Casein kinase 2-mediated synaptic GluN2A up-regulation increases N-methyl-D-aspartate receptor activity and excitability of hypothalamic neurons in hypertension

Abstract

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.

FIGURE 1.

Changes in the decay kinetics of NMDAR-EPSCs of PVN neurons in SHRs. A, representative current traces showing original and normalized NMDAR-EPSCs of labeled PVN neurons from one SHR and one WKY rat. B and C, summary data showing the amplitude (B) and τ_w (C) of NMDAR-EPSCs of PVN neurons from SHRs and WKY rats (n = 7 neurons in each group). *, p < 0.05, compared with the WKY group. Error bars, S.E.

FIGURE 2.

Changes in GluN2A- and GluN2B-mediated NMDAR-EPSCs of PVN neurons in SHRs. A, representative current traces showing NMDAR-EPSCs inhibited by 0.6 μm Ro25-6981 and 300 nm Zn²⁺ in labeled PVN neurons from one SHR and one WKY rat. The remaining NMDAR-EPSCs in the SHRs were completely blocked by a high concentration (50 μm) of AP5. B and C, summary data showing inhibition of NMDAR-EPSCs of labeled PVN neurons by Ro25-6981 and Zn²⁺ in SHRs and WKY rats (n = 7 in each group). D, original current traces showing NMDAR-EPSCs blocked by 5 μm AP5 in labeled PVN neurons from one SHR and one WKY rat. E and F, summary data showing inhibition of NMDAR-EPSCs by 5 μm AP5 in labeled PVN neurons in SHRs (n = 9) and WKY rats (n = 7). *, p < 0.05, compared with the WKY group. #, p < 0.05, compared with the respective base-line control value. Error bars, S.E.

FIGURE 3.

GluN2A-mediated postsynaptic NMDAR currents of PVN neurons are increased in SHRs. A, representative traces show the effect of 5 μm AP5 on NMDAR currents produced by puff application of 100 μm NMDA to labeled PVN neurons from one WKY rat and one SHR. B and C, group data show inhibition of puff NMDA-induced NMDAR currents by 5 μm AP5 in labeled PVN neurons from SHRs and WKY rats (n = 7 in each group). *, p < 0.05, compared with the WKY group. #, p < 0.05, compared with the respective base-line control value. Error bars, S.E.

FIGURE 4.

Presynaptic GluN2A-containing NMDARs are increased in PVN neurons in SHRs. A, original traces and summary data show the effect of 300 nm Zn²⁺ on the frequency and amplitude of mEPSCs of labeled PVN neuron in SHRs (n = 7). B, raw traces and group data show the effect of 5 μm AP5 on the frequency and amplitude of mEPSCs of labeled PVN neuron in SHRs (n = 7). C, original traces and summary data show the effect of 0.6 μm Ro25-6981 on the frequency and amplitude of mEPSCs of labeled PVN neuron in SHRs (n = 7). *, p < 0.05, compared with the base-line control value. Error bars, S.E.

FIGURE 5.

GluN2A-NMDARs contribute to increased firing activity of PVN neurons in SHRs. A, original recordings showing the differential effects of 0.6 μm Ro25-6981 and 5 μm AP5 on the firing activity of one labeled PVN neuron in a SHR. B, summary data showing the effects of Ro25-6981 and AP5 on the firing activity of labeled PVN neurons in SHRs (n = 7). *, p < 0.05, compared with the base-line control value. Error bars, S.E.

FIGURE 6.

GluN2A- and GluN2B-mediated NMDAR-EPSCs in PVN neurons in SHRs subjected to CGx. A, original recordings and group data show the effect of CGx on the ABP in SHRs (Sham, n = 6; CGx, n = 8). B–D, representative examples and summary data show the effects of 0.6 μm Ro25-6981 and 5 μm AP5 on NMDAR-EPSCs of labeled PVN neurons in SHRs subjected to CGx and sham surgery (n = 7 in each group). *, p < 0.05, compared with the sham group. #, p < 0.05, compared with the respective base-line control value. Error bars, S.E.

FIGURE 7.

Changes in the mRNA and protein levels of GluN2A and GluN2B subunits in the PVN in SHRs. A, representative agarose gel image shows the presence of the mRNA of GluN2A-C, but not GluN2D, subunits in the PVN (top), and real-time PCR data show changes in the mRNA levels of GluN2A-C in the PVN in SHRs and WKY rats (n = 6 in each group; bottom). B, representative gel images (20 μg of protein/lane) and group data show differences in the total protein levels of GluN2A and GluN2B subunits in PVN tissues in SHRs and WKY rats (n = 6 in each group). C, original gel images (60 μg of protein/lane) and group data show differences in the synaptosomal protein levels of GluN2A and GluN2B subunits in the PVN in SHRs and WKY rats (n = 6 in each group). D, original gel images (40 μg of protein/lane) and group data show differences in the level of phosphorylated GluN2B Ser¹⁴⁸⁰ in the PVN in SHRs and WKY rats (n = 6 in each group). Molecular mass is indicated on the right side of the gel image. GAPDH was probed as a protein loading control. *, p < 0.05, compared with the WKY group. Error bars, S.E.

FIGURE 8.

CK2 inhibition normalizes GluN2A-mediated pre- and postsynaptic NMDAR activity and the phosphorylated GluN2B level in the PVN in SHRs. A–C, representative traces and group data show the effects of 0.6 μm Ro25-6981 and 5 μm AP5 on electrically evoked NMDAR-EPSCs of labeled PVN neurons in brain slices of SHRs treated with 100 μm DRB, 2 μm TBB, or vehicle (n = 7 in each group). D, summary data show the effect of 5 μm AP5 on the frequency of mEPSCs of labeled PVN neurons in brain slices of SHRs treated with 100 μm DRB, 2 μm TBB, or vehicle (n = 7 in each group). E, representative gel images (40 μg of protein/lane) and group data show the effect of DRB on the level of phosphorylated GluN2B Ser¹⁴⁸⁰ in the PVN in SHRs (n = 6 in each group). Molecular mass is indicated on the right side of the gel image. *, p < 0.05, compared with the vehicle group. #, p < 0.05, compared with the respective base-line control value. Error bars, S.E.