Mepacrine inhibits fMLP-induced activation of human neutrophil granulocytes, leukotriene B4 formation, and fMLP binding.

Pulmonary sequestration and activation of polymorphonuclear leukocytes (PMNLs) are characteristic of many forms of acute lung injury. The present experiments were designed to study the effects of mepacrine on human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (fMLP). Mepacrine inhibited fMLP-induced superoxide production and degranulation in a dose-dependent manner with Kd values of 2.3 +/- 0.5 x 10(-7) M and 5.7 +/- 1.3 x 10(-6) M, respectively. Stimulation of PMNLs by 10(-6) M fMLP provoked the formation of barely detectable amounts of leukotriene B4 (LTB4) (< 5 pg/10(7) cells). Pretreatment of the cells with cytochalasin B augmented generation of LTB4 in response to fMLP (339 +/- 79 pg/10(7) cells). LTB4 formation was also inhibited by mepacrine (50% inhibitory concentration 1.0 +/- 0.5 x 10(-6) M). Furthermore, mepacrine inhibited the specific binding of [3H]fMLP to neutrophils with a Ki value of 1.4 +/- 0.4 x 10(-5) M. Mepacrine decreased the receptor binding affinity without altering the number of receptors. These findings demonstrate that the inhibitory effect of mepacrine is response dependent and suggest that this action of mepacrine could, in part, be attributed to a decrease in fMLP receptor affinity.

C-reactive protein inhibits intracellular calcium mobilization and superoxide production by guinea pig alveolar macrophages.

C-reactive protein (CRP) is a prototypical acute-phase reactant, the humoral and plasma concentrations of which rise dramatically after tissue injury or inflammation. The effects of CRP on superoxide production and intracellular calcium mobilization by guinea pig alveolar macrophages challenged with platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and phorbol 12-myristate 13-acetate (PMA) were studied. CRP by itself did not activate alveolar macrophages up to a concentration of 100 micrograms/ml, whereas it inhibited superoxide production in a time- and dose-dependent manner with median inhibitory concentration (IC50) values of 4.2 +/- 0.3, 3.0 +/- 0.2, and 3.2 +/- 0.3 micrograms/ml for PAF (10(-7) M), fMLP (10(-7) M), and PMA (10(-9) M), respectively. When CRP was incubated with the agonists before addition to cells, it inhibited PMA-, PAF-, and to a lesser extent fMLP-induced superoxide production. CRP also attenuated the rise in intracellular free calcium levels evoked by fMLP or PAF in a dose-dependent manner. These findings suggest that CRP may play a role in attenuating tissue damage secondary to activation of alveolar macrophages by inhibiting superoxide generation and mobilization of intracellular free calcium.

Pulmonary surfactant lipids inhibit prostanoid production of guinea pig alveolar macrophages.

Changes in the amount and composition of pulmonary surfactant are important features of the adult respiratory distress syndrome. The goal of the present study was to investigate the effects of natural surfactant material and several of its lipid components on prostanoid production and superoxide generation by guinea pig alveolar macrophages. Natural surfactant (10-500 micrograms/ml) inhibited (up to 65%) prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) production elicited by platelet-activating factor (PAF, 10(-6) M) and arachidonic acid (5 x 10(-6) M) but not fMet-Leu-Phe (10(-7) M). Dioleyl-phosphatidylglycerol (diOPG, 1-100 micrograms/ml) and dioleyl-phosphatidylcholine (diOPC, 1-100 micrograms/ml) prevented fMet-Leu-Phe- and PAF-stimulated prostanoid release in a concentration-dependent fashion with a maximal inhibition of 94%. DiOPC (100 microgram/ml) also inhibited arachidonic acid induced PGE2 production by 67%. Phosphatidylcholine (100 micrograms/ml) and sphingomyelin (10-100 micrograms/ml) significantly attenuated TxB2 production elicited by arachidonic acid. Neither PAF- nor fMet-Leu-Phe-stimulated superoxide production was affected significantly by natural surfactant and its lipid components with the exception of phosphatidylcholine. At a concentration of 100 micrograms/ml, phosphatidylcholine decreased superoxide production by about 57% in response to PAF. These results show that diOPC and diOPG are capable of inhibiting prostanoid production of guinea pig alveolar macrophages in response to inflammatory stimuli and suggest that a decrease in the diOPG and diOPC content of surfactant would lead to enhanced intrapulmonary formation of prostanoids and consequently to the deterioration of pulmonary function in the adult respiratory distress syndrome.

Modulation by nitric oxide of platelet-activating factor-induced albumin extravasation in the conscious rat.

1. The objective of this study was to assess whether or not endogenous nitric oxide (NO) could mediate the hypotensive response to platelet-activating factor (PAF) and modulate PAF-induced microvascular albumin leakage in the conscious rat. 2. PAF (0.19 and 1.9 nmol kg-1, i.v.) evoked dose-dependent hypotension and significantly enhanced albumin extravasation in the large airways, pancreas, stomach and duodenum 15 min after its administration. Inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME, 0.125-2 mg kg-1, i.v.) produced marked dose-dependent increases in albumin accumulation (up to 290%) in large airways, liver, spleen, pancreas, kidney, stomach and duodenum as measured by the extravasation of Evans blue dye. L-NAME (2 mg kg-1) treatment markedly potentiated PAF (1.9 nmol kg-1)-induced albumin extravasation in these tissues, whereas it did not modify the hypotensive response to PAF. 3. Maintenance of mean arterial blood pressure at the level observed following 2 mg kg-1 L-NAME by infusion of noradrenaline (620-790 ng kg-1 min-1) neither affected significantly albumin extravasation nor potentiated the permeability effect of PAF in the vascular beds studied with the exception of large airways, where noradrenaline mimicked the effects of L-NAME. 4. These results indicate that inhibition of endogenous NO formation leads to an increase in albumin extravasation and to potentiation of the vascular permeability effect of PAF, whereas the hypotensive action of PAF seems to be independent of NO formation in the conscious rat. These data suggest an important role for NO in the regulation of albumin extravasation.

Effects of the ETA/ETB receptor antagonist, bosentan on endothelin-1-induced myocardial ischaemia and oedema in the rat.

1. The purpose of this study were to assess the role of ETB receptors in mediating endothelin-1 (ET-1)-induced myocardial ischaemia and oedema in rats and to study the inhibitory action of the novel nonpeptide ETA/ETB receptor antagonist, bosentan on these actions of ET-1. 2. Intravenous bolus injection of ET-1 (1 nmol kg-1) into anaesthetized rats produced marked ST segment elevation of the electrocardiogram without causing arrhythmias. ST segment elevation developed within 30-50 s and persisted for at least 30 min following injection of the peptide. 3. Pretreatment of the animals with bosentan (10 mg kg-1, i.v.) inhibited on average by 96% the ST segment elevation elicited by ET-1 (1 nmol kg-1) compared to the 82% inhibition observed with the ETA receptor-selective antagonist, FR 139317 (2.5 mg kg-1, i.v.). 4. Bolus injection of ET-1 (1 nmol kg-1, i.v.) to conscious chronically catheterized rats evoked a transient depressor response followed by a prolonged pressor effect. Corresponding to changes in blood pressure, a transient tachycardia and a sustained bradycardia were observed. ET-1 (1 nmol kg-1) enhanced albumin extravasation by 119 and 93% in the left ventricle and right atrium, respectively, as measured by the local extravascular accumulation of Evans blue dye. 5. Pretreatment of the animals with bosentan (10 mg kg-1) inhibited by 71 and 90% the depressor and pressor actions of ET-1 (1 nmol kg-1) and the accompanying tachycardia and bradycardia, respectively. FR 139317 (2.5 mg kg-1) attenuated the pressor response to ET-1 and accompanying bradycardia by 75%, without affecting the depressor action and accompanying tachycardia. ET-1-induced albumin extravasation was completely inhibited by bosentan (10 mg kg-1) both in the left ventricle and right atrium, compared to the 86% inhibition observed with FR 139317 (2.5 mg kg-1).6. Like ET-1, the ETB receptor-selective agonist, IRL 1620 (0.3 and 1 nmol kg-1, i.v.) also produced dose-dependent ST segment elevation in anaesthetized rats and enhanced albumin extravasation (up to141% of control) in the left ventricle and right atrium, respectively, in conscious rats. These effects ofIRL 1620 were completely prevented by bosentan (10 mg kg-1).7. These results indicate that ETB receptors, albeit to a lesser extent than ETA receptors, are also involved in mediating ET-1-induced myocardial ischaemia and oedema in the rat, and suggest the therapeutic potential for bosentan in the treatment of ischaemic myocardial diseases.

Acute pro-inflammatory actions of endothelin-1 in the guinea-pig lung: involvement of ETA and ETB receptors.

1. Although recent observations suggest that endothelin-1 (ET-1) may play a role in the pathogenesis of asthma, to date little is known about the effects of ET-1 on parameters other than bronchoconstriction. The objectives of the present experiments were to study whether intravenously administered ET-1 could exert pro-inflammatory actions in the guinea-pig lung and to assess the involvement of endothelin ETA and ETB receptors in these events by using the ETA receptor-selective antagonist, FR 139317, the novel ETA/ETB receptor antagonist, bosentan and the ETB receptor-selective agonist, IRL 1620. 2. Bolus i.v. injection of ET-1 (0.1-1 nmol kg-1) to anaesthetized guinea-pigs evoked dose-dependent increases in mean arterial blood pressure which lasted for 6-12 min. This was accompanied by a dose-dependent haemoconcentration (8-15% plasma volume losses) and increases (up to 546%) in albumin extravasation in the trachea, upper and lower bronchi, but not in the pulmonary parenchyma. Qualitatively similar changes were observed following i.v. injection of the ETB receptor agonist, IRL 1620 (0.3 and 1 nmol kg-1), although IRL 1620 appeared to be about 3 times less potent than ET-1. The ETA receptor-selective antagonist, FR 139317 (2.5 mg kg-1) inhibited the ET-1 (1 nmol kg-1)-induced pressor response, haemoconcentration and albumin extravasation by 75, 77 and 60-70%, respectively, whereas it did not attenuate IRL 1620 (1 nmol kg-1)-induced changes. The ETA/ETB receptor antagonist, bosentan (10 mg kg-1) almost completely inhibited the pressor, haemoconcentration and permeability effects of both ET-1 and IRL 1620. 3. ET-1, but not IRL 1620 (0.1-1 nmol kg-1), produced a dose-dependent neutropenia with relative lymphocytosis and monocytosis, but did not induce influx of neutrophil granulocytes into pulmonary tissues or the bronchoalveolar space. ET-1 (1 nmol kg-1)-induced neutropenia was prevented by pretreatment of the animals with FR 139317 (2.5 mg kg-1), bosentan (10 mg kg-1) or adrenaline (90 nmol kg-1), indicating that ET-1 caused intravascular sequestration of neutrophil granulocytes. 4. ET-1 or IRL 1620 (10(-10)-10(-6) M) alone did not activate alveolar macrophages in vitro, whereas at a concentration of 10(-8) M, ET-1, but not IRL 1620, markedly potentiated superoxide production in response to f-Met-Leu-Phe (10(-9)-10(-7) M) and platelet-activating factor (PAF, 10(-9)-10(-7) M), but not to phorbol 12-myristate 13-acetate (10(-9) M). ET-1 did not affect f-Met-Leu-Phe- or PAF-induced increases in intracellular free calcium concentration. This potentiating effect of ET-1 was abolished by FR 139317(1.5 X 10-7 M).5. We conclude that, in addition to evoking airway contractions, ET-1 exerts pro-inflammatory actions via activation of the ETA and to a lesser extent the ETB receptors, and therefore, might contribute to the airway inflammation present in asthma. These findings also suggest the therapeutic potential of ETA/ETB receptor and perhaps ETA receptor-selective antagonists in this disease.

Endothelin-1-induced myocardial ischaemia and oedema in the rat: involvement of the ETA receptor, platelet-activating factor and thromboxane A2.

1. The objectives of the present experiments were to assess the role of ETA receptors in mediating endothelin-1 (ET-1)-induced myocardial ischaemia and oedema and to study the involvement of platelet-activating factor (PAF) and thromboxane A2 (TxA2) in these actions of ET-1 in rats. 2. Intravenous bolus injection of ET-1 (0.1-2 nmol kg-1) into anaesthetized rats induced ST segment elevation of the electrocardiogram in a dose-dependent manner without causing arrhythmias. ST segment elevation developed within 20-90 s and persisted for at least 10-20 min following administration of ET-1. 3. Pretreatment of the animals with the selective endothelin ETA receptor antagonist, FR 139317 (2.5 mg kg-1, i.v.) inhibited by 86% the ST segment elevation elicited by ET-1 (1 nmol kg-1). Pretreatment with intravenous administration of BM 13505 (1 mg kg-1), a TxA2 receptor antagonist, OKY-046 (10 mg kg-1), a thromboxane synthase inhibitor or the specific PAF receptor antagonist, WEB 2086 (1 mg kg-1) or BN 52021 (10 mg kg-1) markedly suppressed ST segment elevation in response to ET-1. Infusion of indomethacin (3 mg kg-1 bolus plus 2 mg kg-1 h-1) did not significantly affect ET-1-induced ST segment elevation. 4. Bolus injection of ET-1 (1 nmol kg-1, i.v.) to conscious rats resulted in a prolonged pressor effect preceded by a transient depressor response. Corresponding to changes in blood pressure, a small transient tachycardia was followed by a sustained bradycardia. ET-l enhanced albumin leakage by 87 and 120% in the left ventricle and right atrium, respectively, as measured by the extravasation of Evans blue dye.5. The selective ETA receptor antagonist, FR 139317 (2.5 mg kg-1) significantly blunted the pressor action of ET-1 and the accompanying bradycardia without affecting the depressor response. Furthermore,FR 139317 almost completely abolished the permeability effect of ET-l in both vascular beds studied.6. Pretreatment of the animals with BM 13505 (1 mg kg-1), OKY-046 (10mg kg-1), WEB 2086(1 mg kg-1) or BN 52021 (10mg kg-1) significantly reduced ET-1 (1 nmol kg-1)-induced albumin extravasation both in the left ventricle and right atrium. The PAF receptor antagonists, WEB 2086 and BN 52021 were equally potent inhibitors in the left ventricle, whereas BN 52021 appeared to be a more potent inhibitor than WEB 2086 in the right atrium. Pretreatment with indomethacin (3 mg kg-1 plus 2 mg kg-1 h-1) did not modify the permeability response to ET-1. None of these compounds affected significantly ET-l-induced changes in mean arterial blood pressure and heart rate.7. These results indicate that intravenous administration of ET-1 provokes ST segment elevation and myocardial oedema and suggest that these events are mediated, in part, through release of secondary mediators, such as PAF and TxA2 via the activation of ETA receptors.

Nitric oxide modulates vascular permeability in the rat coronary circulation.

1. The objective of the present study was to assess whether inhibition of nitric oxide (NO) production could modulate vascular permeability in the coronary circulation in conscious rats. 2. Intravenous injection of NG-nitro-L-arginine methyl ester (L-NAME, 2 mg kg-1) resulted in a slowly developing hypertension and evoked twofold increases in vascular permeability in the left ventricle and right atrium as measured by the extravasation of Evans blue dye. Maintenance of mean arterial blood pressure at the level observed following L-NAME injection by infusion of noradrenaline (620-820 ng kg-1 min-1) did not induce significant protein extravasation in the coronary circulation. 3. L-NAME treatment markedly enhanced (up to 490%) protein extravasation both in the left ventricle and right atrium in response to platelet-activating factor (PAF, 1.9 nmol kg-1, i.v.) and endothelin-1 (1 nmol kg-1, i.v.). Noradrenaline infusion potentiated (up to 69%) endothelin-1-induced protein extravasation. The permeability effect of PAF was only slightly enhanced by noradrenaline. 4. The present findings indicate that inhibition of endogenous NO synthesis leads to an increase in protein extravasation and to potentiation of the permeability effects of PAF and endothelin-1 in the coronary circulation. These results also suggest that NO may be an important regulator of vascular permeability under physiological and pathological conditions.

Vascular responses to leukotriene B4, C4 and D4 following FPL 55712, indomethacin, saralasin, phentolamine and verapamil in the conscious rat.

The pressor and vascular permeability effects of leukotrienes B4 (LTB4), C4 and D4 were investigated in conscious unrestrained rats. Leukotrienes C4 and D4 (3.2-51 nmol kg-1 i.v.) caused an acute dose-dependent elevation of the mean arterial pressure, which was maximal after 2 min and returned to control levels within 14 min. Heart rate was significantly reduced by the higher doses of LTC4 and LTD4. LTB4 (up to a dose of 51 nmol kg-1) was essentially inactive. These effects of LTC4 and LTD4 were abolished by FPL 55712, a putative antagonist of sulphidopeptide leukotrienes and by verapamil, a calcium channel blocker. Indomethacin, phentolamine or saralasin pretreatment failed to modify the pressor response to LTC4 and LTD4. LTC4 and LTD4 furthermore caused an increase in haematocrit values, which was significantly attenuated by FPL 55712, indomethacin and verapamil. The present findings show that the pressor effect of LTC4 and LTD4 is not related to prostanoid release and can be reversed by calcium channel blockade; whereas the effect on vascular permeability seems to require the presence of both cyclo-oxygenase product(s) and calcium.

Characterization of receptors mediating vascular responses to endothelin-1 in the conscious rat.

1. The present study has determined the receptors mediating the vascular responses (pressor and depressor actions and vascular permeability effect) to endothelin-1 (ET-1) in the conscious rat by using the novel non-peptide ETA/ETB receptor antagonist, bosentan (Ro 47-0203, 4-tert-butyl-N-[6-(2 hydroxyethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyrimidine- 4-yl]benzene-sulphonamide), the ETA receptor-selective antagonist, FR 139317 and the ETB receptor-selective peptide agonist, IRL 1620. 2. Bolus injection of ET-1 (1 nmol kg-1, i.v.) resulted in a prolonged pressor effect (maximum increase in mean arterial blood pressure (MABP) was 47 +/- 3 mmHg, n = 6) preceded by a transient depressor response (maximum decrease in MABP was 17 +/- 1 mmHg). Both these responses were inhibited by bosentan (1-20 mg kg-1, i.v. bolus) in a dose-dependent manner. The maximum inhibition of ET-induced depressor and pressor responses did not exceed 53 and 87%, respectively. FR 139317 (2.5 mg kg-1, i.v.) attenuated the pressor response to ET-1 by 75% without affecting the depressor response. Furthermore, FR 139317, but not bosentan, prolonged the depressor action of ET-1. Corresponding to changes in blood pressure, a small transient tachycardia (delta heart rate 15 +/- 5 beats min-1) followed by a sustained bradycardia (delta heart rate -48 +/- 10 beats min-1, n = 6) was observed following injection of 1 nmol kg-1 ET-1. FR 139317 and bosentan (10 mg kg-1) inhibited ET-1-induced bradycardia by 79% and 71%, respectively.ET-l-induced tachycardia was significantly attenuated by bosentan,but not FR 139317.3. The ETB receptor agonist, IRL 1620 (0.1-2 micro molkg-1, i.v.) produced biphasic dose-dependent changes in MABP with an initial transient fall followed by a prolonged pressor action. The maximum decrease and increase in MABP were 11 +/- 2 and 19 +/- 3 mmHg, respectively (n = 5). These changes in MABP were accompanied by a transient tachycardia (Delta heart rate 9+/- 3 beats min-1) and prolonged bradycardia (Delta heart rate -17+/-11 beats min-1), respectively. Pretreatment of the animals with FR139317 (2.5 mg kg-1, i.v.) did not affect IRL 1620 (1 nmol kg-1)-induced changes in MABP and heart rate, whereas both the depressor and pressor actions of IRL 1620 and the accompanying tachycardia and bradycardia were almost completely inhibited by bosentan (10mgkg-1).4. ET-1 (1 nmol kg-1) enhanced albumin extravasation in the upper and lower bronchi, spleen, kidney,stomach and duodenum (up to 246%) as measured by the extravasation of Evans blue dye. FR 139317(2.5mgkg-1) completely inhibited ET-l-induced protein extravasation in the stomach and duodenum,whereas 40-75% inhibition was observed in the other vascular beds studied. The permeability effect of ET-l was almost completely inhibited by bosentan (10mgkg-1) in all vascular beds studied.5. IRL 1620 (0.4 or 1 nmol kg-1, i.v.) enhanced albumin extravasation (up to 219%) in the upper and lower bronchi, spleen and kidney in a dose-dependent manner. Unlike ET-1, IRL 1620 failed to increase albumin extravasation in the stomach and duodenum.6. The present study demonstrates in the conscious rat that ETA and ETB receptors are responsible for mediating the majority of the pressor response to ET-l and suggest that a small component of the ET-l pressor response might be mediated via a non-ETA, non-ETB receptor, whereas ETB and perhaps a non-ETA, non-ETB receptor may mediate the depressor action of ET-1. Furthermore, the ET-1 induced albumin extravasation is mediated solely via ETA receptors in the stomach and duodenum, whereas both ETA and ETB receptors are involved in the permeability effect of ET-l in the bronchial, splenic and renal vascular beds.

Effects of calcium antagonists on endothelin-1-induced myocardial ischaemia and oedema in the rat.

1. The effects of the calcium channel blockers, verapamil and nifedipine on myocardial ischaemia and oedema evoked by endothelin-1 (ET-1) or IRL 1620, an ETB receptor-selective agonist were studied in anaesthetized and conscious rats. 2. Bolus injection of ET-1 (1 nmol kg-1, i.v.) or IRL 1620 (1 nmol kg-1, i.v.) to conscious chronically catheterized rats evoked a transient depressor response followed by a prolonged pressor effect. Corresponding to changes in blood pressure, a transient tachycardia and a sustained bradycardia were observed. Pretreatment of the animals with verapamil (1 mg kg-1, i.v.) or nifedipine (200 micrograms kg-1, i.v.) produced on average 5 mmHg decrease in mean arterial blood pressure. Both verapamil and nifedipine inhibited by 63 and 44% the pressor actions of ET-1 or IRL 1620 (1 nmol kg-1), respectively, and the accompanying bradycardia. Both verapamil and nifedipine potentiated the magnitude of the depressor action of ET-1 and IRL 1620 without affecting the accompanying tachycardia. Decreasing mean arterial blood pressure with hydralazine (0.2 - 0.3 micromol kg-1, i.v.) to levels comparable to those observed after verapamil or nifedipine had no significant effects on the haemodynamic responses to ET-1 or IRL-1620. 3. Intravenous bolus injection of ET-1 or IRL 1620 (0.1-2 nmol kg-1) into anaesthetized rats produced dose-dependent ST segment elevation of the electrocardiogram without causing arrhythmias. ST segment elevation developed within 30-50s and persisted for at least 10-20 min following injection of the peptides. 4. Pretreatment of the animals with verapamil (1 mg kg-1, i.v.) or nifedipine (200 micrograms kg-1, i.v.) inhibited on average by 79 and 76% the ST segment elevation elicited by ET-1 (1 nmol kg-1), respectively. Verapamil and nifedipine also attenuated IRL 1620 (1 nmol kg-1)-induced ST segment elevation on average by 71 and 74%, respectively. In contrast, no significant inhibition was observed with hydralazine (0.2-0.3 mumol kg-1). 5. Both ET-1 and, to a lesser extent, IRL 1620 (0.1-2 nmol kg-1) evoked albumin accumulation in cardiac tissues in a dose-dependent fashion as measured by the local extravascular accumulation of Evans blue dye in conscious rats. ET-1 and IRL 1620 (1 nmol kg-1) enhanced albumin extravasation by 109 and 82%, and 34 and 44% in the left ventricle and right atrium, respectively. ET-1 or IRL 1620-induced albumin extravasation was completely prevented by verapamil (1 mg kg-1) or nifedipine (200 micrograms kg-1) in these vascular beds. In contrast, hydralazine (0.2-0.3 mumol kg-1) failed to modify the effects of ET-1 or IRL 1620 on albumin extravasation. 6. These results show that verapamil and nifedipine are highly effective in protecting the myocardium against the pro-ischaemic and microvascular permeability enhancing effects of ET-1 and suggest that ETA and constrictor ETB (tentatively termed ETB2) receptors mediating these actions of ET-1 are coupled to calcium influx through dihydropyridine-sensitive calcium channels.

Dexamethasone-induced gastric mucosal damage in the rat: possible role of platelet-activating factor.

1. The aim of the present experiments was to study the possible role of platelet-activating factor (PAF) in mediating gastric mucosal damage induced by dexamethasone in the rat by measuring gastric tissue levels of PAF during dexamethasone-treatment and by investigating the effects of specific PAF receptor antagonists on dexamethasone-induced gastric lesions. PAF-like bioactivity extracted from the rat glandular stomach was determined by a platelet aggregation assay. 2. Dexamethasone treatment (0.4-4 mg kg-1, daily for 1-6 days) produced time- and dose-dependent damage to the glandular mucosa of the stomach as characterized by extensive, uniform hyperaemia with multiple, focal petechiae and erosions. 3. These changes were accompanied by a time-, and dose-dependent increase in PAF content of the glandular stomach. Control rat stomach contained small amounts of PAF (0.14 +/- 0.04 ng per g wet weight), which increased over 40 fold in response to dexamethasone treatment (4 mg kg-1, daily for 6 consecutive days). The presence of PAF-like material in the stomach extract was ascertained by thin-layer chromatography, high performance liquid chromatography and by alkaline hydrolysis. 4. Pretreatment of the animals with one or other of the structurally unrelated PAF receptor antagonists, BN 52021 (10 mg kg-1, i.p.) or BN 50727 (1 mg kg-1, i.p.) significantly reduced dexamethasone-induced gastric damage. In these animals neither petechiae nor erosions were observed. 5. These observations suggest that PAF is a likely endogenous mediator of glucocorticoid-induced gastric mucosal damage in the rat.

Vascular responses to endothelin-1 following inhibition of nitric oxide synthesis in the conscious rat.

1. The objectives of the present experiments were to assess the role of endogenous nitric oxide (NO) in mediating and/or modulating the effects of endothelin-1 (ET-1) on blood pressure and microvascular permeability in conscious rats. 2. Intravenous administration of the NO synthesis inhibitors, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) at a dose (25 mg kg-1 or 2 mg kg-1, respectively) which evoked maximum increase in mean arterial blood pressure (MABP) significantly attenuated (by about 40%) the vasodepressor response and potentiated (by 100-180%) the pressor response to ET-1 (1 nmol kg-1, i.v.) compared to the effects of ET-1 in animals where the peripheral vasoconstrictor effects of L-arginine analogues were mimicked by an infusion of noradrenaline (620-820 ng kg-1 min-1). Similar inhibition of the depressor and potentiation of the pressor actions of ET-1 were observed when the MABP which had been elevated by L-NMMA or L-NAME was titrated to normotensive levels with hydralazine or diazoxide before injection of ET-1. 3. L-NAME (2 mg kg-1) increased the vascular permeability of the large airways, stomach, duodenum, pancreas, liver, kidney and spleen (up to 280%) as measured by the extravasation of Evans blue dye. The permeability of pulmonary parenchyma, skeletal muscle and skin was not affected significantly by L-NAME treatment. Elevation of MABP by noradrenaline infusion did not evoke protein extravasation in the vascular beds studied with the exception of the lung. In the large airways, tissue Evans blue content was similar following noradrenaline infusion and L-NAME.4. Both the pressor and permeability effects of L-NAME (2 mg kg-1) were effectively reversed by L-arginine (300 mg kg- 1) but not by D-arginine (300 mg kg-1 ). The D-enantiomer of L-NAME, D-NAME(2 mg kg-1) had no effect on the parameters studied.5. Protein extravasation was significantly enhanced by ET-1 (1 nmol kg-1) in the upper and lower bronchi, stomach, duodenum, kidney and spleen (up to 285%). This was potentiated by L-NAME(2 mg kg-1), resulting in marked increases in tissue Evans blue accumulation (up to 550%) in these tissues. The effects of L-NAME and ET-1 were additive in the trachea, duodenum, pancreas and liver.Combined administration of L-NAME plus ET-1 significantly increased protein extravasation in the pulmonary parenchyma, where neither L-NAME nor ET-1 alone caused significant increases.6. Noradrenaline infusion (620-820 ng kg-1 min-1) potentiated the permeability action of ET-1(1 nmol kg-1) in the pulmonary circulation, whereas it did not modify ET-1-induced protein extravasation in the other vascular beds.7. These results indicate that endogenous NO mediates, in part, the vasodepressor effect and attenuates the vasopressor action of ET-1 and modulates the effects of ET-1 on vascular permeability. These findings confirm the role of NO in the maintenance of blood pressure and suggest an important role for NO in the regulation of microvascular permeability.

Enhancement by endothelin-1 of microvascular permeability via the activation of ETA receptors.

1. The objective of the present experiments was to assess the involvement of endothelin-A (ETA) receptors in mediating the effects of endothelin-1 on microvascular permeability in conscious rats. 2. Bolus injection of endothelin-1 (0.1 and 1 nmol kg-1, i.v.) resulted in a dose-dependent prolonged pressor effect preceded by a transient depressor response. These changes were accompanied by a dose-dependent loss of plasma volume. Endothelin-1 (1 nmol kg-1) enhanced the vascular permeability of the upper and lower bronchi, kidney, stomach, duodenum and spleen (up to 270%) as measured by the extravasation of Evans blue dye. 3. Pretreatment of the animals with the selective ETA receptor antagonist, BQ-123 (1 mg kg-1, i.v.) significantly blunted the pressor response to endothelin-1 without affecting the depressor response. BQ-123 inhibited by 87% the endothelin-1 (1 nmol kg-1)-induced plasma volume loss. BQ-123 markedly attenuated protein extravasation elicited by endothelin-1 in the upper and lower bronchi and kidney, whereas it completely inhibited the permeability effect of endothelin-1 in the stomach and duodenum. BQ-123 by itself had no significant effect on the parameters studied. 4. The endothelin-1 analogue, [Trp(For)21]-endothelin-1, in which Trp21 is formylated, was as potent a pressor agent as endothelin-1, but had no depressor action. Bolus injection of [Trp(For)21]-endothelin-1 (0.1 and 1 nmol kg-1, i.v.) evoked similar plasma volume losses to those observed following administration of equimolar doses of endothelin-1. Furthermore, 1 nmol kg-1 [Trp(For)21]-endothelin-l evoked increases in protein extravasation similar to endothelin-l, 1 nmol kg-1.5. The present findings suggest that endothelin- 1 enhances microvascular permeability, in part, via the activation of ETA receptors.

Inhibition by calcium channel blockers of the binding of platelet-activating factor to human neutrophil granulocytes.

The inhibitory action of calcium channel blockers on platelet-activating factor (PAF)-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]PAF to neutrophils was studied. Verapamil and diltiazem inhibited PAF (10(-8)-10(-15) M)-induced degranulation and superoxide production in a dose-dependent manner, with pA2 values of 5.6 and 6.1 for verapamil and 5.9 and 6.2 for diltiazem, respectively. Both channel blockers inhibited the specific binding of [3H]PAF to PMNL in dose-dependent fashion, with Ki values of 3.9 +/- 0.6 X 10(-5) M and 3.2 +/- 0.4 X 10(-5) M for verapamil and diltiazem, respectively. Scatchard analysis of the binding data revealed that both calcium channel blockers decreased the receptor binding affinity and slightly increased the number of high-affinity PAF receptors, whereas they did not affect the binding affinity and number of low-affinity receptors. These results indicate that calcium channel blockers can inhibit neutrophil responses elicited by PAF by a mechanism other than inhibition of calcium influx and suggest that the PAF receptor may be closely associated with calcium channels.

Endothelin-1 enhances vascular permeability in the rat heart through the ETA receptor.

Intravenous injection of endothelin-1 (ET-1, 0.1 and 1 nmol/kg) resulted in a dose-dependent increase in vascular permeability in the coronary circulation of conscious rats. The increase was almost completely abolished by the selective ETA receptor antagonist, BQ-123 (1 mg/kg). The ET-1 analogue, [Trp(For)21]ET-1, which is devoid of the depressor but not the pressor activity, evoked changes in protein extravasation similar to those with ET-1. These data indicate that the permeability effect of ET-1 is mediated through the ETA receptor.

C-reactive protein inhibits binding of platelet-activating factor to human platelets.

Serum concentration of C-reactive protein (CRP), a prototypical acute-phase protein rises dramatically in response to tissue injury or inflammation. We report here that CRP (1-20 micrograms/ml) inhibited platelet-activating factor (PAF)-induced aggregation of human platelets in time-, and dose-dependent manner. This inhibitory action of CRP was nearly completely removed by treatment with anti CRP antiserum. At higher concentrations (20-100 micrograms/ml), CRP stabilized platelet membrane against the detergent-like effect of beta-deoxy-lysolecithin. Furthermore, CRP (10 micrograms/ml) diminished specific [3H]PAF binding to platelets and displaced previously bound labeled PAF from platelets. These results suggest that by depressing the bioavailability of PAF, CRP may be an important modulator of platelet activation during acute inflammatory reactions.

Effects of C-reactive protein on human neutrophil granulocytes challenged with N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor.

The effects of C-reactive protein (CRP), the prototypical acute-phase reactant were studied on human polymorphonuclear leukocytes (PMNL) challenged with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF). CRP at 8-64 micrograms/ml concentrations inhibited degranulation and superoxide production by PMNL in time-, and dose-dependent manner and stabilized PMNL membranes against the lytic effect of lysophosphatidylcholine. CRP was also capable of binding PAF and in lesser extent fMLP. Furthermore, CRP, 32 micrograms/ml, diminished specific binding of [3H]-fMLP and [3H]-PAF to PMNL. These findings imply that CRP may play an important protective role during the early phase of acute inflammatory reactions.

Effect of urinary pH and urine flow rate on prostaglandin E2 and kallikrein excretion by the conscious dog.

1. The effects of urine flow rate and urinary pH on renal prostaglandin E2 (PGE2) and kallikrein excretion were investigated in conscious dogs during water deprivation followed by rehydration and under conditions which altered urine pH, but caused similar change in salt and water excretion. 2. When water-restricted dogs were rehydrated in two steps by gavage giving the animals tap water, urine flow increased by 22- and 63-fold with a concomitant increase in PGE2 excretion by 100 and 318%, respectively; whereas urinary kallikrein excretion and urine pH did not change significantly. 3. Oral administration of isotonic sodium chloride solution increased urine flow as well as electrolyte excretion without altering urine pH (6.90 +/- 0.26) and PGE2 excretion. 4. When urine was made alkaline (pH 7.79 +/- 0.09) by oral sodium bicarbonate, urine flow and electrolyte excretion were similar to those observed after oral sodium chloride, while renal PGE2 excretion increased by 66% (P less than 0.05). 5. When urine was made acidic (pH 5.31 +/- 0.14) by oral ammonium chloride, urine flow and electrolyte excretion were similar to the values seen after oral sodium chloride. Urinary PGE2 excretion, however, was reduced by 46% (P less than 0.05). 6. After oral fluid loads a positive correlation could be detected between urine pH and urinary PGE2 excretion (r = 0.854, P less than 0.001). 7. Urinary kallikrein excretion was not significantly altered by any of the three interventions mentioned above. 8. The present results suggest that, in conscious dogs, urine flow as well as urine pH are important determinants of urinary PGE2 excretion rates, but not of kallikrein excretion.