Direct activation of endothelial NO pathway by Ba²⁺ in canine coronary artery

Abstract

1. We have reported that Ba²⁺ causes endothelium-dependent relaxation of canine coronary arteries through NO synthesis in Ca²⁺-free and depolarizing solution. To determine the cellular mechanisms by which the endothelium-dependent relaxation occurs, we used fura-2 fluorometry (F₃₅₀ and F₃₉₀; excitation wavelengths, 350 and 390 nm, respectively) and estimated the intracellular Ba²⁺ concentration in endothelial and vascular smooth muscle cells.

2. Ba²⁺ (10⁻³ M) increased the fura-2 ratio (F₃₅₀/F₃₉₀) recorded from a combined preparation of smooth muscle and endothelium (0.445±0.073, n=4) and contracted the arteries in the presence of 80 mM K⁺ (0.22±0.06 g, n=4).

3. Diltiazem (3×10⁻⁶ M) blocks Ba²⁺ entry into vascular smooth muscle cells via L-type Ca²⁺ channels. In this condition, Ba²⁺ increased the fura-2 ratio in endothelial cells (0.141±0.014, n=5) and relaxed the underlying smooth muscle (0.08±0.01 g, n=5) by a mechanism which was sensitive to 10⁻⁴ M N^G-methyl-L-arginine (L-NMMA).

4. Ba²⁺-induced relaxation was not attenuated with repeated application and was elicited even after endothelium-dependent relaxations in response to 10⁻⁶ M bradykinin were abolished due to tachyphylaxis. Neither 10⁻² M caffeine nor 10⁻⁶ M thapsigargin had effect upon Ba²⁺-induced relaxation.

5. To further rule out changes in intracellular Ca²⁺ as a mechanism of Ba²⁺-induced relaxation, fura-2 fluorescence was measured at the isosbestic wavelengths for Ca²⁺ (360 nm) and Ba²⁺ (370 nm) in endothelium-intact arteries. Ba²⁺ altered F₃₆₀, but not F₃₇₀, suggesting little or no contribution of intracellular Ca²⁺ to the phenomenon of Ba²⁺-induced relaxation.

6. These results suggest that the Ba²⁺-induced relaxation is due to its direct activation of endothelial NO synthesis without mobilization of intracellular Ca²⁺.

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