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PubMed1992

Guanylate cyclase inhibitors: effect on inhibitory junction potentials in esophageal smooth muscle.

Electrical field stimulation (EFS) of nerves intrinsic to the opossum lower esophageal sphincter (LES) produces LES relaxation, an increase in its guanosine 3',5'-cyclic monophosphate (cGMP) content, and hyperpolarization of its circular muscle membrane potential difference. Activation of esophageal nerves produces an analogous hyperpolarization of the circular esophageal smooth muscle. These studies test the hypothesis that cGMP is an intracellular mediator of this hyperpolarization. The transmembrane potential difference of circular smooth muscle cells was recorded with glass microelectrodes. Nerve-mediated smooth muscle hyperpolarization was evoked by EFS (1 ms, 50 V pulses). Forskolin, an activator of adenylate cyclase, and sodium nitroprusside, an activator of guanylate cyclase, produced hyperpolarization. Cystamine and methylene blue, inhibitors of guanylate cyclase, blocked the hyperpolarization elicited by sodium nitroprusside, but not that by forskolin. Both also reversibly abolished the hyperpolarization evoked by EFS. Membrane-permeable derivatives of cGMP produced a concentration-dependent hyperpolarization. These data support the hypothesis that cGMP is an intracellular mediator of nerve-induced esophageal smooth muscle hyperpolarization.

Animals↗
PubMed1995

K+ channel opening mediates hyperpolarizations by nitric oxide donors and IJPs in opossum esophagus.

The ionic mechanisms by which nitric oxide (NO) or a related compound mediates the inhibitory junction potentials (IJPs) of the opossum esophageal circular smooth muscle were studied using microelectrodes and double sucrose gap. The NO donors, 3-morpholino-sydnonimine hydrochloride and sodium nitroprusside, induced 15- to 20-mV hyperpolarizations that reversed near the potassium equilibrium potential as did the IJPs. They inhibited the IJPs and decreased electrotonic potentials (increased conductance) even during restoration of the resting membrane potential by application of depolarizing current. Quinine was more efficacious than apamin in inhibiting the IJPs or NO donor hyperpolarizations, whereas the other K+ channel blockers tested (tetraethylammonium, charybdotoxin, 4-aminopyridine, Cs+, and glibenclamide) were without effect. Glibenclamide abolished the hyperpolarizing effects of the K+ channel opener BRL-34915. Low Cl- Krebs (isethionate substitutions) caused hyperpolarizations, increased electrotonic potentials, and reduced IJPs. The neural blockers, tetrodotoxin, omega-conotoxin GVIA, and N omega-nitro-L-arginine methyl ester, inhibited IJPs but not the responses to NO donors, indicating a postjunctional effect. Methylene blue and cystamine, soluble guanylate cyclase inhibitors, suppressed IJPs and responses to NO donors. We conclude that NO mediates esophageal IJPs, which depend on guanosine 3',5'-cyclic monophosphate elevation and activation of quinine- and apamin-sensitive K+ channels.

Animals↗
PubMed1998

Effect of nitric oxide on calcium-activated potassium channels in colonic smooth muscle of rabbits.

Nitric oxide (NO) hyperpolarizes intestinal smooth muscle cells. This study was designed to determine the mechanism whereby NO activates KCa channels of circular smooth muscle of the rabbit colon. Transmural biopsies of the rabbit colon were stained for NADPH-diaphorase. Freshly dispersed circular smooth muscle cells were studied in the whole cell configuration, as well as in on-cell and excised inside-out patch recording configurations, while KCa current and the activity of KCa channels, respectively, were monitored. NADPH-diaphorase-positive nerve fibers were found in both muscle layers. NO (1%) increased whole cell net outward current by 79% and hyperpolarized resting membrane voltage from -59 to -73 mV (n = 8 cells, P < 0.01). In the on-cell patch recording configuration. NO (0.5% or 1%) in the bath increased NPo of KCa channels; charybdotoxin (125 nM) in the pipette solution blocked this effect. In the excised inside-out patch recording configuration, NO (1%) had no effect on NPo of KCa channels. In the on-cell patch recording configuration, methylene blue (1 microM) or cystamine (5 mM) in the bath solution decreased the effect of NO (1%) on NPo of KCa channels. NPo was increased by 8-bromo-cGMP (8-BrcGMP; 1 mM), a cGMP analog, and zaprinast (100 microM), an inhibitor of cGMP phosphodiesterase. These data suggest that NO increased whole cell outward K+ current by activating KCa channels through a cGMP pathway.

Animals↗
PubMed1981

Receptors for 5-hydroxytryptamine in rabbit isolated ear artery and aorta.

The effects of N,N'-bis-(O-methoxybenzylaminohexyl)-cystamine tetrahydrochloride (BHC), 2-brom-d-lysergic acid diethylamide (BOL) and prazosin on 5-hydroxytryptamine (5-HT) dose-response curves in rabbit ear artery and thoracic aorta were determined. BHC, an irreversible alpha-adrenoceptor antagonist, and prazosin had no substantial effect while BOL shifted the 5-HT dose-response curve to the right in aorta. BHC shifted the curve to the right and reduced maximal response to 5-HT in ear artery. BOL shifted the curve to the right only above 10(-6) M 5-HT in control, but at all concentrations studied in BHC-pretreated ear arteries. In vitro denervation of the ear artery with 6-hydroxydopamine did not significantly alter the 5-HT dose-response curve, nor the effect of BHC on that curve. On the other hand, desipramine decreased the contractile response to high concentrations of 5-HT in non-denervated ear arteries. Prazosin increased the 5-HT threshold and slope of the curve in the ED50 region, but had no inhibitory effect on contractile responses above 3 x 10(-7) M 5-HT or on maximal response. It is concluded that 5-HT acts exclusively on 5-HT receptors in aorta, but on both alpha-adrenergic and 5-HT receptors in ear artery. 5-HT also possesses a small indirect sympathomimetic action at high concentrations in the ear artery. Prazosin has no effect on 5-HT receptors in either vessel and blocks the alpha-adrenoceptor stimulation by 5-HT in ear artery.

Animals↗
PubMed1992

Cysteamine enhances the procoagulant activity of Factor VIII-East Hartford, a dysfunctional protein due to a light chain thrombin cleavage site mutation (arginine-1689 to cysteine).

We have recently identified the molecular defect responsible for cross-reacting material-positive hemophilia A in two unrelated patients in which the substitution of cysteine for arginine-1689 (Factor VIII-East Hartford[FVIII-EH]) abolishes a critical Factor VIII light chain thrombin cleavage site. As other mutant proteins with a cysteine for arginine substitution have been modified in the presence of cysteamine, we have determined the effect of this and other reducing agents on FVIII-EH function. Cysteamine concentrations between 0.1 and 10 mM caused dose- and time-dependent increases in FVIII-EH VIII:C activity, as much as 14-fold (to 35 and 62 U/dl for the two patients tested). Comparable data were obtained in a standard one-stage VIII:C coagulation assay and in a chromogenic substrate assay measuring Factor Xa generation. Thrombin cleavage of the FVIII-EH light chain in the presence of cysteamine was documented by immunoadsorption and analysis. Cystamine and cysteamine-S-phosphate, similar compounds that do not possess a free thiol group, had no effect. Cysteamine augmentation of FVIII-EH VIII:C was abolished by the simultaneous addition of N-ethyl maleimide or iodoacetamide, but these sulfhydryl blocking agents did not prevent the VIII:C increase and light chain cleavage by thrombin if the plasma samples were dialyzed to remove the inhibitors before adding the cysteamine. However, incubation with DTT before iodoacetamide prevented the cysteamine effect after dialysis. These data suggest that when isolated from patient plasma, FVIII-EH cysteine-1689 is present in a disulfide bond. This bond is cleaved by cysteamine to form a new mixed disulfide, a pseudolysine that restores a thrombin cleavage site that is essential for procoagulant function.

Blood Coagulation↗
PubMed2006

A rapid transglutaminase assay for high-throughput screening applications.

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca(2+)-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The K(m) of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 muM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.

Animals↗
PubMed1984

Depletion of bovine pituitary prolactin by cysteamine involves a thiol:disulfide mechanism.

Cysteamine [2-aminoethanethiol (CySH)] reduces measurable PRL concentrations in vivo and in vitro. Since secretion is also inhibited, CySH may block conversion from a poorly assayable hormone storage form(s) to readily assayable, releasable PRL. This would represent a previously unrecognized mechanism for secretory regulation. We undertook the present study to identify the sites involved in the loss of measurable PRL (depletion) induced by cysteamine. The disulfide cystamine was ineffective on secretory granules unless combined with reduced glutathione, indicating the generation of the active CySH-thiol form. Pretreatment of granules with thiol-blocking agents resulted in dose-dependent enhancement of CySH inhibition, achieving nearly complete inhibition with 5 mM iodoacetamide. In contrast, pretreatment with reduced glutathione or dithiothreitol, respectively, impaired or abolished the CySH effect. Similar results were obtained when tissues were exposed to thiols and/or alkaline conditions after CySH treatment; for example, exposure to 50 mM glutathione reduced the lowering of measured pituitary homogenate PRL from 76% to 37%. These data suggest that the mechanism by which CySH causes PRL depletion is mediated by granule disulfides and the -SH of CySH. The regulation of thiol:disulfide equilibria appears to be an important determinant of the detectability of PRL storage forms and of their secretion.

Animals↗
PubMed1994

Bacterial expression and in vitro folding of the beta-subunit of human chorionic gonadotropin (hCG beta) and functional assembly of recombinant hCG beta with hCG alpha.

A bacterial expression system for the beta-subunit of hCG (hCG beta) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG beta in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG beta complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG beta (rhCG beta) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG beta were recovered from 1 liter culture, rhCG beta was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCG alpha and the purified rhCG beta/urinary alpha dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG beta/urinary alpha dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG beta. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG beta are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG beta in bacteria and of folding it in vitro implies that the beta-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

Animals↗
PubMed1995

Asparagine-linked oligosaccharides facilitate human chorionic gonadotropin beta-subunit folding but not assembly of prefolded beta with alpha.

To determine the role of asparagine (N)-linked oligosaccharide chains in protein folding and assembly, the well established hCG-beta in vitro folding and assembly assays were used to analyze how the human CG (hCG) beta-subunit devoid of one or two N-linked glycans folds and assembles under different conditions. Two approaches were used: 1) site-specific mutagenesis of hCG-beta synthesized in Chinese hamster ovary cells transfected with beta-mutants lacking the asparagine glycosylation sites; and 2) enzymatic deglycosylation of hCG-beta synthesized in JAR cells with peptide N-glycosidase F or endoglycosidase H. In both cases, [35S]cysteine-labeled beta-subunits were used as substrates to measure the conversion of the hCG-beta folding intermediate p beta 1 into p beta 2 and assembly of p beta 2 with urinary alpha. Using the mutated substrates from Chinese hamster ovary cells, it was found that 60% of wild-type p beta 1 (two N-linked glycans), 60% of p beta 1 missing the Asn13-linked glycan, 40% of p beta 1 missing the Asn30-linked glycan, and 10% of p beta 1 missing two N-linked glycans were converted to the corresponding p beta 2, respectively. With the enzymatically deglycosylated substrate from JAR cells, 90% of p beta 1 (two N-linked glycans), 70% of p beta 1(1) (one N-linked glycan), and 10% of p beta 1(0) (without N-linked glycan) folded into p beta 2 under cysteamine and cystamine redox conditions with or without protein disulfide isomerase. These data demonstrate that at least one N-linked glycan is required for efficient folding of hCG-beta and that the Asn30-linked glycan is more important than Asn13-linked glycan for hCG-beta folding. It also was shown that the composition of N-linked glycans of hCG-p beta 1 did not change protein folding, since hCG-beta substrates with high mannose oligosacharides folded as efficiently as beta-substrates containing sialylated complex oligosaccharides. Moreover, assembly of the already folded, assembly-component folding intermediate, p beta 2, was not affected by removal of one or both of the N-linked glycans of the beta-subunit. These data thus show that N-linked glycans play their most important role in the folding component of the folding and assembly pathway for hCG-beta.

Animals↗
PubMed1983

Mechanism of enhanced fusion capacity of mouse red cells infected with Plasmodium berghei.

Plasmodium berghei-infected mouse red cells have enhanced fusion capacity as triggered by addition of poly(ethylene glycol) in the presence of Ca2+. The uptake of Ca2+ in P. berghei-infected cells is greater than in normal cells, and the difference in Ca2+ uptake was found to be enhanced in the presence of poly(ethylene glycol). Fusion of normal and P. berghei-infected red cells by poly(ethylene glycol) was significantly inhibited by N-tosyl-L-lysylchloromethyl ketone and phenylmethylsulphonyl fluoride. In addition, ethyleneglycolbis(aminoethylether)tetra-acetate, N-ethylmaleimide, iodoacetamide, cystamine and tetrathionate also prevented fusion in both systems. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone and N-tosyl-L-arginine methyl ester did not inhibit cell fusion. The latter enhanced fusion of infected cells but was without effect on normal cells. These results indicate that a Ca2+-activated thiol proteinase may be involved in membrane fusion in malaria-infected as well as in normal red cells. However, differences in the effect of proteinase inhibitors and substrate on fusion and Ca2+ entry show that the processes leading to fusion may not be identical.

Animals↗
PubMed1990

The importance of epithelial uptake systems in lung toxicity.

The discovery that the herbicide paraquat was selectively accumulated by the lung, both in vivo and in vitro, in comparison with other tissues, provided an explanation for its selective toxicity to the lung. This uptake process is energy dependent and obeys saturation kinetics. A characterization of the process led to the identification of endogenous chemicals that are the natural substrates for the system. Among these are a series of diamines and polyamines, as well as the diaminodisulfide cystamine. It appears that paraquat, because of specific structural similarities to these endogenous polyamines, is mistakenly accumulated by the lung. This uptake process is specifically located in the alveolar Type II cell, the Clara cell, and probably the alveolar Type I cell. With the development of knowledge of the structural requirements of chemicals to be accumulated by this system, it is possible to predict which chemicals will be accumulated by the lung or design molecules that are targeted to the alveolar epithelial and Clara cells. In the wider perspective, this polyamine uptake system has been found on a number of cancerous cells or tissues. With the knowledge of the uptake system in the lung, it should be possible to design drugs that will be specifically concentrated in cells that possess this system.

Animals↗
PubMed1987

Studies on immunoassays of peptide factors. II. Fluorescence enzyme immunoassay for human little-gastrin.

The fluorogenic chymotrypsin substrate N alpha-(4-carboxybutyryl)-L-phenylalanine (4-methyl-7-coumaryl)amide was converted to a thiol-containing compound via its condensation at the carboxyl function with cystamine followed by reduction of the resulting disulfide compound to a cysteamine derivative. By subsequent reaction of the thiol group with N alpha-maleoyl-beta-alanyl-human-little-gastrin-I-[2-17] a fluorogenic substrate-labeled gastrin, fully immunoreactive against antigastrin antisera, was obtained. This tracer was then applied for developing a fluorescence immunoassay based on separation of bound and free tracer followed by chymotryptic digestion of the fluorogenic substrate in the supernatant. The fluorescence intensity of the extracted fluorophore i.e., 7-amino-4-methyl-coumarin, was found to monitor gastrin concentrations in a reproducible manner. With the model peptide hormone human little-gastrin-I the sensitivity of this alternative immunoassay procedure was well documented.

Amino Acids↗
PubMed1986

Cysteine analogues potentiate glucose-induced insulin release in vitro.

In rat pancreatic islets, cysteine analogues, including glutathione, acetylcysteine, cysteamine, D-penicillamine, L-cysteine ethyl ester, and cysteine-potentiated glucose (11.1 mM) induced insulin secretion in a concentration-dependent manner. Their maximal effects were similar and occurred at approximately 0.05, 0.05, 0.1, 0.5, 1.0, 1.0 mM, respectively. At substimulatory glucose levels (2.8 mM), insulin release was not affected by these compounds. In contrast, thiol compounds, structurally different from cysteine and its analogues, such as mesna, tiopronin, meso-2,3-dimercaptosuccinic acid (DMSA), dimercaprol (BAL), beta-thio-D-glucose, as well as those cysteine analogues that lack a free-thiol group, including L-cystine, cystamine, D-penicillamine disulfide, S-carbocysteine, and S-carbamoyl-L-cysteine, did not enhance insulin release at stimulatory glucose levels (11.1 mM); cystine (5 mM) was inhibitory. These in vitro data indicate that among the thiols tested here, only cysteine and its analogues potentiate glucose-induced insulin secretion, whereas thiols that are structurally not related to cysteine do not. This suggests that a cysteine moiety in the molecule is necessary for the insulinotropic effect. For their synergistic action to glucose, the availability of a sulfhydryl group is also a prerequisite. The maximal synergistic action is similar for all cysteine analogues tested, whereas the potency of action is different, suggesting similarity in the mechanism of action but differences in the affinity to the secretory system.

Acetylcysteine↗
PubMed1988

Metabolism of a new radioprotector; S-acetyl-N-glycyl cysteamine. I. Absorption, distribution and excretion metabolites in mice bearing EMT6 tumours.

1. The disposition of S-acetyl-N-glycyl cysteamine (I) labelled with 14C on the cysteamine group (label 1), the glycyl group (label 2) and the acetyl group (label 3) has been studied in mice bearing EMT6 tumours. 2. Label 1 was mainly excreted in urine (63.1% dose in 24 h). Label 2 elimination was both in urine (36.0% dose in 24 h) and in expired air as 14CO2 (12.1% dose in 24 h). Label 3 was essentially eliminated in expired air as 14CO2 (55.4% dose in 24 h). 3. Tissue distribution studies of label 1 and label 2 showed that concentrations in tissues were higher than blood concentration as early as 10 min after administration. Whichever label was used, only little radio-activity was found in EMT6 tumour and brain. 4. Analysis of the urinary elimination products showed the presence of unchanged I and of cystamine, N-acetylcystamine, N-acetyl-S-methyl cysteamine sulphoxide and taurine. I is a prodrug of cysteamine which is released after deacetylation and hydrolysis of the amide bond. A metabolic pathway is proposed for this new radioprotective agent.

Animals↗
PubMed1993

Modulation of 3 alpha-hydroxysteroid dehydrogenase activity by the redox state of glutathione.

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50), purified to homogeneity from rat liver, was strongly inactivated by incubation with a disulfide such as GSSG, L-cystine or L-cystamine, as well as an SH-reagent such as DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), NEM (N-ethylmaleimide) or iodoacetic acid. The inactivation advanced with incubation time. Coenzyme (NADP+) completely protected the enzyme from this inactivation by disulfides, but neither of the substrates (androsterone and benzenedihydrodiol) did. The activity of inactivated enzyme was restored by treatment with thiols such as DTT (dithiothreitol) or GSH. In the GSH/GSSG redox buffer, the enzyme existed in an equilibrium between active (reduced) and inactive (oxidized) forms.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Reassignment of the structure for the antitumor agent RR-150.

7-Cysteaminomitosane (RR-150) has been reported to be superior to mitomycin C against P388 leukemia and B-16 melanoma in mice and is less leukopenic. Studies reported here indicated the absence of a free thiol group in RR-150 and therefore the structure was incorrectly assigned. Reaction of mitomycin A with either 2-aminoethanethiol or cystamine gave the same disulfide, 7-N,7'-N'-dithiodiethylenedimitomycin C, which is the newly proposed structure for RR-150. Attempts to produce 7-cysteaminomitosane by reduction of the disulfide have not succeeded because of its apparent instability.

Antibiotics, Antineoplastic↗
PubMed1998

A study of the influence of different types of radioprotectors on the survival of mice treated with ionizing radiation over a wide dose range.

The subject of this study is the influence of the preventive administration of cystamine, naphthysin, Ukrain, Lymphokinin, Prodigiosan, and polyribonate on the 30-day survival of mice subject to doses of ionizing radiation ranging from LD50/30 to LD100/30. Conventional radioprotectors were shown to prevent the death of experimental animals irradiated with doses ranging from LD70 to LD100/30 but were inefficient as a means of preventing mortality at doses with a medium lethal effect. In contrast, the bioregulators Ukrain and Lymphokinin were more potent at doses ranging from LD50 to LD70/30 (1.5 times more potent than conventional radioprotectors). Polyribonate and Prodigiosan were the least potent. These findings are discussed with respect to the appropriateness of developing pharmacological means which could protect organisms from different degrees of radiation-induced damage. The need to modify the criteria for the selection of potential radioprotectors is emphasized.

Animals↗
PubMed2001

Facilitated electrochemical oxidation of NADH and its model compound at gold electrode modified with terminally substituted electroinactive self-assembled monolayers.

The electrochemical oxidation of NADH and its model compound, N-benzyl-1,4-dihydronicotinamide (DHN), has been studied at gold electrode modified with self-assembled monolayer of terminally substituted thiols/disulfide, i.e., cystamine (CYST), mercaptopropionic acid (MPA) and mercaptoethanol (ME). A substantial decrease in the overpotential (approximately 250 mV) when compared to the bare electrode has been observed for the oxidation of NADH at the monolayer-modified electrodes, containing no so-called redox mediator. The bare electrode shows an ill-defined voltammetric peak for the oxidation of DHN, whereas the monolayer-modified electrodes showed a well-defined voltammetric peak. The monolayer assembly on the gold electrode prevents the fouling of electrode surface by the oxidation products, which favors the oxidation at the less positive potential. The square-wave voltammograms showed a sharp voltammetric signal for the oxidation of NADH at all the monolayer-modified electrodes. All the monolayer-modified electrodes showed a linear current response to change in the NADH concentration in its range of 25-300 microM and their sensitivities were found to be 0.005+/-0.0003, 0.0063+/-0.0002 and 0.0052+/-0.0003 microA/microM for CYST-Au, ME-Au and MPA-Au electrodes, respectively. The hydrodynamic voltammograms obtained at the rotating CYST-Au electrode for the oxidation of NADH and DHN were used to estimate the diffusion coefficient of DHN, and the number of electrons involved in the oxidation process of NADH.

Electrochemistry↗