Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current

Abstract

Astrocytes swell during neuronal activity as they accumulate K+ to buffer the increase in external K+ released from neurons. This swelling activates volume-sensitive Cl- channels, which are thought to be important in regulatory volume decrease and in the response of the CNS to trauma and excitotoxicity. Mitogen-activated protein (MAP) kinases also are activated by cell volume changes, but their roles in volume regulation are unknown. We have investigated the role of tyrosine and MAP kinases in the activation of volume-activated Cl- channels in cultured astrocytes, using whole-cell patch-clamp recording and Western immunoblots. As previously described, hypo-osmotic solution induced an outwardly rectifying Cl- current, which was blocked by NPPB and SITS. This Cl- current did not depend on [Ca2+ ]i because it was still observed when 20 mM BAPTA was included in the pipette, but it did exhibit rundown when ATP was omitted. Inhibition of tyrosine kinases with genistein or tyrphostin A23 (but not the inactive agents daidzein and tyrphostin A1) blocked the Cl- current. The MAP kinase kinase (MEK) inhibitor PD 98059 reversibly inhibited activation of the Cl- current by hypo-osmotic solution. Western immunoblots showed that genistein or PD 98059 blocked activation of Erk-1 and Erk-2 by hypo-osmotic solution in astrocytes. Therefore, activation of tyrosine and MAP kinases by swelling is a critical step in the opening of volume-sensitive Cl- channels.

Fig. 1.

Hypo-osmotic stimulation activates an outwardly rectifying Cl⁻ current in cultured astrocytes.A, Membrane current recorded in response to voltage ramp commands (from −120 to + 80 mV, 2 sec duration, 0.05 Hz) before and during two successive hypo-osmotic (HOS) stimulations (5 min duration, V_H = −60 mV) in a cultured astrocyte with (in mm) 63 Cl⁻, 2 ATP, and 0.5 GTP in the pipette solution. In this and the following figures the top and the bottom traces illustrate the current and the voltage traces, respectively. B, Individual current–voltage (I/V) relations obtained at the times indicated by the numbers inA. C, Mean I/V relations of HOS-induced current obtained by subtracting the peak current recorded during HOS application (2) from that recorded before HOS application (1) (n = 35). D, Mean I/Vrelations of HOS-induced current obtained with either 33 mmCl⁻ (▪, n = 9) or 63 mm Cl⁻ (□, n = 14) in the pipette solution (minus ATP and GTP). Note that HOS-induced current was outwardly rectifying and reversed at approximately −25 mV (downward arrow, D) or −10 mV (upward arrow, D) with either 33 or 63 mm Cl⁻ in the pipette solution, respectively. In this and the following figures, the error bars represent the SEM.

Fig. 2.

I_Cl,vol is blocked by NPPB and SITS. A, Membrane current and conductance changes evoked by a long HOS application (23 min in duration,V_H = −60 mV) recorded in the absence or in the presence of either NPPB (0.1 mm) or SITS (0.5 mm) in the external media [and (in mm) 63 Cl⁻, 2 ATP, and 0.5 GTP in the pipette solution].B, C, Mean I/V relations of HOS-induced current obtained before (□), during (•), and after (+) bath application of either NPPB (B;n = 5) or SITS (C;n = 11). Note that HOS-induced current was strongly reduced by NPPB in a voltage-independent manner and by SITS in a voltage-dependent manner.

Fig. 3.

Activation of I_Cl,volis independent of a change in intracellular Ca²⁺.A, Membrane current evoked by an HOS application (3 min duration, V_H = −60 mV) recorded in the presence of the Ca²⁺ chelator BAPTA (20 mm) with (in mm) 63 Cl⁻, 2 ATP, and 0.5 GTP in the pipette solution. The external concentration of Ca²⁺ was reduced to 0.15 mm.B, Mean I/V relations ofI_Cl,vol recorded in five separate cells under the same conditions as for currents shown in A(n = 5). Note that, in the presence of the Ca²⁺ chelator BAPTA in the pipette solution and a low external concentration of Ca²⁺, the HOS stimulation still evoked an outwardly rectifying current that reversed at approximately −10 mV.

Fig. 4.

I_Cl,vol is regulated by the cellular nucleotides. A, Membrane current evoked by a long HOS application (30 min in duration,V_H = −60 mV) recorded in the presence (1) or in the absence (2) of ATP (2 mm) and GTP (0.5 mm) in the pipette solution. B–D, Graphs showing the changes of the normalized holding current (B), negative conductance (C), and positive conductance (D) of I_Cl,vol versus time during the HOS application (time 0 corresponds to the beginning of the HOS application), either in the presence (1; n = 6) or in the absence (2; n = 10) of ATP and GTP in the pipette solution. The positive and negative conductances were calculated for potentials ranging from +20 to +80 mV and −120 to −60 mV, respectively. The holding current and the negative and positive conductances have been normalized to 1, using their maximal values obtained during the HOS application. Note that activation ofI_Cl,vol was stable in the presence of ATP and GTP. In contrast, when ATP and GTP were omitted from the pipette solution, I_Cl,vol progressively declined.

Fig. 5.

Tyrosine kinases are involved in the activation ofI_Cl,vol. A, Membrane current evoked by four successive HOS stimulations (5 min in duration,V_H = −60 mV) recorded in the presence or in the absence of either daidzein (50 μm) or genistein (50 μm) in the external media [and (in mm) 63 Cl⁻, 2 ATP, and 0.5 GTP in the pipette solution].B, Mean I/V relations ofI_Cl,vol obtained in five cells before (□), during, and after (+) bath application of either daidzein (50 μm, ▪) or genistein (50 μm, •).C, Mean I/V relations ofI_Cl,vol obtained in seven cells before (□), during, and after (+) bath application of either tyrphostin A1 (50 μm, ▪) or tyrphostin A23 (50 μm, •). Note that genistein and tyrphostin A23 strongly and reversibly reduced I_Cl,vol, in contrast to their inactive analogs, daidzein and tyrphostin A1, which had no significant effect on I_Cl,vol.

Fig. 6.

MAP kinase is involved in the activation ofI_Cl,vol. A, Membrane current evoked by five successive HOS stimulations (3 min and 30 sec in duration, V_H = −60 mV) recorded in the presence or in the absence of 50 μm PD 98059 in the external media [and (in mm) 63 Cl⁻, 2 ATP, and 0.5 GTP in the pipette solution]. B, MeanI/V relations of I_Cl,volobtained in six cells before (Control 1, □;Control 2, ▪), during (•), and after (+) bath application of PD 98059. Note that PD 98059 strongly inhibitedI_Cl,vol and that this inhibition was partially reversible during wash.

Fig. 7.

Activation of MAP kinase in response to hypo-osmotic conditions is sensitive to tyrosine kinase and MEK inhibition. Astrocyte cultures were exposed to either iso-osmotic conditions (Iso) or hypo-osmotic conditions (Hypo) with or without other treatments for the time periods indicated. Whole-cell extracts were separated by SDS-Page, transferred to PVDF, and immunoblotted with either an anti-MAP kinase antibody (A and B) or an anti-phosphotyrosine antibody (C).A indicates that the gel shift of p44 Erk-1 and p42 Erk-2 was greater in the Hypo condition, indicating activation. In B and C, the cultures were subjected to a number of different treatments as indicated: PD 98059 (PD, 50 μm), 30 min preincubation followed by stimulation; DMSO 0.1%, 30 min preincubation followed by stimulation; and daidzein (D, 50 μm) and genistein (G, 50 μm), 15 min preincubation followed by stimulation. The decrease in the intensity of theupper bands representing activated Erk-1 and Erk-2 (B) indicates that MAP kinase activity was attenuated in these treatments. In C, the increased tyrosine phosphorylation of Erk-2 in hypo-osmotic solution is apparent at 20 min. PD 98059 and genistein dramatically reduced tyrosine phosphorylation of Erk-2. Other apparently immunoreactive proteins were not affected by these treatments (C).