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The irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine the relationship between receptor occupancy and response at central 5-hydroxytryptamine1A (5-HT1A) serotonin receptors mediating the inhibition of serotonin synthesis in rat cortex and hippocampus. Rats were treated with vehicle or EEDQ (2 or 6 mg/kg) and 24 hr later dose-response curves were constructed for inhibition of 5-hydroxytrytophan (5-HTP) accumulation (after decarboxylase inhibition with NSD-1015) by the selective 5-HT1A agonists 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT) (0.01-3 mg/kg), buspirone (0.1-7.5 mg/kg), and ipsapirone (0.1-6.25 mg/kg) and the 5-HT1A agonist/antagonist BMY 7378 (0.015-5 mg/kg). In vehicle-pretreated rats, a similar maximal inhibition of 5-HT synthesis (range, 52-59%) was observed in both brain areas with 8-OH-DPAT, buspirone, and ipsapirone. These three agonists were also more potent in reducing 5-HTP accumulation in the cortex than in the hippocampus (ED50, 8-OH-DPAT, 14 and 30 microgram/kg; buspirone, 0.42 and 0.63 mg/kg; ipsapirone, 0.44 and 1.26 mg/kg, respectively). In the cortex, EEDQ treatment shifted the dose-response curves for 8-OH-DPAT, buspirone, and ipsapirone 8.6-, 2.0-, and 2.8-fold to the right, respectively. Corresponding rightward shifts in the hippocampus were smaller, 6.0-, 1.6-, and 2.1-fold, respectively. The EEDQ-induced shifts in the dose-response curves were accompanied by reductions in maximal response. In contrast, whereas the maximal inhibition of cortical 5-HTP accumulation by BMY 7378 (55%) was similar to that obtained with the agonists, maximal response in the hippocampus was much smaller (32%). Furthermore, in both brain regions EEDQ reduced the maximal response to BMY 7378 without shifting the dose-response curves. Analysis of the data by the double-reciprocal method of Furchgott, followed by calculation of fractional receptor occupancy for each dose of agonist, revealed a nonlinear relationship between receptor occupancy and response for 8-OH-DPAT, buspirone, and ipsapirone in both brain regions, demonstrating the presence of a large receptor reserve. For BMY 7378, in contrast, linear relationships were obtained. Because 5-HT1A receptor-mediated regulation of 5-HT synthesis appears to be mediated by somatodendritic autoreceptors on 5-HT neurons in the midbrain raphé nuclei, the results suggest that these autoreceptors possess a large receptor reserve for agonists. The relevance of these findings for the mechanism of action of nonbenzodiazepine anxiolytics is discussed.

Incubation of rat striatal slices with forskolin (0.05-10 microM) elicited a dose-dependent increase in the activity of tyrosine hydroxylase (TH) assayed in subsequently solubilized extracts of the enzyme. At low concentrations (33 microM) of the cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride TH activity was increased 2.5 to 3-fold. Kinetic analysis of TH activity as a function of (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride concentration indicated that the enzyme isolated from control slices was composed of multiple species with different Km's for cofactor. Treatment with forskolin (1.5-15 microM) converted the enzyme into a single species with a low Km (28 microM) for (6R)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride. The dopamine (DA) agonist R-(-)-N-n-propylnorapomorphine (0.1 microM) reversed forskolin-induced activation of TH. Concentration-response curves were obtained for inhibition of forskolin-stimulated TH by R-(-)-N-n-propylnorapomorphine and the DA autoreceptor-selective agonists (+)- and (-)-3-(3-hydroxyphenyl)-N-n-propylpiperidine and 3-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl-1)-butyl]indole. R-(-)-N-n-propylnorapomorphine maximally inhibited forskolin-stimulated activity 85%, as indicated by ALLFIT computer analysis of concentration-response curves. (+)-3-(3-hydroxyphenyl)-N-n-propylpiperidine and 3-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl-1)-butyl]indole produced a lower degree of maximal inhibition (54 and 63%, respectively), whereas (-)-3-(3-hydroxyphenyl)-N-n-propylpiperidine was inactive. The D2 DA receptor blocker sulpiride (1 microM) competitively antagonised the effects of all the agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Akin to receptor inactivation with phenoxybenzamine (PBZ) (1 microM, 1 hr), treatment of anterior pituitary cells with 17 beta-estradiol (10 nM, 3 days) right-shifted the dose-response curve for inhibition of prolactin (PRL) secretion by the full agonist R-(-)-N-n-propylnorapomorphine (NPA) and reduced the maximal effect [EC50 (pM) and percent maximal effect: control, 25.4 and 81.2; PBZ, 115.3 and 57.9; 17 beta-estradiol, 358 and 58.6]. PBZ treatment of 17 beta-estradiol-pretreated cultures further reduced the maximal response but did not alter the EC50. Plots of receptor occupancy vs. response indicated a large receptor reserve for NPA (approximately 60%) in control cultures but its abolition by 17 beta-estradiol. 17 beta-Estradiol pretreatment elicited identical rightward shifts (4.5-fold) and similar reductions in maximal PRL inhibition by quinpirole and (+)-3-PPP, although these drugs were partial agonists with dissimilar efficacies relative to NPA (0.61 and 0.12, respectively) at presynaptic striatal D2 receptors. However, receptor inactivation experiments with (+)-3-PPP and quinpirole, and subsequent comparison of receptor occupancy vs. response plots, demonstrated that the relative efficacies of quinpirole and (+)-3-PPP were reversed in the striatum and anterior pituitary. In striatum, half-maximal response to quinpirole and (+)-3-PPP required 6.2 and 30% receptor occupancy, respectively, whereas 25.6 and 9.6% occupancy was required in the pituitary. Pertussis toxin treatment (10 ng/ml, 24 hr) produced large shifts in the dose-response curves for all three agonists (8.4-21.9-fold), but was distinguished from the effects of both PBZ and 17 beta-estradiol by a significant (P < .001) decrease in the slope factor.(ABSTRACT TRUNCATED AT 250 WORDS)

The irreversible dopamine (DA) receptor antagonist N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine the extent of receptor reserve at DA autoreceptors regulating in vivo tyrosine hydroxylase activity. Rats were treated with vehicle or EEDQ (1 X 0.5-2 X 6 mg/kg, subcutaneously) and, 24 hr later, dose response curves were generated for DA agonist reversal of gamma-butyrolactone-induced striatal L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation. Double reciprocal plots were obtained of equieffective doses of agonist required to elicit response at several levels of effect before and after partial irreversible receptor inactivation. A pseudo-dissociation constant (pseudo-KA, in units of dose) and the fraction of receptors remaining active (q) were determined; these values were then used to calculate the relationship between receptor occupancy and response. The ED50 (1 microgram/kg) for the full DA receptor agonist N-propylnorapomorphine (NPA) was shifted 2.8, 4.8-, and 11.3-fold to the right after partial irreversible receptor blockade which left the fraction of receptors remaining active (q) at 0.37, 0.17 and 0.058, respectively. Corresponding maximal reversal of L-DOPA accumulation was 100, 77, and 58%, indicating a nonlinear relationship between receptor occupancy and response for NPA and the presence of a large receptor reserve; maximal and half-maximal responses were calculated to require occupancy of 30 and 3.8% of the total receptor pool, respectively. Dose response curves were also obtained for the DA autoreceptor-selective agents EMD 23,448 and (+)- and (-)-3-PPP before and after EEDQ treatment. In controls, EMD 23,448 and (+)-3-PPP, like NPA, completely reversed striatal gamma-butyrolactone-induced L-DOPA accumulation, whereas the maximal effect of (-)-3-PPP was 52% reversal. After EEDQ treatment (6 mg/kg), EMD 23,448 and (+)-3-PPP showed relatively small shifts in ED50 values. Furchgott analysis demonstrated that all three atypical agents are partial agonists at the DA autoreceptor with efficacies of 0.19 (EMD 23,448), 0.12 [(+)-3-PPP], and 0.05 [(-)-3-PPP] relative to NPA. The presence of a larger receptor reserve at pre-versus postsynaptic D2 DA receptors and the partial agonist character of drugs such as EMD 23,448 and the enantiomers of 3-PPP may account for their autoreceptor selectivity.

Treatment of rats with the serotonin 5-HT1A agonist 8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT, 1 mg/kg s.c.) markedly elevated plasma levels of corticosterone (CORT), adrenocorticotropic hormone (ACTH) and prolactin (PRL); the levels of growth hormone were unaffected. Pretreatment with the irreversible receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 6 mg/kg s.c.) greatly attenuated the increase in plasma CORT produced by 8-OH-DPAT (0.3 mg/kg s.c.). Prevention of EEDQ-induced 5-HT1A receptor inactivation by prior treatment with the reversible mixed 5-HT1A/beta-adrenergic antagonist (+/-)pinodolol (30 mg/kg s.c.) blocked the reduction of the CORT response to 8-OH-DPAT. In contrast, prevention of EEDQ-induced inactivation of 5-HT2, alpha-1- and alpha-2-adrenergic and D1 and D2 dopamine receptors by a cocktail of selective antagonists of these receptors did not block the attenuation of the CORT response to 8-OH-DPAT. Dose-response curves were obtained for 8-OH-DPAT (0.01-3 mg/kg s.c.)-induced elevation of plasma CORT, ACTH and PRL after treatment (24 hr earlier) with vehicle or EEDQ (6 mg/kg s.c.) and analyzed for the extent of receptor reserve. Whereas substantial receptor reserves were observed for the 8-OH-DPAT rise in plasma CORT (80%) and ACTH (50%), no receptor reserve was seen for the increase in plasma PRL. The results are discussed with regard to potential differences in the receptors, G proteins, effectors and/or stoichiometric relationships between these components of the signal transduction pathway, leading to elevation of these plasma hormones after treatment with 8-OH-DPAT.

Treatment of rats with the peptide coupling agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6 mg/kg i.p.) irreversibly reduced the binding of [3H]spiperone ([3H]SPIP) and cis-[3H] piflutixol to striatal D2 and D1 receptors, respectively, by 70 to 75%. In each instance only the receptor density was affected, without a change in the dissociation constant (Kd) of either radioligand. Pretreatment with sulpiride (200 mg/kg i.p.), a selective D2 antagonist, preferentially protected [3H]SPIP sites against N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline-induced inactivation, whereas pretreatment with SCH 23390 (3 mg/kg i.p.), a putative selective D1 antagonist, preferentially blocked the inactivation of cis-[3H]piflutixol binding sites. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline markedly reduced radioligand binding to cortical alpha-1 ([3H]prazosin) and alpha-2 [( 3H]yohimbine) receptors (10-20% of control) but had a lesser effect on serotonin-2 ([3H]SPIP) and serotonin-1 ([3H]5-HT) receptors (30-40% of control). Muscarinic cholinergic ([3H] quinuclidinyl benzilate) and beta adrenergic ([3H]dihydroalprenolol) receptors were only slightly affected. None of these nondopaminergic sites were protected by sulpiride or SCH 23390, with the exception of serotonin-2 and serotonin-1 which were partially protected by the latter. SPIP (0.2 mg/kg i.p.), haloperidol (1 mg/kg i.p.) and pimozide (2 mg/kg i.p.) all selectively protected the D2 receptor, whereas cis-flupenthixol (2 mg/kg i.p.) protected both dopamine receptors; its inactive isomer trans-flupenthixol (20 mg/kg i.p.) protected neither. Bulbocapnine (25 mg/kg s.c.) selectively, but partially, protected the D1 site.(ABSTRACT TRUNCATED AT 250 WORDS)