Vascular endothelial growth factor VEGF-like heparin-binding protein from the venom of Vipera aspis aspis (Aspic viper).

The heparin-binding dimeric hypotensive factor (HF) was purified from Vipera aspis aspis (Aspic viper) venom [Komori, Y. and Sugihara, H. (1990) Toxicon 28, 359-369]. In this study, the amino acid sequence, and structure and function of HF, were elucidated. By electrospray ionization mass spectrometry (ESI-MS), the molecular weight of HF was determined to be 25 072.1. The complete amino acid sequence of HF was determined by Edman sequencing of the S-pyridylethylated HF and its peptides derived from enzymatic digestion. The theoretical molecular mass calculated from the primary structure agrees well with the molecular weight determined by ESI-MS. HF consists of two homogeneous monomers bound covalently. The monomer with an N-terminal blocked by pyroglutamic acid contains 110 amino acid residues, including eight cysteine residues, two of which are considered to be involved in intermolecular disulfide bonds. Sequential homology search revealed that the primary structure of HF is similar to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) with a sequential homology of 45 and 22%, respectively. When injected intradermally into a rat, an increase in capillary permeability was observed with HF or VEGF. On the other hand, only HF exerted a strong hypotensive effect after intravenous injection of samples into a rat. Purified HF has a mitogenic effect on endothelial cells. Through the use of bovine aortic endothelial cells (BAEC), the half-maximal mitogenic concentration of HF was determined to be 5-5. 5 nM (125-138 ng/mL). Similarly, VEGF had a mitogenic concentration at 0.5-1 nM. When incubated with HF and cycloheximide or HF and heparin, the cell growth was inhibited, suggesting that the mechanism of action of HF is similar to that of VEGF.     

Biological study of muscle degenerating hemorrhagic factor from the venom of Vipera aspis aspis (aspic viper)

1. A hemorrhagic factor was isolated from Vipera a. aspis venom by gel filtration, ion-exchange chromatography and affinity chromatography. 2. Molecular weight of the purified factor was determined to be 67,000 Da, which was composed of 552 amino acid residues. 3. The minimum hemorrhagic dose (MHD) was measured to be 0.11 micrograms per mouse with subcutaneous injection. The hemorrhagic activity was inhibited by chelating agents and reductant. 4. The hemorrhagic factor possesses proteolytic activity against dimethylcasein. 5. Serum creatine phosphokinase level was rapidly increased within 30 min following injections of this preparation into the mice thighs. 6. Actin, one of the main components of muscle fiber, is apparently digested by the hemorrhagic factor. The pathological findings are further reported in this paper.     

Isolation and characterization of factor X activator from the venom of Vipera aspis aspis

1. A factor X activator was isolated from the venom of Vipera aspis aspis (Aspic viper) by gel filtration and ion-exchange chromatography. 2. The purified activator has a mol. wt of 75,000 and an isoelectric point of 4.6. Upon reduction, this activator migrated as two bands with mol. wts of 16,000 and 14,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. 3. The activator from V. a. aspis venom shortened activated partial thromboplastin time (APTT) of normal plasma and factor IX-deficient plasma from humans. 4. Factor X incubated with isolated activator and calcium ions drastically shortened APTT of factor X-deficient plasma and expressed hydrolytic activity against synthetic substrates for factor Xa, however no hydrolytic activity was detected with the activator alone, indicating that the activator converted factor X to the active form.     

Sequences and structural organization of phospholipase A2 genes from Vipera aspis aspis, V. aspis zinnikeri and Vipera berus berus venom. Identification of the origin of a new viper population based on ammodytin I1 heterogeneity.

We used a PCR-based method to determine the genomic DNA sequences encoding phospholipases A2 (PLA2s) from the venoms of Vipera aspis aspis (V. a. aspis), Vipera aspis zinnikeri (V. a. zinnikeri), Vipera berus berus (V. b. berus) and a neurotoxic V. a. aspis snake (neurotoxic V. a. aspis) from a population responsible for unusual neurotoxic envenomations in south-east France. We sequenced five groups of genes, each corresponding to a different PLA2. The genes encoding the A and B chains of vaspin from the neurotoxic V. a. aspis, PLA2-I from V. a. zinnikeri, and the anticoagulant PLA2 from V. b. berus are described here. Single nucleotide differences leading to amino-acid substitutions were observed both between genes encoding the same PLA2 and between genes encoding different PLA2s. These differences were clustered in exons 3 and 5, potentially altering the biological activities of PLA2. The distribution and characteristics of the PLA2 genes differed according to the species or subspecies. We characterized for the first time genes encoding neurotoxins from the V. a. aspis and V. b. berus snakes of central France. Genes encoding ammodytins I1 and I2, described previously in Vipera ammodytes ammodytes (V. am. ammodytes), were also present in V. a. aspis and V. b. berus. Three different ammodytin I1 gene sequences were characterized: one from V. b. berus, the second from V. a. aspis, V. a. zinnikeri and the neurotoxic V. a. aspis, and the third from the neurotoxic V. a. aspis. This third sequence was identical with the reported sequence of the V. am. ammodytes ammodytin I1 gene. Genes encoding monomeric neurotoxins of V. am. ammodytes venom, ammodytoxins A, B and C, and the Bov-B LINE retroposon, a phylogenetic marker found in V. am. ammodytes genome, were identified in the genome of the neurotoxic V. a. aspis. These results suggest that the population of neurotoxic V. a. aspis snakes from south-east France may have resulted from interbreeding between V. a. aspis and V. am. ammodytes.     

Effects of dried bonito (katsuobushi) and captopril, an angiotensin I-converting enzyme inhibitor, on rat isolated aorta: a possible mechanism of antihypertensive action

In order to elucidate the mechanism of the antihypertensive action of dried bonito (katsuobushi), we compared the effects of dried bonito extracts with those of captopril, an angiotensin I-converting enzyme (ACE) inhibitor, on aorta preparations isolated from rats. Dried bonito extracts (3 x 10(-4) to 3 x 10(-3) g/ml) more potently relaxed contractions induced by norepinephrine (10(-7) M) than contractions induced by KCl (55.9 mM). Dried bonito extracts (3 x 10(-3) g/ml) slightly inhibited 10(-7) M angiotensin I-induced contractions. In contrast, captopril (10(-8) to 10(-7) M) did not affect 10(-7) M norepinephrine- or 55.9 mM KCl-induced contractions, but a higher concentration of captopril (10(-6) M) very slightly relaxed it. Captopril (10(-8) to 10(-6) M) markedly inhibited 10(-7) M angiotensin I-induced contractions in a concentration-dependent manner. These results suggest that antihypertensive mechanism of action induced by dried bonito involves direct action on vascular smooth muscle in addition to ACE-inhibitory activity.     

Molecular phylogeny of Vipera Laurenti, 1768 and the related genera Macrovipera (Reuss, 1927) and Daboia (Gray, 1842), with comments about neurotoxic Vipera aspis aspis populations

We used mtDNA sequences (cytochrome b and NADH dehydrogenase subunit 2) to reconstruct molecular phylogenies of Vipera sensu lato, Vipera sensu stricto, and Vipera aspis. Three major clades were identified within the Vipera s.l. group: (1) the European vipers, (2) the oriental vipers, consisting of Montivipera (Vipera 2) plus Macrovipera lebetina, and (3) a group of Asian and North African vipers consisting of Daboia russelii, V. palaestinae, and Macrovipera mauritanica. We also distinguished three clades within the monophyletic European Vipera group: V. ammodytes, V. aspis, and V. latastei, and Pelias with monophyly of Vipera 1 uncertain. Within V. aspis, the specimens collected in France formed the sister group of an Italian clade. The "neurotoxic" French population of V. aspis, which has a specific venom profile, separated from other French V. aspis early in the history of this group.     

A Globin Fragment, LVV-Hemorphin-7, Induces [3H]Thymidine Incorporation in a Neuronal Cell Line via the AT4 Receptor

The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C-terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV-hemorphin-7, a fragment of beta globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108-15, LVV-hemorphin-7 and angiotensin IV competed for 125I-angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 x 10(-10) and 1.1 x 10(-9) M for the high-affinity site, respectively, and 6.7 x 10(-8) and 1.5 x 10(-8) M for the low-affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV-hemorphin-7, but not angiotensin IV, elicited a 1.8-fold increase in DNA synthesis in a dose-dependent manner. Furthermore, co-incubation of the cells with an excess of angiotensin IV (10(-6) M) inhibited LVV-hemorphin-7-stimulated DNA synthesis. Therefore, whereas LVV-hemorphin-7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV-hemorphin-7 elicited [3H]thymidine incorporation in NG108-15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV-hemorphin-7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.     

Climate affects embryonic development in a viviparous snake, Vipera aspis

Climatic conditions during embryonic development can exert profound and long-term effects on many types of organisms, but most previous research on this topic has focussed on endothermic vertebrates (birds and mammals). Although viviparity in ectothermic taxa allows the reproducing female to buffer ambient thermal variation for her developing offspring, even an actively thermoregulating female may be unable to provide optimal incubation regimes in severe weather conditions. We examined the extent to which fluctuations in natural thermal conditions during pregnancy affect reproduction in a temperate viviparous snake, the aspic viper (Vipera aspis). Data gathered from a long term field study demonstrated that ambient thermal conditions influenced (1) female body temperatures and (2) gestation length, embryo viability, and offspring phenotypes. Interestingly, thermal conditions during each of the three months of gestation affected different aspects of reproduction. Hotter weather early in gestation (June) increased ventral scale counts (=number of body segments) of neonates; hotter weather mid-gestation (July) hastened development and thus the date of parturition; and hotter weather late in gestation (August) reduced the incidence of stillborn neonates. The population that we studied is close to the northern limit of the species' range, and embryonic thermal requirements may prevent Vipera aspis from extending into cooler conditions further north.     

Protease nexin II interactions with coagulation factor XIa are contained within the Kunitz protease inhibitor domain of protease nexin II and the factor XIa catalytic domain

Protease nexin II, a platelet-secreted protein containing a Kunitz-type domain, is a potent inhibitor of factor XIa with an inhibition constant of 250-400 pM. The present study examined the protein interactions responsible for this inhibition. The isolated catalytic domain of factor XIa is inhibited by protease nexin II with an inhibition constant of 437 +/- 62 pM, compared to 229 +/- 40 pM for the intact protein. Factor XIa is inhibited by a recombinant Kunitz domain with an inhibition constant of 344 +/- 37 pM versus 422 +/- 33 pM for the catalytic domain. Kinetic rate constants were determined by progress curve analysis. The association rate constants for inhibition of factor XIa by protease nexin II [(3.35 +/- 0.35) x 10(6) M(-1) s(-1)] and catalytic domain [(2.27 +/- 0. 25) x 10(6) M(-1) s(-1)] are nearly identical. The dissociation rate constants are very similar, (9.17 +/- 0.71) x 10(-4) and (7.97 +/- 1.1) x 10(-4) s(-1), respectively. The rate constants for factor XIa and catalytic domain inhibition by recombinant Kunitz domain are also very similar: association constants of (3.19 +/- 0.29) x 10(6) and (3.25 +/- 0.44) x 10(6) M(-1) s(-1), respectively; dissociation constants of (10.73 +/- 0.84) x 10(-4) and (10.36 +/- 1.3) x 10(-4) s(-1). The inhibition constant (K(i)) values calculated from these kinetic parameters are in close agreement with those measured from equilibrium binding experiments. These results suggest that the major interactions required for factor XIa inhibition by protease nexin II are localized to the catalytic domain of factor XIa and the Kunitz domain of protease nexin II.     

Effects of angiotensin I and II and their interactions with some prostanoids in perfused human umbilical arteries

In perfused human umbilical arteries both angiotensin I and II induced vasoconstriction with a monophasic response. Angiotensin I and II induced vasoconstrictions at doses greater than or equal to 10(-8) M and 10(-9) M respectively. Captopril inhibited the angiotensin I response while the angiotensin II receptor blocker Sar1-Ala8 AII inhibited the effect of both angiotensins. PGI2 attenuated the angiotensin II response in a dose dependent pattern. PGE2 and PGF2 alpha in concentrations below the critical levels for creating pressure responses per se, also attenuated the angiotensin II response. The cyclooxygenase inhibitor indomethacin potentiated the angiotensin II response indicating that endogenous production of prostanoids is of importance in the modulation of angiotensin effects.     

Angiotensin I-converting enzyme from guinea pig lung and serum. A comparison of some kinetic and inhibition properties.

The angiotensin I-coverting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) was isolated from both guinea pig lung and serum; Km and V values were determined using both angiotensin I and hippurylhistidylleucine as substrates. Km values for the lung enzyme were 3.1 mM for hippurylhistidylleucine hippurylhistidylleucine and 0.076 mM for angiotensin I. Inhibition studies were performed and I50 values were obtained with the following inhibitors: angiotensin II (lung, 1.9 - 10(-5) M; serum, 1.7 - 10(-5) M), bradykinin (lung, 2.6 - 10(-6) M; serum, 2.1 - 10(-6) M), and pyrrolidone-Lys-Trp-Ala-Pro (lung, 7.9 - 10(-8) M; serum, 5.6 - 10(-8) M). Both enzymes were glycoproteins and were inhibited by concanavalin A. A maximum inhibition of 35% initial enzymatic activity was observed for both enzymes at a concanavalin A concentration of 4 - 10(-4) M suggesting that the sugar moieties of each enzyme are similar. Both enzymes required NaCl for activity and were inhibited by EDTA. A comparison of kinetic and inhibition properties indicates that both enzymes are quite similar.     

Mechanism of increased angiotensin-converting enzyme activity stimulated by platelet-activating factor

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 x 10(-10) to 1 x 10(-6) M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 x 10(-6) M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca2+ influx and inositol 1,4,5-trisphosphate (IP3) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP3. To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca2+ influx and IP3 release stimulated by 10(-6) M of PAF were suppressed by about 60-70%. ACE activity was also inhibited up to 70% in the presence of PAF (10(-10) - 10(-6) M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activity in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP3-mediated Ca2+ release from intracellular stores.     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

Inhibition of some hepatic lysosomal glycosidases by ethanolamines and phenyl 6-deoxy-6-(morpholin-4-yl)-beta-D-glucopyranoside.

The hepatic lysosomal glycosidases alpha-glucosidase and beta-glucuronidase were inhibited in vitro and in vivo by mono- and diethanolamines. The in vivo inhibition is dose dependent and occurs at a value less than LD50. Phenyl 6-deoxy-6-(morpholin-4-yl)-beta-D-glucopyranoside inhibited alpha-glucosidase both in vitro and in vivo. The treatment of the enzymes in vitro by ethanolamine exhibited a reversible inhibition of the mixed and competitive types for alpha-glucosidase and beta-glucuronidase, respectively. Diethanolamine showed a reversible inhibition of the competitive type for both enzymes. It is a potent inhibitor for beta-glucuronidase, in vitro, whose inhibition constant (Ki) is 5 x 10(-5) M. Phenyl 6-deoxy-6-(morpholin-4-yl)-beta-D-glucopyranoside is a potent inhibitor only for hepatic alpha-glucosidase with a Ki value of 1.6 x 10(-5) M. The pattern of the pH dependence of enzymic activity was not affected by ethanolamine inhibition. The magnitude of the inhibition of enzymes is dependent on the structure of the inhibitor.     

The inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes.

A novel proteinase inhibitor, Lentinus proteinase inhibitor, has been purified from the fruiting bodies of the edible mushroom, Lentinus edodes, by buffer extraction and affinity chromatography on immobilized anhydrotrypsin. The protein simultaneously inhibits bovine beta-trypsin and alpha-chymotrypsin at independent sites, with apparent dissociation constants of 3.5 x 10(-10) M and 4 x 10(-8) M, respectively. The purified protein is eluted as two well-separated peaks on reversed-phase HPLC, one of which is inhibitory-active and the other inactive, and they are interconvertible under folding/unfolding conditions. Among the mammalian and microbial serine proteinases examined, including human enzymes of blood coagulation and fibrinolysis, activated factor XI was inhibited by the Lentinus proteinase inhibitor. Chemical modification studies suggest involvement of one or more arginine residues in the inhibition of trypsin. The complete primary structure composed of 142 amino acids with an acetylated N-terminus was determined by protein analysis. The theoretical molecular mass (15999.2) from the sequence is close to the experimental value of 15999.61 +/- 0.61 determined by mass spectrometry. Although there are no apparently homologous proteinase inhibitors in the protein database, there is a rather striking similarity to the propeptide segment of a microbial serine proteinase, as well as to the N-terminal region of the mature enzyme.     

Hydrocortisone inhibits rat basophilic leukemia cell mediator release induced by neutrophil-derived histamine releasing activity as well as by anti-IgE.

We determined the ability of hydrocortisone to inhibit rat basophilic leukemia cell mediator release induced by anti-IgE and by neutrophil-derived histamine-releasing activity (HRA-N). Serotonin release induced by HRA-N and anti-IgE was inhibited by 78 +/- 5 and 70 +/- 4%, respectively (IC50 7.5 x 10(-7)M) by hydrocortisone (10(-5)M). HRA-N does not cause arachidonic acid metabolism, however, anti-IgE induced the generation of PGD2 and leukotriene (LT)C4, and the generation of both mediators was inhibited by 10(-5)M hydrocortisone (IC50 = 4.8 x 10(-7)M, and 3.6 x 10(-9)M, respectively). Inhibition required at least 5 to 6 h of hydrocortisone exposure and was maximal after 22 h. The observed effects of hydrocortisone could be reproduced by human recombinant lipocortin-I (5 x 10(-7)M). Hydrocortisone, 10(-5)M, was a less potent inhibitor of calcium ionophore A23187-mediated serotonin release and PGD2 and LTC4 generation (inhibition of 20 +/- 2, 17 +/- 10, and 37 +/- 10%, respectively). Inasmuch as A23187-induced stimulation is not dependent on receptor coupling, the enhanced ability of hydrocortisone to inhibit IgE- and HRA-N-mediated events as compared with A23187 suggests that one possible site of action of hydrocortisone may be interruption of receptor-effector signals. In the presence of arachidonic acid, hydrocortisone-treated cells released as much LTB4 and PGD2 as control cells, however, serotonin release and LTC4 generation were inhibited 50 and 55%, respectively. Thus, these data suggest that hydrocortisone has three possible sites of action: 1) inhibition of phospholipase A2 activity, 2) inhibition of glutathione-s-transferase, and 3) inhibition of serotonin release by a third mechanism, possibly by interrupting the coupling of receptor and effector systems.     

Characterization of urinary proteinase inhibitors with segments of amino acids sequences identical to sequences of pancreatic secretory trypsin inhibitor

1. Slow migrating proteinase inhibitors were isolated from pathological human urine. 2. The N-terminal amino acid sequence including 23 amino acids was identical to the one in pancreatic secretory trypsin inhibitor. 3. The slow migrating proteinase inhibitors occurred in 3 forms with different electrophoretic mobility. 4. Time of flight mass spectrometry showed that the Mw of one of the forms was 6241 while the Mw of another form was 5923. 5. The Ki of complexes with trypsin was determined to be 1 x 10(-10) M, with chymotrypsin and plasmin Ki was 1 x 10(-7) M. Elastase, kallikrein and thrombin were not inhibited.     

Angiotensin II contributes to arterial compliance in congestive heart failure

Arterial compliance is determined by structural factors, such as collagen and elastin, and functional factors, such as vasoactive neurohormones. To determine whether angiotensin II contributes to decreased arterial compliance in patients with heart failure, this study tested the hypothesis that administration of an angiotensin-converting enzyme inhibitor improves arterial compliance. Arterial compliance and stiffness were determined by measuring carotid artery diameter, using high-resolution duplex ultrasonography, and blood pressure in 23 patients with heart failure secondary to idiopathic dilated cardiomyopathy. Measurements were made before and after intravenous administration of enalaprilat (1 mg) or vehicle. Arterial compliance was inversely related to both baseline plasma angiotensin II (r = -0.52; P = 0.015) and angiotensin-converting enzyme concentrations (r = -0.45; P = 0.041). During isobaric conditions, enalaprilat increased carotid artery compliance from 3.0 +/- 0.4 to 5.0 +/- 0.4 x 10(-10) N(-1). m(4) (P = 0.001) and decreased the carotid artery stiffness index from 17.5 +/- 1.8 to 10.1 +/- 0.6 units (P = 0.001), whereas the vehicle had no effect. Thus angiotensin II is associated with reduced carotid arterial compliance in patients with congestive heart failure, and angiotensin-converting enzyme inhibition improves arterial elastic properties. This favorable effect on the pulsatile component of afterload may contribute to the improvement in left ventricular performance that occurs in patients with heart failure treated with angiotensin-converting enzyme inhibitors.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

Methyl xanthine phosphodiesterase inhibitors behave as prostaglandin antagonists in a perfused rat mesenteric artery preparation.

The methyl xanthines, theophylline, caffeine and 3-isobutyl-1 methyl xanthine (MIX) inhibited the pressure responses to noradrnealine, angiotensin II and potassium ions in the isolated perfused mesenteric vascular bed of the male rat. The ID50s for inhibition of responses to noradrenaline were 1.85 mug/ml (0.83 x 10(-5) M) for MIX, 18 mug/ml (1 x 10(-4)M) for theophylline and 133 mug/ml (6.8 x 10(-4) M) for caffeine. Similar ID50 concentrations were found for responses to angiotensin II and potassium. We have previously found that substances which inhibit the three pressor agents equally may be prostaglandin (PG) synthesis inhibitors or PG antagonists. Xanthine itself, cyclic AMP and dibutyrl cyclic AMP had no inhibitory effects on the preparation up to concentrations of 10-2 M. Partial inhibition of PG synthesis by indomethacin shifted the % inhibition/log concentration curve to the left, while addition of exogenous PGE2 shifted it to the right. In preparations completely inhibited by sufficient indomethacin added to the perfusate to block PG synthesis, and then restored by adding 1 or 5 ng/ml PGE2 in addition to the indomethacin, the methyl xanthines again inhibited responses suggesting that they were PG antagonists rather than inhibitors of synthesis or release. In preliminary experiments MIX also inhibited effects of PGF2alpha on rat uterus and PGE1 on guinea pig ileum. Effective concentrations of theophylline were similar to the therapeutic levels in human plasma. PG antagonists may be a major action of methyl xanthines requiring reinterpretation of many experiments which have attributed their effects to PDE inhibition. PGs may also be involved in regulating PDE action.