Nongenomic effects of steroids on the nicotinic acetylcholine receptor.

A fast signaling mode of natural and synthetic steroids is exerted on some ion channels and cell-surface receptors. This activity contrasts with their classic mode of action, via intracellular receptors. Early studies from our laboratory demonstrated that spin-labeled androstanol and cholestane interact with the nicotinic acetylcholine receptor (AChR) and that lipid mobility at the lipid belt surrounding the AChR is reduced relative to that of the bulk membrane lipid. The occurrence of discrete and independent sites for phospholipids and sterols, both accessible to fatty acids, was subsequently disclosed in the native membrane. Synthetic and natural glucocorticoids were found to act as noncompetitive inhibitors of AChR function. The influence of different substituent groups in the cyclepentane perhydrophenanthrene ring on the channel-shortening potency of various steroids has also been assayed in muscle-type AChR, and we found a certain selectivity of this effect. Some organochlorine pesticides are xenoestrogens, that is, environmental agents capable of disrupting endocrine system signaling. We determined their effects on the AChR membrane using novel fluorescence techniques.

Effect of chemical modification of extracellular histidyl residues on the channel properties of the nicotinic acetylcholine receptor.

We have examined the effect of chemical modification with diethyl pyrocarbonate (DEP) on the properties of acetylcholine (ACh)-activated channels in the cloned muscle-cell line BC3H-1. After protein modification, patch-clamp recordings showed alterations in the kinetics of the nicotinic acetylcholine receptor (AChR) channel. The major effect was observed in the channel mean open time, which was reduced up to about 12-fold at 466 microM DEP. The specificity of the effect was first established through comparison with both untreated cells and cells treated with inactivated DEP. Consistent with an increase in the number of unprotonated histidine residues (pKa = 6.0), this effect increased concomitantly with the pH of the reaction medium, being faster at pH 8 than at pH 6. The changes were dependent on time and DEP concentration, with an apparent EC50 = 114 microM. Modified channels also showed an increase in the number of events per burst of openings together with a decrease in burst durations. The amplitude of the channel-closed time component of about 1 ms increased with respect to the longest-duration-closed component. The number of alpha-bungarotoxin sites was slightly reduced after the modification, without affecting ligand binding affinity. The results suggest that DEP affects extracellular histidine residues involved in the ion translocation function of the AChR, but not its toxin-recognition ability. DEP could, therefore, induce a dissociation between toxin and agonist binding, as is often observed in neuronal AChR.

Effects of long-chain fatty acids on the channel activity of the nicotinic acetylcholine receptor.

We have analyzed the effect of free fatty acids on the function of the acetylcholine receptor (AChR) at the single-channel level, using the patch-clamp technique. Long-chain fatty acids, in the presence of albumin as a carrier, were applied to intact cells or to the cytoplasmic surface of excised membrane patches. In the latter case, AChR channels underwent immediate changes in their behaviour and only very brief opening events were apparent. This could be accounted for by a four-fold reduction in the channel mean open time, with no significant changes occurring in the conductance. An increase in the duration of intermediate closed intervals and a decrease in the burst duration were also observed. The modification appeared not to be critically dependent on the degree of saturation of fatty acyl chains. Addition of free fatty acids in the absence of albumin, as well as treatment of the excised membrane patches with phospholipase A2, resulted in complete inhibition of AChR channel activity. In intact cells, fatty acids could reach and affect AChR channels in the plasmalemma under the patch pipette when added from outside the patch-clamped area. The fatty acid-modified AChR was still able to undergo desensitization. The open channel probability was higher than 0.8 for 100 microM agonist, decreasing to 0.4 in the fatty acid-modified receptors. The results are discussed within the framework of the hypothesis that some lipophilic compounds exert their action on the AChR-lipid interface (the "annulus") (see Barantes, 1993).

Myogenic differentiation of the muscle clonal cell line BC3H-1 is accompanied by changes in its lipid composition.

Phospholipid and neutral lipid composition was studied in the course of myogenic differentiation of the clonal cell line BC3H-1. Total phospholipid content increased during differentiation, predominantly in the major classes of choline and ethanolamine glycerophospholipids. The contents of other lipids, such as triacylglycerols, diminished more than 50% during this period. The content and distribution of fatty acids also underwent marked differentiation-dependent changes. The polyunsaturated (tetrapenta- and hexaenoic) fatty acid species of several phospholipid classes diminished during differentiation, especially those in choline, serine and inositol glycerophospholipids. Most noticeable were the changes in phosphatidylserine; long-chain fatty acids having 20 to 22 carbon atoms and 4 to 6 double bonds decreased from about 30 to about 10 mol%. Although increased levels of saturation in other phospholipid fatty acyl chains appear to accompany the myogenic changes of BC3H-1 cells, some unsaturated fatty acids, such as oleic acid (18:1), increased by as much as 80% during the same period, suggesting the activation of a delta 9 desaturase. Sphingomyelin contained only saturated and monoenoic fatty acids and exhibited a four- to five-fold decrease in its content of monoenoic acyl groups. Diacylglycerols became enriched in arachidonate and docosahexaenoate. The amount of cholesterol and its esters increased slightly during differentiation of BC3H-1 cells. The data show that several metabolic pathways change during myogenic differentiation of the BC3H-1 clonal cell line, particularly de novo biosynthetic pathways, elongation/desaturation reactions, and acyl chain turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic cholinergic receptor of rat cerebral cortex. Location and characterization of ligand binding site-carrying peptides in synaptosomal membranes and isolated neuronal perikarya.

A careful examination of the location and biochemical properties of the tryptic peptides identified by site-specific labelling of the muscarinic cholinergic receptor (mAChR) of rat cerebral cortex has been carried out. In brain synaptosomal membranes and isolated neuronal perikarya, mAChR labelled with [3H]propylbenzilylcholine mustard (PrBCM) was tryptically cleaved to peptides of Mr 50,000, 30,000. 18,000 and a limiting fragment of Mr 8000. All of these binding site-carrying fragments, characterized in terms of their content of carbohydrates and thiol groups, were quantitatively recovered as membrane-bound peptides. The delipidated [3H]PrBCM-labelled tryptic limiting fragment was found to be highly hydrophobic and insoluble in aqueous media. Experiments performed with proteinase on the tryptic limiting fragment suggest the existence of an ester linkage between the ligand and the peptide. The results strongly support the hydropathicity profile which predicts the location of the muscarinic receptor protein with respect to the membrane bilayer.

Distance between the propylbenzilylcholine mustard attachment site and carbohydrates and thiol groups in muscarinic acetylcholine receptor protein from rat cerebral cortex.

When rat cerebral-cortex membranes were labelled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), a single protein of Mr 68,000 was found to carry the atropine-sensitive covalent label. After trypsinolysis of the receptors solubilized in 0.075% SDS, the resulting fragments were submitted to size analysis in combination with wheat-germ agglutinin (WGA)-Sepharose and organomercurial-agarose chromatography. Peptides of Mr 75,000, 50,000, 30,000, 18,000 and 8000 were specifically released from the receptor. All fragments above Mr 8000 were able to bind WGA-Sepharose and therefore the peptide of Mr 18,000 was taken as the upper limit of the distance between the antagonist and the glycan moieties. The limit fragment of Mr 8000 carried chemical groups which were modified by N-ethylmaleimide and reacted with an immobilized organomercurial. About 65-80% of the labelled receptors were adsorbed on concanavalin A-Sepharose with low affinity, generating two further components after sequential application to WGA-Sepharose. About 50% of the receptors were susceptible to neuraminidase treatment, with a concomitant slight modification of the SDS/polyacrylamide-gel-electrophoretic pattern.