G protein dependent alterations in [125I]iodocyanopindolol and +/- cyanopindolol binding at 5-HT1B binding sites in rat brain membranes.

Several manipulations that affect G protein/receptor coupling also alter the binding of [125I]iodocyanopindolol ([125I]ICYP) and [corrected] +/- cyanopindolol (+/- CYP) to rat brain 5-HT1B binding sites in radioligand binding assays. Inclusion of 5 mM MgSO4 in these assays results in a small but significant increase in the affinity of [125I]ICYP (from KD = 0.046 nM to KD = 0.037 nM). In contrast, 100 microM Gpp(NH)p, GTP, or GDP reduce [125I]ICYP affinity (KD = 0.056 nM with GTP) while ATP and GMP are less effective. +/- CYP affinity for 5-HT1B sites labeled by [3H]dihydroergotamine [( 3H]DE) also displays a small but significant reduction (from Ki = 1.4 nM to Ki = 3.5nM) by the inclusion of 100 microM GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT1B specific binding of [125I]ICYP. The NEM induced reduction in [125I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [125I]ICYP to 5-HT1B sites is dependent on G proteins. The effects of Mg2+ ion, guanine nucleotides, NEM and G protein reconstitution on [125I]ICYP and +/- CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT1B sites.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states.

[3H]Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation [Bmax] = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of [3H]DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, [3H]DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. [3H]DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at [3H]DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Competition by the agonists 5-HT and RU 24969 at 5-HT1B sites are often best described by two site fits. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of [3H]DE. This suggests that the 5-HT1B site exists in two interconvertable agonist affinity states and is yet another member of the G-protein-linked receptor family. In agreement with previous studies using other radioligands, no 5-HT1B sites can be detected in bovine, porcine or human hippocampus membranes.

Ascorbic acid prevents nonreceptor "specific" binding of [3H]-5-hydroxytryptamine to bovine cerebral cortex membranes.

[3H]-5-Hydroxytryptamine ([3H]-5-HT) decomposes rapidly when exposed to air in solution at physiological pH if antioxidants are not present. The decomposition products appear to bind to two saturable sites on brain membranes (apparent Kd values = 1-2 and 100-1000 nM). This binding mimics "specific" ligand/receptor binding in that it is inhibited by 10 microM unlabeled 5-HT. This inhibition is not competitive, but rather is due to the prevention of [3H]-5-HT breakdown by excess unlabeled 5-HT. Unlike genuine ligand/receptor binding, the binding of [3H]-5-HT breakdown products is essentially irreversible and does not display a tissue distribution consistent with binding to authentic 5-HT receptors. [3H]-5-HT decomposition can be eliminated by the inclusion of 0.05 to 5 mM ascorbic acid. At these concentrations ascorbic acid is not deleterious to reversible [3H]-5-HT binding. When [3H] 5-HT exposure to air occurs in the presence of brain membranes, the apparent antioxidant activity of brain membranes themselves affords protection against [3H]-5-HT degradation equal to ascorbic acid. This protection is effective below final [3H]-5-HT concentrations of 10 nM. Above 10 nM [3H]-5-HT, addition of ascorbic acid or other antioxidants is necessary to avoid the occurrence of additional low affinity (apparent Kd = 15-2000 nM) binding sites that are specific but nonetheless irreversible. When care is taken to limit [3H]-5-HT oxidation, the only reversible and saturable specific binding sites observed are of the 5-HT1 high affinity (Kd = 1-2 nM) type. Radioligand oxidation artifacts may be involved in previous reports of low affinity (Kd = 15-250 nM) [3H]-5-HT binding sites in brain membrane preparations.