Prostacyclin release stimulated by thrombin or bradykinin in porcine endothelial cells cultured from aorta and umbilical vein

Drazan

Newby

1989

Primary cultures of pig aortic endothelial cells produced 6‐keto‐prostaglandin F1α (6‐keto PGF1α), the stable breakdown product of prostacyclin, both in the resting state and in response to bradykinin. The rise in 6‐keto‐PGF1α production induced by bradykinin (1–100 nm) was concentration‐dependent. Treating endothelial cells with the inhibitor of soluble guanylate cyclase, methylene blue (0.1–20 μm) produced an irreversible reduction in resting and bradykinin (0.1 μm)‐stimulated production of 6‐keto‐PGF1α with an IC50 of 0.5 ± 0.1 μm. Treating endothelial cells with haemoglobin (10 μm) had no effect on resting or bradykinin (0.1 μm)‐stimulated production of 6‐keto‐PGF1α. Two stimuli that elevate the level of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) in endothelial cells, 8‐bromo cyclic GMP (30 μm) and atriopeptin II (0.1 μm), each had no effect on resting or bradykinin (0.1 μm)‐stimulated production of 6‐keto‐PGF1α. Furthermore, treating endothelial cells with either 8‐bromo cyclic GMP (30 μm) or atriopeptin II (0.1 μm) had no effect on the ability of methylene blue (20 μm) to inhibit resting or bradykinin (0.1 μm)‐stimulated production of 6‐keto‐PGF1α. Adding arachidonic acid (1 μm) to endothelial cells led to a marked stimulation of 6‐keto‐PGF1α production. Treating cells with either methylene blue (20 μm) or the cyclo‐oxygenase inhibitor, flurbiprofen (10 μm), inhibited both resting and arachidonic acid (1 μm)‐induced production of 6‐keto‐PGF1α. In pig aortic endothelial cells methylene blue appears to block prostacyclin production by a mechanism independent of inhibition of soluble guanylate cyclase. Care should be exercised when using methylene blue as a selective inhibitor of endothelium‐derived relaxing factor due to its additional ability to block production of the other endothelium‐derived vasodilator, prostacyclin.

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Section: Discussionsupporting

confidence: 79%

Methylene blue but not changes in cyclic GMP inhibits resting and bradykinin‐stimulated production of prostacyclin by pig aortic endothelial cells

Drazan

Newby

1989

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“…and Brotherton (1986) used only primary cultures but Ganz et al (1986) used subcultured cells. It has been shown that endothelial cell responsiveness declines with time in culture (Pearson et al, 1983; Ager & coli endotoxin had no effect on the cyclic Martin, 1984;Needham et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

Endothelium‐derived relaxing factor and atriopeptin II elevate cyclic GMP levels in pig aortic endothelial cells

White

Henderson

1988

125

Two directly‐acting stimulants of soluble guanylate cyclase, glyceryl trinitrate (0.1 μm) and sodium azide (10 μm), and a receptor‐mediated stimulant of particulate guanylate cyclase, atriopeptin II (10 nm), each elevated the cyclic GMP content of primary cultures of pig aortic endothelial cells without affecting the cyclic AMP content. Two receptor‐mediated stimulants of adenylate cyclase, glucagon (1 μm) and isoprenaline (10 μm), had no effect on the cyclic AMP or cyclic GMP content of these cells, but the directly acting stimulant, forskolin (30 μm), induced a small increase in cyclic AMP content. Three agents that release endothelium‐derived relaxing factor (EDRF); bradykinin (0.1 μm), ATP (10 μm) and ionophore A23187 (0.1 μm), each markedly elevated the cyclic GMP content of pig aortic endothelial cells, but acetylcholine (1 μm) had no effect. None of these agents had any effect on cyclic AMP content. Two agents that potentiate the actions of EDRF; M & B 22948 (100 μm) and superoxide dismutase (30 units ml−1), each elevated the cyclic GMP content of pig aortic endothelial cells without affecting the cyclic AMP content. Pretreating cells with catalase (100 units ml−1) did not affect the rise in cyclic GMP content induced by superoxide dismutase (30 units ml−1). Pretreatment of pig aortic endothelial cells with haemoglobin (10 μm) reduced the resting content of cyclic GMP and blocked the increase in cyclic GMP content induced by glyceryl trinitrate (0.1 μm), sodium azide (10 μm), bradykinin (0.1 μm) ATP (10 μm), ionophore A23187 (0.1 μm), M & B 22948 (100 μm) and superoxide dismutase (30 units ml−1), but not that induced by atriopeptin II (10 nm). Pretreatment of pig aortic endothelial cells with an inhibitor of soluble guanylate cyclase, methylene blue (20 μm), had no effect on the resting content of cyclic GMP. Methylene blue (20 μm) blocked the increase in cyclic GMP content induced by glyceryl trinitrate (0.1 μm), M & B 22948 (100 μm) and bradykinin (0.1 μm), but not that induced by atriopeptin II (10 nm). The data show that soluble guanylate cyclase, particulate guanylate cyclase and adenylate cyclase are present in pig aortic endothelial cells. They further suggest that EDRF, produced spontaneously or in response to vasoactive agents, elevates endothelial cyclic GMP content by stimulating soluble guanylate cyclase. It is possible that this may serve as a feedback loop by which the endothelial cell modulates EDRF production.

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“…Platelet activating factor and thrombin were also unable to promote increased albumin transfer despite having been shown to induce a shape change in and promote increased permeability of bovine pulmonary artery endothelial cells (Grigorian & Ryan, 1987;Minnear et al, 1988;De-Michele et al, 1988). Of these two agents only thrombin is known to have other effects on pig aortic endothelial cells e.g., stimulation of prostacyclin production (Pearson et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

Modulation of arterial endothelial permeability: studies on an in vitro model

Gudgeon

1989