Endothelial modulation of vasoconstrictor responses in the perfused rabbit ovarian vascular bed.

The purpose of this study was to investigate the effects of endothelial denudation, inhibitors of nitric oxide (NO) and prostanoid synthesis on vasoconstrictor responses in the perfused rabbit ovarian vascular. The experiments were conducted using an in vitro perfusion system, where the ovarian vascular bed (en bloc) was perfused with Krebs' solution delivered at a constant flow rate using a peristaltic pump. Changes in perfusion pressure, which reflected peripheral resistance, were measured. Results showed that noradrenaline (NA) (10(-9) to 10(-6) mol) induced reproducible dose-dependent vasoconstrictor responses. Vasoconstrictor effects of low doses of noradrenaline were not affected by perfusion of the vascular bed with CHAPS (4.7 mg/ml for 30 s) to remove the endothelium. The same treatment however, significantly reduced responses induced by the higher doses of noradrenaline, thus depressing the maximum response. KCl-induced vasoconstriction was not affected by CHAPS. L-N(G)-nitroarginine (L-NOARG) (10(-5) mol/l) enhanced NA-induced vasoconstriction. D-NOARG, the inactive isomer of L-NOARG and aminoguanidine, an inhibitor of inducible nitric oxide synthase reduced rather than enhanced noradrenaline-induced responses. Methylene blue (3 x 10(-5) mol/l) and LY 83583 (10(-5) mol/l) produced a potentiation of NA-induced vasoconstrictor responses. Indomethacin (3 x 10(-6) mol/l) did not significantly enhance NA-induced vasoconstriction. The nonselective endothelin antagonist, SB 209670 (10(-7) and 10(-6) mol/l) did not inhibit the vasoconstriction to NA. In conclusion, results are interpreted to suggest that NA-induced vasoconstriction in the perfused rabbit ovarian vascular bed is accompanied by a release of NO and possibly endothelium-derived contracting factor. There was no evidence for a modulation of vasoconstrictor responses by products of arachidonic acid metabolism or endothelins released from the endothelium.

Chronic hypoxia differentially alters the responses of pulmonary arteries and veins to endothelin-1 and other agents.

The effects of chronic hypoxia on the responses of rat large pulmonary arteries and veins to vasoactive agents have been examined. Endothelin-1-induced contractions of pulmonary arteries and pulmonary veins were reduced by chronic hypoxia. In contrast, chronic hypoxia augmented sarafotoxin 6c-induced contractile responses in pulmonary veins but not in pulmonary arteries. Chronic hypoxia augmented the constrictor effect of phenylephrine in pulmonary arteries, but not in pulmonary veins. The thromboxane receptor agonist, U46619 (9,11-dideoxy-9alpha,11alpha-epoxy-methanoprostaglandin++ + f2alpha) contracted pulmonary arteries and pulmonary veins, and although maximal responses were not altered in chronically hypoxic preparations, the EC50 value in pulmonary arteries was increased following chronic hypoxia. The relaxant effects of acetylcholine and isoprenaline on pulmonary arteries were potentiated by chronic hypoxia. In contrast, ionomycin-mediated relaxations of pulmonary arteries and pulmonary veins were reduced, while sodium nitroprusside-induced relaxation of pulmonary arteries and veins were not altered by chronic hypoxia. Previous studied have looked primarily at the effects of chronic hypoxia on pulmonary arteries. This data provides evidence that chronic hypoxia also causes selective changes in the reactivity of large pulmonary veins.

Source(s) of activator calcium for noradrenaline-induced vasoconstriction in the perfused rabbit isolated ovarian vascular bed: a role for tyrosine kinase.

The effects of Ca2+ withdrawal and agents affecting Ca2+ translocation on alpha1-adrenoceptor-mediated vasoconstrictor responses in the perfused rabbit ovarian vascular bed were studied. Noradrenaline-induced vasoconstriction was lost in a Ca(2+)-free Krebs' solution, and the rate of loss of the response was accelerated by EGTA (2 mM). Noradrenaline-induced vasoconstriction and SDZ NVI 085-induced vasoconstriction were concentration-dependently inhibited by verapamil and nifedipine. These agents were, however, more effective against KCl-induced responses. Cyclopiazonic acid, an intracellular Ca(2+) depletor, concentration-dependently inhibited noradrenaline-induced responses and abolished the response in Ca(2+)-free Krebs' solution. GF 109203X and polymyxin B, inhibitors of protein kinase C (PKC), had no significant effect on noradrenaline-induced responses. Tyrosine kinase inhibitors, genistein and erbstatin, inhibited noradrenaline-induced vasoconstriction in the perfused rabbit ovarian vascular bed. The results would suggest that both extracellular Ca2+ and intracellular Ca2+ participate in noradrenaline-induced vasoconstrictor responses in the perfused rabbit ovarian vascular bed. The results would also suggest that tyrosine kinase and not protein kinase C activation has a role in such effects.

Evidence for oxygenation-induced endothelin release from isolated lungs of chronically hypoxic rats.

In lungs from chronically hypoxic (CH, 3 weeks at 10% inspired O2) rats, oxygenation (20% O2, 5% CO2, 75% N2; PO2 121 mmHg) of the perfusate increases pulmonary perfusion pressure (PPP) and lung weight (LW). Hypoxic perfusate (95% N2, 5% CO2; PO2 5.5 mmHg) had no effect on PPP in lungs from CH rats. Indomethacin and nitro-L-arginine (L-NOARG) augmented the oxygen-induced increase in PPP. In contrast, the free radical scavengers superoxide dismutase (SOD) plus catalase delayed the onset of oxygen-induced vasoconstriction, while the endothelin (ET)B receptor antagonist BQ788 inhibited it. The ET(A) receptor antagonist BQ123 did not affect the PPP changes. This suggests a role for endogenous endothelins and ET(B) receptors in mediating the oxygenation-induced pulmonary vasoconstriction. Indomethacin had no effect on oxygen-induced lung weight (LW) changes while BQ788 and L-NOARG reduced the LW increase. This evidence shows that ET(B) receptor activation and NO generation are involved in the LW changes. In conclusion, oxygenation of the perfusate in isolated lungs from CH rats leads to pulmonary vasoconstriction which involves endothelins and activation of ET(B) receptors. In addition, increased NO production associated with ET(B) receptor activation is the prime stimulus for observed LW increase.

Cooling-induced contraction in ovine airways smooth muscle.

The mechanism of cold-induced bronchoconstriction is poorly understood. This prompted the present study whose aim was to determine the step-wise direct effect of cooling on smooth muscle of isolated ovine airways and analyse the role of calcium in the mechanisms involved. Isolated tracheal strips and bronchial segments were suspended in organ baths containing Krebs' solution for isometric tension recording. Tissue responses during stepwise cooling from 37 to 5 degrees C were examined. Cooling induced a rapid and reproducible contraction proportional to cooling temperature in ovine tracheal and bronchial preparations which was epithelium-independent. On readjustment to 37 degrees C the tone returned rapidly to basal level. Maximum contraction was achieved at a temperature of 5 degrees C for trachea and 15 degrees C for bronchiole. Cooling-induced contractions (CIC) was resistant to tetrodotoxin (1; 10 micrometer), and not affected by the muscarinic antagonist atropine (1 micrometer) or the alpha-adrenergic antagonist phentolamine (1 micrometer), or the histamine H1-antagonist mepyramine (1 micrometer) or indomethacin (1 micrometer). Ca2+ antagonists (nifedipine and verapamil) and Mn2+ raised tracheal but not bronchiolar tone and augmented CIC. Incubation in Ca2+-free, EGTA-containing Krebs' solution for 5 min had no effect on CIC, although it significantly reduced KCl-induced contraction by up to 75%. Cooling inhibited Ca2+ influx measured using 45Ca2+ uptake. Caffeine (100 micrometer) significantly inhibited CIC. The results show that cooling-induced contractions do not appear to involve activation of nerve endings, all surface reception systems or Ca2+ influx. However, CIC is mainly dependent on release of intracellular Ca2+.

Cooling-induced bronchoconstriction: the role of ion-pumps and ion-carrier systems.

The mechanism of cold-induced bronchoconstriction is poorly understood. Our previous results show that cooling-induced contractions (CIC) do not involve activation of cell surface receptor systems nor innervation nor Ca2+ uptake. However, the results show that CIC is mainly dependent on intracellular Ca2+ [32]. Isolated tracheal strips and bronchiolar segments were suspended in organ baths containing Krebs' solution for isometric tension recording. Tissue responses during stepwise cooling from 37 to 5 degrees C were examined. Cooling ovine tracheal strips and bronchiolar ring segments to 20 degrees C caused a rapid contraction which decreased slowly until it reached the basal level in approximately 30 min. There is a significant inhibition of 45Ca2+ efflux at 20 degrees C to those incubated at 37 degrees C. This prompted this study whose aim was to determine the role of ion-pump and ion-carrier systems on cooling mechanisms. Inhibition of the Na+/K+ pump with ouabain (10 micrometer) evoked contraction in tracheal and bronchiolar preparations. When cooling was superimposed on this contraction the cooling-induced contractions were reduced in the bronchiolar segments. In the tracheal strips, at temperatures down to 15 degrees C ouabain converted the contractions to a relaxation, but further cooling to 10 and 5 degrees C resulted in contractions that were similar to control at 5 degrees C. Staurosporine, a protein kinase inhibitor (1 micrometer) enhanced CIC in trachea and bronchiole. Vanadate, a Ca2+-ATPase pump inhibitor (1 mm) potentiated CIC in the two preparations. Trifluoperazine and W-7, calmodulin antagonists (10 and 100 micrometer) enhanced CIC in tracheal preparations but not in the bronchiolar segments. Thapsigargin and cyclopiazonic acid (CPA), inhibitors of sarcoplasmic reticulum (SR) Ca2+-ATPase pump (1 and 10 micrometer) potentiated CIC in tracheal but not in bronchiolar preparations. Amiloride, Na+/H+ and Na+/Ca2+ exchange system inhibitor (1 mm) abolished CIC in both trachea and bronchiole. These results show a strong relationship between cooling and the activity of ion transport systems and indicate that CIC is due to inhibition of calcium removal mechanisms as a result of inhibition of these ion-pump and ion carrier systems.

Biphasic relaxant response of ovine trachealis muscle to electrical field stimulation: influence of cooling.

Electrical field stimulation of ovine trachealis muscle produced neurogenic atropine-sensitive contractions under resting conditions. However, when the tissues were precontracted with 5-hydroxytryptamine in the presence of atropine, electrical field stimulation induced a frequency-dependent tetrodotoxin-sensitive relaxation. The relaxation was biphasic, consisting of fast and slow phases. The fast component was attenuated by propranolol, indicating an action on beta-adrenoceptors. The slow phase was attenuated by capsaicin and, therefore, involved release of a peptide. These results showed that excitatory responses in ovine trachealis muscles are cholinergically mediated, while both adrenergic and peptidergic components mediate electrically induced relaxation in the trachea. We also examined the influence of lowering bath temperature to 20 degrees C on electrically evoked responses. These were significantly reduced by cooling. At 20 degrees C, under resting conditions, the time-to-peak tension was lengthened, and the amplitude of the contractile responses was significantly (p < 0.05) reduced. In the same preparation, carbachol-induced contractions were not reduced by cooling, indicating that the reduction in electrically induced contractions was probably due to a reduction in transmitter release. Cooling also abolished the fast inhibitory phase (adrenergic in nature) without significantly inhibiting the slow (non-adrenergic, non-cholinergic) component. Propranolol (1 micromol/l) and capsaicin (100 micromol/l) did not affect significantly the slow relaxation observed during cooling. It was concluded that cooling inhibited cholinergically mediated, electrically induced contractions and selectively abolished the adrenergic component of electrically induced relaxant responses.

Sodium nitroprusside-induced cGMP-independent vasodilator responses in the perfused rabbit ovarian vascular bed.

Sodium nitroprusside (SNP) (10(-9)-10(-6) mol) and carbachol (10(-9)-10(-6) mol) induced dose-dependent vasodilatation in the perfused rabbit isolated ovarian vascular bed. Carbachol, but not SNP-induced vasodilatation was abolished by treatment with CHAPS (4. 7 mg ml-1, 30 s) to remove the endothelium. Carbachol-induced responses were also significantly attenuated by LY 83583 (10(-5) M) and methylene blue (3x10(-5) M). L-NOARG (10(-5) M) reduced carbachol-induced vasodilatation. None of these compounds affected SNP-induced vasodilator responses. Both SNP- and carbachol-induced vasodilatation were attenuated by raising the [K+] in the Krebs' solution to 40 mM. The responses were also reduced by TEA (20 mM) but not by glibenclamide. It was therefore concluded that SNP induced cGMP-independent vasodilator responses in the perfused rabbit ovarian vascular bed. This vasodilator response involved membrane hyperpolarisation since it was lost in high [K+] Krebs' solution. (c) 1998 The Italian Pharmacological Society.

Evidence for endothelin involvement in the pulmonary vasoconstrictor response to systemic hypoxia in the isolated rat lung.

We investigated the effect of systemic hypoxia (Krebs-Henseleit solution gassed with 5% CO2/95% N2) on an isolated, perfused rat lung. Hypoxia resulted in a slowly developing sustained increase in pulmonary perfusion pressure (PPP) accompanied by an increase in lung weight (LW). The endothelin (ET) receptor antagonists BQ123 (3 and 10 microM), BQ788 (3 microM) and bosentan (1.5 and 5 microM) all attenuated the hypoxia-induced increases in LW and PPP. In addition, phosphoramidon (1 microM), an ET-converting enzyme inhibitor, also significantly attenuated the hypoxia-induced increases in PPP and LW. The use of two agents that alter peptide secretion, phalloidin (10 and 50 nM) and colchicine (100 nM), and the peptide synthesis inhibitor cycloheximide (5 microM) all significantly attenuated the hypoxia-induced increases in PPP and LW. The increase in PPP and LW after the onset of hypoxia was accompanied by an increase in perfusate levels of ET-1 compared with normoxic time-matched controls. The results show that in this model, systemic hypoxia is capable of causing a sustained vasoconstriction and increased LW. The fact that these increases can be attenuated by an ET-converting enzyme inhibitor, ET receptor antagonists and agents that block peptide synthesis and secretion, together with the increase in perfusate levels of ET-1, suggests that ET production and release contribute to the changes seen.

A pharmacological study on the role of nitric oxide in the pathogenesis of experimental allergic encephalomyelitis.

OBJECTIVE AND DESIGN: The study examines the effects of nitric oxide synthase (NOS) inhibitors on the development of neurological EAE and the levels of nitrite in the central nervous system (CNS) during established disease. MATERIALS: EAE was induced in male Lewis rats (200-250 g). TREATMENT: Rats received NG-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg body weight) day 7 to 12 post-inoculation (P.I.), 7-nitroindazole (10 mg/kg) day 7 to 11 P.I. or aminoguanidine (200 or 400 mg/kg) day 1 to 12 P.I. METHODS: Neurological symptoms were assessed and CNS cytosol nitrite and protein levels measured. Results were analysed using the Mann Whitney U-test and the Fischer exact probability test. RESULTS: Symptoms of EAE were associated with a significant elevation in CNS nitrite (p < 0.001). Treatment with NOS inhibitors caused a marked reduction in nitrite levels (p < 0.01). However, in some experiments, vehicle administration also reduced CNS nitrite content (p < 0.05). Although neurological disease was suppressed in EAE-sensitised rats receiving L-NAME (2 +/- 0.3 vs 3 +/- 0.3 mean peak severity +/- SEM) and 7-nitroindazole (1 +/- 0.3 vs 3 +/- 0.3, p < 0.01) comparable inhibition was achieved by respective vehicle treatment (p < 0.01). In contrast, neither aminoguanidine nor corresponding vehicle altered disease development. CONCLUSIONS: Drug-induced reductions in CNS nitrite levels during EAE are not always associated with a suppression of neurological symptoms, which suggests NO is not of primary importance in disease pathogenesis. In addition, the study emphasises the strict requirement for appropriate controls when assessing the efficacy of drugs on the course of EAE.

Investigation of the contributions of nitric oxide and prostaglandins to the actions of endothelins and sarafotoxin 6c in rat isolated perfused lungs.

1. The aims of the study were to assess the contribution of prostaglandins and nitric oxide (NO) to the effects of endothelin (ETs) and sarafotoxin 6c (SX6c) in perfused rat lungs. This was carried out by using indomethacin, a cyclo-oxygenase inhibitor and NG-nitro-L-arginine (L-NOARG), a NO synthase inhibitor. Responses were studied under basal perfusion conditions and in other experiments after the elevation of vascular tone with the thromboxane-mimetic, U46619. The sub-types of ET receptors involved were characterized by use of ET receptor antagonists and cross-tachyphylaxis. 2. Pulmonary perfusion pressure (PPP), lung weight and pulmonary inflation pressure (PIP), were continuously recorded. Although L-NOARG (100 microM) did not alter basal parameters it markedly augmented the vasoconstriction and lung weight increases induced by ET-1 (50-400 pmol) or SX6C (25-200 pmol) while vasoconstrictor responses to phenylephrine were not affected by L-NOARG. 3. L-NOARG markedly potentiated the bronchoconstriction induced by ET-1 or SX6C whereas it had no effect on responses to carbachol. 4. When vascular tone was elevated, low doses (1.25-40 pmol) of ET-1, ET-3 and SX6C produced falls in PPP. The vasodilator potencies were SX6C > ET-1 = ET-3. The ETA receptor antagonist, BQ123, did not affect these depressor responses whereas the mixed ETA/ETB antagonist, bosentan, blocked them. 5. Indomethacin (10 microM) partially inhibited vasodilator response to ET-1, whereas it had no effect on SX6C-induced vasodilation. 6. L-NOARG plus indomethacin completely blocked ET-1 induced vasodilation, whereas responses to SX6C were blocked by L-NOARG alone. 7. Repeated injections of submaximal doses of ET-1 or SX6C caused tachyphylaxis to vasodilator responses. Subsequent injections of SX6C or ET-1 did not elicit depressor responses showing cross tachyphylaxis had occurred. 8. These findings indicate that under basal conditions the pulmonary vasoconstrictor, lung weight and bronchoconstrictor responses to ET-1 and SX6C are attenuated by evoked release of nitric oxide (NO). When vascular tone was elevated, lower doses of ETs and SX6C produced vasodilatation. These vasodilator responses are indirect, those to SX6C being mediated via NO production, whereas those to ET-1 involve both NO and prostanoid(s). Tachyphylaxis and ET antagonist experiments indicate that the same receptor subtype is involved in mediating the vasodilatation and that this is of the ETB type located on the endothelium. However the post-receptor vasodilator events triggered by ET-1 or SX6C appear to be different.

Characterization of alpha-adrenoceptor subtype(s) mediating vasoconstriction in the perfused rabbit ovarian vascular bed.

1. alpha 1-Adrenoceptor agonists, noradrenaline, phenylephrine, methoxamine, oxymetazoline and SDZ NVI 085 but not alpha 2-adrenoceptor agonists, UK 14304, tizanidine or clonidine evoked dose-dependent vasoconstriction of the isolated perfused rabbit ovarian vascular bed. The rank order of agonist potency was noradenaline > oxymetazoline > phenylephrine > SDZ NVI 085 > methoxamine. 2. Prazosin (10(-8) M - 10(-5) M) displaced agonist dose-response curves to the right. The pA2/pKB values ranged between 7.27 and 7.66 against noradrenaline, phenylephrine, methoxamine and SDZ NVI 085 and were not significantly different from each other. Prazosin was however significantly less potent against oxymetazoline (pA2 6.38). Yohimbine (10(-6) M - 10(-5) M) was not very effective against any of the agonists. 3. WB 4101 (10(-8) M - 10(-5) M) displaced agonist dose-response curves to the right. The pA2/ pKB values ranged between 7.08 and 7.93 against noradrenaline, phenylephrine, methoxamine and SDZ NVI 085. WB 4101 was significantly less potent against oxymetazoline (pKB 6.85). 4. SZL-49 (5 x 10(-6) M) but not chloroethylclonidine (3 x 10(-5) M) significantly reduced vasoconstrictor responses to all the agonists. 5. Electrical field stimulation of the ovarian bed produced frequency-dependent vasoconstrictor effects which were abolished by 6-OHDA. The responses were also antagonized in a concentration-dependent by prazosin (10(-7) M - 10(-5) M) and WB 4101 (3 x 10(-8) M - 3 x 10(-7) M). Yohimbine reduced the response to electrical stimulation by 20% at 10(-5) M. The vasoconstrictor effect was also inhibited by SZL-49 but not by chloroethylclonidine. 6. These results would suggest that the vasoconstrictor responses of the ovarian vascular bed to adrenergic agonists and to electrical stimulation are mediated via the alpha 1A-adrenoceptor subtype.

Actions of endothelins and sarafotoxin 6c in the rat isolated perfused lung.

1. Endothelin (ET) receptors within the vasculature and airways were studied in a rat perfused lung model in which pulmonary perfusion pressure (PPP), pulmonary inflation pressure (PIP) and lung weight were continuously monitored. 2. The vascular potencies of ETs (ET-1 > ET-2 > ET-3) suggest an action via ETA receptors. This was confirmed by use of the antagonist, BQ123 (2 microM). The vasoconstrictor effects of sarafotoxin 6c (SX6C) also indicated the presence of ETB receptors. 3. Lung weight increases induced by ETs appeared to be a consequence of their vasoconstrictor potencies. The mixed ET receptor antagonist, bosentan (5 microM), markedly attenuated the responses of ET-1 and SX6C on PPP and lung weight, further implicating activation of both ETA and ETB receptors in these responses. 4. Endothelin-1 (ET-1) induced an accumulation of albumin-bound Evans blue dye in orthogradely perfused lungs. Retrograde perfusion attenuated the extravasation and increase in lung weight due to ET-1 but significantly augmented those induced by SX6C. 5. The bronchoconstrictor actions of ETs (ET-1 = ET-2 = ET-3) and SX6C suggest this is an ETB-mediated response. However SX6C was more potent than ETs and the dose-response curve was significantly steeper and achieved a higher maximum. 6. Indomethacin did not affect the vascular or bronchial responses to ET-1 or SX6C. 7. These findings indicate that rat pulmonary vasculature contains both ETA and ETB receptors. Retrograde perfusion suggests that ETB receptors are located arterially whereas ETA receptors are predominantly venous in distribution. Differences in the bronchoconstrictor potency of SX6C (compared to ETs) and the antagonism by bosentan may indicate ETB receptor heterogeneity in the airways.

Differential effects of agents on bronchial and vascular tone and lung weight in the rat isolated perfused lung.

A ventilated perfused model of rat lung is described enabling continuous simultaneous measurement of pulmonary perfusion pressure (PPP), pulmonary inflation pressure (PIP) and lung weight. The preparation was stable for 2h before oedema formation commenced. Phenylephrine injected into the pulmonary artery produced selective increases in PPP (ED50 4.0 +/- 0.7 nmol, n = 5) without affecting airway tone or lung weight. Carbachol under basal perfusion conditions caused bronchoconstriction (ED50 4.25 +/- 0.4 nmol, n = 7) without affecting PPP and lung weight. Bradykinin (BK) caused increases in PPP (ED50 22 +/- 3 nmol, n = 8), lung weight and PIP (ED50 35.6 +/- 3.6 nmol, n = 8). Low doses of BK (25-50 nmol) caused increases in lung weight which were reversible. Higher doses (100-200 pmol) caused irreversible increases in lung weight which albumin-bound dye infusion showed were associated with albumin accumulation. Manipulation of venous outflow pressure revealed that the changes in lung weight induced by BK were potentially explicable as sequelae of vascular hydrostatic pressure changes possibly resulting from venoconstriction. It is concluded that this model is useful for studying drug actions on bronchial tone, pulmonary vasculature tone and vascular permeability.

Characterization of Gaddum's substance R.

1. When the isolated small intestine of the rat is perfused via the mesenteric artery, an oxytocic principle (Gaddum's substance R) is released which is detectable in the perfusate after 30 min and is present in samples collected 8 h later. 2. The oxytocic activity of substance R is lost after boiling but is unaffected by treatment with thioglycolate. Furthermore, atropine, methysergide and indomethacin failed to antagonize uterine contractions to substance R. 3. Neither substance R nor urinary kallikrein alone induce a contraction of the guinea-pig isolated ileum. However, in the presence of kininogen both substance R and urinary kallikrein produce a slow and prolonged contraction of the guinea-pig ileum. 4. The oxytocic and kininogenase properties of both substance R and urinary kallikrein are inhibited by Trasylol. 5. Soy bean trypsin inhibitor (SBTI) selectively inhibited both the oxytocic and the kininogenase activities of substance R but not those of urinary kallikrein. 6. Gel filtration of substance R resolved a single peak of oxytocic activity with an estimated molecular weight of 40 kDa. 7. We conclude that substance R is a kininogenase enzyme which may be distinguished from plasma kallikrein by its molecular weight and from urinary kallikrein by its susceptibility to SBTI. The exact nature of this enzyme remains to be elucidated.

Metabolism of estrogens by rabbit blastocysts: formation of estrogen glucosides and preferential conversion of estrone to estradiol-17 beta.

When day 6 rabbit blastocysts were cultured (3 embryos/mL) in medium 199 containing 3.68 microM estradiol-17 beta (E2), 40% of E2 was metabolized in 24 h, at a rate of 18 pmol/embryo(b)/h, yielding 4 major metabolite fractions. Two of them were identified to be estrogen glucosides: 17 beta-hydroxyestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(2)3G) (12 pmol/b/h) and 17-oxoestra-1,3,5(10)-trien-3-yl beta-D-glucopyranoside (E(1)3G) (0.5 pmol/b/h). If the blastocysts were cultured in 3.68 microM E1 medium, 75% of E1 was metabolized in 24 h (34.1 pmol/b/h); most of it appears as E2 (8 pmol/b/h), E(1)3G (16 pmol/b/h), and E(2)3G (6 pmol/b/h). Thus, the 17 beta-hydroxysteroid dehydrogenase activity in the rabbit blastocysts catalyzes mainly in the direction of the E1----E2 conversion, with little or no E2----E1. This may be responsible in part for the faster metabolism of E1 than E2 by the rabbit blastocyst. In comparison with the rat, mouse, and hamster blastocyst, the rabbit embryo shows an additional capability to conjugate large amounts of estrogens into glucosides by steroid glucosyltransferase.

Endothelial-dependent relaxant effects of vaso-active intestinal polypeptide and arachidonic acid in rat aortic strips.

The relaxant actions of vaso-active intestinal polypeptide (VIP), acetylcholine (ACh), histamine and papaverine have been compared using circular muscle strips of rat aorta contracted with noradrenaline (NA). Arachidonic acid (AA) in a low dose (6.7 X 10(-7M) also relaxed the aorta. The relaxant actions of all these substances except papaverine were abolished by removal of the endothelial cells. Higher doses of AA (6.7-13.4 X 10(-6M) contracted aortic strips in the absence of NA but the con tractile effect "faded" while AA was still present in the bathing fluid. De-endothelialisation abolished this "fade" portion of the response leaving a sustained contracture. Indomethacin inhibited the contractile effect of AA revealing a weak inhibitory effect. However, it did not affect the relaxations induced by VIP, ACh, histamine or papaverine. ETYA abolished the relaxant actions of all these substances except papaverine. The results are consistent with the hypothesis that VIP, ACh and histamine relax the rat aorta via an endothelial-dependent mechanism which may involve the synthesis of a lipoxygenase product.