Vascular relaxation mediated by hydroxylamines and oximes: their conversion to nitrites and mechanism of endothelium dependent vascular relaxation

Hydroxylamines (R-NHOH) and oximes (R = NOH) relax rat aortic rings independent of the presence of the endothelium. The relaxation is inhibited by methylene blue, an inhibitor of soluble guanylate cyclase and by hemoglobin, an inhibitor of the endothelium dependent relaxing factor (EDRF). Both the oximes and hydroxylamines generate NO/NO2- ions on treatment with iodine in glacial acetic acid. However, there is no correlation between relaxation and NO/NO2- formation. Compared to hydroxylamines, the oximes are less potent relaxing agents and not efficiently converted to NO/NO2- ions. We suggest that endothelium dependent relaxation is associated with a hydroxylamine like compound and is not directly related to NO.     

NW-nitro L-arginine benzyl ester, a potent irreversible inhibitor of endothelium dependent relaxation

In the rat aorta preparation, we have used the nitro-arginine derivative, NW-nitro L-arginine benzyl ester (NABE), as a probe to investigate the relationship between L-arginine and the endothelium derived relaxing factor (EDRF). We find NABE to be a potent endothelium dependent vasoconstrictor and inhibitor of relaxation. The effect of NABE is irreversible and not removed by subsequent washing. The vasoconstrictor effect of NABE differs from other EDRF inhibitors like NG-monomethyl L-arginine (L-NMMA) in that it is not antagonized by pre-treatment with excess L-arginine. In contrast, like other EDRF inhibitors, at high concentration NABE exhibits vasodilation which is antagonized by methylene blue. We suggest that the previous reports on the antagonism between L-arginine and the putative EDRF inhibitors like L-NMMA are due to their structural similarities rather than to externally added L-arginine acting as a substrate for EDRF synthesis.     

Endothelium dependent vascular relaxation by arginine containing polypeptides

The vascular endothelium appears obligatory for the expression of the vasodilating property of most polypeptides. A number of polypeptides were studied on the rat aortic ring preparation which was pre-contracted with phenylephrine and only basic polypeptides containing one or more arginine residues elicited relaxation which was endothelium dependent. These peptides included melittin and poly-L-Arg. The basic polypeptide poly-L-Lys also elicited endothelium dependent relaxation, but to a lesser extent than arginine containing polypeptides. Two basic polypeptides, apamin and mastoparan do not promote endothelium dependent relaxation. The former contains arginine between disulfide bonds and in the latter arginine is absent. Basic amino acids and dipeptides which contain arginine, and also polyamines did not elicit relaxation even at high concentrations (10(-3) M). The relaxation elicited by melittin, poly-L-Arg and poly-L-Lys was inhibited by ETYA, NDGA, p-bromophenacyl bromide and not by indomethacin. Methylene blue, an inhibitor of soluble guanylate cyclase, also abolished the relaxation. We suggest that arginine containing peptides may relax vascular smooth muscle by acting directly on the vascular smooth muscle (eg: atriopeptins) and/or or by eliciting release of a relaxing factor(s) from the endothelium.     

Role of the endothelium and arginine peptides on the vaso-motor response of porcine internal mammary artery

The long-term patency of the internal mammary artery (IMA) graft is of considerable interest owing to its extensive use in myocardial revascularization. The aim of the present study was to elucidate the role of endothelium in modulating the responses of the porcine IMA to several vasoactive drugs. Isolated ring segments of porcine IMA contracted in a reproducible and dose dependent manner to phenylephrine, potassium chloride and the thromboxane mimic U46619, but the responses to serotonin, histamine and ATP were significantly less prominent. Both acetylcholine and bradykinin elicited endothelium-dependent relaxation which was not inhibited by indomethacin, but by methylene blue, an inhibitor of soluble guanylate cyclase. These two endothelium-dependent drugs and two endothelium-independent relaxing drugs, nitroprusside and nitroglycerin relaxed the IMA in a dose dependent manner which was associated with an elevation of cyclic GMP. The endothelium dependent vasodilator peptides such as bradykinin contain L-arginine in their sequence. Benzoyl derivatives of L-arginine but not L-arginine relaxed the IMA in a dose dependent manner. These data confirm and extend exploratory studies performed with a simpler vascular model which indicate that the precursor of endothelium derived relaxing factor (EDRF) is an arginine moiety.     

Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.     

Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects.

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.     

Vasodilatory properties of mono-L-arginine-containing compounds

Benzoyl derivatives of L-arginine, unlike arginine, elicited relaxation of pre-contracted rat aortic rings in a concentration dependent manner. The most potent relaxing agent was N-alpha-benzoyl-L-arginine ethyl ester. The relaxation was abolished by methylene blue, but not by indomethacin. When incubated with rat aortic rings, the benzoyl derivatives exhibited colorimetric reactions characteristic of citrulline and nitrite ion. This indicates the presence of a peptidyl arginine deiminase like activity in rat aorta. Citrulline had no vasodilatory property. The other product of the iminase reaction is ammonia which through oxygenase pathway may generate nitric oxide, the proposed endothelium derived relaxing factor(EDRF). Our results suggest that an as yet unidentified arginine derivative from the endothelium may be the biological precursor of EDRF.     

Carboxypeptidase inhibitors from Ascaris suum: the primary structure

The carboxypeptidase A inhibitor from Ascaris suum was isolated from aqueous extracts by affinity chromatography toward immobilized carboxypeptidase A. The amino acid sequence is DQVRKCLSDT10DCTNGEKCVQ20KNKICSTIVE30IQRCEKEHFT40IPCKSNNDCQ50VWAHEKICN K60LPWGL65 . The carboxypeptidase A inhibitor is not homologous with the chymotrypsin/elastase or trypsin inhibitors from Ascaris, but shows homology in a 9-residue internal sequence with the 37/39-residue carboxypeptidase inhibitors from tomato and potato. The carboxy-terminal 5 (4) residues in the three inhibitors are similar, suggesting a common mechanism of inhibition.     

Alpha-adrenergic regulation of the secretion of an anticomplementary factor in mouse saliva.

Mouse saliva contains a potent inhibitor of complement activity. The secretion of this inhibitor appears to be regulated by action on alpha-adrenergic receptors for two reasons. First, an alpha-agonist (norepinephrine) elicited saliva with a 260-fold higher specific activity of the inhibitor than that obtained with a cholinergic agent (pilocarpine). Second, the alpha-agonist elicited saliva having 43-foldgreater specific activity than that obtained following administration of a beta-adrenergic agonist (isoproterenol). This anticomplementary factor probably proteolytically degrades one or more of the complement components since it is inhibited by several protease inhibitors. The salivary anticomplementary factor is more potent than trypsin, chymotrypsin, thrombin, or Kallikrein. The anticomplementary factor has a pattern of inhibition like that of Kallikrein but unlike those of trypsin or chymotrypsin.     

Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme

Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.

Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum).

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.     

Inhibition mechanism of a peanut trypsin-chymotrypsin inhibitor, B-III: determination of the reactive sites for trypsin and chymotrypsin

Peanut inhibitor B-III was found to form two types of complexes with trypsin, T2I and TI, by gel filtration HPLC. Two cleaved peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in the trypsin modified inhibitor (TM-B-III*R*S) (J. Biochem. 93, 479-485 (1983] were resynthesized by the complex formation with 2 mol of trypsin. These results suggest that the two peptide bonds may be the reactive sites for trypsin. TM-B-III*R*S inhibited bovine trypsin as well as native B-III but had little chymotrypsin inhibitory activity. The two peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in B-III were cleaved partly by prolonged incubation with a catalytic amount of chymotrypsin. But gel filtration HPLC of the chymotrypsin-inhibitor complex showed the formation of only CI complex. Incubation of TM-B-III*R*S with an equimolar amount of chymotrypsin resulted in the resynthesis of only the Arg(10)-Arg(11) bond. These findings suggest that Arg(10)-Arg(11) may be a true reactive site for chymotrypsin. An inhibition mechanism of B-III against trypsin and chymotrypsin was proposed from the results obtained by the present studies.     

Endothelium Dependent and Independent Relaxation of Aortic Rings from Watanabe Heritable Hyperlipidemic Rabbits after Exposure to a Free Radical Generating System

The effects of the xanthine oxidase/hypoxanthine free radical generating system on endothelium dependent and independent relaxation were compared in aortic rings from New Zealand white rabbits and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits with mild atherosclerosis. Studies were carried out in young (3 months) and mature (18 months) animals. Plasma cholesterol levels were significantly higher in both 3 and 18 month WHHL animals. Endothelium independent relaxation to SNP did not differ between groups. However, the attenuation of relaxation to carbachol after xanthine oxidase/hypoxanthine treatment tended to be less in WHHL. This reached significance at 18 but not 3 months. We propose that this could be due to increases in levels of endogenous scavenger enzymes in these WHHL rabbits.     

Characterization of alternative molecular forms of xanthine oxidase in the mouse

1. Two major forms of xanthine oxidase are demonstrated for the mouse. On polyacrylamide-gel electrophoresis the duodenal form migrates faster towards the anode than that of the liver. Both forms also differ in their (NH(4))(2)SO(4) precipitation patterns and sucrose-density-gradient molecular-weight determinations. 2. The liver form is fully converted into the duodenal form by incubation at 37 degrees C with 2.5mg of crude trypsin/ml for 1(1/2)h, without loss of activity. The trypsin-treated liver form behaves like the normal duodenal form as characterized by electrophoresis, (NH(4))(2)SO(4) precipitation patterns, and sucrose-density-gradient molecular-weight determinations. 3. Partial conversion is also brought about by purified trypsin and chymotrypsin, but not with beta-carboxypeptidase or lipase. The conversion is inhibited by soya-bean trypsin inhibitor. 4. In embryo mice the duodenal form is similar to the liver form on electrophoresis. 5. These studies indicate, as might be expected, that the duodenal form is a modified version of the liver enzyme, probably caused by proteolytic alteration.     

Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes.

A technique utilizing Pregnant Mare's Serum Gonadotropin and Human Chorionic Gonadotropin treatment of hens (Gallus domesticus), followed by manual ovulation of the excised follicles, was developed to obtain a large number of mature ova. The intact ova were used to test whether acrosin, partially purified from the spermatozoa of the cock (Gallus domesticus), partially purified rabbit testicular acrosin and commercial preparations of several hydrolytic enzymes could dissolve the inner vitelline membrane. Enzymes were applied to pieces of filter paper placed on the ovum. Cock acrosin and endopeptidases such as trypsin, chymotrypsin, collagenase and elastase hydrolyzed the membrane whereas exopeptidases such as leucine aminopeptidase and carboxypeptidase A did not. Phospholipase A, sulfatase, hyaluronidase, beta-glucuronidase and rabbit testicular acrosin also failed to hydrolyze the membrane. Cock acrosin hydrolysis of the ovum surface was inhibited by soybean trypsin inhibitor. The surface of the ovum over the germinal disc region was hydrolyzed more quickly by cock acrosin than the surface over other regions of the ovum. Acrosin from cock sperm caused the release of trichloroacetic acid soluble material absorbing at 280 nm from sonicated preparations of inner vitelline membranes. Hydrolysis was greatest at pH 8.0 and was inhibited by soybean trypsin inhibitor.     

Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors.

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.     

Stability and specificity of extracellular protein inhibitor for trypsin from Actinomyces janthinus 118]

Some properties of protein inhibitor for trypsin (TI) from Act. janthinus 118 were studied. It was shown that TI has an antitrypsin activity within a wide pH range with a maximum at about 9,5. At 4 degrees and 20 degrees C TI is stable for 24 hours within the pH range of 6,0--11,0. At 100 degrees C TI is more stable in the slightly acid region of pH than at neutral or alkaline conditions. Trypsin and chymotrypsin inactivate the inhibitor for 8 hours. TI inhibits trypsin, fibrinolysin, subtilisin, pronase and terrilytin, but have no effect on chymotrypsin, thrombin, papain and pepsin. The dissociation constants for the trypsin-inhibitor complex were found to be 1,7.10-8 M, 4,1.10-9 M and 2,4.10-10 M, with casein, p-nitroanilide benzoylarginine and tosylarginine methyl ester used as substrates, respectively. The corresponding dissociation rate constants for the subtilisin-inhibitor complex were equal to 1.10-9 M and 4.10-10 M with casein and carbobenzoxy-L-alanyl-L-alanyl-L-leucin p-nitroanilide used as substrates, respectively.     

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).     

Inhibitory effects of L-NG-nitro-arginine on the synthesis of EDRF and the cerebroarterial response to vasodilator nerve stimulation

Treatment with L-NG-nitro-arginine (L-NA) inhibited the brady-kinin-induced relaxation, mediated via EDRF, in dog coronary artery strips partially contracted with prostaglandin F2 alpha; the inhibition was prevented by L-, but not D-, arginine. Relaxation caused by nitroglycerin was not altered by L-NA. The release of EDRF, as assayed using dog coronary artery strips without endothelium, from perfused femoral artery segments with endothelium in response to acetylcholine and substance P was significantly reduced by treatment of the femoral artery with L-NA. The inhibitory effect was reversed by L-arginine. Relaxant responses of dog cerebral artery strips with and without endothelium to electrical stimulation of non-adrenergic, non-cholinergic nerves were suppressed by L-NA, whereas relaxation of coronary arteries with and without endothelium by the stimulation of adrenergic nerves was not influenced. The L-NA-induced inhibition was reversed by L-arginine. It is speculated that L-NA inhibits the synthesis of EDRF, as does L-NG-monomethyl arginine, and NO-like substance(s) produced plays an important role in transferring information from vasodilator nerves to smooth muscle in cerebral arteries.