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Alkaline buffers release EDRF from bovine cultured aortic endothelial cells
Author(s) -
Mitchell Jane A.,
Nucci Gilberto,
Warner Timothy D.,
Vane John R.
Publication year - 1991
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1991.tb09783.x
Subject(s) - chemistry , endothelium derived relaxing factor , bradykinin , prostacyclin , arginine , monensin , calcium , sodium , chromatography , biochemistry , in vitro , receptor , organic chemistry , amino acid
1 Release of endothelium‐derived relaxing factor (EDRF) and prostacyclin (PGI 2 ) from bovine cultured aortic endothelial cells (EC) was measured by bioassay and radioimmunoassay, respectively. 2 Bradykinin (BK, 3–30 pmol), adenosine diphosphate (ADP, 2–6 nmol) or the sodium ionophore monensin (40–100 nmol) injected through a column of EC released EDRF. l ‐Arginine free base (FB; 10–20 μmol) or d ‐arginine FB (10–20 μmol) injected through the column of EC released similar amounts of EDRF and also caused an increase in pH of the Krebs solution perfusing the EC from 7.5–8.0 to 8.6–9.5. Sodium carbonate (Na 2 CO 3 ) an alkaline buffer which caused the same changes in the pH of the Krebs solution also induced the same release of EDRF. The hydrochloride salts of l ‐ or d ‐arginine did not cause either release of EDRF when injected through the column of EC or increases in the pH of the Krebs solution. 3 Inhibitors of either diacylglycerol lipase (RHC 80267) or kinase (R59022) inhibited the release of EDRF induced by BK or ADP but potentiated the release induced by l ‐arginine FB, monensin (40–100 nmol) or alkaline buffer (Na 2 CO 3 ). R59022 and RHC 80267 infused through the EC increased the basal release of EDRF. 4 When calcium chloride was omitted from the Krebs solution the release of EDRF induced by alkaline buffer (Na 2 CO 3 ; pH 8.6–9.5) or l ‐arginine FB (10–20 μmol) was selectively inhibited when compared to that induced by BK (3–30 pmol) or ADP (2–6 nmol). This inhibition was reversed when calcium (2.5 m m ) was restored. 5 N G ‐monomethyl‐ l ‐arginine (NMMA; 30 μ m ) inhibited release of EDRF induced by BK (10–30 pmol) or alkaline buffers (Na 2 CO 3 or d ‐arginine FB; pH 8.6–9.5). This inhibition was partially reversed by l ‐but not d ‐arginine FB or HCl (30–100 μ m ). 6 Prostacyclin was released when BK (10 pmol), ADP (2 nmol) or arachidonic acid (30 nmol) were injected through the column of EC. However, monensin (40 nmol) or alkaline buffers (pH 8.6–9.5) did not release detectable amounts of PGI 2 as measured by radioimmunoassay for 6‐oxo‐prostaglandin F 1α.7 Thus alkalinisation of the external bathing solution can release EDRF from cultured EC by a mechanism which does not involve receptor activation and which depends on the presence of extracellular calcium.