17 beta-Estradiol potentiates kainate-induced currents via activation of the cAMP cascade

Abstract

Evidence for nongenomic actions of steroids is now coming from a variety of fields of steroid research. Mechanisms of steroid action are being studied with regard to the membrane receptors and the activation of second messengers. The present study investigated the mechanism for the rapid effect of estrogen on acutely dissociated hippocampal CA1 neurons by using the whole-cell, voltage-clamp recording. Under the perforated patch configuration, 17 beta-estradiol potentiated kainate-induced currents in 38% of tested neurons. The potentiation was stereospecific, rapid in onset, and reversible after the removal of the steroid. Dose-response curves show that the potentiation by 17 beta-estradiol was evident at a concentration as low as 10 nM and saturated at 10 microM. 17 beta-Estradiol did not affect the kinetics (i.e., affinity and cooperativity) and reversal potential of kainate-induced currents. This suggests that the potentiation did not result from direct interaction with kainate receptors nor the activation of ion channels other than kainate receptor-channels. The potentiation by 17 beta-estradiol was similar to the enhancement of kainate-induced currents evoked by 8-bromo-cAMP, and was modulated by an inhibitor of phosphodiesterase (IBMX). The estrogen potentiation was blocked by a specific blocker of PKA (Rp-cAMPS). Under standard recording configuration, the effect was significantly affected by intracellular perfusing with GDP-beta-S or GTP-gamma-S. The data suggest that the potentiation of kainate-induced currents by 17-beta-estradiol was likely a G-protein(s) coupled, cAMP-dependent phosphorylation event. By involvement of this non-genomic mechanism, estrogen may play a role in the modulation of excitatory synaptic transmission in the hippocampus.

Fig. 1.

Comparison of kainate-induced currents using conventional and perforated whole-cell patch recording techniques on acutely dissociated CA1 hippocampal neurons. A, Two cells were recorded under the conventional whole-cell recording configuration. A 20 msec pressure application of kainate (100 μm) to the dendrites of these CA1 neurons evoked an inward current, the amplitude of which declined to 50% of its initial value within 20 min (open triangles). One cell (open circles) was tested with 17β-estradiol (100 nm). The application of 17β-estradiol (solid circles) significantly retarded the decline in the amplitude of kainate-induced currents. Also in A, the amplitude of kainate-induced currents, obtained using the perforated patch recording, was stable for a period of up to 25 min (open squares). Individual current traces recorded under the conventional whole-cell patch configuration in the absence (B) and presence (C) of 17β-estradiol, and under perforated whole-cell patch configuration (D), are shown to the right of the graph.

Fig. 2.

17β-Estradiol potentiated the kainate-induced current. The illustrated example shows the effects of 17β- and 17α-estradiol on kainate-induced currents recorded from an individual hippocampal neuron. The amplitude of kainate-induced currents was increased in the presence of 17β-estradiol (100 nm) but not 17α-estradiol (100 nm). The potentiation was rapid in onset (2–3 min from its application) and reversible after removal of the chemical. Actual current traces selected at specific time points (filledcircles) are displayed to the right of the graph.

Fig. 3.

Potentiation of kainate-induced currents by 17β-estradiol was dose-dependent. A, The effect of 17β-estradiol was not evident at concentrations below 10 nm and appears saturated at 10 μm (n = 5). B, Dose–response curves for various concentrations of kainate before (○) and after (•) 17β-estradiol (100 nm) application show that 17β-estradiol increased the amplitude of kainate-induced currents with no effect on the values of EC₅₀ and n_H (n = 5).

Fig. 4.

Current–voltage curves obtained in the absence (open circles) and presence (filledcircles) of 17β-estradiol (100 nm). Both relationships are linear with a nearly identical reversal potential; however, the slopes were significantly different. 17β-Estradiol (100 nm) increased the conductance to kainate (100 μm) from 1.8 to 2.5 nS, as determined from the slope of the current–voltage relation. Individual kainate-induced currents used to construct the plot are shown to the right of the graph.

Fig. 5.

The enhancement of kainate-induced currents by cAMP, 8-bromo-cAMP (500 μm) increased the amplitude of kainate-induced currents, whereas 8-bromo-cGMP (500 μm) had no obvious effect on kainate-induced current. The enhancement evoked by 8-bromo-cAMP was similar to that of 17β-estradiol in the latency and magnitude. Sample current traces before and after 8-bromo-cAMP and 8-bromo-cGMP application are shown to the right of the graph.

Fig. 6.

Potentiation of kainate-induced currents by 17β-estradiol was mediated by cAMP. A, Application of IBMX (100 μm) enhanced the effect of 17β-estradiol (100 nm). B, The presence of 17β-estradiol at its saturating concentration (10 μm) almost occluded the enhancement induced by 8-bromo-cAMP (500 μm), even though both agents could potentiate the kainate-induced currents when applied alone (data not shown). Sample current traces selected at specific time points (filled circles) are shown to the right of the graphs.

Fig. 7.

Involvement of cAMP-dependent protein kinase.A, The administration of Rp-cAMPS (50 μm) completely eliminated the potentiation normally observed with the application of 17β-estradiol (100 nm). Sample current traces selected at specific time points (filled circles) are shown to the right of the graph.

Fig. 8.

Effect of G-protein(s) on the 17β-estradiol potentiation of kainate-induced currents. Isolated hippocampal neurons were recorded under the conventional whole-cell configuration and dialyzed with GDP-β-S (500 μm), GDP alone, or GTP-γ-S (500 μm). As illustrated inA, the potentiation induced by 17β-estradiol shortly after membrane rupture was suppressed as GDP-β-S diffused into the cell (46 ± 11%; n = 3), whereas in B, intracellular perfusion with GDP alone had no effect on the kainate-induced currents (n = 3). C, The intracellular administration of GTP-γ-S prolonged the potentiation by 17β-estradiol (n = 4).