Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin

Abstract

Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke by increased transport of Na from blood into brain across an intact BBB. We previously showed that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently, we found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchange protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB Na/H exchange activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion-fixed rat brain revealed that Na/H exchanger isoforms 1 and 2 (NHE1 and NHE2) are present in BBB endothelial cells. Using microspectrofluorometry and the pH-sensitive dye BCECF, we found that hypoxia (2% O(2), 30 min), aglycemia (30 min), and AVP (1-200 nM, 5 min) significantly increased CMEC Na/H exchange activity, assessed as Na-dependent, HOE-642-sensitive H(+) flux. We found that AVP stimulation of CMEC Na/H exchange activity is dependent on intracellular Ca concentration and is blocked by V(1), but not V(2), vasopressin receptor antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation.

Fig. 1.

Evaluation of Na/H exchange (NHE) protein isoforms in cerebral microvascular endothelial cells. A and B: lysates of bovine cerebral microvascular endothelial cells (CMEC, 5 μg) and freshly isolated rat cerebral microvessels (10 μg) were subjected to Western blot analysis (duplicate lanes are shown). A: blots were incubated with primary antibody to NHE1 protein (rabbit polyclonal antibody XB17 for CMEC and mouse monoclonal antibody 4E9 for microvessels) and then with secondary antibody, and bands were visualized by enhanced chemiluminescence. B: blots were incubated with primary antibody to NHE2 protein (rabbit polyclonal antibody AB3038 for CMEC and microvessels or NHE2 antibody in the presence of NHE control peptide) and then incubated with secondary antibody, and bands were visualized by enhanced chemiluminescence. Western blots are representative of 4 similar results for NHE1 and 3 similar results for NHE2. C and D: CMEC monolayers grown on collagen-coated glass slides were fixed and then incubated with NHE1 rabbit polyclonal primary antibody (XB17) or NHE2 polyclonal primary antibody (Millipore) and then with biotin-conjugated secondary antibody. Bound antibodies were visualized using diaminobenzidine (brown). Control slides were incubated with secondary antibody only. Slides were counterstained with hematoxylin for visualization of nuclei (blue). Images are representative of 3 similar results for NHE1 and 2 similar results for NHE2.

Fig. 2.

Immunoelectron microscopy localization of NHE proteins in rat brain microvascular endothelial membranes. Rat brains were perfusion fixed and then labeled with NHE1 primary antibody at dilutions of 1:1,000 and 1:2,000 (A and B, respectively) or NHE2 primary antibody at dilutions of 1:500 and 1:1,000 (C and D, respectively) and then with gold particle-conjugated secondary antibody. Images are representative micrographs. Vessel lumens are at the top of each image; astrocyte and neuronal elements are below the basal lamina underlying the endothelium. Arrowheads show locations of gold particles. EC, endothelial cell; N, nucleus. Scale bars, 0.2 μm.

Fig. 3.

NHE1 and NHE2 distribution between luminal and abluminal membranes of rat brain microvascular endothelial cells: quantitation of immunoelectron micrograph gold particles. Immunoelectron micrographs generated using NHE1 or NHE2 antibodies were evaluated for relative distribution of NHE isoforms in luminal (L) and abluminal (A) membranes of brain microvascular endothelial cells in perfusion-fixed rat brains. Values are means ± SE of 26 and 24 microvessels for NHE1 antibody dilutions of 1:1,000 and 1:2,000, respectively, and 56 and 81 microvessels for NHE2 antibody dilutions of 1:500 and 1:1,000, respectively.

Fig. 4.

Evaluation of CMEC NHE activity as Na-dependent, HOE-642-sensitive H⁺ flux. NH₄ prepulse technique was used to evaluate NHE activity in CMEC monolayers grown on collagen-coated glass coverslips. A: representative NH₄ prepulse experiment showing Na-dependent, HOE-642-sensitive pH recovery from NH₄ (20 mM) prepulse-induced intracellular acidification. Data are averages of 30 cells in the field of view. All HEPES-buffered media contain 147 mM Na, except Na-free HEPES. NHE inhibitor HOE-642 was present at 10 μM. At the end of each experiment, cells were exposed to pH 5.5–8.5 media containing high-K⁺-nigericin to calibrate pH. B: dependence of CMEC H⁺ efflux inhibition on dose of HOE-642. CMEC were assessed for NH₄ prepulse-induced H⁺ flux in the presence of 0–80 μM HOE-642. HOE-642 inhibited Na-dependent H⁺ flux, with IC₅₀ of ∼7 μM. Values are means ± SE of 3 separate dose-response experiments.

Fig. 5.

Effects of AVP, hypoxia, and aglycemia on CMEC NHE activity measured as HOE-642-sensitive H⁺ flux. A: confluent CMEC monolayers grown on glass coverslips were exposed for 30 min to glucose or glucose-free medium (left), hypoxic (∼2% O₂) or normoxic (middle) medium, or 0 or 100 nM AVP (right), and NHE activity was assessed. Values are means ± SE of 4 experiments each for control and aglycemia, 3 experiments each for control and hypoxia, and 6 and 3 experiments for control and 100 nM AVP, respectively. *Significantly different from control: P < 0.03, P < 0.05, and P < 0.0001 for aglycemia, hypoxia, and AVP, respectively (Student's t-test). B: CMEC monolayers were exposed to 0–200 nM AVP for 5 min, and NHE activity was assessed as HOE-642-sensitive H⁺ flux. AVP was also present during the assay. Values are means ± SE of 3 experiments for each condition. *Significantly different from control (i.e., 0 nM AVP): P < 0.013, P < 0.002, P < 0.0001, and P < 0.0001 for 10, 50, 100, and 200 nM AVP, respectively.

Fig. 6.

Effects V₁ and V₂ vasopressin receptor agonists and antagonists on NHE activity measured as HOE-642-sensitive H⁺ flux. A: CMEC monolayers grown on coverslips were exposed to 0 or 100 nM AVP, the V₁ vasopressin receptor agonist [Phe²,Orn⁸]-vasotocin (Orn VP, 0 or 100 nM), or the V₂ vasopressin receptor agonist [deamino-Cys¹,d-Arg⁸]-VP (DDAVP, 0 or 100 nM) for 5 min, and NHE activity was assessed in the presence of AVP or the agonists. Values are means ± SE of 10, 5, 5, and 6 experiments for control, AVP, DDAVP, and Orn VP, respectively. *Significantly different from control: P < 0.0001 for 100 nM AVP and 100 nM Orn VP. B: bovine CMEC monolayers grown on glass coverslips were pretreated for 5 min in medium containing 0 or 100 nM AVP and the V₁ vasopressin receptor antagonist des-Gly⁹[phenylacetyl¹,d-Tyr(Et)²,Lys⁶,Arg⁸]-VP (PhaaEt VP, 0 or 100 nM) or the V₂ vasopressin receptor antagonist [d(CH₂)₅¹,d-Ile²,Ile⁴,Arg⁸,Ala-NH₂⁹]-VP (d-Ile VP, 0 or 100 nM), and NHE activity was assessed in the presence of AVP ± antagonists. Values are means ± SE; n = 6 and 3 for controls without and with AVP, respectively; n = 12 and 10 for PhaaET VP without and with AVP, respectively; and n = 8 and 5 for d-Ile VP without and with AVP, respectively. *Significantly different from respective control (i.e., −AVP): P < 0.0001 for both with 100 nM AVP and 100 nM d-Ile VP with 100 nM AVP. C: CMEC were pretreated with 100 nM AVP + 0–100 nM PhaaEt VP or d-Ile VP, and NHE activity was assessed in the presence of the same concentrations of AVP and antagonists. Values are means ± SE for 38 individual experiments for d-Ile VP dose response and 46 separate experiments for PhaaET VP dose response. *Significantly different from NHE activity in the presence of AVP without antagonist: P < 0.0001 for all concentrations of antagonist tested. NHE activity in the presence of AVP with the V₂ vasopressin receptor antagonist d-Ile VP was not significantly different from NHE activity in the presence of AVP alone at any concentration of d-Ile tested.

Fig. 7.

Ca dependence of AVP-stimulated CMEC NHE activity measured as HOE-642-sensitive H⁺ flux. CMEC monolayers were exposed to medium containing 0 or 5 μM BAPTA-AM at 37°C for 20 min and then to BAPTA-AM-free medium for 5 min. CMEC were then treated for 5 min with 0 or 100 nM AVP in BAPTA-AM-free medium and finally assayed for 5 min with AVP-containing, BAPTA-AM-free medium. Values are means ± SE of 5 and 6 experiments for control with and without AVP, respectively (both without BAPTA) and 5 and 3 experiments for cells treated with BAPTA with and without AVP, respectively. *Significantly different from control without AVP: P < 0.0001 for control with AVP.

Fig. 8.

Estradiol inhibition of AVP-stimulated CMEC NHE activity. A: CMEC were pretreated with 0–100 nM 17β-estradiol (E₂) 1 day before use, and NHE activity was measured as HOE-642-sensitive H⁺ flux. Values are means ± SE of 3, 3, 4, and 4 experiments for control, 1 nM E₂, 10 nM E₂, and 100 nM E₂, respectively. *Significantly different from control without E₂: P < 0.0026 for 1, 10, and 100 nM E₂. B: CMEC monolayers on glass coverslips were treated with 0–100 nM E₂ 1 day before use. On the day of the experiment, cells were exposed to 100 nM AVP for 10 min, and NHE activity was measured as HOE-642-sensitive H⁺ flux in the presence of AVP and 1–100 nM E₂. Values are means ± SE of 3 experiments for each condition. *Significantly different from control: P < 0.0001 for 100 nM AVP. #Significantly different from AVP without E₂: P < 0.0001 for 1, 10, and 100 nM E₂ with AVP, respectively.