M2-muscarinic receptors: how does ligand binding affinity relate to intrinsic activity?

In this study we looked for evidence regarding a correlation between M2-muscarinic receptor binding affinity and ligand intrinsic activity. Guanine nucleotide-binding protein-coupled receptors have been shown to exist in both a high affinity and a low affinity, agonist state. The agonist [3H]Oxotremorine-M, was used to determine the affinity of compounds for the high affinity state and the antagonist, [3H]N-methylscopolamine, plus GppNHp, was used to determine the affinity for the low agonist state. The magnitude of the difference in the affinity a compound has for the high versus the low agonist state of the receptor has been related to the intrinsic activity of the compound. NMS/Oxo-M ratios were established for muscarinic agonists, partial agonists and antagonists. NMS/Oxo-M ratios varied from 1695 for the agonist carbachol to 1.9 for the antagonist AFDX-166 with intermediate values for the partial agonists oxotremorine-M, pilocarpine and RS86 (233, 36 and 17 respectively). Intrinsic activity was assessed by receptor-mediated Gi-protein GTPase activity. Indeed, a close correlation (r=0.92) was found between the NMS/Oxo-M ratios of the ligands on the one hand, and their ability to activate the M2-receptor coupled Gi-protein on the other.

The effects of radical stress and N-ethylmaleimide on rat hepatic alpha 1-adrenergic receptors.

Liver plasma membranes of non-treated rats or rats treated with carbon tetrachloride were prepared. Liver plasma membranes obtained from non-treated rats were preincubated with cumene hydroperoxide, N-ethylmaleimide or hydrogen peroxide in the absence or presence of Fe2+. The effects of in-vivo or in-vitro treatment on hepatic alpha 1-adrenergic receptors were assessed in a [3H]prazosin binding assay. All treatments except preincubation with hydrogen peroxide alone caused a decrease in the number of alpha 1-adrenergic receptors without a change in the affinity of [3H]prazosin. The decrease in receptor number was accompanied by an increase in the level of lipid peroxidation in the plasma membranes. The results indicate that the hepatic alpha 1-adrenergic receptor is vulnerable to free radical stress.

Mu-, delta- and kappa-opioid receptor-mediated inhibition of neurotransmitter release and adenylate cyclase activity in rat brain slices: studies with fentanyl isothiocyanate.

We investigated the effects of [D-Ala2,D-Leu5]enkephalin (DADLE). [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) (0.01-1 microM) and bremazocine (0.001-0.3 microM) on the electrically evoked release of radiolabelled neurotransmitters and on the dopamine (DA)-stimulated cyclic AMP efflux from superfused rat brain slices. The differential inhibitory effects of these agonists on the evoked neurotransmitter release indicate that the opioid receptors mediating presynaptic inhibition of [3H]noradrenaline (NA, cortex), [14C]acetylcholine (ACh, striatum) and [3H]DA (striatum) release represent mu, delta and kappa receptors, respectively. In agreement with this classification, preincubation (60 min) of the slices with the delta-opioid receptor-selective irreversible ligand, fentanyl isothiocyanate (FIT, 0.01-1 microM), antagonized the inhibitory effects of DADLE and DPDPE on striatal [14C]ACh release only. On the other hand, the D-1 DA receptor-stimulated cyclic AMP efflux from striatal slices appeared to be inhibited by activation of mu as well as of delta receptors. In this case, the reversible mu antagonist, naloxone (0.1 microM), fully antagonized the inhibitory effect of the mu agonist, DAGO, without changing the effect of the delta agonist DPDPE but was ineffective as an antagonist in slices pretreated with FIT (1 microM). The inhibitory effect of DAGO on the electrically evoked [3H]NA release was antagonized by naloxone whether the receptors were irreversibly blocked by FIT or not. These data not only further support the existence of independent presynaptic mu-, delta- and kappa-opioid receptors in rat brain but also evidence strongly that mu and delta receptors mediating the inhibition of DA-sensitive adenylate cyclase could share a common binding site (for naloxone and FIT) and, therefore, may represent constituents of a functional opioid receptor complex.