R. Benjamin Free

Ring E analogs of methyllycaconitine (MLA) as novel nicotinic antagonists.

We have prepared ring E analogs of the diterpenoid alkaloid methyllycaconitine. These compounds have been assayed for nicotinic activity and were found to act as functional antagonists on adrenal nicotinic receptors.

Structure–activity studies with ring E analogues of methyllycaconitine on bovine adrenal α3β4* nicotinic receptors

The development of new agents that selectively interact with subtypes of neuronal nicotinic receptors (nAChRs) is of primary importance for the study of physiological processes and pathophysiological conditions involving these receptors. Our laboratory has evidence that simple ring E analogues of methyllycaconitine (MLA) act as antagonists to bovine adrenal alpha3beta4* nAChRs. The following studies were designed to characterize the concentration-response effects of several ring E analogues of MLA in order to assess structural requirements involved with their inhibitory activity on bovine adrenal alpha3beta4* nAChRs. Ring E analogues with various substitutions on the ring E nitrogen were tested for their ability to inhibit nicotinic stimulated adrenal catecholamine release and [3H]epibatidine binding to a bovine adrenal membrane preparation. Several N-alkyl derivatives inhibited secretion with IC50 values in the low micromolar range. The N-phenpropyl analogue was the most potent of the analogues tested (IC50, 11 microM) on adrenal secretion. Competition binding studies suggest a noncompetitive interaction of the analogues with bovine adrenal nAChRs. These studies identify several structural features of ring E analogues of MLA which significantly affect their inhibitory activity on bovine adrenal alpha3beta4* nAChRs.

[3H]Epibatidine binding to bovine adrenal medulla: evidence for α3β4* nicotinic receptors

Abstract In these studies, [ 3 H]epibatidine is used as the radioligand to characterize nicotinic acetylcholine receptors (nAChRs) from bovine adrenal medulla. Specific binding reaches equilibrium within 30 min, and is saturable with a K d value of 0.5 nM. The affinities of several cholinergic agents were determined, including nicotine ( K i , 0.2 μM), cytisine ( K i , 0.4 μM), carbachol ( K i , 4.7 μM), dihydro-β-erythrodine ( K i , 33.6 μM), d -tubocurarine ( K i , 0.4 μM), 1,1-dimethyl-4-phenyl-piperazinium ( K i , 0.8 μM), decamethonium ( K i , 234 μM) and methyllycaconitine ( K i , 1.3 μM). These values are similar to reported values for recombinant α3β4 nAChRs in transfected cell lines. These studies demonstrate [ 3 H]epibatidine binding to an easily obtainable adrenal membrane preparation and support the characterization of adrenal nAChRs as α3β4* nAChRs.

Receptor protection studies to characterize neuronal nicotinic receptors: tubocurarine prevents alkylation of adrenal nicotinic receptors

Our laboratory has evidence that multiple nicotinic acetylcholine receptor subtypes regulate bovine adrenal catecholamine release. In the following studies, receptor protection assays were used to differentiate adrenal nicotinic receptor subpopulations. Under alkylating conditions, bromoacetylcholine (30 microM) reduced nicotinic receptor-stimulated adrenal catecholamine secretion by approximately 80%. When 100 microM tubocurarine was present during alkylation, nicotine-stimulated secretion was reduced by less than 30%. Hexamethonium (500 microM), decamethonium (500 microM), mecamylamine (50 microM), pentolinium (50 microM), adiphenine (50 microM), methyllycaconitine (1 microM) and alpha-bungarotoxin (1 microM) afforded no protection when present during alkylation. When the pharmacology of residual, tubocurarine-protected receptors was investigated, the EC50 value for nicotines stimulatory effects on secretion significantly increased from 4.0 (2.5-6.5) microM in control cells to 9.1 (7.2-11.4) microM in tubocurarine-protected cells. In addition, the IC50 value for tubocurarines inhibitory effects on release significantly decreased from 0.7 (0.5-0.9) microM in control cells to 0.3 (0.2-0.4) microM in tubocurarine-protected cells. These studies support the use of protection assays to characterize nicotinic receptor subpopulations.

Pharmacological characterization of recombinant bovine α3β4 neuronal nicotinic receptors stably expressed in HEK 293 cells

Abstract In these studies, [ 3 H]epibatidine is used as the radioligand to characterize recombinant bovine α3β4 nicotinic acetylcholine receptors (nAChRs) expressed in HEK 293 cells. Specific binding reaches equilibrium quickly and is saturable with a K d value of 0.66 nM. The affinities of the several cholinergic agents were determined, including nicotine ( K i , 0.5 μM), cytisine ( K i , 0.5 μM), carbachol ( K i , 4.1 μM), dihydro-(β)-erythroidine ( K i , 43.5 μM), d-tubocurarine ( K i , 0.1 μM), 1,1-dimethyl-4-phenylpiperazinium ( K i , 0.5 μM), decamethonium ( K i , 175 μM) and methyllycaconitine ( K i , 0.4 μM). These studies show that the pharmacological characteristics of recombinant bovine α3β4 nAChRs are similar to native bovine α3β4* nAChRs, and indicate that the α5 subunit, if present in the native nAChRs, does not affect ligand affinity.

Surface and intracellular nicotinic receptors expressed in intact adrenal chromaffin cells: direct measurements using [3H]epibatidine.

The presence and importance of assembled, intracellular neuronal nicotinic acetylcholine receptors (nAChRs) has not been established in native systems. In these studies [3H]epibatidine binding techniques were used to characterize surface and intracellular sites expressed in intact bovine adrenal chromaffin cells in culture. Permeant (300 microM nicotine) and impermeant (5 mM carbachol) cholinergic agents were used to define specific [3H]epibatidine binding to total (surface and intracellular) sites and surface sites, respectively. Intracellular [3H]epibatidine binding sites were characterized after eliminating surface binding sites via alkylation. Equilibrium binding to all sites was reached within 30 min at room temperature. Homologous (epibatidine) competition experiments on total (surface and intracellular) binding sites demonstrated a significant fraction of the high affinity sites were localized to intracellular compartments. Saturation binding assays to surface and intracellular sites revealed K(d) values of 1.9+/-1.1 and 3.6+/-1.9 nM, respectively. These binding studies document the existence of a significant population of high affinity, intracellular binding sites in native neuronal cells and support their characterization as assembled, alpha3beta4* nAChRs. Although the intracellular nAChRs represent approximately 70% of the total, high-affinity nAChRs expressed in cultured chromaffin cells, they do not appear to be involved in functional recovery after nAChR down-regulation.

Effects of sulfhydryl modification on adrenal nicotinic acetylcholine receptors: disulfide integrity is not essential for activation.

The importance of disulfide bridges in muscle nicotinic receptors is well established; however, for neuronal nicotinic receptors, the effects of sulfhydryl modification are less definitive. In these studies the effects of treatment with the mild reducing agent, dithiothreitol, on adrenal nicotinic receptors are described. We have found that following dithiothreitol treatment, adrenal chromaffin cells retained the ability to be stimulated by a variety of nicotinic receptor agonists including nicotine, acetylcholine, cytisine, epibatidine, and bromoacetylcholine. However, with dithiothreitol treatment, changes in apparent affinities were seen with two agonists, epibatidine and bromoacetylcholine. These effects of dithiothreitol on apparent affinities were concentration-dependent and reversible upon treatment with an oxidizing agent. Dithiothreitol treatment also produced effects on secretion that were independent of nicotinic receptor activation. Our results are unlike those in other tissues containing nicotinic receptors and suggest that subunit composition of nicotinic receptors influences the functional outcome of sulfhydryl modification.

Receptor protection studies comparing recombinant and native nicotinic receptors: Evidence for a subpopulation of mecamylamine-sensitive native α3β4* nicotinic receptors

Studies involving receptor protection have been used to define the functional involvement of specific receptor subtypes in tissues expressing multiple receptor subtypes. Previous functional studies from our laboratory demonstrate the feasibility of this approach when applied to neuronal tissues expressing multiple nicotinic acetylcholine receptors (nAChRs). In the current studies, the ability of a variety of nAChR agonists and antagonists to protect native and recombinant alpha3beta4 nAChRs from alkylation were investigated using nAChR binding techniques. Alkylation of native alpha3beta4* nAChRs from membrane preparations of bovine adrenal chromaffin cells resulted in a complete loss of specific [(3)H]epibatidine binding. This loss of binding to native nAChRs was preventable by pretreatment with the agonists, carbachol or nicotine. The partial agonist, cytisine, produced partial protection. Several nAChR antagonists were also tested for their ability to protect. Hexamethonium and decamethonium were without protective activity while mecamylamine and tubocurarine were partially effective. Addition protection studies were performed on recombinant alpha3beta4 nAChRs. As with native alpha3beta4* nAChRs, alkylation produced a complete loss of specific [(3)H]epibatidine binding to recombinant alpha3beta4 nAChRs which was preventable by pretreatment with nicotine. However, unlike native alpha3beta4* nAChRs, cytisine and mecamylamine, provide no protection for alkylation. These results highlight the differences between native alpha3beta4* nAChRs and recombinant alpha3beta4 nAChRs and support the use of protection assays to characterize native nAChR subpopulations.