Role of Na ⁺ -K ⁺ -2Cl ^- Cotransporter 1 in Phenylephrine-Induced Rhythmic Contraction in the Mouse Aorta: Regulation of Na ⁺ -K ⁺ -2Cl ^- Cotransporter 1 by Ca ²⁺ Sparks and K _Ca Channels

Background/Aims: Vasoconstrictor-induced rhythmic contraction of arteries or veins has been observed both in vivo and in vitro. Many studies have reported that gap junctions, ryanodine receptors, Na ⁺ , K ⁺ -ATPase and other factors are involved in vasoconstrictor-induced rhythmic contraction in vascular smooth muscle. However, the mechanism is still not completely understood. Methods: We used vessel tension measurements, intracellular recordings and intracellular Cl ^- concentration ([Cl ^- ] _i ) measurements to investigate the mechanism underlying phenylephrine (PE)-induced rhythmic contraction in the mouse aorta. Results: We found that Na ⁺ -K ⁺ -2Cl ^- cotransporter 1 (NKCC1) inhibitor bumetanide abolished PE-induced rhythmic contraction. The Cl ^- channel blockers DIDS and niflumic acid initially augmented the amplitude of PE-induced rhythmic contraction but later inhibited the rhythmic contraction. The large Ca ²⁺ -activated K ⁺ channel blocker TEA and iberiotoxin increased the amplitude of PE-induced rhythmic contraction. The voltage-dependent Ca ²⁺ channel blocker, nifedipine, and a Ca ²⁺ -free solution abolished PE-induced rhythmic contraction. The inhibitor of ryanodine receptors in the sarcoplasmic reticulum, ryanodine, inhibited PE-induced rhythmic contraction. Moreover, bumetanide hyperpolarized the membrane potential of vascular smooth muscle cells in a resting state or after PE pre-treatment. Bumetanide, niflumic acid, ryanodine, iberiotoxin, nifedipine and Ca ²⁺ -free buffer significantly suppressed the PE-induced [Cl ^- ] _i increase. Conclusion: These data indicate that NKCC1 is involved in the formation of PE-induced rhythmic contraction, and we also provide a method with which to indirectly observe the NKCC1 activity in isolated intact mouse thoracic aortas.