Aldosterone rapidly induces endothelial exocytosis. (A) Time-course study: HAEC were treated with control, thrombin 1 U/ml, or aldosterone 10⁻⁹ M. Cell media was analyzed for vWF as described previously (n = 3 ± SD; *, P < 0.01 vs. control). (B) Aldosterone activation of exocytosis does not depend on new RNA transcription. HAEC were treated with actinomycin D 2.5 μM or DMSO for 1 h, and then stimulated with aldosterone 10⁻⁹ M or ethanol control for 1 h. Cell media was analyzed for vWF as described (n = 3 ± SD; *, P < 0.01 vs. control).

Aldosterone induces endothelial exocytosis. (A) Dose-response study for vWF release: HAEC were incubated with aldosterone for 1 h. The amount of vWF released from cell into the media was measured by an ELISA (n = 3 ± SD; *, P < 0.05 vs. control). (B) Aldosterone activates interleukin-8 exocytosis. Endothelial cells were treated with TNF-a for 16 h to load IL-8 into granules. Endothelial cells were then stimulated with control (EtOH), thrombin, or aldosterone for 1 h. Cell media was analyzed for IL-8 with an ELISA as above (n = 3 ± SD; *, P < 0.05 vs. control). (C) Tissue plasminogen activator: aldosterone does not activate TPA release. Endothelial cells were treated with control, thrombin, or aldosterone for 1 h. Cell media was analyzed for TPA with an ELISA as described (n = 3 ± SD; P > 0.05 vs. control for all levels of aldosterone). (D) Specificity: aldosterone and estrogen activates vWF exocytosis. HAEC were treated with control, thrombin, aldosterone, estrogen, progesterone, or hydrocortisone for 1 h. Cell media was analyzed for vWF as described (n = 3 ± SD; *, P < 0.01 vs. control).

Mineralocorticoid receptor mediates aldosterone activation of exocytosis: pharmacological data. (A) Specificity: spironolactone inhibits aldosterone-induced vWF exocytosis. HAEC were pretreated with control or spironolactone for 1 h, and then treated with aldosterone 10⁻⁹ M for 1 h. Cell media was analyzed for vWF as described previously (n = 3 ± SD; *, P < 0.01 vs. media + aldosterone). (B) Spironolactone does not affect thrombin-induced exocytosis. HAEC were pretreated with EtOH (control vehicle) or spironolactone 10⁻⁷ M for 1 h, and then treated with thrombin 1 U/ml for 1 h. Cell media was analyzed for vWF as described (n = 3 ± SD; *, P < 0.01 vs. control). (C) Inhibitors of other steroid hormone receptors do not affect aldosterone-induced vWF exocytosis. HAEC were pretreated with estrogen receptor inhibitor tamoxifen 10⁻⁷ M, progesterone receptor inhibitor mifepristone 10⁻⁷ M, and spironolactone 10⁻⁷ M for 1 h, and then treated with aldosterone 10⁻⁹ M for 1 h. Cell media was analyzed for vWF as described (n = 3 ± SD; *, P < 0.01 vs. media + aldosterone). (D) Inhibitors of other steroid hormone receptors alone do not affect vWF exocytosis in the absence of aldosterone. HAEC were pretreated with estrogen receptor inhibitor tamoxifen 10⁻⁷ M, progesterone receptor inhibitor mifepristone 10⁻⁷ M, or aldosterone 10⁻⁹ M for 1 h. Cell media was analyzed for vWF as described (n = 3 ± SD; *, P < 0.01 vs. control).

Mineralocorticoid receptor mediates aldosterone activation of exocytosis: genetic data. (A) Expression of MR mRNA by RT-PCR of HAEC and HeLa cells. (B) Expression of MR protein by immunoblotting in HAEC, HUVEC, and HeLa cells (Top) and in HAEC, and mouse inner medullary collecting duct (mIMCD). Equal amounts 3.0 μg of cell lysates were loaded per lane. (C) Knockdown of mineralocorticoid receptor in endothelial cells. (D) Knockdown of mineralocorticoid receptor decreases effect of aldosterone on exocytosis. Endothelial cells were transfected with a control siRNA oligonucleotide or an oligonucleotide directed against MR; cells were treated or not with aldosterone 10⁻⁹ M or vehicle (EtOH) for 1 h and vWF release measured (n = 3 ± SD; *, P = 0.01 vs. control siRNA + aldo.).

Aldosterone activates leukocyte-endothelial interactions ex vivo. (A) Images of leukocyte adherence to endothelial cells. HAEC were pretreated or not with spironolactone (10⁻⁷M) for 1 h, and then aldosterone (10⁻⁹M), thrombin 1 U/ml, or TRAP (10⁻⁶M) were added for 1 h. HL-60 promyelocytic leukemia cells were then loaded with BCEF-AM and cocultured with the HAEC. The cell mixtures were incubated at 4 °C for 15 min, washed twice with HBSS, and then imaged with a digital fluorescent camera. (B) Quantitation of above, measuring the effect of aldosterone (aldo.) and spironolactone (spiro.) on HL-60 adherence to HAEC (n = 4 wells with 3 different fields from each well ± SD. *, P < 0.001 compared with control). (C) Images of leukocyte adherence to endothelial cells. HAEC were treated with aldosterone (10⁻⁹ M) for 1 h with or without antibody to P-selectin (10⁻³ M) for 15 min, and then cocultured with HL-60 cells and imaged as described. (D) Quantitation of above, measuring the effect of antibody to P-selectin on aldosterone or calcium ionophore (A23187) stimulated HL-60 adherence to HAEC (n = 4 wells with 3 different fields from each well ± SD; *, P < 0.001 compared with control).