Dopamine receptor occupancy in vivo: measurement using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivates a variety of monoamine neurotransmitter receptors. In this report, protection against EEDQ-induced inactivation of D-1 and D-2 DA receptors by DA antagonists and agonists was used to obtain a measure of occupancy of these receptors in vivo by such drugs. Rats were pretreated with drugs and then given EEDQ (10 mg/kg, i.p.). Twenty-four hours after the EEDQ injections, the animals were decapitated and the number of receptors remaining was measured using conventional receptor binding assays. The D-1 antagonist SCH 23390 potently protected D-1 sites from EEDQ-induced inactivation in a dose-dependent manner. Similarly, NO-756, another D-1 antagonist, selectively protected D-1 sites from inactivation. Conversely, haloperidol, a relatively selective D-2 antagonist, protected D-2 sites from inactivation. Likewise, a number of antipsychotic DA antagonists also protected D-2 sites from inactivation. Clozapine, fluperlapine, and (+) butaclamol were effective at protecting both D-1 sites and D-2 sites. In addition, the D-1 agonist SKF 38393 protected D-1 sites from EEDQ-induced inactivation, whereas the D-2 agonist quinpirole protected D-2 sites. (-) Apomorphine, a mixed D-1/D-2 agonist, protected both sites. Thus, this type of method provides a simple means of evaluating the occupation of DA receptors by DA antagonists and agonists in vivo.     

A kinetic and binding study of the reactivity of Escherichia coli ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

Escherichia coli H+-ATPase (ECF1) was inactivated in a time- and concentration-dependent manner by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a selective carboxyl group reagent. Among the subunits of ECF1, only the beta subunit was modified by EEDQ. The reaction of 1 mol of EEDQ per mol of ECF1 resulted in total inactivation, in spite of the fact that the enzyme possesses three beta subunits.     

Inactivation of the renal outer cortical brush-border membrane D-glucose transporter by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

The inactivation of the renal outer cortical brush-border membrane D-glucose transporter by the covalent carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is studied by monitoring its effects on sodium-dependent phlorizin binding to the active site of the carrier. In the presence of EEDQ, this component of phlorizin binding decreases exponentially and irreversibly with time. The order of this inactivation reaction is very close to 1, indicating that EEDQ modifies the transporter at a single essential site. This site can be partially protected by glucose and by other substrates of the transporter and completely protected by phlorizin, a nontransported competitive inhibitor. By contrast, sodium, a co-transported activator, has no protective effect. The concentration dependence of the protection provided by glucose and phlorizin indicates that the site of action of EEDQ is at or closely related to the substrate binding site on the carrier. The effects of EEDQ on the transporter are mimicked by another carboxyl specific reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The rate of inactivation of the transporter by EEDQ increases dramatically with decreasing pH, consistent with the hypothesis that the rate-limiting step in the inactivation process is a reaction with an essential carboxyl group. The properties of this group indicate, however, that it is distinct from the carboxyl group proposed by others as forming (a part of) the sodium binding site of sodium-coupled sugar carriers.     

Reactivity of gastric (H+ + K+)-ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

The K+-dependent ATPase and p-nitrophenyl phosphatase activity of, and formation of phosphoenzyme by, hog parietal cell membranes were inhibited in a time- and concentration-dependent manner by the carboxyl-activating reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The kinetics of inactivation was pseudo first order and was similar to the EEDQ-catalyzed incorporation of [14C]glycine ethyl ester. The most likely mechanism is the EEDQ-dependent formation of inter- and intramolecular amide bonds. Cross-linking between the subunits of the ATPase occurs with EEDQ treatment. The presence of K+ on the luminal face of the enzyme is able to prevent EEDQ inhibition of K+ ATPase activity (but not intermolecular cross-linking), whereas ATP enhanced the rate of inactivation. EEDQ reaction with the enzyme therefore allows investigation of K+- and ATP-dependent states of the enzyme.     

Influence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline modification on proton translocation and membrane potential of reconstituted cytochrome-c oxidase support "proton slippage".

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.     

Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [3H]SCH23390 and [3H]forskolin binding in rat striatum

The binding of [3H]forskolin to a homogeneous population of binding sites in rat striatum was enhanced by NaF, guanine nucleotides and MgCl2. These effects of NaF and guanylylimidodiphosphate (Gpp(NH)p) were synergistic with MgCl2, but NaF and Gpp(NH)p together elicited no greater enhancement of [3H]forskolin binding. These data suggest that [3H]forskolin may label a site which is modulated by the guanine nucleotide regulatory subunit which mediates the stimulation of adenylate cyclase (NS). The D1 dopamine receptor is known to stimulate adenylate cyclase via NS. In rat striatum, the Bmax of [3H]forskolin binding sites in the presence of MgCl2 and NaF was approximately two fold greater than the Bmax of [3H]SCH23390-labeled D1 dopamine receptors. Incubation of striatal homogenates with the protein modifying reagent EEDQ elicited a concentration-dependent decrease in the binding of both [3H]SCH23390 and [3H]forskolin, although EEDQ was approximately 14 fold more potent at inactivating the D1 dopamine receptor. Following in vivo administration of EEDQ there was no significant effect on [3H]forskolin binding sites using a dose of EEDQ that irreversibly inactivated greater than 90% of D1 dopamine receptors. These data suggest that EEDQ is a suitable tool for investigating changes in the stoichiometry of receptors and their second messenger systems.     

Differential protection and recovery of 5-HT1A receptors from N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivation in regions of rat brain

The effect of N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) on 5-HT_1A receptors was studied in Sprague Dawley rats. A single dose of EEDQ (4 mg/kg body wt., i.p.) significantly inactivated 5-HT_1A receptors, as measured by [³H]8-hydroxy-2-[di-n-propylamino]-tetralin ([³H]8-OH-DPAT), in cortex (64%, p < 0.0001) and hippocampus (48%, p < 0.0001). A significant (p < 0.01) increase in the affinity of 5-HT_1A receptors for radioligand was observed in both regions. A dose dependent protection of cortical 5-HT_1A receptors from EEDQ inactivation with pre-treatment of different doses of 8-OH-DPAT (4–20 mg/kg) was observed, along with recovery of affinity of [³H]8-OH-DPAT for 5-HT_1A receptors in both regions. Although, a dose of 4 mg/kg of 8-OH-DPAT failed to attenuate the effect of EEDQ on hippocampal 5-HT_1A receptors, a significant protection of these receptors was observed with 10 and 20 mg/kg of 8-OH-DPAT. Displacement studies revealed that EEDQ has more affinity for cortical (Ki = 101.3 ± 11.8 nM) than hippocampal (Ki = 133.5 ± 25.8 nM) 5-HT_1A receptors. A time dependent natural recovery of 5-HT_1A receptors from inactivation by a single dose of EEDQ (4 mg/kg) was observed more in cortex compared to hippocampus over a period from 1 day to 14 days. The results of this study suggest that 8-OH-DPAT inhibited EEDQ inactivation of cortical and hippocampal 5-HT_1A receptors in a concentration dependent manner. The synthesis and turnover of 5-HT_1A receptors differ in cortex and hippocampus, as evident by earlier recovery in the cortex.     

Effect of aging on recovery of striatal dopamine receptors following n-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) blockade

The recovery rate of striatal dopamine receptors following blockade by N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) $\text{in vivo}$ is reduced by 25–35% in striata from senescent Wistar rats when compared to mature counterparts. No differences in binding affinity for [³H]-spiperone were observed for the different age groups at various times after EEDQ injection. These results suggest that loss of striatal dopamine receptors during aging may be due to a decreased biosynthetic rate.     

Mitochondrial nicotinamide nucleotide transhydrogenase: nonidentical modification by N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline at the NAD(H) binding site

The energy-linked nicotinamide nucleotide transhydrogenase (TH) purified from bovine heart mitochondria is inhibited by the carboxyl group modifiers, N,N'-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ). With either reagent, complete activity inhibition corresponds to modification of one carboxyl group per 2 mol (monomers) of this dimeric enzyme, suggesting half-site reactivity toward DCCD and EEDQ [D. C. Phelps, and Y. Hatefi (1984) Biochemistry 23, 4475-4480; 6340-6344]. It has also been shown in the former reference that DCCD appears to modify TH at the NAD(H)-binding site. The present paper presents data suggesting that EEDQ also binds at or near the NAD(H)-binding domain of TH, but at a site not identical to that of DCCD: TH modified with and inhibited approximately 85% by EEDQ could be further labeled with [14C]DCCD to the extent of 70% of the maximum in the same time period that unmodified TH was modified by [14C]DCCD to near saturation (1 mol DCCD/TH dimer); DCCD-modified TH did not bind to NAD-agarose, while EEDQ-modified TH showed partial affinity for NAD-agarose; 5'-AMP completely protected TH against modification by DCCD, but showed only a weak protective effect against EEDQ; by contrast, NMNH, which is a TH substrate and binds to the NADH site, did not protect TH against DCCD, but completely protected the enzyme against attack by EEDQ. The results are consistent with the possibility that DCCD modifies TH where the 5'-AMP moiety of NAD(H) binds, while EEDQ modifies the enzyme where the NMN(H) moiety of NAD(H) resides.     

Interactions of Neurospora crassa plasma membrane H+-ATPase with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The carboxyl group activating reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) interacts with the Neurospora plasma membrane H+-ATPase in at least three different ways. This reagent irreversibly inhibits ATP hydrolysis with kinetics that are pseudo-first-order at several concentrations of EEDQ, and an appropriate transform of these data suggests that 1 mol of EEDQ inactivates 1 mol of the H+-ATPase. Inhibition probably involves activation of an ATPase carboxyl group followed by a nucleophilic attack by a vicinal nucleophilic functional group in the ATPase polypeptide chain, resulting in an intramolecular cross-link. The enzyme is protected against EEDQ inhibition by MgATP in the presence of vanadate, a combination of ligands that has previously been shown to "lock" the H+-ATPase in a conformation that presumably resembles the transition states of the enzyme phosphorylation and dephosphorylation reactions, but is not protected by the substrate analogue MgADP, which is consistent with the notion that one or both of the residues involved in the EEDQ-dependent inhibitory intramolecular cross-linking reaction normally participate in the transfer of the gamma-phosphoryl group of ATP, or are near those that do. The ATPase is also labeled by the exogenous nucleophile [14C]glycine ethyl ester in an EEDQ-dependent reaction, and the labeling is diminished in the presence of MgATP plus vanadate. However, peptide maps of [14C]glycine ethyl ester labeled ATPase demonstrate that the labeling is not related to the EEDQ inhibition reaction in any simple way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of solubilization on the inhibition of the p-type ATPase from maize roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.     

Behavioral recovery after irreversible inactivation of D-1 and D-2 dopamine receptors

Irreversible inactivation of both D-1 and D-2 dopamine (DA) receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in complete loss of stereotypy response to R-(-)-N-propylnorapomorphine (NPA; 0.1-1.0 mg/kg, s.c.) 24 hr later. Stereotyped sniffing recovered much more rapidly than oral behaviors. The D-2 antagonist sulpiride (200 mg/kg) and the putatively nonselective antagonist cis-flupenthixol (2 mg/kg), administered prior to EEDQ, prevented the loss of NPA-induced sniffing but only partially protected against loss of oral behaviors 24 hr later. Complete protection of both behaviors was seen after pretreatment with a combination of sulpiride and the selective D-1 antagonist SCH 23390 (1 mg/kg); pretreatment with the selective D-1 antagonist SCH 23390 alone, however, did not modify the rate of recovery of either behavioral response. The results suggest that either different populations of DA receptors mediate expression of these behaviors or stimulation of a small fraction of the total DA receptor pool may be sufficient to elicit sniffing but not oral responses. Furthermore, maintaining a normal complement of D-2 rather than D-1 receptors appears to be a critical determinant for the elicitation of these behaviors.     

Radioiodinated 2'-iodospiperone: a new radioligand for in vivo dopamine receptor study

In vivo dopamine receptor binding of the newly synthesized ligand, 125I-2'-iodospiperone (125I-2'-ISP), was studied in mouse brain. The highest accumulation was found in the striatum. Analysis of the striatal homogenate showed the 125I-2'-ISP to be metabolically stable. Furthermore, this striatal binding was saturable and displaced only by dopaminergic drugs. On the other hand, the accumulation in the cortex was as low as that of the cerebellum and uneffected by the administration of serotoninergic drugs and dopaminergic drugs; results assessed by macroautoradiographic studies. Thus, the newly synthesized 125I-2'-ISP presented high affinity for dopamine receptors in vivo and therefore, holds great potential for the in vivo dopamine receptor studies, provided 123I becomes readily available.     

Behavioral control by striatal adenosine A2A‐dopamine D2 receptor heteromers

G protein‐coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A_2A receptors (A_2AR) and dopamine D₂ receptors (D₂R) predominantly form A_2AR‐D₂R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A_2AR and D₂R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain‐related differences, a new D₂R‐deficient mouse with the same genetic background (CD‐1) than the A_2AR knock‐out mouse was generated. Locomotor activity, pre‐pulse inhibition (PPI) and drug‐induced catalepsy were then evaluated in wild‐type, A_2AR and D₂R knock‐out mice, with and without the concomitant administration of either the D₂R agonist sumanirole or the A_2AR antagonist SCH442416. SCH442416‐mediated locomotor effects were demonstrated to be dependent on D₂R signaling. Similarly, a significant dependence on A_2AR signaling was observed for PPI and for haloperidol‐induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A_2AR‐D₂R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders.     

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D2 receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [3H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [3H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [3H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [3H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D2 receptor and should prove useful in studies on functional reconstitution of the receptor.