Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells

Abstract

N-methyl-D-aspartate (NMDA) receptors are widely expressed in the central nervous system. However, their presence and function at extraneuronal sites is less well characterized. In the present study, we examined the expression of NMDA receptor subunit mRNA and protein in human pulmonary artery (HPA) by quantitative polymerase chain reaction (PCR), immunohistochemistry and immunoblotting. We demonstrate that both GluN1 and GluN2 subunit mRNAs are expressed in HPA. In addition, GluN1 and GluN2 (A-D) subunit proteins are expressed by human pulmonary artery smooth muscle cells (HPASMCs) in vitro and in vivo. These subunits localize on the surface of HPASMCs and form functional ion channels as evidenced by whole-cell patch-clamp electrophysiology and reduced phenylephrine-induced contractile responsiveness of human pulmonary artery by the NMDA receptor antagonist MK801 under hypoxic condition. HPASMCs also express high levels of serine racemase and vesicular glutamate transporter 1, suggesting a potential source of endogenous agonists for NMDA receptor activation. Our findings show HPASMCs express functional NMDA receptors in line with their effect on pulmonary vasoconstriction, and thereby suggest a novel therapeutic target for pharmacological modulations in settings associated with pulmonary vascular dysfunction.

Figure 1

NMDA receptor mRNA expression in human pulmonary artery. RT-PCR was performed using RNA isolated from human pulmonary artery. The presence of mRNAs for GluN1 and all GluN2 subunits (2A–D) was detected in human pulmonary artery. Human brain RNA was used as a positive control.

Figure 2

NMDA receptor expression by HPASMCs in vivo. Human lung tissue containing pulmonary artery was stained with NMDA receptor subunit specific antibodies. PECAM-1 was used as a marker for vascular endothelial cells (EC). Antibody against PECAM-1 revealed immunoreactivity within the interior surface of pulmonary artery. Immunoreactive staining for both GluN1 (B) and four GluN2 subunits (2A–D) (C–F) was observed in pulmonary artery smooth muscle cells (SMC). Control experiments with primary antibody omitted showed no immunoreactivity in either pulmonary artery endothelial cells or PASMCs (G). Scale bar = 50 μM.

Figure 3

NMDA receptor expression in cultured HPASMCs. Immunofluorescence staining was performed on permeabilized HPASMCs. Network-like immunoreactive staining was observed for both GluN1 and all four GluN2 (2A–D) subunits. Control experiments with primary antibody omitted showed no immunoreactivity. Results are representative images taken from at least three separate experiments. Scale bar = 50 μM.

Figure 4

Full-length NMDA receptors are expressed by cultured HPASMCs. Cultured HPASMCs were lysed and immunoprecipitated with GluN1- or GluN2- subunit specific antibodies or control non-immune IgG, and the pellets were analyzed by Western blot. Immunopositive bands containing full-length GluN1 (A) and GluN2 (2A–D) (B–E) protein were detected in immune pellets from lysates of PASMCs but not in the IgG control conditions studied in parallel. Cropped images are shown for conciseness. Full-length blots are represented in Supplementary Fig. S9. Results are representative of three separate experiments.

Figure 5

Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs (A,B). GluN1 co-localized with GluN2B (A) and GluN2D (B) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. (C,D) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x-axis and GluN2 red fluorescent intensity along the y-axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. (E) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. (F) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p < 0.01, n = 16). (G) NMDA and glycine treatment evoked increased AUC compared with baseline (*p < 0.05, n = 16).

Figure 6

Serine racemase and VGLUT1 expression in HPASMCs. HPASMCs showed positive immunoreactive staining for serine racemase (A) and VGLUT1 (C), respectively. Immunopositive bands containing serine racemase and VGLUT1 protein were also detected in whole cell lysates from cultured HPASMCs ((B,D), respectively). Cropped images are shown for conciseness. Full-length blots are represented in Supplementary Fig. S13. Results are representative images taken from at least three separate experiments. Scale bar = 50 μM.

Figure 7

NMDA receptors regulate phenylephrine-induced pulmonary vasoconstriction. Precision-cut human lung slices containing pulmonary vessels were exposed to normoxic (21% O₂) or hypoxic (5% O₂) conditions overnight. The cells were pre-treated with 10 µM MK801 or vehicle (DMSO) for 1 h prior to adding varying concentrations of phenylephrine (PE), as indicated. (A) Phenylephrine induced pulmonary vasoconstriction in a dose-dependent manner in both normoxic and hypoxic conditions. (B) MK801 had little effect on maximal vasoconstriction (E_max) under normoxic conditions, but significantly attenuated maximal response to phenylephrine under hypoxic conditions (*p = 0.0231). (C) MK801 had little effect on the sensitivity of the pulmonary vessels to phenylephrine-measured by Log EC₅₀ of the dose–response curve-in both conditions. (D) MK801 had little effect on area under the curve (AUC) of the phenylephrine dose–response curve in both conditions. (Data are representative of: Normoxic-Vehicle, n = 13 donors; Normoxic-MK801, n = 6 donors; Hypoxic-Vehicle, n = 13 donors; Hypoxic-MK801, n = 6 donors).

Figure 8[replace with the revised figure]

NMDA receptors regulate endothelin-1 (ET-1)-induced pulmonary vasoconstriction in murine precision-cut lung slices. ET-1 induced pulmonary vasoconstriction under a normoxic (21% O₂) condition in a dose-dependent manner (A). Pretreatment with 10 µM MK801 or 50 µM AP5 significantly attenuated ET-1-induced maximal vasoconstriction (E_max) (B), the sensitivity of the pulmonary vessels to ET-1 measured by Log EC₅₀ of the dose–response curve (C), and the area under the curve (AUC) (D). *p < 0.05, ***p < 0.0001, n = 3.