Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled.

1 Release of endothelium derived relaxing factor (EDRF) and prostacyclin (PGO2) from endothelial cells (EC) cultured from bovine aortae was measured by bioassay and radioimmunoassay, respectively, during infusions (10min) of bradykinin (BK), adenosine diphosphate (ADP), arachidonic acid (AA), alkaline buffers and the free-bases (FB) of L-arginine or D-arginine. Release of EDRF from the luminally perfused rabbit aorta was also measured during infusions (10 min) of acetylcholine (ACh), substance P and ADP. 2 Bradykinin (10 or 30 nM) infused through the column of EC induced release of both EDRF and PGO2, neither of which was maintained for the duration of the infusion. 3 ADP (1.6 or 4pM) infused through the column of EC induced release of a EDRF which was maintained for the duration of the infusion and a release of PGO2 which lasted for a much shorter period. 4 Arachdonic acid (30 or 90 pM) infused through the column of EC caused a sustained release of EDRF and PGI2, both of which outlasted the infusion of AA.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Different patterns of release of endothelium‐derived relaxing factor and prostacyclin

Mitchell¹,

Nucci²,

Warner³

et al. 1992

“…When administered as bolus injections, L-arginine (1-30 mmol) produced a drop in basal tone (6.2 + 0.5 mmHg; n = 3) the magnitude of which was independent of the dose applied. In view of the observation that EDRF and PGI2 release appear to be coupled in aortic endothelial cells (de Nucci et al, 1988;D'Orleans-Juste et al, 1989) we examined the combined effect of L-NOArg (100puM) and flurbiprofen (10pM) on the vasodilator response to BK. This combination, like the action of L-NOArg alone, only reduced the duration of the BK-induced vasodilatation (data not shown).…”

Section: Effects Of L-nitro Argininementioning

confidence: 99%

“…In addition to EDRF, BK is also known to release prostacyclin (PGI2), a potent vasodilator in the rat and guinea-pig perfused heart (Schror et al, 1978;Belo & Talesnik, 1982). In vascular endothelial cells the BK-induced release of PGI2 is coupled to that of EDRF and is mediated by the activation of kinin B2 receptors (de Nucci et al, 1988;D'Orleans-Juste et al, 1989). Thus the possibility of PGI2 being released by BK in the rat heart has also been investigated.…”

Section: Introductionmentioning

confidence: 99%

Effects of bradykinin in the rat isolated perfused heart: role of kinin receptors and endothelium‐derived relaxing factor

Baydoun

Woodward

1991

1 The effects of bradykinin (BK) in the microcirculation of the isolated perfused heart of the rat were examined. The kinin receptors mediating the effects of BK were characterized and the role of endothelium-derived relaxation factor (EDRF) and prostacyclin investigated. Gossypol (1-15pM), a presumed inhibitor of EDRF biosynthesis, caused a marked drop in perfusion pressure followed by vasoconstriction. These changes in coronary tone were accompanied by an irreversible depression of cardiac contractility and heart rate. Over the same concentration range gossypol abolished the vasodilator action of BK (1.Onmol), however it also blocked the endothelium-independent vasodilator response to sodium nitroprusside (30nmol) and the vasoconstrictor effect of endothelin-1 (1Opmol) which suggests non-specific toxic actions of gossypol. 6 Bolus injections of BK (0.001-1.Onmol) failed to elevate basal levels of prostacyclin (PGI2) as shown by assaying for its stable metabolite 6-keto-PGF<,,. In addition, BK-induced vasodilatation was not blocked by flurbiprofen (2,pM) or BW755C (7.5 pM) which are inhibitors of the arachidonic acid pathway.When added with L-NOArg (100pM), flurbiprofen (10pM) did not potentiate the inhibitory action of L-NOArg on the BK response. 7 These results show that the vasodilator action of BK in the rat heart is dependent on the activation of the kinin B2 receptors but independent of PGI2 release. Although a conclusive role for EDRF could not be established, this study has questioned the suitability of several agents commonly used as inhibitors of EDRF-mediated responses.

“…Bovine aortic EC were harvested and grown on cytodex 3 microcarrier beads (Pharmacia/LKB, Ltd.) in Dulbecco's modified Eagle's medium (DMEM, Flow Laboratories) supplemented with 4 mM L-glutamine and 10% (v/v) foetal calf serum (Gibco) as described previously (de Nucci et al, 1988). The approximate cell number was 3.5 x 107ml-' of beads.…”

Section: Preparation Of Endothelial Cells or Smooth Muscle Cellsmentioning

confidence: 99%

Conversion of glyceryl trinitrate to nitric oxide in tolerant and non‐tolerant smooth muscle and endothelial cells

Salvemini

Pistelli

Vane

1993

Exposure of smooth muscle cells (SMC) to glyceryl trinitrate (GTN, 75–600 μm) for 30 min led to a concentration‐dependent increase in nitrite (NO2−), one of the breakdown products of nitric oxide (NO). This was not affected by 30 min pretreatment of the cells with 0.5 mm of sulphobromophthalein (SBP) an inhibitor of glutathione‐S‐transferase (GST), by metyrapone or SKF‐525A inhibitors of cytochrome P450. These experiments were confirmed by organ bath studies using rabbit aortic strips denuded of endothelium and contracted with phenylephrine. Thus, a 30 min incubation of the strips with 0.5 mm SPB, metyrapone or SKF‐525A did not affect the relaxations in response to GTN (10−10−10−6 m). Potentiation of the anti‐platelet effect of GTN (44 μm) by endothelial cells (EC, 40 × 103 cells) was not affected by prior incubation of EC with SBP, metyrapone or SKF‐525A (all at 0.5 mm). Potentiation of the antiplatelet activity of GTN (11–352 μm) by small numbers of SMC (24 × 103 cells) or EC (40 × 103 cells) treated with indomethacin (10 μm) was attenuated when the SMC or EC were treated in culture with a high concentration of GTN (600 μm) for 18 h beforehand (referred to as ‘tolerant’ cells). In addition, tolerant SMC produced far less NO2− than non‐tolerant SMC. Exposure of non‐tolerant SMC or EC (105 cells) to GTN (200 μm) for 3 min increased (3–4 fold) the levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP). This increase was much less (≤ 1 fold) in the tolerant SMC or EC (105 cells). The basal levels of cyclic GMP were similar in normal or tolerant SMC or EC. Sodium nitroprusside (80 μm) or atrial natriuretic factor (ANF, 10−7 m) increased the levels of cyclic GMP in normal or tolerant SMC or EC to the same extent. The anti‐platelet effects of GTN (44 μm) were potentiated by the sulphydryl donor N‐acetylcysteine (NAC, 0.5 mm). Incubation of GTN (150–1200 μm) for 30 min with NAC (0.1–1 mm) led to a concentration‐dependent increase in NO2− formation. The reduced ability of tolerant SMC or EC to potentiate the anti‐platelet activity of GTN was restored by NAC (0.5 mm). These anti‐aggregatory effects were abolished by concurrent co‐incubation with oxyhaemoglobin (10 μm) indicating that they were due to NO release. Thus, in SMC or EC, metabolism of GTN to NO does not depend on glutathione‐S‐transferase or the cytochrome P450 system. Furthermore, when compared to normal cells, tolerant SMC or EC metabolize GTN to NO less effectively as assessed by the reduced capacity to potentiate the antiplatelet effects of GTN, to release NO2− and to increase the level of cyclic GMP. This decrease in NO formation shows that tolerance to GTN is mainly due to impaired biotransformation of GTN to NO. NAC, by directly forming NO from GTN, compensates for this failing mechanism.