Inhibition of the cardiac inotropic effect of phenylephrine by a tetramine disulfide with irreversible alpha-adrenoceptor blocking activity.

The newly synthesized alpha-adrenoceptor blocking drug BHC (N,N'-bis[6-(10-methoxybenzyl-amino)-a-hexyl]cystamine) was found to block irreversibly the positive inotropic effect of the sympathomimetic drug phenylephrine on the isolated rat left atrium. BHC was used to test the adrenoceptor interconversion hypothesis which claims that low temperature converts inotropic beta-adrenoceptors in rat atrium and frog ventricle to alpha-adrenoceptors. There was no evidence of adrenoceptor 'interconversion.' In the rat atrium low temperature did not increase the BHC antagonism of phenylephrine and did not cause BHC to inhibit the inotropic effect of noradrenaline or isoprenaline. In the perfused frog heart low temperature did not lead BHC to inhibit the inotropic effect of phenylephrine, adrenaline, or isoprenaline.     

Irreversible alpha adrenoceptor blocking effects and reversible muscarinic blocking and nicotinic blocking effects of tetramine disulfides on the heart

Inotropic effects of phenylephrine, carbachol, and butyrylcholine were used in the rabbit left atrium to evaluate respectively alpha adrenoceptor blocking, muscarinic blocking, and nicotinic blocking effects of tetramine disulfides ((RNH(CH2)nNH(CH2)2S-)2 x 4HX). The alpha adrenoceptor blocking potencies of newly synthesized derivatives R = 3',4'-(OH)2-benzyl, n = 5-9, were similar to those of compounds R = 2'-OCH2-benzyl, n = 5-7. Muscarinic blocking and nicotinic blocking potencies of tetramine disulfides were correlated with alpha adrenoceptor blocking potency. Compounds R = 3',4'-(OH)2-benzyl had relatively low muscarinic blocking potencies and compounds R = 2'-OCH3-benzyl had relatively low nicotinic blocking potencies.     

Potentiation and inhibition of nicotinic effects on striated muscle by the tetramine disulfide benextramine

In low concentrations (0.3-3 muM) the tetramine disulfide benextramine (BHC; N,N'-bis[6-(o-methoxybenzylamino)-n-hexyl]cystamine) potentiated the contracture of the isolated frog rectus abdominis muscle caused by acetylcholine but in the presence of physostigmine or in a higher concentration (10 muM) it inhibited. Benextramine only inhibited the contracture caused by carbachol or butyrylcholine. The all-carbon analog of benextramine only inhibited the effect of acetylcholine. The inhibitory effects of benextramine and its carbon analog were noncompetitive and readily reversible but the potentiating effect of benextramine was not readily reversible.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Interaction of the neuromuscular blocking drug atracurium with muscarinic acetylcholine receptors.

On isolated rat heart atria, atracurium competitively antagonized the negative chronotropic effect of methylfurmethide, shifting the concentration-response curve to the right without diminishing the agonist's maximal effect; Kd calculated from dose ratios was 3.0 mumol/l. On the longitudinal muscle of rat ileum, atracurium antagonized the effect of methylfurmethide in a non-competitive manner; at 50 mumol/l atracurium, the maximum response to methylfurmethide was diminished by about 50%. Atracurium antagonized the binding of (3H)quinuclidinyl benzilate [3H)QNB) to muscarinic binding sites in the atria, ileal longitudinal muscle and cerebellum with IC50 values of 5-8 mumol/l, and in brain cortex of 25 mumol/l. Atracurium was little efficient, however, in antagonizing the binding of N-(3H-methyl) scopolamine [3H)NMS) to muscarinic binding sites. Complete blockade was not achieved at concentrations up to 1 mmol/l. Concentrations required to diminish the binding by 50% were 10 - 1000 times higher for (3H)NMS than for (3H)QNB. Atracurium brought about the dissociation of (3H)QNB-receptor complexes, but its effect was considerably stronger at a concentration of 30 mumol/l than at 1 mmol/l. Atracurium slowed down the dissociation of (3H)QNB-receptor complexes observed after the addition of atropine. The effects of atracurium on the dissociation of (3H)NMS-receptor complexes were similar to those on (3H)QNB-receptor complexes, but a high concentration of atracurium (1 mmol/l) produced a transient increase in (3H)NMS binding preceding its subsequent dissociation. Although the observations of the antagonism by atracurium of the effect of methylfurmethide on the heart atria, and of the inhibition of the specific binding of (3H)QNB to the atria, ileal smooth muscle, cerebellum and brain cortex are compatible with the assumption of a competitive interaction, the discrepancy between the effects of atracurium on the binding of (3H)QNB and (3H)NMS indicates that atracurium does not bind to the same binding site as (3H)QNB and (3H)NMS. It appears that most effects of atracurium on muscarinic receptors are allosteric and that both negative and positive cooperatives play a role in interactions between atracurium and muscarinic ligands.     

Quinuclidinone O-Alkynyloximes with muscarinic agonist activity.

A series of quinuclidinone O-alkynyloximes (14-19) were synthesized and evaluated in radioligand displacement assays for binding affinities to M1-M3 muscarinic receptors. Radioligand displacement assays were carried out using [3H] oxotremorine-M and [3H] pirenzepine on rat cortical tissue and [3H] N-methylscopolamine on rat heart and submandibulary glands. Two alkynyloximes 15 and 18 had pirenzepine/oxotremorine M ratios which were indicative of muscarinic agonist and partial agonist activity, respectively. They were tested for their mnemonic effects in mice using the swimming escape task and found to attenuate scopolamine induced impairment of the task in mice at 2mg/kg. The results show that the O-alkynyloxime moiety linked to azacycles of appropriate size and rigidity (for example quinuclidine and tropane) is a potentially useful muscarinic pharmacophore that can be exploited for the design of muscarinic agonists.     

Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T(m)) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated alpha-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees C as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution.     

Neuromuscular blocking agents and spontaneous sympathetic activity.

The action of neuromuscular blocking agents on the spontaneous sympathetic activity has been quantitated. "On line" spectrum analysis has been applied to the action potential of pre- and post ganglionic nerves of the coeliac plexus. The activity, the frequency spectrum and their changes after the injection of clinical and high doses of decamethonium, D-tubocurarine, succinylcholine, gallamine and pancuronium are determined.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

GYKI-12743 a new postsynaptic vascular alpha-adrenoceptor antagonist.

Using the pithed rat preparation it has been proven that GYKI-12743 exerted its alpha-adrenergic blocking action only at the postsynaptic vascular level in the cardiovascular system. This new molecule failed to antagonize the presynaptic alpha 2-adrenoceptors of the cardiac sympathetic nerve endings. Thereby it was possible to demonstrate the first vasoselective postsynaptic adrenoceptor antagonist which potentially might be interesting in the cardiovascular therapy.     

Ligand-specific changes in M3 muscarinic acetylcholine receptor structure detected by a disulfide scanning strategy

G protein-coupled receptor (GPCR) function can be modulated by different classes of ligands including full and inverse agonists. At present, little is known about the conformational changes that agonist ligands induce in their target GPCRs. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced structural changes in a series of cysteine (Cys)-substituted mutant M 3 muscarinic acetylcholine receptors. One of our goals was to study whether the cytoplasmic end of transmembrane domain V (TM V), a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change, similar to the adjacent region of TM VI. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated 20 double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492). These receptors were transiently expressed in COS-7 cells and subsequently characterized in pharmacological and disulfide cross-linking studies. Our cross-linking data, in conjunction with a three-dimensional model of the M 3 muscarinic receptor, indicate that M 3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors. The present study provides novel information about the dynamic changes that accompany M 3 receptor activation and how the receptor conformations induced (or stabilized) by full versus inverse muscarinic agonists differ from each other at the molecular level. Because all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from the present study should be of broad general relevance.     

Bombyxin-II and its disulfide bond isomers: synthesis and activity.

Bombyxin-II, an insulin superfamily peptide of the silkmoth Bombyx mori, and its disulfide bond isomers have been synthesized by two ways of stepwise, semi-regioselective disulfide bond formation. The disulfide bond CysA20-CysB22 or CysA7-CysB10 was formed first, and then the two other disulfide bonds were formed by iodine oxidation. The conditions for the iodine oxidation were improved to suppress oxidative degradation of unprotected Trp residues. With these conditions, bombyxin-II was synthesized in high yields (26% and 32%). Its disulfide bond isomers were also obtained. Specific activity of the products indicates that the disulfide bond CysA20-CysB22 is important to the bombyxin activity.     

Ribostamycin inhibits the chaperone activity of protein disulfide isomerase.

In the process of screening of proteins binding to ribostamycin in bovine liver using the affinity column chromatography, we found that ribostamycin inhibited the chaperone activity of protein disulfide isomerase (PDI), but it did not inhibit the isomerase activity. PDI was identified by SDS-PAGE, Western blotting, and N-terminal amino acid sequence analysis. A 100:1 molar ratio of ribostamycin to PDI was almost sufficient to completely inhibit the chaperone activity of PDI. The binding affinity of ribostamycin to purified bovine PDI was determined by the Biacore system, which gave a K(D) value of 3.19 x 10(-4) M. This suggests that ribostamycin binds to region distinct from the CGHC motif of PDI. This is the first report to describe the inhibitor of the chaperone activity of PDI.     

DsbG, a protein disulfide isomerase with chaperone activity

DsbG, a protein disulfide isomerase present in the periplasm of Escherichia coli, is shown to function as a molecular chaperone. Stoichiometric amounts of DsbG are sufficient to prevent the thermal aggregation of two classical chaperone substrate proteins, citrate synthase and luciferase. DsbG was also shown to interact with refolding intermediates of chemically denatured citrate synthase and prevents their aggregation in vitro. Citrate synthase reactivation experiments in the presence of DsbG suggest that DsbG binds with high affinity to early unstructured protein folding intermediates. DsbG is one of the first periplasmic proteins shown to have general chaperone activity. This ability to chaperone protein folding is likely to increase the effectiveness of DsbG as a protein disulfide isomerase.