Electric fields caused by blood flow modulate vascular endothelial electrophysiology and nitric oxide production

Abstract

Endothelial cells are exposed to a ubiquitous, yet unexamined electrical force caused by blood flow: the electrokinetic vascular streaming potential (EVSP). In this study, the hypothesis that extremely low frequency (ELF) electric fields parameterized by the EVSP have significant biological effects on endothelial cell properties was studied by measuring membrane potential and nitric oxide production under ELF stimulation between 0 and 2 Hz and 0-6.67 V/m. Using membrane potential and nitric oxide sensitive fluorescent dyes, bovine aortic endothelial cells (BAECs) in culture were studied in the presence and absence of EVSP-modeled electric fields. The transmembrane potential of BAECs was shown to depolarize between 1 and 7 mV with a strong dependency on both the magnitude and frequency of the isolated ELF field. The findings also support a field interaction with a frequency-dependent tuning curve. The ELF field complexly modulates the nitric oxide response to adenosine triphosphate stimulation with potentiation seen with up to a sevenfold increase. This potentiation was also frequency and magnitude dependent. An early logarithmic phase of NO production is enhanced in a field strength-dependent manner, but the ELF field does not modify a later exponential phase. This study shows that using electric fields on the order of those generated by blood flow influences the essential biology of endothelial cells. The inclusion of ELF electric fields in the paradigm of vascular biology may create novel opportunities for advancing both the understanding and therapies for treatment of vascular diseases.

Figure 1

Diagram of apparatus for bovine endothelial cell monolayer electric stimulation.

Figure 2

DiBAC4(3) calibration curve. Equation for best-fit line: ΔF/F0=0.3(Ψ) + 8.98 (n=6). The x-intercept (ΔF/F0 = 0) is the resting membrane potential of the BAECs.

Figure 3

EVSP-induced frequency-dependent depolarization. (A): Direct current (n=6) and 0.5 Hz (n=6) stimulation show no significant difference at the three field strengths (1.67 V/m, p=0.35; 3.33 V/m, p= 0.08; 6.67 V/m, p=0.42). At 1 Hz stimulation (n=9), a ~ 3 mV depolarization occurs, (*) with a lower depolarization at 6.67 V/m (vs. 1.67 V/m, p = 0.01; vs. 3.33 V/m, p = 0.02). At 2 Hz stimulation (n=9), a ~6 mV depolarization occurs, (**) with a higher depolarization at 3.33 V/m (vs. 1.67 V/m, p=0.001; vs. 6.67 V/m, p=0.0001). (B): Equations for best-fit lines: 1.67 V/m, 3.61×ln(t)+3.37, R²=0.99; 3.33 V/m, 4.93×ln(t)+3.79, R²=0.99; 6.67 V/m, 3.08×ln(t)+2.72, R²=0.99.

Figure 4

NO signal with ATP stimulation and varying calcium conditions without electric stimulation. (A): NO signal in the presence of Ca²⁺. (B): Early logarithmic phase during the first 30 min of (A). Equation for best-fit line: NO=0.20×1n(t) )+0.03, R²=0.97. (C): A late exponential phase seen in the final 90 min. Equation for best-fit line: NO=0.434×e^(0.134t), R²=0.99. (D): ATP stimulation with [Ca²⁺]e (n=3) and ATP stimulation without [Ca²⁺]e (n=4) or with calcium channel blockade (n=3).

Figure 5

EVSP effect on the NO signal in the presence of 1.1 mM [Ca²⁺]e. (A): Unstimulated control (n=3) and ATP stimulation (n=3) and EVSP+ATP stimulation at 3.33 V/m (n=3) and 6.67 V/m (n=3). (B): Relationship of early log phase to EVSP field strength. Equations of best-fit lines: ATP only, NO= 0.20×ln(t) + 0.03, R² = 0.97; 3.33 V/m + ATP, NO=0.52×ln(t)+0.02, R² = 0.98; 6.67 V/m + ATP, NO=0.94×ln(t)+0.11, R²=0.97. (C): Exponential phases of the three curves with the log phase value at t=30 min subtracted.

Figure 6

EVSP effect on the NO signal in Ca²⁺-free and calcium channel-blocked conditions. Unstimulated (n=3), ATP only (n=4), 1.67 V/m + ATP (n=3), 3.33 V/m + ATP (n=3), and 6.67 V/m + ATP (n=3) at (A): 1 Hz and (B): 2 Hz. (C): EVSP stimulation with the NiCl² blockade (n=3), 3.33 V/m +ATP (n=2), and 6.67 V/m +ATP (n=2).