Optimization of isolated perfused/ventilated mouse lung to study hypoxic pulmonary vasoconstriction

Abstract

Hypoxic pulmonary vasoconstriction (HPV) is a compensatory physiological mechanism in the lung that optimizes the matching of ventilation to perfusion and thereby maximizes gas exchange. Historically, HPV has been primarily studied in isolated perfused/ventilated lungs; however, the results of these studies have varied greatly due to different experimental conditions and species. Therefore, in the present study, we utilized the mouse isolated perfused/ventilated lung model for investigation of the role of extracellular Ca(2+) and caveolin-1 and endothelial nitric oxide synthase expression on HPV. We also compared HPV using different perfusate solutions: Physiological salt solution (PSS) with albumin, Ficoll, rat blood, fetal bovine serum (FBS), or Dulbecco's Modified Eagle Medium (DMEM). After stabilization of the pulmonary arterial pressure (PAP), hypoxic (1% O2) and normoxic (21% O2) gases were applied via a ventilator in five-minute intervals to measure HPV. The addition of albumin or Ficoll with PSS did not induce persistent and strong HPV with or without a pretone agent. DMEM with the inclusion of FBS in the perfusate induced strong HPV in the first hypoxic challenge, but the HPV was neither persistent nor repetitive. PSS with rat blood only induced a small increase in HPV amplitude. Persistent and repetitive HPV occurred with PSS with 20% FBS as perfusate. HPV was significantly decreased by the removal of extracellular Ca(2+) along with addition of 1 mM EGTA to chelate residual Ca(2+) and voltage-dependent Ca(2+) channel blocker (nifedipine 1 μM). PAP was also reactive to contractile stimulation by high K(+) depolarization and U46619 (a stable analogue of thromboxane A2). In summary, optimal conditions for measuring HPV were established in the isolated perfused/ventilated mouse lung. Using this method, we further confirmed that HPV is dependent on Ca(2+) influx.

Keywords: endothelial nitric oxide synthase; hypoxia; mouse lung; pulmonary artery; pulmonary vasoconstriction.

Figure 1

Comparison of HPV using different pretone agents in PSS perfusate with 4% albumin in the isolated perfused/ventilated mouse lung. Flow rate was regularly controlled with 2 mL/min. After stabilization of pulmonary arterial pressure (PAP) for 40-60 min, treatment of hypoxia or drug was applied. Hypoxic (1% O2) and normoxic (21% O₂) gas mixture were applied for 5 min intervals via rodent ventilator. (A) Hypoxia (1% O2) alone did not induce vasoconstriction without a pretone agent. (B) Angiotensin II (Ang-II, 0.2 μg/100 μL) did not affect the HPV response. (C and D) In the presence of U46619 (1 μM, C, or 2 μM, (D), baseline PAP was dose-dependently increased and the amplitude of HPV was gradually increased. Mean ± SEM is shown as a bar graph (right panel). *Indicates statistically significant difference from the control value (P< 0.05). Lungs are isolated from two to four mice.

Figure 2

Effects of PSS with 4% Ficoll as a perfusate to induce HPV in the isolated perfused/ventilated mouse lung. After basal PAP was stabilized, the application of hypoxia or pretone agent or change of flow rate was performed. (A) Hypoxia alone (1% O2) did not induce vasoconstriction without a pretone agent. Increasing flow rate of perfusate (from 2 to 3 mL/min) induced the increase of the basal PAP, but not the amplitude of HPV. (B) When Ang-II (0.2 mg/100 ml) as a pretone agent was applied, a strong, transient increase of PAP was induced. After applying Ang-II to return to the basal PAP, hypoxic exposure was applied in the lung. The amplitude of HPV was slightly increased in the presence of angiotensin II but had no significant difference. Mean ± SEM is shown as a bar graph (right panel). Lungs are isolated from two to three mice.

Figure 3

Effects of PSS with rat blood as a perfusate to induce HPV in the isolated perfused/ventilated mouse lung. The blood mixed with perfusate (40 mL PSS and 10 mL whole blood of rat) was used as a perfusate. (A) HPV (1% O₂) was repetitively induced in the absence of a pretone agent. (B) Effects of Ang-II (1 mg/100 ml) as a pretone agent on basal PAP and HPV. After the 3rd hypoxic challenge via ventilator in the isolated lung, the injection of Ang- II was applied into the PAP. Application of angiotensin II induced a transient increase of basal PAP; however, the amplitude of HPV did not significantly increase in the presence of Ang-II. (C) Comparison between before (control) and after AII treatment is shown as a bar graph. Bar graph shows mean ± SEM. Lungs are isolated from eight mice.

Figure 4

Effects of DMEM with 5%-10% FBS as a perfusate on HPV in the isolated perfused/ventilated mouse lung. After basal PAP was stabilized, hypoxic exposure (1% O₂) was repeatedly performed with six challenges. (A) When DMEM with 10% FBS was applied as a perfusate, HPV was strongly induced but the amplitude of HPV or hypoxia-induced increase in PAP was spontaneously decreased after applying the 1^st challenge of hypoxia. (B) The amplitudes of HPV using DMEM with 5% FBS were spontaneously decayed. After Ang-II (0.2 mg/100 ml) was applied, HPV was not significantly increased. Bar graphs are shown as mean ± SEM (right panels).

Figure 5

Effects of PSS with 20% FBS as a perfusate on HPV in isolated perfused/ventilated mouse lung. (A) Representative trace of HPV is shown. Consistent and repetitive HPV was observed with PSS perfusate with 20% FBS. The amplitudes of HPV were increased by the application of Ang-II (0.2 mg/100 ml). (B) Bar graphs are shown as mean ± SEM. The amplitude of HPV from 1^st to 6^th trials of hypoxia without a pretone agent (left panel) and the amplitude of HPV with Ang-II (right panel) are shown as a bar graph. (C) Representative trace of HPV for 180 min. When hypoxia (1% O2) was applied via ventilator, the amplitude of HPV was transiently increased for the first 5 min, and then secondary increase was slowly shown and maintained. Lungs are isolated from two to six mice.

Figure 6

Involvement of extracellular Ca²⁺ influx through VDCC in HPV using PSS perfusate with 20% FBS in the isolated perfused/ventilated mouse lung. (A) Removal of extracellular Ca²⁺ (Ca-free) significantly decreased the basal PAP and inhibited HPV. After restoration of extracellular Ca²⁺ in the perfusate to 1.8 mM Ca2+ again, HPV was recovered as before (left panel). Bar graphs are shown as mean ± SEM (right panel). **P < 0.01 vs. open and grey bars. (B) Application of Ca²⁺-free (-Ca) solution during hypoxic conditions (1% O₂) decreased the amplitude of the hypoxia-induced increase in PAP (left panel). 1 mM EGTA was included in the Ca2+-free solution. Bar graphs are shown as mean ± SEM (right panels). +Ca, solution including 1.8 mM Ca²⁺. **Indicates statistically significant difference from the control value (P < 0.01). Lungs are isolated from three to six mice. (C) Effect of Nif on basal PAP and HPV was shown as a representative trace (left panel). After the 3^rd challenge of hypoxic exposure, Nif (1 mM) was applied in the perfusate. The 4^th to 6^th challenges of hypoxia were performed in the presence of Nif. Application of Nif decreased amplitudes of HPV. Bar graphs are shown as mean ± SEM (right panel). **Indicates statistically significant difference from the control value (P < 0.01). Lungs are isolated from six mice.

Figure 7

Comparison of 40K+-induced contraction, HPV, and vasoconstrictive response to 90 minutes-hypoxic ventilation among mice deleted selectively for endothelial nitric oxide synthase (NOS3^-/-), caveolin-1 (Cav1^-/-), and NOS3^-/-/Cav1^-/- in the isolated perfused/ventilated mouse lung. (A) 40K+-induced contraction (or increase in PAP) was significantly enhanced by eNOS deletion (NOS3^-/-) or eNOS/Cav1 deletion (NOS3^-/-/Cav1^-/-) in comparison to WT control mice. (B) Basal PAP in the NOS3^-/- or NOS3^-/-/Cav1^-/- mice was significantly higher than balsa PAP in control or Cav1^-/- mice. HPV was also enhanced by eNOS/ Cav1 deletion; however, neither Cav1 nor eNOS deletion alone had any effect on HPV. (C) Summarized data (mean ± SEM) showing the basal PAP (left panel), the amplitude of 40K-mediated increase in PAP (middle panel) and hypoxia-induced increase in PAP (right panel) in isolated perfused/ventilated lungs of WT, NOS3^-/-, Cav1^-/- and NOS3^-/-/Cav1^-/- mice. *P < 0.05, **P < 0.01 vs. WT control. (D) Effect of hypoxic treatment for 90 min was shown as a representative data in WT control, NOS3^-/-, and NOS3^-/-/Cav1^-/- mice. Horizontal broken lines indicate the level of the basal PAP in WT control mice. (E) Summarized data (mean ± SEM) showing the time courses of prolonged (80 min) hypoxia-induced increase in PAP in WT control (open circles), NOS3^-/-, and NOS3^-/-/Cav1^-/- mice. *P < 0.05, **P < 0.01 vs. WT control mice. Lungs are isolated from three to six mice.