The α2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries

Abstract

The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the α2 isoform of the Na,K-pump (α2), Na+, Ca2+-exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating α2 in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca2+]i was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 μM ouabain uncoupled them. Downregulation of α2 reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of α2 in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endothelium-dependent hyperpolarizing factor-like component of the response was significantly diminished in α2-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the α2-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on α2-downregulated arteries. In the presence of a low NE concentration the α2-downregulated arteries had higher [Ca2+]i and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca2+-sensitivity. The α2-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K+ was not affected. Our results demonstrate the importance of α2 for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone.