Thapsigargin Differentiates Between Edrf and Prostacyclin Release Induced by Shear-Stress or Agonists

Abstract

The aggonist-mediated simultaneous release of endothelium-derived relaxing factor(EDRF) and prostacyclin (PGI2) is initiated by a mobilization of intracellular calcium ([Ca2+]i). It is, however, as yet unclear how this increase in [Ca2+]i relates to the physiologically more important, release of EDRF induced by shear stress. Recently the tumor-promoting sesquiterpene lactone, thapsigargin, was shown irreversibly to inhibit the re-uptake of CA2+ into inositol-1,4,5-triphosphate (InsP3)-sensitive Ca2+ stores by blocking the Ca2+-ATPase located in the endoplasmic reticulum of various cells, including platelets and polymorphocyclear cells (Thastrup et al., 1990, Takemura et al., 1991). By employing this Ca2+-ATPase inhibitor, we have now investigated further the effects of inhibition of intracellular Ca2+ sequestration on the release of EDRF and PGI2 from bovine aortic endothelial cells (BAEC). The cells (grown on microcarrier beads with 2 ml corresponding to 6 X 107 BAEC) were packed into a jacketed chromatography column and perfused (5 ml/min) with warmed (370C), oxygenated (95% O2/5% CO2) Kreb’s solution. The effluent superfused an endothelium denuded rabbit aortic ring preconstricted to 2-3g tension with 10 nM U46619 (9α, 11α-methaneoepoxy-prostaglandin F2a) for the detection and quantification of EDRF release. PGI2 release was determined by radioimmunoassay for 6-keto-prostaglandin Fla and changes in [Ca2+]i were monitored with Fura-2/AM loaded BAEC in suspension. EDRF release induced by shear stress was defined as the degree of relaxation (16.7 ± 3% of induced tone; n=5) caused by an infusion of superoxide dismutase (SOD; 10 U/ml) through the column of BAEC (t.c). This relaxation was further enhanced (10α25%; n=5) by bolus injections of agonists such as ADP (9 nmol), ionomycin (60 pmol), or poly-L-lysine (550 pmol). Infusions t.c of thapsigargin (1 µM) caused a sustained release of EDRF (29.8 ± 8.1% relaxation; n=5) which declined after stopping the infusion. Thereafter, the agonist-induced release of EDRF was abolished, whereas, the shear stress-induced release remained unaffected (n=5). The EDRF activity after both types of release was blocked by infusions t.c of hemoglobin (10 µM; n=3). The basal release of PGI2 from the BAEC (1.04 ± 0.27 ng/min; n=5) was significantly enhanced (3-6 ng/min) by bolus injections t.c of any of the three agonists. Infusions of thapsigargin t.c also caused a substantial increase in PGIZ release (5.04 ± 1.99 ng/min), but thereafter, as with EDRF, challenges by the agonists failed to cause any further rise in PGIZ release. The resting [Ca2+]i level in Fura-2/AM-load-ed BAEC (5X 105 cells/ml) was 70-100 nM. ADP (1 µM) produced a typical biphasic change in [Ca2+]i i.e a marked transient (< 2 min) rise (peak 400-1000 nM) followed by a plateau phase (200-300 nM) lasting for more than 10 min (n=4). Thapsigargin (1 µM) elicited an initial sharp increase in [Ca2+]i (Peak 800-1200 nM) which declined slowly and remained elevated throughout the experiment at approximately 500 nM. Subsequent exposure to ADP produced no further increase in [Ca2+]i (n=4). It appears, therefore, that by emptying the InsP3 sensitive and insensitive Ca2+ pools, thapsigargin selectively blocks the agonist-stimulated release of EDRF and PGI2, suggesting a fundamental difference in the control by CA2+ of release of both substances by shear stress or agonists. (Supported by a grant from Glaxo Group Research Ltd.)