Reactive hyperemia occurs via activation of inwardly rectifying potassium channels and Na+/K+-ATPase in humans

Abstract

Rationale: Reactive hyperemia (RH) in the forearm circulation is an important marker of cardiovascular health, yet the underlying vasodilator signaling pathways are controversial and thus remain unclear.

Objective: We hypothesized that RH occurs via activation of inwardly rectifying potassium (KIR) channels and Na(+)/K(+)-ATPase and is largely independent of the combined production of the endothelial autocoids nitric oxide (NO) and prostaglandins in young healthy humans.

Methods and results: In 24 (23±1 years) subjects, we performed RH trials by measuring forearm blood flow (FBF; venous occlusion plethysmography) after 5 minutes of arterial occlusion. In protocol 1, we studied 2 groups of 8 subjects and assessed RH in the following conditions. For group 1, we studied control (saline), KIR channel inhibition (BaCl2), combined inhibition of KIR channels and Na(+)/K(+)-ATPase (BaCl2 and ouabain, respectively), and combined inhibition of KIR channels, Na(+)/K(+)-ATPase, NO, and prostaglandins (BaCl2, ouabain, L-NMMA [N(G)-monomethyl-L-arginine] and ketorolac, respectively). Group 2 received ouabain rather than BaCl2 in the second trial. In protocol 2 (n=8), the following 3 RH trials were performed: control; L-NMMA plus ketorolac; and L-NMMA plus ketorolac plus BaCl2 plus ouabain. All infusions were intra-arterial (brachial). Compared with control, BaCl2 significantly reduced peak FBF (-50±6%; P<0.05), whereas ouabain and L-NMMA plus ketorolac did not. Total FBF (area under the curve) was attenuated by BaCl2 (-61±3%) and ouabain (-44±12%) alone, and this effect was enhanced when combined (-87±4%), nearly abolishing RH. L-NMMA plus ketorolac did not impact total RH FBF before or after administration of BaCl2 plus ouabain.

Conclusions: Activation of KIR channels is the primary determinant of peak RH, whereas activation of both KIR channels and Na(+)/K(+)-ATPase explains nearly all of the total (AUC) RH in humans.

Keywords: blood flow regulation; hyperpolarization; ischemia; vasodilation.

Figure 1. Representative Tracing of Baseline and Reactive Hyperemia

Representative tracing (n=1) of the last 30 seconds of rest and the first minute of the reactive hyperemia response in control (saline; A) conditions and with inhibition of K_IR channels via BaCl₂ (B). Tracings are shown for the electrocardiogram (ECG), intra-arterial pressure (I.A. Press.), and venous occlusion plethysmography (VOP) output from which heart rate, mean arterial pressure, and forearm blood flow, respectively, are calculated and/or derived. Notes: The vertical scale for VOP is 4 times greater during rest (pre-occlusion) than during reactive hyperemia (post-occlusion). Vertical deflections indicate balancing of the plethysmography signal to maintain a consistent baseline.

Figure 2. Protocol 1: Independent effects of K_IRchannel inhibition (Group 1)

A. Forearm blood flow (FBF) response following 5 minutes of arterial occlusion in the following conditions: control (black circles), independent K_IR channel inhibition (BaCl₂; dark grey triangles), combined K_IR channel and Na⁺/K⁺-ATPase inhibition (BaCl₂+ouabain; light grey squares), and combined inhibition of K_IR channels, Na⁺/K⁺-ATPase, NO and PGs (BaCl₂+ouabain+L-NMMA+ketorolac; white diamonds) conditions. BaCl₂ significantly inhibited the response for 75 seconds and there was little additional effect of ouabain, or L-NMMA+ketorolac. *P<0.05 vs BaCl₂; †P<0.05 vs BaCl₂+ouabain; ‡ P<0.05 vs BaCl₂+ouabain+L-NMMA+ketorolac. B. Peak reactive hyperemic FBF was significantly attenuated from control by BaCl_2, and ouabain had no additional effect whereas there was a slightly greater reduction with the addition of L-NMMA+ketorolac. *P<0.05 vs Control; †P<0.05 vs BaCl₂. C. Similarly, total reactive hyperemic FBF (area under curve) was significantly reduced from control by BaCl_2, and ouabain had no additional effect whereas L-NMMA+ketorolac further reduced this response. *P<0.05 vs Control; †P<0.05 vs BaCl₂.

Figure 3. Protocol 1: Independent effects of Na⁺/K⁺-ATPase inhibition (Group 2)

A. Forearm blood flow (FBF) response following 5 minutes of arterial occlusion in control (black circles), independent Na⁺/K⁺-ATPase inhibition (Ouabain; dark grey triangles), combined Na⁺/K⁺-ATPase and K_IR channel inhibition (Ouabain+BaCl₂; light grey squares), and combined inhibition of Na⁺/K⁺-ATPase, K_IR channels, NO and PGs (Ouabain+BaCl₂+L-NMMA+ketorolac; white diamonds) conditions. Ouabain did not affect initial FBF, but thereafter reduced FBF from control until 90 seconds post-cuff deflation. The addition of BaCl₂ further attenuated FBF for 30 seconds, whereas addition of L-NMMA+ketorolac had no further effect. *P<0.05 vs Ouabain; †P<0.05 vs Ouabain+BaCl₂; ‡P<0.05 vs Ouabain+BaCl₂+L-NMMA+ketorolac. B. Peak reactive hyperemic FBF was not affected by ouabain. Infusion of BaCl₂ significantly reduced peak FBF from control, and L-NMMA+ketorolac had no further impact. *P<0.05 vs Control; †P<0.05 vs Ouabain. C. Total reactive hyperemic FBF (area under curve) was significantly reduced from control by ouabain, and BaCl₂ had an additional effect whereas L-NMMA+ketorolac did not. *P<0.05 vs Control; †P<0.05 vs Ouabain.

Figure 4. Protocol 2: Effects of combined inhibition of nitric oxide and prostaglandins

A. Forearm blood flow (FBF) response following 5 minutes of arterial occlusion in control (black circles), combined inhibition of NO and PG synthesis (L-NMMA+ketorolac; light grey squares), and combined inhibition of NO, PGs, K_IR channels and Na⁺/K⁺-ATPase (L-NMMA+ketorolac +BaCl₂+ouabain; white diamonds) conditions. L-NMMA+ketorolac attenuated the response from control only from 30-60 seconds post-cuff deflation. The addition of BaCl₂+ouabain significantly reduced FBF for 30 seconds and thereafter had no further effect. *P<0.05 vs L-NMMA+ketorolac; †P<0.05 vs L-NMMA+ketorolac +BaCl₂+ouabain. B. Peak reactive hyperemic FBF was not affected by L-NMMA+ketorolac and was significantly attenuated by L-NMMA+ketorolac +BaCl₂+ouabain. *P<0.05 vs Control; †P<0.05 vs L-NMMA+ketorolac. C. Similar to peak, total reactive hyperemic FBF (area under curve) was not affected by L-NMMA+ketorolac and was significantly attenuated by L-NMMA+ketorolac +BaCl₂+ouabain. *P<0.05 vs Control; †P<0.05 vs L-NMMA+ketorolac.

Figure 5. Summary: Effects of inhibition of K_IRchannels, Na⁺/K⁺-ATPase, nitric oxide and prostaglandins on peak and total reactive hyperemia

Combined results from the three experimental protocols are presented for relative impact (%Δ) on both peak (A) and total (B) reactive hyperemic forearm blood flow (FBF) in each experimental condition (BaCl₂: n=8; Ouabain: n=8; BaCl₂+ouabain: n=16; L-NMMA+ketorolac: n=8; BaCl₂+ouabain+L-NMMA+ketorolac: n=24). BaCl₂ reduced peak FBF and this attenuation was unchanged with the addition of ouabain or L-NMMA+ketorolac. Neither ouabain alone nor L-NMMA+ketorolac attenuated peak FBF. BaCl₂ and ouabain both independently reduced total FBF and in combination (BaCl₂+ouabain), the reduction was enhanced. There was no additional reduction by L-NMMA+ketorolac, nor did L-NMMA+ketorolac independently reduce total FBF. *P<0.05 vs zero; †P<0.05 vs BaCl₂; ‡P<0.05 vs Ouabain