Nitric-oxide-mediated zinc release contributes to hypoxic regulation of pulmonary vascular tone

Abstract

The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 microm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT(-/-) mice. Data from NO-sensitive fluorescence resonance energy transfer-based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT(-/-) mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.

Figure 1. Hypoxia causes active vasoconstriction of intra-acinar arteries in the isolated perfused mouse lung

Confocal images of intra-acinar arteries of the IPL from a Tie2-GFP mouse at baseline, after 10 min of hypoxia, and after 10 min of recovery (A). The 9.7 +/− 2.0 % change in perfusion pressure was accompanied by a 9.2 ± 1.1% (P < 0.001) decrease in vessel diameter (≤40 μm) in response to hypoxia (B). N=5, with 3–5 vessels per experiment.

Figure 2. Isolated rat pulmonary microvascular endothelial cells (RPMEC) contract in response to hypoxia

RPMEC embedded in a collagen matrix showed a −30% ± 15.1% decrease in surface area upon exposure to hypoxia and a 25% ± 18.9% increase during recovery. In contrast, rat aortic endothelial cells (RAEC) did not respond to hypoxia (A). Single cell mechanics were analyzed by tracking material displacement between time lapse images to create the 2D density increment (Density Analysis) and continuous vector field (Strain Analysis) shown in the right hand panels. The black area shows the area where the cell is located. Positive principal strains, representing extension, are coded blue, and negative principal strains, representing compression, are coded red. The pure tension (Strain Analysis, blue vectors) exerted by the cell as it contracted during hypoxia resulted in a decrease in collagen density in the area of retraction (Density Analysis, blue areas). The medial increases in collagen density (Density Analysis, red areas) are a result of localized compression of the matrix (Strain Analysis, red arrows). Relaxation during the normoxic recovery period was accompanied by radial decreases in collagen density (Density Analysis, blue areas) in the area immediately adjacent to the cell body as it spread and pulled for anchorage (Strain Analysis, blue arrows). The regions surrounding the cell showed an increase in collagen density (Density Analysis, red areas) resulting from radial compression forces (Strain Analysis, red arrows) exerted by the cell pushing against the collagen matrix.

Figure 3. Hypoxia increases labile zinc in the isolated perfused lung (IPL)

Confocal microscopy was used to image FluoZin-3 fluorescence in reconstructed vasculature of mouse IPL. FluoZin-3 fluorescence was increased during hypoxia in the MT +/+ lungs (A, upper panel) whereas IPL of MT-null mice showed no significant change (A, middle panel). NOS inhibition (L-NAME, 1mM) prevented hypoxia-induced changes in fluorescence in MT +/+ lungs (A, lower panel). Results were reproducible in separate experiments on 3–5 mice per condition (B). * Emissions increased above baseline levels, P < 0.001. Calibration bar equals 50 μm.

Figure 4. Zinc chelation attenuates hypoxic pulmonary vasoconstriction (HPV) in the isolated perfused lung (IPL)

Panels A and C show representative pressure tracings from a mouse and rat IPL, respectively. TPEN (25 μM) attenuated (P < 0.05) HPV in both species (n=5 for mice, B and n=6 for rats, D).

Figure 5. Hypoxia regulates FRET.MT and cygnet-2 function in sheep pulmonary artery endothelial cells (SPAEC)

Representative spectral reports for single cells (A and C) show the decrease in energy transfer following hypoxic exposure as evidenced by an increase in the emission intensity of the donor (cyan) and a decrease in that of the acceptor (yellow). The mean data (± SD) for three separate experiments (2–5 cells per experiment) are expressed as a percent change from the baseline FRET ratio (I_{535 nm}/I_{480 nm}, B and D). NOS inhibition (L-NAME) attenuated (P < 0.01) the effects of hypoxia on FRET.MT and cygnet-2 (B and D).

Figure 6. Hypoxia regulates both FRET.MT and cygnet-2 function in isolated perfused mouse lung

Representative images from a single experiment using FRET.MT illustrate the separation of the two emitted signals (cyan and yellow) following spectral unmixing based on individual calibration spectra for each protein (A). The spectral reports from single experiments with each reporter (B and C) show decreases in energy transfer following hypoxia, as evidenced by an increase in the emission intensity of the donor (cyan, 480 nm) and a decrease in that of the acceptor (yellow, 535 nm). The mean change in energy transfer (I₅₃₅/I₄₈₀) for the FRET.MT reporter was −24.5 ± 5.4% (n=5), and −26.4 ± 5.7% (n=6) for cygnet-2 (D).

Figure 7. Metallothionein (MT) null mice have a blunted HPV

P < 0.05 and n=5 per group.

Figure 8. NO-mediated vasoconstriction in the isolated perfused lung (IPL)

Eliminating the vasodilatory effects of NO on the hypoxic pressor response via pharmacological inhibition of sGC (ODQ) and NO-sensitive large conductance Ca²⁺-activated potassium channels (BK_Ca2+, charybdotoxin, ChTx) enhanced HPV (P < 0.01) in the mouse IPL. Under these conditions, the NO donor, DETAnonate caused further constriction (P < 0.05). a, different from hypoxia. b, different from hypoxia/ODQ/ChTx. c, different from hypoxia and hypoxia/ODQ/ChTx (n=5).