Role of Na+-K+-2Cl- Cotransporter 1 in Phenylephrine-Induced Rhythmic Contraction in the Mouse Aorta: Regulation of Na+-K+-2Cl- Cotransporter 1 by Ca2+ Sparks and KCa Channels

Abstract

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 Ca²⁺-activated K⁺ channel blocker TEA and iberiotoxin increased the amplitude of PE-induced rhythmic contraction. The voltage-dependent Ca²⁺ channel blocker, nifedipine, and a Ca²⁺-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 Ca²⁺-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.

Negative News: Cl−and HCO3−in the Vascular Wall

“…Instead, bestrophins are required for the rhythmic contractile pattern known as vasomotion (23). This is consistent with the oscillatory Cl Ϫ conductance observed during vasomotion (79), and with the sensitivity of vasomotion to extracellular Cl Ϫ substitution (12) and the NKCC inhibitor bumetanide (142). The effects of Cl Ϫ substitution on arterial tone vary: acute reduction of extracellular [Cl Ϫ ] has been found to potentiate agonist-induced contractions (39-41, 85, 119) and cause VSMC depolarization (111,119), indicating that Cl Ϫ channels are open under the experimental conditions and allow Cl Ϫ efflux down the enhanced electrochemical gradient, whereas long-term exposure to Cl Ϫ -free FIGURE 1.…”

Role of the Cation-Chloride-Cotransporters in Cardiovascular Disease

“…Further studies have provred that this is accomplished through the channel opening of NKCC1 which increases the [Cl − ] i above the electrochemical equilibrium which depolarizes the VSMC through activation of voltage-gated Ca 2+ [ 87 ]. Consistent with previous findings, more recent studies in intact isolated thoracic aortas demonstrated that NKCC1 is involved in phenylephrine-induced rhythmic contraction in the mouse aorta and that NKCC1 is regulated by calcium sparks [ 88 ].…”

Role of the Cation-chloride-cotransporters in Cardiovascular Disease

“…[78]. Consistent with previous findings, more recent studies in intact isolated thoracic aortas demonstrated that NKCC1 is involved in phenylephrine-induced rhythmic contraction in the mouse aorta and that NKCC1 is regulated by calcium sparks [79].…”