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. 2014:2014:873590.
doi: 10.1155/2014/873590. Epub 2014 Nov 13.

Inhibition of brain swelling after ischemia-reperfusion by β-adrenergic antagonists: correlation with increased K+ and decreased Ca2+ concentrations in extracellular fluid

Dan Song  1 , Junnan Xu  1 , Ting Du  1 , Enzhi Yan  1 , Leif Hertz  2 , Wolfgang Walz  3 , Liang Peng  1
Affiliations

Inhibition of brain swelling after ischemia-reperfusion by β-adrenergic antagonists: correlation with increased K+ and decreased Ca2+ concentrations in extracellular fluid

Dan Song et al. Biomed Res Int. 2014.

Abstract

Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl-, and water cotransporter NKCC1 and by β1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the β-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically β1-adrenergic pathways, indicating failure of K+-mediated, but not β1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making β1-adrenergic activation the only stimulus and its inhibition effective against edema.

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Figures

Figure 1
Figure 1
Schematic illustration of stimulation of ERK phosphorylation by β-adrenergic receptors in astrocytes. Isoproterenol (ISO) binds to β-adrenergic receptors. At high concentrations (>1 μM), the activation of the receptors induces β 1-adrenergic (red arrows), PKA-dependent “Gs/Gi switching,” which in turn induces an enhancement of intracellular Ca2+ concentration by Ca2+ release from intracellular stores. The latter activates Zn-dependent metalloproteinases (MMPs) and leads to shedding of growth factor(s). The released epidermal growth factor (EGF) receptor ligand stimulates phosphorylation of the EGF receptor in the same and adjacent cells. The downstream target of the EGF receptor, extracellular regulated kinase (ERK), shown in blue, is phosphorylated via the Ras/Raf/MEK pathway. As shown in yellow ovals the ERK phosphorylation by isoproterenol at high concentration can be inhibited by H-89, an inhibitor of protein-kinase A (PKA); by PTX, an inhibitor of Gi protein; by BAPTA/AM, an intracellular Ca2+ chelator; by GM6001, an inhibitor of Zn-dependent metalloproteinases; by AG1478, an inhibitor of the receptor-tyrosine kinase of the EGF receptor; by siRNA against β-arrestin 1 and less completely by siRNA against β-arrestin 2; and by U0126, an inhibitor of MEK, which directly phosphorylates ERK. In contrast, at low concentration (100 nM) β 2-adrenergic (green arrows) activation of the receptors activates Src via recruitment of β-arrestin 2. Src in turn stimulates ERK phosphorylation and phosphorylates EGF receptors at different sites than β 1-adrenergic stimulation, without involvement of the receptor-tyrosine kinase. Its ERK1/2 phosphorylation is secondary to MEK activation, which may be induced by direct activation of Raf or Ras by Src. The ERK phosphorylation by isoproterenol at low concentration can be inhibited by siRNA against β-arrestin 2; by PP1, a Src inhibitor; and by the MEK inhibitor U0126. The effect of most of these inhibitors on MCAO-induced edema was investigated and tabulated in Table 3.
Figure 2
Figure 2
Uptake of K+ into primary cultures of astrocytes measured with the aid of the fluorescent drug PBFI-AM as described in Section 2. After incubation of PBFI-AM-loaded cells in saline solution for 45 min and subsequent wash, the cells were perfused either in similar solution with 2 mM CaCl2 or in a solution without CaCl2 for 2 min. From the start of the graphs they were perfused in a solution to which an additional 5 mM KCl or 10 μM of the β 1-adrenergic agonist dobutamine had been added at zero time (with a corresponding reduction of NaCl concentration when K+ was added). Results for K+ addition are averages from 60 individual cells on three coverslips and those after addition of dobutamine are from 38 cells. SEM values are indicated by vertical bars. *Statistically significant (P < 0.05) difference from +5 mM K+ with 2 mM Ca2+ group at the same time period and from addition of dobutamine in either the presence or absence of Ca2+.
Figure 3
Figure 3
Ouabain induced inhibition of net influx of 42K into primary cultures of mouse astrocytes. The uptake was determined during a 1.00 min incubation, which provides initial uptake rates. The graph marked by diamonds was obtained in a slightly modified Dulbecco's medium containing 1.8 mM Ca2+ (for further details see Section 2) and that marked by X in a corresponding medium containing no CaCl2. The inhibition was calculated from the ratios between uptake rates in the absence of ouabain and that at the ouabain concentration in question. The calculated inhibitions were analyzed as a function of the ouabain concentration by GraphPad Prism 6, assuming that they are biphasic, which gave the best fit. SEM values for inhibition of cultures incubated at 1.8 mM Ca2+ were obtained from the standard errors given in the Graph Pad analysis for, respectively, high and low affinity. For cultures incubated without Ca2+ SEM value for the lower concentrations (high affinity) were similarly based on the Graph Pad analysis. A similar procedure could not be used for the low-affinity component, since the total inhibition did not approach 100%, and the computed standard error accordingly was excessively high. Therefore no SEM values are shown for the two highest concentrations, but they must resemble those at the lower concentrations, since the original uptake readings had approximately similar uncertainties at all concentrations. The apparent stimulation at low ouabain concentrations in the absence of Ca2+ reflects an ouabain-mediated stimulation of uptake, normally occurring at low nanomolar concentrations [17]. In both graphs 6 cultures from 2-3 different batches had been used for each point.
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