Endothelial-derived hyperpolarization contributes to acetylcholine-mediated vasodilation in human skin in a dose-dependent manner

Abstract

Cutaneous acetylcholine (ACh)-mediated dilation is commonly used to assess microvascular function, but the mechanisms of dilation are poorly understood. Depending on dose and method of administration, nitric oxide (NO) and prostanoids are involved to varying extents and the roles of endothelial-derived hyperpolarizing factors (EDHFs) are unclear. In the present study, five incremental doses of ACh (0.01-100 mM) were delivered either as a 1-min bolus (protocol 1, n = 12) or as a ≥20-min continuous infusion (protocol 2, n = 10) via microdialysis fibers infused with 1) lactated Ringer, 2) tetraethylammonium (TEA) [a calcium-activated potassium channel (KCa) and EDHF inhibitor], 3) L-NNA+ketorolac [NO synthase (NOS) and cyclooxygenase (COX) inhibitors], and 4) TEA+L-NNA+Ketorolac. The hyperemic response was characterized as peak and area under the curve (AUC) cutaneous vascular conductance (CVC) for bolus infusions or plateau CVC for continuous infusions, and reported as %maximal CVC. In protocol 1, TEA, alone and combined with NOS+COX inhibition, attenuated peak CVC (100 mM Ringer 59 ± 6% vs. TEA 43 ± 5%, P < 0.05; L-NNA+ketorolac 35 ± 4% vs. TEA+L-NNA+ketorolac 25 ± 4%, P < 0.05) and AUC (Ringer 25,414 ± 3,528 vs. TEA 21,403 ± 3,416%·s, P < 0.05; L-NNA+ketorolac 25,628 ± 3,828%(.)s vs. TEA+L-NNA+ketorolac 20,772 ± 3,711%·s, P < 0.05), although these effects were only significant at the highest dose of ACh. At lower doses, TEA lengthened the total time of the hyperemic response (10 mM Ringer 609 ± 78 s vs. TEA 860 ± 67 s, P < 0.05). In protocol 2, TEA alone did not affect plateau CVC, but attenuated plateau in combination with NOS+COX inhibition (100 mM 50.4 ± 6.6% vs. 30.9 ± 6.3%, P < 0.05). Therefore, EDHFs contribute to cutaneous ACh-mediated dilation, but their relative contribution is altered by the dose and infusion procedure.

Keywords: calcium-activated potassium channels; endothelium; laser-Doppler flowmetry; nitric oxide; skin.

Fig. 1.

Representative skin blood flow response from one subject to a 1-min bolus infusion of 10 mM acetylcholine (ACh) at microdialysis sites receiving lactated Ringer (black) and tetraethylammonium (TEA; gray). Dashed lines represent baseline skin blood flow for each site.

Fig. 2.

Peak (A) and area under the curve (AUC) (B) cutaneous vascular conductance (CVC) in response to bolus infusions of ACh at the four microdialysis sites. Drugs include tetraethylammonium (TEA), N-nitro-l-arginine (L-NNA), and ketorolac (Keto). Data are means ± SE. *P < 0.05 from Ringer site; †P < 0.05 from TEA site; ‡P < 0.05 from L-NNA+Keto site.

Fig. 3.

Representative skin blood flow response from one subject to continuous infusions of 0.01, 0.1, 1, 10, and 100 mM ACh at the four microdialysis sites. Drugs include tetraethylammonium (TEA), N-nitro-l-arginine (L-NNA), and ketorolac (Keto).

Fig. 4.

Plateau cutaneous vascular conductance to five continuous infusions of acetylcholine (ACh). Drugs include tetraethylammonium (TEA), N-nitro-l-arginine (L-NNA), and ketorolac (Keto). Data are means ± SE. *P < 0.05 from Ringer site; †P < 0.05 from TEA site; ‡P < 0.05 from L-NNA+Keto site.