BK channel activation in chronic vasodilation by thiazide-like diuretics. Role ot the beta-1 auxilary

MARTÍN, PEDRO; ASUAJE, AGUSTÍN; ENRIQUE, NICOLÁS; MILESI, MARIA VERÓNICA; REBOLLEDO, ALEJANDRO

Foz do Iguacu

Congreso; XXI Congreso Latinoamericano de Farmacologí­a de la ALF; 2016

Sociedad Latinoamericana de Farmacologia (ALF)

Introduction: Arterial hypertension is one of the major risk factors for cardiovascular disease. Treatment with thiazide-like diuretics is based on the decrease in blood volume and direct vasodilation. The goal of this study was to determine the vasodilator mechanism of the diuretic hydrochlorothiazide (HCTZ), whose molecular target is still unknown. Previous studies indicated that activation of the large-conductance Ca2+- and voltage-operated K+ channel (BK) may be involved in this effect. This channel is formed by 4 alpha-subunits that may (or may not) be accompanied by accessory subunits, depending on the cell type, that confer specific pharmacological characteristics to the channel. In particular, the accessory beta-1 subunit is mainly expressed in smooth muscle cells (SMCs).Methods: Using the patch-clamp technique we analyzed the effect of HCTZ on BK channel activity expressed (with and without the beta-1 subunit) in HEK293 T (an heterologous expression system) and in SMCs from human umbilical artery (HUASMCs) as native system.Results: Using the whole-cell configuration in HEK cells transfected with the BK channel accompanied by beta-1 subunit we observed that addition of HCTZ to the extracellular solution increased the current amplitude in a concentration-dependent manner, with an EC50 of 28.4 µM (pD2=4.546 ± 0.211, n: 5-8). However, HCTZ did not change the channel activity when it was evaluated in the same cells in the inside-out configuration, where cell integrity is lost (% increase in current at + 90mV: 6.3 ± 15.0, 12.8 ± 38.1, - 17.8 ± 12.7% to HCTZ 10, 30 and 100 µM, respectively, NS; n: 3-7), suggesting an indirect action of the diuretic in channel activation. Furthermore, we checked the effect of HCTZ in whole-cell currents in HEK cells transfected with the BK channel without the beta-1 subunit. In this condition HCTZ did not change the BK current (% increase in current at +40 mV: 13.9 ± 15.7 and 6.9 ± 5.1% to HCTZ 100 and 300 µM, respectively, NS; n: 3-4). Interestingly , these results indicate that, despite being an indirect activation, HCTZ requires the presence of beta-1 subunit to stimulate the BK channel. Finally, these experimental results were repeated in HUASMCs, where the channel is naturally expressed with the beta-1 subunit and is further modified by post-transcriptional regulation. Consistent with the results of the heterologous system, the extracellular application of 10 µM HCTZ caused significant activation of BK current recorded in whole-cell configuration (528 ± 215 to 1379 ± 132 pA at +40mV, n: 4, p 0.05).Conclusion: These results suggest that the vasodilatory effects of HCTZ could be due to an indirect activation of the BK channel which depends of the beta-1 subunit expression.Financial support: PICT-2014-0603, ANPCYT, Argentina.The use of HUA was approved by the ethics and research committee of the Hospital General Zonal de Agudos San Roque / Gonnet, and the written informed consent of each subject were obtained.