Acidosis dilates brain parenchymal arterioles by conversion of calcium waves to sparks to activate BK channels

Abstract

Rationale: Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BK(Ca)) channels by local Ca(2+) signals (Ca(2+) sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known.

Objective: The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BK(Ca) channels in this response.

Methods and results: Internal diameter and vascular smooth muscle cell Ca(2+) signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), vascular smooth muscle cells exhibited primarily RyR-dependent Ca(2+) waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca(2+) wave activity, and dramatically increased Ca(2+) spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BK(Ca) channel or RyR blockers, in a nonadditive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BK(Ca) channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of K(ATP) channels or nitric oxide synthase.

Conclusions: These results support the novel concept that acidification, by converting Ca(2+) waves to sparks, leads to the activation of BK(Ca) channels to induce dilation of cerebral PAs.

Figure 1

Mouse parenchymal arterioles express functional BK_Ca channels and RyRs but the myogenic response is not counterbalanced by Ca²⁺ spark driven BK_Ca currents under basal conditions. Typical recordings of the internal diameter of pressurized parenchymal arterioles (40 mm Hg) during the perfusion of the BK_Ca blocker paxilline 1 μmol/L (A), RyR blocker ryanodine 10 μmol/L (B), or BK_Ca channel agonist NS11021 3 μmol/L (C). (D) Summary data showing change in luminal diameter expressed as percentage change in baseline diameter with the number of experiment indicated in parentheses. *P < 0.05 (E) typical recordings of the internal diameter showing the vasoconstriction induced by noncumulative addition of caffeine in papaverine 100μmol/L in absence (left) or presence (right) of ryanodine 10 μmol/L. (F) summary data showing change in luminal diameter expressed as percentage of baseline diameter. (n = 5).

Figure 2

Spontaneous Ca²⁺ signals in intact cerebral arteries. (A) Representative recordings showing Ca²⁺ sparks in pressurized (80 mm Hg) mouse pial artery (middle cerebral artery, ~100 μm diameter) and Ca²⁺ waves in pressurized (40 mm Hg) mouse parenchymal arterioles (~ 15 μm diameter). Squares on the photographs are regions of interest, where F/F0 is measured. Spontaneous Ca²⁺ signals (traces below photographs) were detected as F/F₀ from regions of interest marked as colored squares in the top panels. (B) Progression of a Ca²⁺ wave from one end of the cell to the other (bracketed cell from a parenchymal arteriole). Images are color coded as indicated by the color bar. Images were acquired every 35.9 ms (for display, every twentieth image is shown). (C) Summary data of the percent of cells exhibiting Ca²⁺ sparks and Ca²⁺ waves in pial arteries and parenchymal arterioles.

Figure 3

Acidic pH reshapes the intracellular Ca²⁺ dynamic from Ca²⁺ waves to Ca²⁺ sparks. (A) Spontaneous Ca²⁺ signals recorded from the same regions of interest of a pressurized parenchymal arteriole at pH = 7. 4 and pH = 7.0. (B) Compiled data showing Ca²⁺ spark frequency (sparks/cell/s, black bars) and the percentage of cells presenting sparks (% of cells with spark, grey bars) at different pHs. At pH 7.2, both the percentage of cells with Ca²⁺ sparks and spark frequency became significantly different from values observed at pH 7.4 (P < 0.05).

Figure 4

Normocapnic and hypercapnic acidosis dilate pressurized parenchymal arterioles by activating Ca²⁺ spark-driven BK_Ca channel activity. (A) Typical recording of the internal diameter of a pressurized parenchymal arteriole (40 mm Hg) during the perfusion of acidic aCSF under normocapnic conditions, without and with the RyR blocker ryanodine (10 μmol/L). (B) Effect of BK_Ca channel blocker paxilline (1 μmol/L) on acidic pH-induced dilation under normocapnic conditions. (C) Effect of RyR blocker ryanodine (10 μmol/L) and BK_Ca channel blocker paxilline (1 μmol/L) on acidic pH-induced dilation under hypercapnic conditions. (D) Summary data showing vasodilator responses related to initial level of myogenic tone (0%) and fully relaxed diameter (100%; 0 Ca²⁺, papaverine 100 μmol/L), with the number of experiments indicated in parentheses. *P < 0.05

Figure 5

Hypercapnic-, normocapnic- and NS11021-induced dilations are decreased in Slo-KO mouse. (A) Typical recordings of the internal diameter of pressurized parenchymal arterioles (40 mm Hg) from a wild type mouse (Kcnma1^+/+, upper trace) or a mouse lacking the α subunit of the BK_Ca channel (Kcnma1^−/−, lower trace) during the perfusion of acidic aCSF under hypercapnic or normocapnic conditions, or in presence of the BK_Ca channel agonist NS11021, in the absence or presence of the BK_Ca channel blocker paxilline. (B) Summary data showing vasodilator responses related to initial level of myogenic tone (0%) and fully relaxed diameter (100%; 0 Ca²⁺, papaverine 100 μmol/L), with the number of experiments indicated in parentheses. *P < 0.001

Figure 6

Acidic pH-induced dilation of PAs is not affected by glibenclamide. (A) Typical recording of the internal diameter of a pressurized (40 mm Hg) parenchymal arteriole (PA) during the perfusion of acidic aCSF under normocapnic conditions with and without the K_ATP blocker glibenclamide (1 μmol/L). (B) Absence of effect of K_ATP channel agonist cromakalim on a parenchymal arteriole. (C) Vasodilation of a pressurized (60 mm Hg) third order mesenteric artery (Mesenteric A) induced by cumulative addition of Cromakalim (10⁻⁸ to 3.10⁻⁸ mol/L). Glibenclamide (1 μmol/L) completely blocked the cromakalim-induced dilation of mesenteric artery. (D) Cromakalim concentration response relationships in parenchymal arterioles (n = 5) and mesenteric arteries (n = 5). Dilation is related to initial level of myogenic tone (0%) and fully relaxed diameter (100%).

Figure 7

NOS-inhibition does not alter dilations induced by acidic pH_o in normocapnic and hypercapnic conditions. (A) Typical recording of the internal diameter of a pressurized parenchymal arteriole (40 mm Hg) during the perfusion of acidic aCSF induced by reduced bicarbonate concentration (normocapnia) with and without the eNOS inhibitor L-NAME (100 μmol/L). (B) Summary data (n = 4). (C) Typical recording of the internal diameter of a pressurized parenchymal arteriole (40 mm Hg) during the perfusion of acidic aCSF induced by increased pCO₂ (hypercapnia) with and without the eNOS inhibitor L-NAME (100 μmol/L). (D) Summary data (n = 6). Dilation is expressed as percent of maximum dilation (0 Ca²⁺, papaverine 100 μmol/L).

Figure 8

Proposed mechanism for RyRs- and BK_Ca-dependent, acidosis-induced dilation of brain parenchymal arterioles. By decreasing the P₀ of RyRs in VSMCs, protons reshape intracellular Ca²⁺ signaling from waves to sparks which activate BK_Ca channels and then cause dilation.