Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice.

(6R)-5,6,7,8-Tetrahydro-biopterin (H(4)B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H(4)B synthesis. A relative shortage of H(4)B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H(4)B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BLxCBA) were compared. H(4)B levels were determined by high-performance liquid chromatography and NO synthase activity by [(3)H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H(4)B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with N(G)-nitro-L-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H(4)B, the differences between the 2 groups disappeared. Our findings demonstrate that H(4)B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H(4)B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.

WEB 2086 inhibits neutrophil dependent increases in coronary resistance in blood perfused rabbit heart.

OBJECTIVE: The aim was to investigate the mechanism of the pressor action of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine in the blood perfused Langendorff preparation of the isolated rabbit heart; and in particular to establish whether the response was dependent on the presence of neutrophils and whether the release of platelet activating factor contributed to the pressor effect. METHODS: An isolated rabbit heart was perfused with blood from an anesthetised support rabbit. Formyl-methionyl-leucyl-phenylalanine was injected intra-arterially proximal to the isolated heart and measures of cardiac performance recorded. In some experiments the support animal was depleted of neutrophils by pretreatment with mechlorethamine while in others the specific platelet activating factor receptor antagonist WEB 2086 was given to the support animal before formyl-methionyl-leucyl-phenylalanine. Differential blood cell counts were determined throughout the course of each experiment. Each heart was examined histologically after the experiment. RESULTS: Formyl-methionyl-leucyl-phenylalanine caused a significant rise in perfusion pressure which was virtually abolished by leucocyte depletion of the support animal. The response could also be reduced by about 80% with intravenous WEB 2086. Histological examination of the perfused hearts showed that the number of accumulated neutrophils was very variable and not correlated with the rise in perfusion pressure. There was no significant difference between control hearts and those receiving WEB 2086. CONCLUSIONS: The results confirm previous reports that the response to formyl-methionyl-leucyl-phenylalanine is neutrophil dependent and show that this model of a blood perfused heart can be used successfully to examine the response to a leucocyte dependent stimulus. The results also suggest that the response to formyl-methionyl-leucyl-phenylalanine may not only be due to physical obstruction of the coronary circulation or "neutrophil plugging", but may also be due to the release of platelet activating factor.

Potent vasodilator activity of calcitonin gene-related peptide in human skin.

We have recently shown that the novel neuropeptide calcitonin gene-related peptide, CGRP, is a potent vasodilator. In this paper we report a detailed study of the effects of CGRP in human skin. CGRP induces a clearly defined, long-lasting erythema. We have measured the effect of CGRP on blood flow in human skin using a laser Doppler technique and have demonstrated increased local blood flow that persists for a number of hours. We compared the response of CGRP with other known vasodilators [histamine, prostaglandin (PG) E2, PGI2, substance P, and vasoactive intestinal peptide (VIP)] in the skin, and in all subjects the erythema induced by CGRP was more persistent than that induced by the other mediators tested. Except at high doses the local vasodilatation induced by CGRP was not associated with a wheal and flare as seen with histamine, substance P, and VIP. CGRP is an extremely potent vasodilator and if released into the circulation, or locally from peripheral nerve endings, it could have a role in the regulation of blood flow in both physiologic and pathologic conditions; CGRP may be the endogenous mediator of the flare in the triple response. A deficiency in CGRP secretion or action could be an important component of peripheral vascular disease. Some flushing reactions (e.g., those associated with medullary thyroid carcinoma) may result from circulating CGRP.

Hypothalamic hypertensive factor: an inhibitor of nitric oxide synthase activity.

Human and rat plasma and rat hypothalamus contain a cytochemically detectable substance, the concentration of which rises with an increase in salt intake. The plasma concentration of this material is also raised in essential hypertension and in the spontaneously hypertensive rat (SHR), the Milan hypertensive rat, and the reduced renal mass (RRM) hypertensive rat. In the normal rat, the greatest concentration is found in the hypothalamus of the SHR and the RRM hypertensive rat. The physicochemical characteristics of this cytochemically detectable hypothalamic hypertensive factor (HHF), including chromatographic behavior and molecular weight range, suggest that it may share features common to a substituted guanidine that is present in established nitric oxide synthase (NOS) inhibitors. It was therefore decided to determine the effect on NOS activity of the HHF obtained from mature SHR. The ability of HHF to inhibit NOS activity was studied on (1) NOS extracted from bovine aorta, rat brain, and human platelets by measuring the conversion of radiolabeled L-arginine to L-citrulline and (2) rat liver NOS measured indirectly with a cytochemical technique based on the stimulation of soluble guanylate cyclase activity in hepatocytes by NO. HHF showed a biphasic inhibitory action on platelet NOS activity that was greater with HHF obtained from SHR than from Wistar-Kyoto rats. HHF also had a biphasic inhibitory effect on hepatocyte NOS activity that was more potent when obtained from SHR. It is proposed that the increase in HHF, a novel form of NOS inhibitor that is elevated in SHR, may be involved in the rise in arterial pressure.

Preparation, purification, and structure elucidation of slow-reacting substance of anaphylaxis from guinea pig lung.

The work that we have described had originally three main aims: (a) to design a new purification system for SRS-A from which we would obtain pure material for the structural analysis: (b) to define the functional groups in the pure material by spectrophotometric, chemical, and enzymic inactivation methods; and (c) to deduce the complete covalent structure by an accepted spectroscopic method capable of defining structure in atomic detail. These aims have been achieved. The structure of SRS-A, the physiologically more relevant example of the SRSs that were studied, because it was derived immunologically from an animal model of an acute hypersensitivity reaction, has been rigorously defined. Of paramount importance in the determination of this structure was the mass spectrometric analysis of the intact molecule. Degradative and comparative studies are not capable of unequivocally defining structure. For example, the mass spectrum clearly showed the absence of an amide or similar C-terminal blocking groups or, as has been suggested, a sulfone in the molecule; such conclusions could not be drawn from comparative chromatographic data even on multiple systems. Mass spectrometric analysis of the intact molecule could overcome these problems by allowing the complete covalent structure to be collated from the information obtained from each fragmentation. The use of stable isotopes and accurate mass measurement removed possible ambiguities in the interpretation, and the sensitivity and specificity of mass spectrometry made it the method of choice for the structural analysis.

Coronary artery constriction by the isoprostane 8-epi prostaglandin F2 alpha.

1. This study was undertaken to compare the effects of 8-epi prostaglandin F2 alpha (8-epi PGF2 alpha) to those of prostaglandin F2 alpha (PGF2 alpha) and U46619, a thromboxane mimetic, on ovine, bovine and porcine coronary arteries. 2. 8-epi PGF2 alpha constricted porcine and bovine coronary arteries in a concentration-dependent manner with EC50 values of 689.0 +/- 229.3 and 1361.0 +/- 272.3 nM, respectively, but had no effect on ovine coronary arteries. 3. U46619 was a potent vasoconstrictor of porcine, ovine and bovine coronary arteries with EC50 values of 33.0 +/- 23.5, 373.3 +/- 69.7 and 254.1 +/- 134.3 nM, respectively. Emax values were significantly greater than those obtained with 8-epi PGF2 alpha. 4. PGF2 alpha constricted procine and bovine coronary arteries in a concentration-dependent manner with EC50 values of 1631.0 +/- 207.6 and 3644.0 +/- 344.8 nM, respectively, but had no effect on ovine coronary arteries. 5. Concentration-dependent constriction to U46619 in porcine coronary arteries was competitively inhibited by SQ29548 (10(-8) M to 10(-7) M) and BM13505 (10(-8) M to 10(-6) M) with no decrease in maximal responses. 6. Concentration-dependent constriction to 8-epi PGF2 alpha in porcine coronary arteries was inhibited in a concentration-dependent manner by SQ29548 (10(-8) M to 10(-7) M) and BM13505 (10(-8) M to 10(-6) M). However, the inhibition was associated with a decrease in maximal response. 7. Maximal responses of porcine coronary artery to U46619 (1 microM) and 8-epi PGF2 alpha (30 microM) were inhibited in a concentration-dependent manner by SQ29548 with IC50 values 99 +/- 12.36 nM and 46.5 +/- 18.67 nM, respectively. 8. Although ovine coronary arteries did not constrict to 8-epi PGF2 alpha pre-incubation of these vessels with 8-epi PGF2 alpha caused a rightward shift of the U46619 response curve in a concentration-dependent manner. 9. Pre-incubation of porcine coronary arteries with 8-epi PGF2 alpha competitively inhibited responses to U46619 with a Schild slope of 0.99 and a pA2 of 6.13. 10. We conclude that 8-epi PGF2 alpha is a vasoconstrictor within porcine and bovine coronary arteries, with a potency approximately twice that of PGF2 alpha but 5-20 times lower than U46619. The data suggest that 8-epi PGF2 alpha is acting as a partial agonist on the TP-receptor in the coronary vasculature.

The vasoconstrictor effect of 8-epi prostaglandin F2alpha in the hypoxic rat heart.

1. 8-epi prostaglandin (PG) F2alpha, a vasoconstrictor isoprostane, is synthesized under conditions of oxidative stress. This study was undertaken to investigate the vasoconstrictor effect of 8-epi PGF2alpha in the coronary circulation before and after a period of oxidative stress. 2. The effects of the isoprostane 8-epi PGF2alpha and the thromboxane mimetic U46619 were compared in the isolated rat heart perfused in the Langendorff mode at a constant pressure of 80 mmHg. 3. In normal hearts U46619 caused a dose-related reduction in coronary flow (ED50 4.7+/-2.2 nmol). In contrast, 8-epi PGF2alpha had no effect. 4. After reducing perfusion pressure to 20 mmHg for 30 min and reperfusing at 80 mmHg, the dose-response curve to U46619 was unaffected. In contrast, 8-epi PGF2alpha caused a dose-dependent drop in coronary flow (ED50 52.6+/-12.7 nmol), producing a similar maximal reduction to U46619. 5. Similarly, after perfusion with xanthine and xanthine oxidase for either 15 or 30 min there was little change in the response to U46619 in comparison to control hearts. In contrast, 8-epi PGF2alpha caused a reduction in coronary flow similar to that produced by U46619, the magnitude of the response being related to the length of xanthine/xanthine oxidase perfusion. 6. Responses to both U46619 and 8-epi PGF2alpha after xanthine/xanthine oxidase perfusion were blocked by the selective thromboxane receptor antagonist SQ29548 10(-7) M. 7. These results show that oxidative stress in the isolated perfused rat heart reveals a potent vasoconstrictor effect of the isoprostane 8-epi PGF2alpha by an action on the thromboxane receptor. 8. The data also suggest that, since 8-epi PGF2alpha is a partial agonist at the thromboxane receptor, thromboxane receptor reserve is increased by oxidative stress.

Endothelin induces potent microvascular constriction.

Endothelin is a recently discovered peptide produced by endothelial cells. It has been shown to have potent constrictor effects on major arteries in vitro and to raise rat blood pressure in vivo. The present experiments show that endothelin has a potent constrictor action on the microvasculature. Blood flow changes were measured by a xenon clearance technique in rabbit skin. Endothelin, when injected intradermally into rabbit skin, decreased local blood flow in a dose-dependent manner. Endothelin reduced basal skin blood flow and reversed the increased blood flow induced by a vasodilator. These results show that endothelin, administered extravascularly, has potent vasoconstrictor activity. This adds further support to the suggestion that endothelin may have an important role in the physiological control of blood flow and pressure.

Comparative studies on immunologically and non-immunologically produced slow-reacting substances from man, guinea-pig and rat.

1 Slow-reacting substance of anaphylaxis (SRS-A) was produced by antigen challenge of passively sensitized human lung and actively sensitized guinea-pig lung. 2 A slow-reacting substance (SRS) was prepared from the peritoneal fluid of rats treated with calcium ionophore A23187. 3 These substances were extensively purified by charcoal adsorption, Sephadex G-15 gel filtration, ether extraction and reverse phase high pressure liquid chromatography. 4 The three substances are pharmacologically, chemically and chromatographically indistinguishable. 5 Our data suggest that the same SRS entities are released from a variety of tissues and that these acidic lipids may have a wider physiological significance than just anaphylaxis.

Leukotriene release by endometrium and myometrium throughout the menstrual cycle in dysmenorrhoea and menorrhagia.

Endometrium and myometrium were collected at hysterectomy from 21 women with measured menstrual blood loss. Eight women complained of dysmenorrhea and the remaining 13 had pain-free periods. Specimens were obtained throughout the menstrual cycle (menstrual, n = 5; follicular, n = 4; early luteal, n = 3; mid-luteal, n = 5; late luteal, n = 4). Leukotriene C4, leukotriene D4 and leukotriene E4 release were examined using a short-term incubation technique. Endometrial leukotriene release, which was always significantly greater than myometrial release, changed throughout the menstrual cycle and the highest concentrations were found during menstruation. Endometrial, but not myometrial, leukotriene concentrations were significantly higher in tissues obtained from women with a complaint of dysmenorrhoea compared with those in tissue from pain-free women. No correlation was found between leukotriene release in either endometrium or myometrium and menstrual blood loss (range 15-457 ml).

Leukotriene release by human fetal membranes, placenta and decidua in relation to parturition.

Complete placentas and membranes were obtained from women after uncomplicated singleton pregnancies. Six were collected after labour at term, six after preterm labour and six after elective Caesarean section at term. Leukotriene C4 (LTC4), leukotriene D4 (LTD4) and leukotriene E4 (LTE4) release was examined using a short-term incubation technique. Release by amnion was significantly higher than that by the other tissues for the three modes of delivery. Comparison of LTC4, LTD4 and LTE4 release by individual tissues with regard to the type of delivery showed no significant difference.

Regional variations in endothelin-1 and its receptor subtypes in human coronary vasculature: pathophysiological implications in coronary disease.

Endothelin-1 is a potent vasoconstrictor peptide and mitogen for vascular smooth muscle cells. Increased plasma or tissue levels of endothelin-1 have been described after myocardial infarction and in atherosclerosis, suggesting that this peptide may play a pathophysiological role in various coronary syndromes. Here, we have studied regional variations in ET-1 and its receptors in control and atherosclerotic human coronary vasculature using standard immunohistochemistry and in vitro autoradiography. ET-1 immunoreactivity was associated with luminal endothelial cells and smooth muscle cells at regions of atherosclerosis. ET(A) receptors were present on smooth muscle cells of coronary arteries and on cardiac myocytes. Medial ET(B) receptor binding at the proximal region of coronary arteries was weak, but increased significantly towards distal regions of this vessel (p<0.005 in control and p<0.0005 in ischaemic heart disease). Microvascular endothelial cells in the adventitia of coronary arteries, myocardial microvessels and the endocardial endothelium expressed the ET(B) receptor exclusively. The receptor variations revealed in this study provide supporting evidence that ET-1 is associated with (1) vascular smooth muscle and endothelial cell proliferation, including areas of intimal hyperplasia and regions of neovascularization (2) increased ET-1-induced reactivity of distal portions of the human coronary artery, (3) ET-1-mediated constriction of myocardial microvessels. These results provide new insights into different potential roles for this peptide in healthy and diseased human coronary vasculature.

Neuropeptides and leukotriene release: effect of peptide histidine isoleucine and secretin in platelet activating factor-stimulated rat lung.

We have previously demonstrated that vasoactive intestinal peptide and calcitonin gene-related peptide inhibit leukotriene release from platelet activating factor-stimulated rat lung. We now report evidence that Peptide Histidine Isoleucine and Secretin, two naturally occurring peptides which are structurally homologous to vasoactive intestinal peptide, show a significant ability to inhibit leukotriene release in the same animal model. A preliminary hypothesis about the mechanism of this inhibition by the neuropeptides is discussed.

Neuropeptides and leukotriene biosynthesis: the effect of calcitonin, peptide histidine valine-42, helodermin, neuropeptide Y and galanin.

The effect of five neuropeptides was tested on platelet activating factor (PAF)-stimulated peptidoleukotriene biosynthesis in rat lungs. Calcitonin and peptide histidine valine-42 (PHV-42) were found to inhibit leukotriene C4 (LTC4) and D4 (LTD4) biosynthesis to an extent similar to the previously reported effects of calcitonin gene-related peptide (CGRP) and peptide histidine isoleucine (PHI) respectively. Helodermin potently inhibited the biosynthesis of all three peptidoleukotrienes. Neuropeptide Y and galanin, both present in airways, enhanced the levels of LTD4 and had no effect on LTC4 or leukotriene E4 (LTE4) levels. The theoretical implications of these findings are discussed.

Coronary vasodilatation induced by calcitonin gene-related peptide in the anaesthetised pig.

The ability of CGRP to increase blood flow in the coronary circulation of the anaesthetized pig was studied in a constant pressure perfusion model. Human alpha-CGRP, when infused close-arterially into the left anterior descending coronary artery perfused at constant pressure, produced a marked and prolonged dose-related increase in coronary flow, at doses above 10 pmol min-1. The gradient of the flow/pressure curves at each dose increased with an increase in pressure, indicating a drop in the resistance of the coronary bed. No significant change was observed in heart rate, left ventricle pressure, mean arterial pressure or cardiac output.

The myotropic and plasma-calcium modulating effects of calcitonin gene-related peptide (CGRP).

Human and rat calcitonin gene-related peptides cause a dose-related contraction of guinea pig ileum, which is antagonised by an anti-histamine, mepyramine, and an anticholinergic compound, hyoscine. Both peptides also cause a positive inotropic and a positive chronotropic effect in the rat isolated auricle and these responses are antagonised by propranolol, a B adrenoceptor blocker. Further, the peptides lower plasma calcium levels in both rats and rabbits in a dose-related manner resembling calcitonin; in the rabbit, but not in the rat, the initial calcium lowering effect is succeeded by hypercalcaemia at higher doses, while in the chick, only the parathyroid hormone-like calcium-raising effect is seen.

The role of cyclic AMP in the inhibition of leukotriene biosynthesis by neuropeptides.

Certain neuropeptides, including vasoactive intestinal peptide, inhibit peptidoleukotriene release from platelet activating factor-stimulated rat lung. We have now shown that vasoactive intestinal peptide will also inhibit peptidoleukotriene release from platelet activating factor-stimulated or ovalbumin-challenged guinea pig lung, but not from calcium ionophore-stimulated rat or guinea pig lung. In rat lung a pre-incubation with the peptide prior to addition of platelet activating factor was required for the effect to be maximal. When vasoactive intestinal peptide was substituted with cyclic AMP, the inhibitory effect was reproduced. In addition, pre-incubation with MDL 12330A, an inhibitor of adenylate cyclase, reduced the inhibitory effect of vasoactive intestinal peptide on platelet activating factor-stimulated leukotriene C4 biosynthesis. We suggest that the inhibition of platelet activating factor-stimulated peptidoleukotriene release in rat lung by vasoactive intestinal peptide involves the events prior to phospholipase A2 activation and requires cyclic AMP as a mediator.

Interactions between second messengers: cyclic AMP and phospholipase A2- and phospholipase C-metabolites.

The article reviews several new findings on the interactions between phospholipase A2- and phospholipase C-derived metabolites and cyclic AMP, in view of the developments recently achieved in studies on intracellular signal transduction. A complex network of multi-directional regulative mechanisms in the airways and inflammatory blood cells is briefly outlined.

CGRP: a novel neuropeptide from the calcitonin gene is the most potent vasodilator known.

The calcitonin gene has been shown to give rise to another peptide, calcitonin gene-related peptide (CGRP). The structure of human CGRP has been determined by mass spectrometry. Calcitonin gene-related peptide-containing neurons have been detected in a number of species, particularly in association with heart and blood vessels. Pharmacological studies have shown that CGRP is a potent vasodilator in a number of vessels and vascular beds, including the skin and coronary circulation. Investigations in humans have also reported the potent vasodilator action of CGRP. The evidence from these studies suggests that CGRP is an important regulator of vascular tone and blood flow.