Enrique L.M. Ochoa

Receptor hydrophobic protein fraction from intestinal smooth muscle binding muscarinic ligands

1. 1. After the binding of [3H]atropine to the circular smooth muscle layer of the bovine intestine most of the radioactivity was extracted with chloroform methanol (2:1, v/v) and after column chromatography in Sephadex LH20 the radioactivity appeared associated with a special fraction of hydrophobic protein (i.e. Peak 3) out of five peaks obtained from the same tissue. 2. 2. This protein peak, when added to the organic extract, bound [3H]atropine and [14C]acetylcholine but not (+)-[14C]tubocurarine. In double labelling experiments [3H]atropine appeared bound to Peak 3 and [14C]serotonin to another peak of hydrophobic protein (i.e. Peak 2). 3. 3. Preliminary auto- and cross-protection experiments using unlabelled atropine (to protect the muscarinic receptor) and Dibenamine were carried out to further study the specificity of the binding. The autoprotection experiments confirmed the results of [3H]atropine binding. Its control showed that 5·10−6 M Dibenamine blocked about 85% of the binding of [3H]atropine to peak 3. Cross-protection experiments using [14C]serotonin or [14C]histamine as ligands showed no radioactivity associated with any protein peak. 4. 4. The results obtained suggest that Peak 3 of hydrophobic protein represents a fraction containing cholinergic receptors of the muscarinic type and that this is different from the serotonin and histamine receptor. This interpretation is also supported by some quantitative considerations of our findings.

Isolation of a proteolipid binding 3H-isoproterenol from the heart ventricle of the cat☆

Abstract A chloroform-methanol extract from cat heart ventricle was incubated with 10 −8 M d,1–7- 3 H-norepinephrine, d,1–7 3 H-isoproterenol or 3 H-ouabain and chromatographed on a Sephadex LH20 column. 5 proteolipid peaks were eluted of which the first showed binding for catecholamines but not for 3 H-ouabain. Upon subcellular fractionation of the ventricular muscle the membrane fraction showed a 2–3 fold increase in specific activity for 3 H-isoproterenol. This binding was inhibited by propranolol but not by phentolamine. It is suggested that the adrenergic proteolipid of the ventricle is localized in the plasma membrane of the muscle cells.

Arg-Lys-Asp-Val-Tyr (thymopentin) accelerates the cholinergic-induced inactivation (desensitization) of reconstituted nicotinic receptor.

Summary1.The thymic hormone thymopoietin blocks neuromuscular transmission and was proposed (Goldstein, 1974) as a modulator of synaptic conductivity.2.The cholinergic-induced inactivation of nicotinic receptor reconstituted into asolectin lipid vesicles was studied in the presence and in the absence of thymopentin, a synthetic pentapeptide corresponding to positions 32–36 of thymopoietin.3.The present data show that thymopentin accelerates desensitization of the nicotinic acetylcholine receptor, supporting the aforementioned physiological role proposed for thymopoietin.4.They also suggest that the hormone itself and/or a yet unidentified hormine-derived peptide fragment may act as an endogenous ligand for nicotinic acetylcholine receptor desensitization.

Reconstitution of solubilized atrial cholinergic muscarinic receptors in liposomes

The reconstitution of solubilized bovine atrial cholinergic muscarinic receptor into liposomes made of exogenous lipids has been achieved by polyethyleneglycol precipitation. Of the different lipid mixtures used, soybean lecithins were shown to be the best on the basis of receptor recovery. The receptor reconsituted into soybean lecithins liposomes exhibited ligand binding properties very similar to those of the native receptor. The dissociation constant of [3H]-N-methyl-scopolamine ([3H]NMS) was 0.46 and 0.30 nM as determined by equilibrium and kinetics experiments respectively. The potency of a range of muscarinic ligands in displacing [3H]NMS binding was atropine > methyl-atropine > scopolamine > pirenzepine oxotremorine > gallamine > carbamylcholine > pilocarpine bethanechol. The Hill slopes of the displacement curves were near 1 for the antagonists and smaller than 1 for the agonists and for gallamine. The agonist binding may be modulated by guanine nucleotides. These results indicate that soybean lecithins fulfill the lipid requirements for the reconstitution of the atrial muscarinic receptor.

The heterogeneity and nucleotide modulation of cholinergic muscarinic receptors are restored by poly (ethylene) glycol-6000 precipitation of solubilized atrial receptors

The membrane bound muscarinic receptor exhibits heterogeneity of binding sites for agonists that are modulated by guanine nucleotides. These properties are generally lost upon receptor solubilization by several detergents, but may be recovered after rapid detergent removal by poly (ethylene) glycol-6000 precipitation of the digitonin plus cholate solubilized atrial muscarinic receptor. The results are discussed in terms of muscarinic receptor interactions in the intact membrane.

Ultrastructure of reconstituted membranes containing the muscarinic receptor

In this paper the demonstration is made that membrane vesicles (liposomes) containing the muscarinic receptor can be formed by polyethylene glycol (PEG) precipitation of detergent extracts of bovine atrial membranes. The incorporation of the muscarinic receptor in these vesicles may be related to the restoration of the heterogeneity and nucleotide modulation of muscarinic agonist binding by PEG precipitation of atrial detergent extracts, previously reported. Vesicles are also formed when detergent solubilized asolectin lipids, alone or in combination with membrane detergent extracts, are precipitated by PEG. The structure of the vesicles seems depend on their lipid and protein composition and the procedure employed for the removal of the dispersing medium. These results indicate that PEG precipitation could be used for the reconstitution of the muscarinic receptor into the liposomes of exogenous lipids.

The nicotinic acetylcholine receptor from Discopyge tschudii: Purification, characterization and reconstitution into liposomes

The nicotinic acetylcholine receptor from Discopyge tschudii electroplax was purified by affinity chromatography on Affi-Gel 401 using bromoacetylcholine as the ligand. Its specific activity was about 4000 pmol 125I-alpha-bungarotoxin/mg protein. SDS-polyacrylamide gel electrophoresis revealed four bands of apparent molecular weights: 41,200, 49,500, 60,000 and 66,300. The amino acid composition of each individual subunit was determined. Native membranes, rich in nicotinic receptor exhibited carbamylcholine-catalysed cation transport (blocked by curare and desensitized by prior incubation with the cholinergic agonist). The functional activity of the purified material could be reconstituted into soybean lecithin liposomes. Our data show that the Discopyge tschudii nicotinic receptor is similar to that from Torpedo californica.

Incorporation of the nicotinic acetylcholine receptor into liposomes by rapid removal of detergent using poly (ethylene) glycol 6000

The poly (ethylene) glycol 6000 (PEG) precipitation procedure was applied for incorporating the nicotinic acetylcholine receptor from D. tschudii electric organ into Asolectin liposomes. The incorporated receptor had its toxin binding sites oriented towards the external bulk phase and bound [(125)I]?-bungarotoxin with the same rate of toxin binding and cholinergic agonist equilibrium low affinity constant as its membrane bound counterpart. A characteristic feature of those vesicles, however, was the absence of the affinity transitions which normally correlate with the receptor densensitization phenomenon, and a poor carbamylcholine-stimulated ion flux as compared to that exhibited by vesicles prepared by the cholate dialysis procedure. When ether-purified PEG was used, the incorporated receptor showed affinity transitions and carbamylcholine-induced cation influx into vesicles. Thin layer chromatography and quantitative densitometry analysis showed that the vesicles prepared by the PEG precipitation method contained higher amounts of free fatty acids than those prepared by the cholate-dialysis procedure. Results are interpreted as an impairment of receptor function by impurities contained in commercial preparations of PEG and/or free fatty acids possibly generated by PEG impurities from Asolectin lipids. The proposed mechanisms are modifications in vesicle geometry and perturbations in lipid-protein interactions known to be essential for normal nicotinic receptor operation. The PEG precipitation procedure has the potential for rapid reconstitution of the nicotinic receptor and other membrane proteins that cannot be solubilized by easily dialyzable detergents provided purified PEG is used.

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of ?-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked. By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself. The present data and those by Earnest et al. (Biochemistry22, 5523-5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.

Characteristics of a presynaptic plasma membrane Ca2+-ATPase activity from electric organ

Ca2(+)-ATPase activity was measured in electric organ synaptosomal homogenates and their derived presynaptic plasma membranes using a low ionic strength medium, low in Ca2+ and Mg2+, and devoid of K+. The enzyme activity showed a high apparent affinity for Ca2+ (KCa:0.5 microM) and was: (1) 5-fold stimulated by 120 nM calmodulin, (2) highly sensitive to LaCl3 inhibition, and (3) not affected by 20 mM NaN3 or 0.1 mM ouabain. The addition of Mg2+ promoted the disappearance of Ca2(+)-ATPase activity. Incubation of synaptosomal homogenates in the above-mentioned assay medium with [gamma -32P]ATP resulted in the appearance of a 140 kDa band as revealed by SDS-gel electrophoresis. Labeling of this band with 32P was inhibited by 1 mM EGTA or 10 mM NH2OH, indicating that the isotope incorporation required the presence of Ca2+ and the formation of an acyl-phosphate derivative. The results indicate that the Ca2(+)-ATPase activity from synaptosomal homogenates had characteristics corresponding to those of the enzyme that catalyzes an outward transport of Ca2+ in nerve terminals. Preincubation of synaptosomes in Ca2+ plus K+, a depolarizing procedure, induced a large and rapid decrease in the Ca2(+)-ATPase activity, possibly mediated via Ca2+ entry through voltage-gated Ca2+ channels. Furthermore, the muscarinic cholinergic agonist oxotremorine (at 15 microM concentration) did not significantly affect either the enzyme activity or the intensity of the Ca2(+)-dependent 32P incorporation into the 140 kDa band, suggesting that the enzyme is not coupled to muscarinic binding sites.