Photolabeling the Torpedo nicotinic acetylcholine receptor with 4-azido-2,3,5,6-tetrafluorobenzoylcholine, a partial agonist.

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

Sheep brain pseudocholinesterase: inhibition kinetics of the partially purified enzyme by some substrate analogues.

Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20-80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290,000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.

2,2,4-Trimethylpentane induces Ca2+ release from the sarcoplasmic reticulum terminal cisterns.

Using quin2, the effects of aliphatic hydrocarbons on the system of Ca(2+)-induced Ca2+ release in isolated membranes of rabbit skeletal muscle terminal cisterns have been studied. The hydrocarbons were inserted into the membranes by means of hydrocarbon-containing liposomes. 2,2,4-Trimethylpentane (isooctane) caused a rapid release of 70-75% of Ca2+ taken up by the terminal cistern vesicles during the Ca(2+)-pump operation. This effect was inhibited by the caffeine-induced Ca2+ release blockers--Mg2+, ruthenium red and tetracaine. The same was observed with a decrease in the concentration of ATP that is known to activate the terminal cistern Ca2+ channels. The effect of 2,2,4-trimethylpentane on the longitudinal cistern fractions practically devoid of Ca(2+)-channels was insignificant. Heptane, hexane and octane caused a slow release of 5-10% of the accumulated Ca2+ from the terminal cistern vesicles; no such effect was induced by decane.

Inhibition of rat uterine contractions by rat and human CGRP.

Effects of rat and human calcitonin gene-related peptide (r alpha CGRP and h beta CGRP, respectively) upon uterine contractile force were investigated using uterine horns from nonpregnant rats, r alpha CGRP and h beta CGRP were equipotent (pD2 = 8.85-9.09) in inhibiting spontaneous and electrically evoked uterine contractions. r alpha CGRP was relatively ineffective in inhibiting potassium-induced contractures of preparations from stilbestrol-pretreated rats. The use of selective antagonists established that r alpha CGRP did not release prostanoids, or release or act at receptors for catecholamines and histamine. The effects of the peptides were not significantly modulated by estrogen levels since pD2 values were similar (8.56-8.86) in field-stimulated preparations from rats in proestrus/estrus or metestrus/diestrus.

[Effect of choline compounds on rosette formation in mouse lymphocytes]. / O vliianii nekotorykh soedinenii kholina na rozetkoobrazovanie limfotsitov u myshei.

The effects of some choline compounds (acetylcholine, benzoylcholine, phosphorylcholine) on immune rosette-formation were studied in experiments on 150 BALB/c mice immunized with sheep red blood cells. Low concentration of the choline-containing substances (from 10(-14) to 10(-12) M) stimulated immune rosette-formation, whereas high concentrations (from 10(-8) to 10(-5) M) inhibited it. The blockade of lymphocyte cholinoreceptors with atropine reversed the action of phosphorylcholine on the processes of rosette-formation. Interaction of immune and mediator receptors is discussed.

[Effects of benzoylcholine on ChE activity in rabbit and rat brain and erythrocytes (author's transl)].

Cholinesterase (ChE) hydrolyzing acetylcholine (ACh) in vivo can be classified into two groups. The ChE localizing in brain and erythrocytes is known as ChE and hydrolyzes ACh and acetyl-beta-methylcholine (MeCh) but not benzoylcholine (BzCh). The ChE localizing in liver and serum is termed pseudo ChE and hydrolyzes ACh and BzCh but not MeCh. Effects of BzCh, a specific substrate of pseudo ChE on true ChE in brain mitochondria and erythrocytes of rabbit and rat were studied. The ChE activities in rabbit brain with ACh and MeCh as substrates were decreased to 1/4 and 1/3 of the control activities by addition of 10 mM BzCh, respectively. The pS curve for ChE in rabbit brain and erythrocytes with ACh and MeCh as substrates markedly decreased by addition of 3 mM of BzCh. The inhibitory effect of BzCh was reversible and competitive, as assessed by a Lineweaver-Burk plot method. BzCh protected by the irreversible inactivating effect of true ChE by DEP. These results suggest that BzCh is not hydrolyzed by true ChE but does have an affinity for the active center of true ChE.

Kinetics of local anesthetic esters and the effects of adjuvant drugs on 2-chloroprocaine hydrolysis.

A rapid, reliable method for the determination of 2-chloroprocaine in serum was developed. The method, using double-beam ultraviolet spectroscopy, provides rapid, accurate analysis of 2-chloroprocaine in the range of 5.5 to 111 microM (1.5--30 microgram/ml), as documented by comparison with the accepted gas chromatographic procedure. The contribution of 4-amino-2-chlorobenzoic acid, the principal metabolite of 2-chloroprocaine, to the total absorbance at 300 nm was examined and found to be negligible. Using the ultraviolet spectrophotometric method, values of the Michaelis-Menton constant (Km) and maximal reaction velocity (Vmax) for hydrolysis of procaine and 2-chloroprocaine by homozygous typical, heterozygous, and homozygous atypical plasma cholinesterase were determined. The Kms for the three genotypes were 5.0, 6.2, and 14.7 microM, respectively, for procaine, and 8.2, 17, and 103 microM, respectively for 2-chloroprocaine. The Vmaxs for the three genotyps were similar for all esters. Vmax for procaine was 18.6 +/- 0.9 nmol/min/ml serum, while Vmax for 2-chloroprocaine was 98.4 +/- 2.1 nmol/min/ml serum. At high concentrations, 2-chloroprocaine acts as an inhibitor of its hydrolysis. The inhibitory effects of lidocaine, bupivacaine, neostigmine, and succinyldicholine on 2-chloroprocaine hydrolysis for homozygous typical and atypical variants, respectively, were studied. Competitive inhibition was demonstrated for all four drugs. However, at clinically significant concentrations, only neostigmine and bupivacaine produced high degrees of inhibition. The competitive inhibition constants (K1) for the typical and atypical variants, respectively, were 3.3 +/- 0.3 microM and 15.1 +/- 4.8 microM for neostigmine, and 4.2 +/- 0.3 microM and 36.9 +/- 9.8 microM for bupivacaine.

Purification and properties of human serum esterase.

Human serum esterase was purified by affinity column chromatography on a column of covalently linked p-trimethylammoniumanilinium dichloride to Sepharose 4B. The purified preparation hydrolysed both benzoylcholine and tributyrin. p-Trimethylammoniumanilinium dichloride inhibited non-competitively the hydrolysis of tributyrin and inhibited competitively the splitting of benzoylcholine. The Km value was 0.62 . 10(-3) M for tributyrin and 0.4 . 10(-3) M for benzoylcholine. Antiserum to this purified esterase was prepared in rabbit and it was found that the antiserum did not inhibit esterase activities of human liver, muscle and adipose tissue, although it could inhibit completely the esterase activities of human serum.

L-Tryptophan-induced depression of the pressor response to clinidine in anaesthetized rats.

1. The interaction of serotonin precursor L-tryptophan with the pressor responses of the anaesthetized rat to the intravenous injection of clonidine, adrenaline and angiotensin has been studied. 2. Pretreatment of rats with L-tryptophan (100 mg/kg) depressed the pressor response to clonidine but had no effect on the responses elicited by adrenaline or angiotensin. 3. The L-tryptophan-induced depression of the clonidine response was prevented by pretreating rats with either Rö 4-4602, carbidopa, BW 172C58, methysergide or by pithing. 4. Intravenous infusions of serotonin depressed the pressor responses to clonidine, adrenaline and angiotensin in both intact anaesthetized and pithed rats. 5. It is concluded that the depressant action of L-tryptophan is dependent on its conversion within the periphery to serotonin. This action is also dependent on or mediated by the sympathetic nervous system.