but had no effect on the rate of dissociation (K-1) of the radioligand-receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the third intracellular loop of the delta-opioid receptor inhibits signaling by opioid receptors and other G protein-coupled receptors

To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the delta-opioid receptor (delta-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In delta-i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the delta- and the mu-opioid (delta-OR and mu-OR) and attenuated opioid and alpha2-adrenergic receptor (alpha2AR)-dependent [35S]guanosine-5'-O-(3-thio)triphosphate binding. Furthermore, delta-i3L expression resulted in inhibition of delta-, mu-OR-, and alpha2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the beta2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of delta-i3L expression were selective for Gi/Go proteins. Moreover, although delta-i3L expression also attenuated drastically phospholipase C accumulation and Ca2+ release following mu- and delta-OR stimulation, it failed to inhibit carbachol-mediated stimulation of inositol phosphate accumulation in M1-muscarinic receptor-expressing human embryonic kidney 293 cells. Finally, we also examined the effects of delta-i3L expression on the regulation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway. Our results demonstrate that, although ERK activation by mu- and delta-ORs is attenuated by the presence of delta-i3L, ERK activation mediated by alpha2AR remained unaffected. Collectively, our data demonstrate that the delta-i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins.     

Relationship between rate and extent of G protein activation: comparison between full and partial opioid agonists.

Opioid agonists acting at their receptors alter intracellular events by initiating activation of various types of Gi/Go proteins. This can be measured by the binding of the stable GTP analog [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS). In this study agonist efficacy is defined by the degree to which an opioid stimulates the binding of [(35)S]GTPgammaS. This allows for a definition of full and partial agonists; a full agonist causing a greater stimulation of [(35)S]GTPgammaS binding than a partial agonist. The hypothesis that the rate of agonist-stimulated [(35)S]GTPgammaS binding is dependent upon agonist efficacy was tested using membranes from C6 glioma cells expressing mu- or delta-opioid receptors. At maximal concentrations the rate of agonist-stimulated [(35)S]GTPgammaS binding followed the efficacy of mu-agonists in stimulating [(35)S]GTPgammaS binding, i.e., [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin > morphine > meperidine > butorphanol > nalbuphine. At submaximal concentrations of mu- or delta-full agonists the [(35)S]GTPgammaS association rate was also reduced, such that the rate of [(35)S]GTPgammaS binding correlated with the extent of [(35)S]GTPgammaS bound, whether this binding was stimulated by a full agonist or a partial agonist. Agonists also stimulated [(35)S]GTPgammaS dissociation, showing that binding of this stable nucleotide was reversible. Comparison of the delta-agonists [D-Ser(2),Leu(5)]-enkephalin-Thr and (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, a compound with slow dissociation kinetics, showed the measured rate of G protein activation was not influenced by the agonist switching between receptors. The results are consistent with the idea that the active state(s) of the receptor induced by full or partial agonists is the same, but the number of activated receptors determines the rate of G protein activation.     

Heterogeneity of high-affinity nicotinic [3H]acetylcholine binding sites

Studies were conducted on high-affinity, nicotinic binding of 3H-labeled acetylcholine ([3H]ACh) to membrane preparations derived from the TE671 human clonal line and the PC12 rat pheochromocytoma. For comparative purposes and to extend results obtained by others in previous studies, [3H]ACh binding to membrane preparations derived from rat brain and from the electric tissue of Torpedo californica also was characterized. In each case, specific [3H]ACh binding (KD values of about 10 nM) could be fit by linear Scatchard and logit-log curves (slope of the latter of about 1.0) indicating that binding occurred to a single class of noninteracting sites, except that a better fit to PC12 cell membrane binding data was obtained using a two-site model. Quantitation of high-affinity binding sites for [3H]ACh and radiolabeled alpha-bungarotoxin and studies of unlabeled toxin competition for [3H]ACh binding indicated that toxin and agonist sites on TE671 cell or Torpedo membranes were closely related, but that toxin and agonist sites had limited or no physical overlap on PC12 cell or rat brain membranes. Generally, drugs of the bisonium and bis-choline series were more effective inhibitors of [3H]ACh binding to TE671 cell or Torpedo membranes, but nicotine and cytisine interacted with PC12 cell or rat brain sites with highest affinity. These results suggest that nicotinic agonists bind with high affinity to specific, membrane-bound sites on presumptive nicotinic ACh receptors expressed on each of these preparations. Moreover, the data are consistent with structural heterogeneity of these nicotinic receptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bidirectional allosteric effects of agonists and GTP at alpha(2A/D)-adrenoceptors

Agonists and GTP exert reciprocal effects on the stability of the G protein-coupled receptor/G protein complex, implying bidirectional control over the receptor/G protein interface. To investigate this relationship, we compared the ability of a series of hydroxyl-substituted phenethylamine and imidazoline agonists to stimulate [(35)S]guanosine 5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) binding in membranes from alpha(2A/D)-adrenergic receptor-transfected PC12 cells with the magnitude of the GTP-induced reduction in agonist affinity in [(3)H]rauwolscine-binding studies. Agents previously described as full and partial agonists in functional studies showed similar relative efficacies in promoting GTP binding (r = 0.97) as well as similar relative potencies (r = 0.94). Efficacy among agonists for promotion of [(35)S]GTPgammaS binding was closely correlated with the relative influence of GTPgammaS on agonist binding (r = 0.97), consistent with a bidirectional allosteric influence by agonists and GTP on receptor/G protein complexation. In an additional series of tolazoline derivatives, a range in efficacy from full agonism to strong inverse agonism was observed, depending on the presence or absence of hydroxyl substituents. Together these results suggest that agonist-induced repositioning of transmembrane helices via their hydroxyl interactions is a critical determinant of the stability of the receptor/G protein complex and therefore of agonist efficacy.