Cooperativity has been investigated as the mechanistic basis for effects observed with cardiac muscarinic receptors in washed membranes from Syrian hamsters. Specifically, N-[3H]methylscopolamine labeled only 66-75% of the sites labeled by [3H]quinuclidinylbenzilate at apparently saturating concentrations of each radioligand. Also, receptors labeled by N-[3H]methylscopolamine revealed three states of affinity for agonists, both in native membranes and following irreversible blockade of about 80% of the sites by propylbenzilylcholine mustard; in both preparations, guanylylimidodiphosphate (GMP-PNP) effected an apparent interconversion of sites from higher to lower affinity for agonists and from lower to higher affinity for the antagonist. Excellent and mechanistically consistent descriptions of the data were obtained in terms of a model comprising cooperative and noncooperative forms of the receptor; the former was described by a variant of the Adair equation, and the latter was included to account for low-affinity sites that survived treatment with the mustard. If differences in apparent capacity derive from negative cooperativity in the binding of N-[3H]methylscopolamine, the cooperative form of the receptor was at least trivalent in native membranes; otherwise, constraints imposed by the effects of GMP-PNP at the concentrations of radioligand used in the assays dictate that the cooperative form of the receptor was at least tetravalent. In contrast, a divalent receptor is sufficient with the data from alkylated membranes, in accord with the reduced likelihood of interactions between functional sites within an oligomeric array. A model is presented wherein the receptor interconverts spontaneously between two or more states differing in their cooperative properties. The effects of GMP-PNP can be rationalized as a shift in the equilibrium between the different states.

An expanded version of the mobile receptor model has been assessed in studies on the binding of N-[3H]methylscopolamine and [35S]GTPgammaS to cardiac muscarinic receptors and their attendant G proteins in ventricular membranes from hamster. The model comprises two pools of receptor, one of which lacks G proteins, and a heterogeneous population of G proteins that compete for the receptor within the G protein-containing pool. To guide the formulation of the model itself and to define the various parameters, data were combined from assays performed under various conditions with native membranes and following irreversible blockade of about 80% of the receptors with propylbenzilylcholine mustard. Multiple G proteins are indicated primarily by multiple states of affinity evident in the dose-dependent effect of guanyl nucleotides on the binding of carbachol; G protein-free receptors are indicated by sites of low affinity for carbachol that survive treatment with the mustard. The expanded model generally succeeds where more frugal schemes have been inadequate, but it nevertheless fails to yield a mechanistically consistent description of the data. Guanyl nucleotides and partial alkylation do not affect the inhibitory potency of carbachol in a manner consistent with their supposed effect on the equilibrium between uncoupled and G protein-coupled receptors. As inferred from the model, G proteins are lost upon alkylation of the receptor, and their numbers are regulated by guanyl nucleotides. Parameters estimated via N-[3H]methylscopolamine are wholly inconsistent with the same parameters estimated via [35S]GTPgammaS. The failure of the model suggests that multiple states of affinity may not arise from a ligand-regulated equilibrium between free receptors and G proteins on the one hand and one or more RG complexes on the other.

Recent work indicates that the therapeutic action of lithium may be mediated through perturbation of postreceptor second messenger systems. To elucidate further the postreceptor cellular sites of action(s) of lithium, the effect of chronic lithium treatment on various components of the receptor-activated phosphoinositide pathway was investigated. We found that chronic administration of lithium (0.2% LiCl, 21 days) to adult male rats did not significantly affect phosphoinositide hydrolysis in cerebral cortical slices induced by carbachol (1 mM) or NaF (10 mM). Nor did the same treatment alter the carbachol (1 mM) potentiation of guanosine 5'-(gamma-thio)triphosphate (30 microM) stimulation of phosphoinositide hydrolysis (an index of receptor/G protein coupling) in cortical membranes. Immunoblotting studies revealed no changes in the levels of G alpha q/11 immunoreactivity in the cortex after chronic lithium treatment. The levels of protein kinase C, as revealed by specific binding of [3H]phorbol dibutyrate ([3H]PDBu), were significantly reduced in the cytosolic fraction and increased in the particulate fraction of rat cortex after chronic lithium, whereas the KD of [3H]PDBu binding remained relatively constant. A small and insignificant decrease in the density of [3H]inositol 1,4,5-trisphosphate binding was also found in the cortex. The above data suggest that chronic lithium treatment affects neither the muscarinic cholinergic-linked phosphoinositide turnover nor the putative G protein alpha subunit (G alpha q/11) responsible for phospholipase C activation. However, a possible translocation and activation of protein kinase C activity may be significant in the therapeutic effect of this mood-stabilizing agent.

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