INTRODUCTION

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Hypoxia-induced pulmonary artery hypertension is characterized by elevated pulmonary artery pressure, increased pulmonary vascular resistance, and pulmonary vascular remodeling (1). With acute hypoxia there is an increase in pulmonary artery pressure and pulmonary vascular resistance that is reversible when returned to room air. If hypoxia continues there is a further rise in pulmonary artery pressure and pulmonary vascular resistance because of both a polycythemia-related increase in blood viscosity and because of a decrease in vascular lumen caused by remodeling. The architectural changes in the pulmonary vasculature include extension of smooth muscle into more distal vessels which are normally nonmuscular, an increase in the muscularity of muscular arteries, and a decrease in the number of countable alveolar arteries. The stimulus for pulmonary artery smooth muscle proliferation is not fully understood, but it appears to involve release of growth factors and other vasoactive substances that are activators of Na⁺/H⁺ exchange (2). Increased Na⁺/H⁺ exchange with an intracellular alkalization is an early event in cell proliferation. This intracellular alkalization by stimulation of Na⁺/H⁺ exchange appears to play a permissive role in the pulmonary artery smooth muscle cell (PASM) proliferation of vascular remodeling. The Na⁺/H⁺ exchanger is a cell membrane protein that exchanges Na⁺ ions for H⁺ ions in an electroneural fashion. The exchanger is active in pH regulation, volume regulation, and smooth muscle cell growth (3, 4). PASM have an active Na⁺/H⁺ exchange mechanism even in the presence of bicarbonate (5, 6). Exposure of PASM to growth factors results in rapid activation of Na⁺/H⁺ exchange (7, 8). Growth-factor-induced PASM proliferation is inhibited by dimethyl amiloride, a potent Na⁺/H⁺ exchange inhibitor (8).

Amiloride, a potassium-sparing diuretic, inhibits the activity of the Na⁺ channel, the Na⁺/H⁺ exchanger, and the Na⁺/ Ca²⁺ exchanger. There are approximately 1,000 analogs of amiloride that vary in their specificity and potency of inhibition of these transport processes. Dimethyl amiloride (DMA) and ethylisopropyl amiloride (EIPA) are amiloride analogs that, compared with amiloride, are potent and specific inhibitors of Na⁺/H⁺ exchange (8) and inhibitors of vascular smooth muscle growth (9, 10).

Rats exposed to 14 d of normobaric hypoxia develop elevated pulmonary artery pressures and pathologic changes of vascular remodeling (11). We wondered if DMA, specific inhibitors of Na⁺/H⁺ exchange, would inhibit the pulmonary vascular remodeling in these animals. DMA was continuously infused intraperitoneally by minipumps during the 14 d of hypoxic exposure. At the end of 14 d, pulmonary hemodynamics were measured and lung histology was examined for evidence of pulmonary vascular remodeling.

	METHODS

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Experimental Animals and Animal Facilities

Sprague-Dawley rats weighing 200 to 300 g were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed separately and allowed free access to food and water.

Hypoxia Chambers

The chronic hypoxia chamber was a 219-L Lucite box capable of housing eight animals. Normobaric hypoxia was produced by venting room air (2 L/min) balanced with N₂ (2 L/min) through the chronic chamber (mixed with fans). This resulted in an FI_O2 of 0.10. CO₂ absorbent (barium hydroxide lime, USP; Warren E. Collins, Braintree, MA) was added to keep the CO₂ content < 1.0%. FI_O2 of 0.10 produces an arterial PO₂ of less than 60 mm Hg (12). Gas tensions were sampled daily during chronic hypoxia experiments.

Preparation and Placement of Catheters, Hemodynamic Measurements, Thermodilution Cardiac Outputs, and Arterial Blood Gas Analysis

Hemodynamic measurements on the rats were performed under general anesthesia with intraperitoneally administered ketamine (40 mg/ kg) and diazepam (5 mg/kg) while the rats breathed room air to reverse any acute hypoxic vasoconstriction. Pulmonary artery pressure was measured with a silastic catheter (0.012-inch ID, 0.025-inch OD) introduced via a right neck cutdown to the external jugular vein and advanced to the pulmonary artery as previously described (13, 14). The catheter was connected to a transducer (Model 049924-507; Cobe Laboratories, Lakewood, CO) positioned at the midthorax of the animal. A Gould monitor (Model RS3400; Gould Inc., Glen Burnie, MD) was connected to the transducer and the position of the catheter was confirmed by the wave form of the pressure tracing. Cardiac output was measured by thermodilution, using a 1.5 French thermodilution catheter of our design placed in the thoracic aorta via a right carotid artery cutdown, as previously described (13). For determination of hematocrit, blood was centrifuged in microcapillary tubes for 7 min and the hematocrit was read directly.

Histologic Preparation

The animals were killed by an overdose of interperitoneally administered ketamine and diazepam. The heart, lungs, and trachea were removed en bloc. A polyethylene catheter (PE 205) was placed in the pulmonary artery through the right ventricle. An additional catheter (PE 205) was placed in the trachea. The lungs were fixed by simultaneously infusing 10% buffered formalin through the right ventricle at 100 cm H₂O and the trachea at 23 cm H₂O. The trachea was then tied off and the heart was removed. The lungs were submerged in 10% buffered formalin for at least 48 h before the right lower lobe was embedded in paraffin. Elastin stains were performed on sections 5 µm thick. The degree of pulmonary remodeling was assessed by measuring the percent wall thickness of vessels indexed to terminal bronchioles and intra-acinous vessels (vessels indexed to respiratory bronchioles or alveolar ducts), and the ratio of the thick-walled vessels to total number of intra-acinous vessels visualized (percent thick vessels). Vessels were considered thick-walled if they contained an internal and external lamina for greater than 50% of the circumference of the vessel as previously described (15). The percent wall thickness was measured by an ocular micrometer. Percent wall thickness was expressed as the medial wall thickness (the distance between the internal and external lamina) divided by the diameter of the vessel (distance between the external lamina) × 100. Percent thick-walled vessels was expressed as the number of thick-walled intra-acinous vessels divided by the number of thick- plus thin-walled vessels × 100.

The degree of right ventricular hypertrophy was measured by the ratio of the right heart to left heart weight. The right ventricular free wall (RV) was removed. The right ventricle and the left ventricle plus the septum (LV + S) were dried separately at 90° C and weighed at 48 and 72 h. There was no further reduction in the weight between 48 and 72 h. The %RV/LV + S was expressed as the RV dry weight divided by the LV + S dry weight × 100.

Experimental Design

Animals were placed in the Lucite chamber for 14 d and exposed to either room air or 10% oxygen with constant infusion of DMA, EIPA, saline, or DMSO (carrier for EIPA) or without infusion. The animals had a subcutaneous minipump (Model 2ml2; Alza Corp., Palo Alto, CA) placed 24 h prior to study to deliver a continuous infusion of DMA (3 mg/kg/d, n = 7), EIPA (3 mg/kg/d, n = 9), DMSO (70% in saline, 2 ml/14 d, n = 9), or saline (2 ml/14 d, n = 4). At the end of 14 d, the animals were removed from the hypoxic chamber and catheters were placed under anesthesia for measurement of pulmonary hemodynamics while the animals breathed room air. Animals were then killed and lungs and hearts were removed for histologic examination.

To assess the effect of DMA on acute hypoxic vasoconstriction, rats were given an infusion of DMA 3 mg/kg/d for 2 d, then exposed to 10% O₂ for 30 min. On the day of hypoxic exposure, catheters were placed under anesthesia with ketamine and diazepam as described earlier, but then the rats were allowed to awaken for 4 h before hemodynamics were measured. Pulmonary artery pressure and cardiac output were then measured while each animal was in a small holding chamber breathing air and then 10% O₂ for 30 min.

Statistical Methods

Analyses were performed using Statview 4.5 (Abacus Concepts, Inc., Berkeley, CA). The pulmonary hemodynamic measurements, percent wall thickness, and percent thick vessels after hypoxic challenge were compared by analysis of variance (ANOVA) and subsequent multiple comparisons by the Scheffe test. Significance was set at p < 0.05. All values were expressed as mean ± standard error of the mean.

	RESULTS

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Pulmonary Hemodynamics and Arterial Blood Gas Determinations

There was no difference in pulmonary artery pressure, cardiac index, or total pulmonary vascular resistance (pulmonary artery pressure divided by cardiac index) among normoxic control animals without continuous infusion, normoxic control animals with continuous infusion of saline, or normoxic control animals with continuous infusion of DMA. Likewise, there were no differences in the parameters measured between hypoxic control animals with or without constant infusion of saline. Hypoxic and normoxic control animals in figures and tables represent controls without constant infusion pumps.

After 14 d of hypoxia, pulmonary artery pressure and total pulmonary vascular resistance index (TPVRI) rose significantly (p < 0.05) (Figure 1 and Table 1). Cardiac index was unchanged (p = 0.40). In animals treated with 10% O₂ plus DMA for 14 d (hypoxia + DMA) the pulmonary artery pressure and TPVRI were significantly reduced (p < 0.05) versus animals exposed to hypoxia without DMA, but it was still higher than normoxic control (p < 0.05) (Table 1). Cardiac index was unchanged (p = 0.40). Likewise, animals treated with 10% O₂ plus EIPA (hypoxia + EIPA), the pulmonary artery pressure and the TPVRI were significantly reduced (p < 0.05), but they were still higher than normoxic control (p < 0.05). EIPA is insoluble in water and is delivered in DMSO. DMSO infusion did not affect the TPVRI with hypoxia (0.09 ± 0.006 mm Hg/ml/min/kg, p = 0.66).

View larger version (18K): [in this window] [in a new window]   Figure 1.   Pulmonary artery pressures (mm Hg) in rats after 14 d in the chamber with normoxia without constant infusions (normoxic control, n = 6), 10% O2 (hypoxic control, n = 9), 10% O2 with infusion of dimethyl amiloride (hypoxia + DMA, n = 7), or 10% O2 with infusion of ethylisopropyl amiloride (hypoxia + EIPA, n = 9). *p < 0.05 versus normoxic control; #p < 0.05 versus hypoxic control.

Blood gases drawn on anesthetized rats 1 h after removal from the chronic hypoxia chamber showed normoxemia and a metabolic acidosis (PCO₂, 36 ± 2; pH, 7.31 ± 0.01) versus normoxic anesthetized control rats (PCO₂, 40 ± 1; pH, 7.38 ± 0.03) (Table 2). DMA and EIPA were without effect on the blood gases compared with hypoxic control rats (Table 2).

Right Ventricular Hypertrophy and Hematocrit

Animals exposed to 14 d of hypoxia developed significant ventricular hypertrophy as measured by the ratio of the RV/LV + S dry weight (normoxic control, 22 ± 1% versus hypoxic control, 46 ± 3%; p < 0.05) (Figure 2). RV/LV + S ratio was significantly reduced in animals treated with DMA and EIPA as compared with hypoxic control (p < 0.05), but it was still higher than in normoxic control (p < 0.05) (Figure 2). Hematocrit was significantly increased after hypoxia (normoxic control, 42 ± 1% versus hypoxic control, 59 ± 1%, p < 0.05) and was unchanged with treatment with DMA or EIPA (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Right ventricular hypertrophy measured as right ventricular dry weight (RV) divided by left ventricular plus septum dry weight (LV + S) × 100 in rats after 14 d in the chamber with normoxia without constant infusions (normoxic control, n = 6), 10% O2 (hypoxic control, n = 9), 10% O2 with infusion of dimethyl amiloride (hypoxia + DMA, n = 7), or 10% O2 with infusion of ethylisopropyl amiloride (hypoxia + EIPA, n = 9), *p < 0.05 versus normoxic control; #p < 0.05 versus hypoxic control.

View larger version (18K): [in this window] [in a new window]   Figure 3.   Hematocrit (percent) in rats after 14 d in the chamber with normoxia without constant infusions (normoxic control, n = 6), 10% O2 (hypoxic control, n = 9), 10% O2 with infusion of dimethyl amiloride (hypoxia + DMA, n = 7), or 10% O2 with infusion of ethylisopropyl amiloride (hypoxia + EIPA, n = 9). *p < 0.05 versus normoxic control.

Pulmonary Vascular Remodeling

In animals exposed to 14 d of hypoxia there was significant pulmonary vascular remodeling, measured as percent wall thickness of intra-acinous vessels (%WT-IA) and the percentage of thick-walled intra-acinous vessels (%Thick), as compared with normoxic control animals (normoxic control: %WT-IA, 4.0 ± 0.03; %Thick, 23 ± 3 versus hypoxic control: %WT-IA, 6.5 ± 0.3; %Thick, 48 ± 4; p < 0.05). Treatment with DMA (Figure 4) and EIPA resulted in significant reduction in pulmonary vascular remodeling (p < 0.05) (Table 3), but it was not completely attenuated. There was no difference in %WT of vessels indexed to terminal bronchioles between the groups.

View larger version (97K): [in this window] [in a new window]   View larger version (106K): [in this window] [in a new window]   View larger version (108K): [in this window] [in a new window]   Figure 4.   Representative photomicrographs of pulmonary vascular remodeling. Elastin stains on 5-µm-thick sections of paraffin-embedded lung (original magnification, ×480). Arrowheads indicate intra-acinous pulmonary vessels. (A) Normoxic control. Pulmonary vessels with a thickened medial layer (distance between internal and external lamina) are infrequent in control rats; majority of vessels are small and thin-walled. (B) Hypoxic control (14 d in the chamber at 10% O2. Numerous thick-walled pulmonary vessels are present after 14 d of hypoxia. (C ) Hypoxic control + DMA (14 d in the chamber at 10% O2 with constant infusion of diemthyl amiloride). In rats treated with DMA, intra-acinous pulmonary vessels are present, but the majority are not thickened (no visible internal and external lamina).

Exposure to Acute Hypoxia

To evaluate the effect of DMA on hypoxic vasoconstriction, awake rats were exposed to acute hypoxia for 30 min with (n = 12) and without (n = 10) constant infusion of DMA (3 mg/kg/ d) by osmotic minipump for 2 d prior to and during exposure. DMA had no effect on the magnitude of hypoxic vasoconstriction (pulmonary artery pressure, mm Hg: acute hypoxia, 19 ± 1 versus acute hypoxia + DMA, 19 ± 1; p = 0.44) (Figure 5).

View larger version (46K): [in this window] [in a new window]   Figure 5.   Pulmonary artery pressure (mm Hg) in rats after 30 min of 10% O2 without (control, n = 5) or with infusion of dimethyl amiloride (DMA, n = 6). Room air (solid columns); 10% O2 (hatched columns); *p < 0.05 versus room air.

	DISCUSSION

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In rats exposed to chronic hypoxia for 14 d there was a significant elevation in pulmonary artery pressure (Figure 1), TPVRI (Figure 2), right ventricular hypertrophy (Figure 3), and pulmonary vascular remodeling (Table 1) with no change in cardiac index. We found that treatment with the Na⁺/H⁺ exchange inhibitor, DMA during 14 d of hypoxia significantly reduced these responses to chronic hypoxia (Figures 1-4 and Table 1).

DMA, however, did not completely block the response to chronic hypoxia as the pulmonary artery pressure, TPVRI, and right ventricular hypertrophy were still higher in animals treated with DMA during hypoxia than in normoxic control animals. This residual rise in pulmonary artery pressure, despite treatment with DMA, may be in part attributed to other changes besides vascular remodeling that occur during chronic hypoxia. Meyrick and Reid (1) have studied the effect of polycythemia in hypoxic pulmonary hypertension in the rat. They have shown that the pulmonary artery pressure in hypoxic polycythemic rats (10% O₂ for 10 d) was approximately 36 mm Hg (measured on room air to remove hypoxic vasoconstriction). In rats that underwent hemodilution to keep them normocytic, the pulmonary artery pressure was approximately 25 mm Hg compared with approximately 15 mm Hg in normoxic normocyte control animals. Also it is hard to know how much of the residual remodeling shown in Table 3 is due to ineffectiveness of DMA/EIPA on hypoxia-induced pulmonary hypertension and how much is due to polycythemia causing secondary pulmonary hypertension with remodeling. Rabinovitch and colleagues (16) showed that banding of the left pulmonary artery in rats to prevent pressure rise during hypoxia from reaching the vascular bed decreased the remodeling in the banded side compared with the unbanded side. The dose of DMA may also have been too low, but it was as high as technically feasible with the minipumps.

DMA is an amiloride analogue with high specificity for Na⁺/H⁺ exchange inhibition. Amiloride is pyrazinoyl guanidine which has a pyrazine ring with amino groups on the 3- and 5-positions and a chloro group in the 6-position. Substitutions of hydrophobic groups on the 5-position such as dimethyl group confers high specificity and potency for the inhibition of Na⁺/H⁺ exchange. Although the potency of inhibition of Na⁺/H⁺ exchange for DMA is 12 times amiloride (9), other nonspecific effects of amiloride analogs may be responsible for the inhibition of pulmonary vascular remodeling with chronic hypoxia. Amiloride has been shown to intercalate DNA (17), inhibit DNA topoisomerase II (17), interact with guanine nucleotide regulatory proteins, which attenuates hormonal adenylate cyclase (18), and inhibit growth factor receptor tyrosine kinase activity (19). These effects may also play a role in the inhibition of pulmonary vascular remodeling, but these effects have not been studied with the DMA.

Other possible mechanisms for DMA to reduce pulmonary vascular remodeling were through inhibition of acute hypoxic vasoconstriction, an effect on intravascular volume or alterations in acid-base status. To test for inhibition of acute hypoxic vasoconstriction we treated rats with constant infusion of DMA for 2 d prior to and during a 30-min hypoxic challenge. There was no difference in pulmonary vasoconstriction during hypoxia between rats with and those without DMA treatment (Figure 4), suggesting that DMA does not inhibit pulmonary vascular remodeling by reducing vasoreactivity. Also there was no change in cardiac output that would indicate a significant decrease in intravascular blood volume. Acidosis can potentiate hypoxic vasoconstriction and alkalosis can inhibit it (20). Arterial blood gases taken on room air 1 h after removal from 14 d of hypoxia (Table 2) showed no difference in the acid-base balance between hypoxic controls, hypoxia plus DMA, and hypoxia plus EIPA, indicating there was no superimposed contraction alkalosis from any possible diuretic effects of DMA or EIPA. A mild metabolic acidosis was present in all the groups removed from 2 wk in 10% O₂ chamber. This acidosis was likely due to HCO₃ loss during chronic hypoxia. Rats acutely placed in 10% O₂ hyperventilate with the arterial PCO₂ falling to 20 mm Hg and the arterial pH rising to 7.50 (21). However, after chronic exposure in the hypoxic chamber the arterial PCO₂ remained at 20 mm Hg but the pH fell to 7.4 (12). We suspect the metabolic acidosis in the rats in this study that is seen on blood gases taken after coming out of the hypoxic chamber is due to loss of the hypoxic stimulus to ventilation, a rise in PCO₂ and thus a fall in arterial pH below 7.4 because of the previous compensatory loss of HCO₃. Nevertheless, DMA and EIPA did not change the presence of the metabolic acidosis (Table 2) and thus unlikely decreased chronic pulmonary hypertension via an effect on pH.

Because DMA may inhibit pulmonary vascular remodeling through a mechanism other than Na⁺/H⁺ exchange inhibition, we also tested EIPA. EIPA is an amiloride analogue with an ethylisopropyl group substitution in the 5-position and 133 times the potency for Na⁺/H⁺ exchange inhibition as compared with amiloride. We found similar decreases in pulmonary artery pressure (Figure 1), TPVRI (Figure 2), right ventricular hypertrophy (Figure 3), and pulmonary vascular remodeling (Table 1), suggesting that inhibition of Na⁺/H⁺ exchange is important for inhibition of pulmonary vascular remodeling. Kranzhofer and colleagues (22) have used another amiloride analogue (3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate, Hoe 694) and EIPA. They have shown a suppression of neointimal thickening and smooth muscle cell proliferation after arterial injury in the rat with both compounds. The position isomer of Hoe 694, lacking Na⁺/H⁺ exchange-blocking properties, had no effect on neointimal thickening (22). This adds further data to support the contention that inhibition of Na⁺/H⁺ exchange is involved in vascular smooth muscle proliferation. It may be that inhibition of Na⁺/H⁺ exchange leads to inhibition of other factors such as tyrosine kinase (23) or pulmonary artery pressure kinases (24) that in turn inhibit smooth muscle growth.

Other investigators have found that amiloride analogues attenuate mitogenic responses to growth factors in other cell types (25, 26) and decrease neointima formation in carotid arteries after balloon injury (22, 23). However, not all studies have found that Na⁺/H⁺ exchange stimulation with intracellular alkalization is necessary for cell proliferation. Some cell types will proliferate even with complete inhibition of Na⁺/H⁺ exchange and some Na⁺/H⁺ exchanger-deficient cell types will proliferate normally in the presence of bicarbonate (2). However, for vascular smooth muscle cells there appears to be mounting evidence that Na⁺/H⁺ exchange is important for cell proliferation (27), and this appears to be mediated by both calcium and protein kinase-C-dependent and independent pathways (4).

In summary, DMA and EIPA, amiloride analogues that are specific for inhibition of Na⁺/H⁺ exchange, inhibit pulmonary hypertension and pulmonary vascular remodeling when administered as a constant infusion during 14 d of chronic hypoxia. The hydrophobic 5-position substituted amiloride analogues hold promise as a treatment for pulmonary vascular remodeling with pulmonary hypertension.