Role of epoxyeicosatrienoic acids as autocrine metabolites in glutamate-mediated K+ signaling in perivascular astrocytes

Abstract

Epoxyeicosatrienoic acids (EETs), synthesized and released by astrocytes in response to glutamate, are known to play a pivotal role in neurovascular coupling. In vascular smooth muscle cells (VSMC), EETs activate large-conductance, Ca(2+)-activated K(+) (BK) channels resulting in hyperpolarization and vasodilation. However, the functional role and mechanism of action for glial-derived EETs are still to be determined. In this study, we evaluated the effect of the synthetic EET analog 11-nonyloxy-undec-8(Z)-enoic acid (NUD-GA) on outward K(+) currents mediated by calcium-activated K(+) channels. Addition of NUD-GA significantly increased intracellular Ca(2+) and outward K(+) currents in perivascular astrocytes. NUD-GA-induced currents were significantly inhibited by BK channel blockers paxilline and tetraethylammonium (TEA) (23.4 ± 2.4%; P < 0.0005). Similarly, NUD-GA-induced currents were also significantly inhibited in the presence of the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin along with a combination of blockers against glutamate receptors (12.8 ± 2.70%; P < 0.05). No changes in outward currents were observed in the presence of the channel blocker for intermediate-conductance K(+) channels TRAM-34. Blockade of the endogenous production of EETs with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) significantly blunted (dl)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD)-induced outward K(+) currents (P < 0.05; n = 6). Both NUD-GA and t-ACPD significantly increased BK channel single open probability; the later was blocked following MS-PPOH incubation. Our data supports the idea that EETs are potent K(+) channel modulators in cortical perivascular astrocytes and further suggest that these metabolites may participate in NVC by modulating the levels of K(+) released at the gliovascular space.

Fig. 1.

Representative whole cell current profiles from cortical perivascular astrocytes. A: differential interference contrast (DIC) image of a Lucifer yellow-filled astrocyte in direct contact to an intraparenchymal arteriole. Calibration bar, 10 μm. Representative voltage-dependent (B) and linear membrane currents (C) in response to a ramp protocol from −120 to +80 mV.

Fig. 2.

Effect of endogenous epoxyeicosatrienoic acids (EETs) on outward currents induced by activation of metabotropic glutamate receptors (mGluR) in perivascular astrocytes. (dl)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD)-induced (100 μM) outward currents in the absence (A) and presence (B) of the CYP epoxygenase substrate inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) (20 μM) (solid black line: control, solid gray line: t-ACPD, dashed line: subtracted current) are shown. C: summary data of averaged peak currents (pA/pF) at V_m = 80 mV induced by t-ACPD in the presence and absence of MS-PPOH and the large-conductance, Ca²⁺-activated K⁺ (BK) channel blocker paxilline. D: representative traces of single BK channel activation induced by t-ACPD in the presence and absence of MS-PPOH. E: changes in single channel open probability (NPo) induced by t-ACPD in the presence and absence of MS-PPOH (*P < 0.05, n = 4).

Fig. 3.

Calcium and electrophysiological responses of cortical astrocytes to the EET agonist 11-nonyloxy-undec-8(Z)-enoic acid (NUD-GA). A: NUD-GA-induced calcium transients in cortical astrocytes, representative traces and fluorescence images are shown. B: summary data of NUD-GA-induced changes in averaged peak F/F₀ (***P < 0.0001, n = 11) and calcium oscillation frequency (**P < 0.01, n = 11). C and D: representative NUD-GA-induced outward voltage-dependent and linear membrane currents in response to a ramp protocol from −120 to +80 mV in perivascular astrocytes, respectively. The corresponding subtracted currents for C and D are shown below. E: summary data of peak currents (pA/pF) at +80 mV under control conditions, in the presence of NUD-GA, in the presence of 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and in the presence of 14,15-EEZE and NUD-GA (*P < 0.005, n = 7). F: summary data of peak currents (pA/pF) at +80 mV in the presence and absence of the calcium chelator BAPTA (**P < 0.05, n = 5). Calibration bar, 10 μm.

Fig. 4.

Contribution of BK channels to NUD-GA-induced outward currents. A: representative current profiles of perivascular astrocytes in the presence and absence of the BK channel blocker paxilline (2 μM) and tetraethylammonium (TEA, 1 mM) in response to a ramp protocol from −120 to +80 mV (P < 0.001, n = 11). Subtracted currents are shown below. B: representative NUD-GA-induced outward K⁺ currents from an astrocyte in the presence of the BK channel blocker paxilline and TEA. Corresponding subtract currents are shown below. C: summary data of peak currents at +80 mV in the presence (*P < 0.05, n = 7) and absence of BK channel blockers.**P < 0.01; ***P < 0.001 D: left, immunolabeling against BK channels (green) and the astrocyte marker glial fibrillary acidic protein (GFAP) (red) (scale bar = 10 μm); right, representative single channel activity from an astrocytic endfoot in the absence and presence of NUD-GA, O, open state and C, closed state. E: left, averaged current-voltage relationship for BK channels expressed in astrocytic endfeet; right, NUD-GA-induced increased in NPo (**P < 0.01, n = 4).

Fig. 5.

Lack of contribution of intermediate conductance (IK) channels to NUD-GA-induced outward currents. A: representative ramp profile of a perivascular astrocyte in the presence and absence of the IK channel blocker TRAM-34 (1 μM). B: representative ramp profile of a perivascular astrocyte in the presence and absence of NUD-GA in the presense of TRAM-34.

Fig. 6.

Contribution of small conductance (SK) channels to NUD-GA-induced outward currents. A: representative ramp profiles of perivascular astrocytes in the presence and absence of the SK channel blocker apamin (300 nM) (P < 0.05, n = 7), subtracted currents are shown below. B: representative ramp profiles of perivascular astrocytes in the presence of tetradotoxin (TTX) (0.5 μM) and glutamate receptor blockers kyneurenic acid (1 mM), LY-367385 (50 μM), and 2-methyl-6-phenylethynylpyridine hydrochloride (MPEP, 100 μM) with or without apamin. C: NUD-GA-induced outward currents in the presence of apamin and combined inhibitors against ionotropic and metabotropic GluR. D: representative calcium trace showing apamin-induced calcium oscillations in astrocytes. E: summary data of apamin-induced changes in averaged peak F/F₀ and calcium oscillation frequency from cortical astrocytes (**P < 0.01, n = 7). F: summary data of averaged peak currents at +80 mV in the presence and absence of NUD-GA, combined inhibitors against ionotropic and metabotropic GluR and the SK channel blocker apamin. *P < 0.05.