Roles and Mechanisms of Dopamine Receptor Signaling in Catecholamine Excess Induced Endothelial Dysfunctions

Abstract

Endothelial dysfunction may contribute to pathogenesis of Takotsubo cardiomyopathy, but mechanism underlying endothelial dysfunction in the setting of catecholamine excess has not been clarified. The study reports that D1/D5 dopamine receptor signaling and small conductance calcium-activated potassium channels contribute to high concentration catecholamine induced endothelial cell dysfunction. For mimicking catecholamine excess, 100 μM epinephrine (Epi) was used to treat human cardiac microvascular endothelial cells. Patch clamp, FACS, ELISA, PCR, western blot and immunostaining analyses were performed in the study. Epi enhanced small conductance calcium-activated potassium channel current (I_SK1-3) without influencing the channel expression and the effect was attenuated by D1/D5 receptor blocker. D1/D5 agonists mimicked the Epi effect, suggesting involvement of D1/D5 receptors in Epi effects. The enhancement of I_SK1-3 caused by D1/D5 activation involved roles of PKA, ROS and NADPH oxidases. Activation of D1/D5 and SK1-3 channels caused a hyperpolarization, reduced NO production and increased ROS production. The NO reduction was membrane potential independent, while ROS production was increased by the hyperpolarization. ROS (H2O2) suppressed NO production. The study demonstrates that high concentration catecholamine can activate D1/D5 and SK1-3 channels through NADPH-ROS and PKA signaling and reduce NO production, which may facilitate vasoconstriction in the setting of catecholamine excess.

Keywords: Takotsubo syndrome; dopamine receptor; endothelial dysfunction, nitric oxide.; small conductance calcium-activated potassium channel.

Figure 1

Expression of dopamine receptors in HCMECs. A. qPCR data showing the expression level (normalized to GAPDH) of D1- (DRD1), D2- (DRD2), D3- (DRD3), D4- (DRD4) and D5- (DRD5) dopamine receptors in HCMECs, n=5. B. Representative immunostaining with D1 dopamine receptor antibody showing expression of D1 receptor protein in HCMECs.

Figure 2

D1/D5 receptor activation enhanced I_SK1-3 in HCMECs. I_SK1-3 (apamin-sensitive currents) was isolated by apamin (1 μM), a specific blocker for SK1-3 channels. A. Representative traces of I_SK1-3 at +80 mV (holding potential is -40 mV) in absence (Ctr) and presence of epinephrine (Epi,100 μM for 1 h) and epinephrine plus 10 μM SCH23390 (Epi+SCH). B. Current-voltage relationship (I-V) curves of I_SK1-3 in absence (Ctr) and presence of 10 μM epinephrine for 1 h (Epi-10) and 100 μM epinephrine for 1 h (Epi-100) and 100 μM epinephrine plus 10 μM SCH23390 (Epi+SCH). C. Mean values of I_SK1-3 at +80 mV in absence (Ctr) and presence of 10 μM epinephrine for 1 h (Epi-10) and 100 μM epinephrine for 1 h (Epi-100) and 100 μM epinephrine plus 10 μM SCH23390 (Epi+SCH). D. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) or presence of SKF38393 (SKF, 10 μM for 1 h) or fenoldopam (Fen, 5 μM for 1 h) or SKF38393 plus SCH23390 (SKF+SCH) or fenoldopam plus SCH23390 (Fen+SCH). E. I-V curves of I_SK1-3 in each group. F. Mean values of I_SK1-3 at +80 mV in each group. The n numbers represent number of measured cells. *p<0.05 versus Ctr determined by one-way ANOVA with Holm-Sidak post-test.

Figure 3

Protein kinase A is involved in SKF38393 effect on SK1-3 channel current in HCMECs. A. Representative traces of I_SK1-3 at +80 mV (holding potential is -40 mV) in absence (Ctr) and presence of SKF38393 (SKF, 10 μM for 1 h) and SKF38393 plus 10 μM H89, a PKA inhibitor (SKF+H89). B. Current-voltage relationship (I-V) curves of I_SK1-3 in each group. C. Mean values of I_SK1-3 at +80 mV in each group. D. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) or presence of Sp-8-Br-cAMPS (cAMP, 10 μM for 1 h). E. I-V curves of I_SK1-3 in each group. F. Mean values of I_SK1-3 at +80 mV in each group. The n numbers represent number of measured cells. *p<0.05 versus Ctr, #p<0.05 versus SKF determined by one-way ANOVA with Holm-Sidak post-test (C) or unpaired t-test (F).

Figure 4

ROS contributes to SKF38393 effect on I_SK1-3 in HCMECs. A. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) and presence of SKF38393 (SKF, 10 μM for 1 h) and SKF38393 plus 1 mM NAC (SKF+NAC). B. Current-voltage relationship (I-V) curves of I_SK1-3 in each group. C. Mean values of I_SK1-3 at +80 mV in each group. The n numbers represent number of measured cells. *p<0.05 versus Ctr, #p<0.05 versus SKF determined by one-way ANOVA with Holm-Sidak post-test.

Figure 5

ROS is downstream of PKA in D1/D5 receptor signaling. A. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) and presence of Sp-8-Br-cAMPS (cAMP, 10 μM for 1 h) and cAMP plus 1 mM NAC (cAMP+NAC). B. Current-voltage relationship (I-V) curves of I_SK1-3 in each group. C. Mean values of I_SK1-3 at +80 mV in each group. D. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) or presence of hydrogen peroxide (H2O2, 100 μM for 1 h) and H2O2 plus 10 μM H89 (H2O2+H89). E. I-V curves of I_SK1-3 in each group. F. Mean values of I_SK1-3 at +80 mV in each group. The n numbers represent number of measured cells. *p<0.05 versus Ctr determined by one-way ANOVA with Holm-Sidak post-test.

Figure 6

NADPH oxidases are involved in SKF38393 and cAMP effects on I_SK1-3 in HCMECs. A. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) and presence of SKF38393 (SKF, 10 μM for 1 h) and SKF plus 10 μM NADPH oxidase inhibitor DPI (SKF+DPI). B. Current-voltage relationship (I-V) curves of I_SK1-3 in each group. C. Mean values of I_SK1-3 at +80 mV in each group. D. Representative traces of I_SK1-3 at +80 mV in absence (Ctr) and presence of Sp-8-Br-cAMPS (cAMP, 10 μM for 1 h) and cAMP plus DPI (cAMP+DPI). E. I-V curves of I_SK1-3 in each group. F. Mean values of I_SK1-3 at +80 mV in each group. The n numbers represent number of measured cells. *p<0.05 versus Ctr determined by one-way ANOVA with Holm-Sidak post-test.

Figure 7

Changes of cell membrane potential in HCMECs. Cell membrane potentials were measured with patch clamp whole configuration (current clamp mode). A. Mean values of cell membrane potential (RP) in absence (Ctr) and presence of 10 μM SKF38393 (SKF), SKF 38393 plus 1 μM apamin (SKF+Apa), 5 μM fenoldopam (Fen), fenoldopam plus apamin (Fen+Apa), 100 μM hydrogen peroxide (H2O2) and H2O2 plus apamin (H2O2+Apa). B. Representative traces of RP in each group. The numbers in brackets represent number of measured cells. *p<0.05 versus Ctr determined by one-way ANOVA with Holm-Sidak post-test.

Figure 8

Nitric oxide and ROS generation influenced by D1/D5 receptor and SK1-3 channels in HCMECs. Nitric oxide (NO) concentration in culture medium of HCMECs was measured by ELISA and ROS level in HCMECs was measured by FACS. A. NO concentration in culture medium of HCMECs treated with vehicle (Ctr), 100 μM epinephrine (Epi), epinephrine plus 10 μM SCH23390 (Epi+SCH), 10 μM SKF38393 (SKF), 5 μM fenoldopam (Fen), SKF 38393 plus 1 μM apamin (SKF+Apa), SKF38393 plus SCH23390 (SKF+SCH),10 μM NS309+1 μM TRAM-34 (NS309+TR), 10 mM KCl (KCl) and100 μM H₂O₂ and (H₂O₂). B. ROS levels of HCMECs treated with vehicle (Ctr), 100 μM epinephrine (Epi), epinephrine plus 10 μM SCH23390 (Epi+SCH), 10 μM SKF38393 (SKF), 5 μM fenoldopam (Fen), SKF 38393 plus 1 μM apamin (SKF+Apa), SKF38393 plus SCH23390 (SKF+SCH). C. Representative FACS measurements showing ROS levels of HCMECs of each group. “Blank” means no ROS dye was applied. *p<0.05 versus Ctr, #p<0.05 versus Epi, &p<0.05 versus SKF determined by one-way ANOVA with Holm-Sidak post-test.