INTRODUCTION

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HYPOCAPNIC ALKALOSIS is widely used in treating severe neonatal and pediatric pulmonary hypertension (3, 8, 26). This practice is based on studies showing that alkalosis causes acute pulmonary vasodilation (9, 12, 17, 18, 34-36, 41). However, the mediator(s) of alkalosis-induced pulmonary vasodilation remains uncertain (1, 12, 18, 25, 41, 42). Furthermore, responses to alkalosis have generally been measured in the lungs or vessels from normal animals after vasomotor tone was acutely raised (9, 12, 17, 18, 34-36, 41). Neither the pulmonary vascular responses to alkalosis nor the mediators involved have been studied in a model of preexistent pulmonary hypertension, the condition for which alkalosis therapy is often used.

In some studies, alkalosis-induced pulmonary vasodilation appeared to be mediated by endothelium-derived modulators such as prostacyclin (18, 41) and nitric oxide (2). However, others (12, 25, 42) found that neither of these modulators played a role. More recently, it has been suggested that activation of K⁺ channels, particularly voltage-dependent K⁺ (K_V) channels, may contribute to alkalosis-induced pulmonary vasodilation (1, 39). Because both endothelium-derived modulator activity and K⁺-channel activation are altered by pulmonary hypertension (11, 21, 28, 33, 37), we hypothesized that the magnitude and/or mediator(s) of alkalosis-induced pulmonary vasodilation would differ between normal and pulmonary hypertensive animals. To test this hypothesis, responses to alkalosis were measured under control conditions and after inhibition of endothelium-derived modulators or K⁺-channel activation in pulmonary arteries from normoxic (Norm) and chronically hypoxic (Hyp) 1-wk-old piglets.

	METHODS

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Preparation. This study was approved by the Institutional Animal Care and Use Committees of the Medical College of Wisconsin and the Zablocki Veterans Affairs Medical Center (Milwaukee, WI). Responses to alkalosis were measured in 186 rings from 35 Norm piglets and 55 rings from 7 Hyp piglets. The Hyp piglets were maintained in 12% O₂ for 3 days before study, and all piglets were euthanized at 1 wk of age. Fike and Kaplowitz (10, 11) previously showed that this relatively brief chronic Hyp exposure leads to reactive pulmonary hypertension (10, 11). At the time of study, the piglets were anesthetized with ketamine (40 mg/kg im), acepromazine (1.5 mg/kg im), and pentobarbital sodium (25 mg/kg ip). Heparin (5,000 units iv) was administered, and the piglets were euthanized by exsanguination. The lungs and heart were removed en bloc, and the lungs were then immersed in cold modified Krebs solution (MKS) and refrigerated at 4°C.

At the time of study, fourth- to fifth-generation pulmonary arterial segments (0.5-mm diameter) were identified and gently dissected from the lungs. The segments were cut into 2-mm-long rings, and small stainless steel hooks were inserted through the lumen. The upper hook was suspended from an isometric force transducer (model 52-9529, Harvard Apparatus, South Natick, MA), and the lower hook was attached to the base of a 10-ml organ chamber (Harvard Apparatus). The organ baths were filled with MKS maintained at 37.5°C and initially bubbled with 6% CO₂ and 21% O₂ to achieve a pH of 7.40. The transducer was mounted on a micromanipulator (model 55020, Stoelting, Chicago, IL), allowing adjustment of resting tension as needed. Vessel tension was constantly measured and recorded on flatbed recorders (model L6514-4, Linseis, Princeton Junction, NJ).

Protocol. In preliminary KCl length-tension experiments (data not shown), we found that the optimal resting tension for 0.5-mm-diameter piglet pulmonary arteries was 1 g. Therefore, the resting tension was set at 1 g in all subsequent experiments. The arteries were allowed to equilibrate at the resting tension for 60-90 min, then responses to three successive challenges with 40 mM KCl were measured to assess vascular smooth muscle integrity. The arteries were washed with fresh MKS and allowed to stabilize at the resting tension for 20 min between each KCl challenge. After the final wash, the arteries were submaximally contracted with the thromboxane mimetic U-46619. In arteries from Norm piglets, a mean concentration of 8.7 × 10⁸ ± 1.1 × 10⁸ M was used for this initial challenge with U-46619 (termed U-46619 challenge 1) because preliminary experiments had shown that 80% of maximal contraction occurred at a concentration of U-46619 between 3 × 10⁸ and 10⁷ M. However, arteries from Hyp piglets were more responsive to U-46619 challenge 1, so a significantly lower mean concentration of U-46619 (2.5 × 10⁸ ± 0.1 × 10⁸ M; P < 0.001) was used. Arteries that failed to exhibit an increase in tension of at least 200 mg/mg vessel wt in response to the third challenge with 40 mM KCl or to U-46619 challenge 1 were excluded from further study.

After a stable response to U-46619 challenge 1 was achieved (20 min), relaxation in response to bradykinin (BK; 10⁹ M) or acetylcholine (ACh; 10⁷ M) was measured to test for endothelial function. Arteries from Norm piglets were assigned to control (Con) or inhibitor-treated groups described in Study groups if BK caused a >40% decrease and/or ACh caused a >25% decrease in the U-46619 challenge 1 response. If BK or ACh caused a <15% decrease in the U-46619 challenge 1 response, the arteries were deemed to have nonfunctional endothelium and were assigned to the endothelium-negative (Endo) group. In arteries from Hyp piglets, relaxation to ACh was abolished (see Study groups) and responsiveness to BK was blunted. Therefore, the arteries from Hyp piglets were deemed to have functional endothelium if BK caused a >30% decrease in the U-46619 challenge 1 response.

After the arteries were tested for endothelial function, the arteries were washed with fresh MKS and assigned to only one study group (Tables 1 and 2) for the remainder of the experiment. The arteries were allowed to stabilize for a further 15-30 min after addition of the inhibitor, and the resting tension was measured. Submaximal contraction was then again induced with U-46619 (termed U-46619 challenge 2). The mean concentration of U-46619 challenge 2 was also significantly higher in arteries from Norm piglets compared with those from Hyp piglets (1.1 × 10⁷ ± 2.0 × 10⁸ and 3.9 × 10⁸ ± 0.2 × 10⁸ M, respectively; P = 0.02). After a stable U-46619 challenge 2-induced increase in tension occurred (20 min), the bath was made alkalotic by changing the gas mixture to 21% O₂-2% CO₂-balance N₂. The pH stabilized at 7.65-7.70 within 5 min, and a stable tension was measured after 20 min. The gas mixture was then returned to 21% O₂ and 6% CO₂, resulting in a rapid return to normal pH (7.40 ± 0.002), and the resultant tension was again measured. At the end of each experiment, arterial rings were blotted dry and weighed. All tension measurements are expressed as milligrams per milligram of vessel weight.

Study groups. The arteries from Norm piglets were assigned to 12 study groups as shown in Table 1. The arteries from Hyp piglets were assigned to the five study groups indicated in Table 2. The concentrations of inhibitors and other criteria used to assign rings to the different groups in this study were based on reports in the literature and our preliminary experiments. In Con arteries from Norm piglets (Norm Con), BK caused a 73.3 ± 4.3% and ACh caused a 56.4 ± 7.0% decrease in the U-46619 challenge 1 response, indicating intact endothelial function. In contrast, arterial rings without functional endothelium (Norm Endo) exhibited decreases of 11.3 ± 2.3 and 11.1 ± 1.6% in response to BK and ACh, respectively. The addition of 10⁴ M N-nitro-L-arginine (L-NNA) to the bath of Norm L-NNA arteries also reduced BK-induced relaxation to 10.7 ± 2.5%, indicating effective inhibition of endothelium-dependent relaxation as previously reported in a study (13) on newborn pulmonary vessels. Arteries treated with either 10⁶ (n = 8) or 10⁵ M (n = 2) [1H-(1,2,4)oxadiazolo(4,3,-a)quinoxaline-1-one] (ODQ) exhibited similar blunting of the responses to BK (mean 13.3 ± 5.4%). In addition, relaxation in response to 10⁴ M sodium nitroprusside was significantly reduced (39.7 ± 9.8% in Con arteries vs. 1.0 ± 1.6% in those treated with 10⁶ M ODQ). Thus, as in previous studies (6, 14), both 10⁶ and 10⁵ M ODQ blocked the nitric oxide-cGMP pathway. Therefore, arteries treated with 10⁶ and 10⁵ M ODQ were combined into a single Norm ODQ group.

Indomethacin (Indo) at 10⁵ M or meclofenamate (Mec) at 10⁵ M has been used to inhibit cyclooxygenase (Cyclo) activity in previous studies (22, 27, 38), and we found that 10⁵ M Mec significantly attenuated arachidonic acid-induced relaxation in preliminary experiments (data not shown). Responses to both BK and alkalosis were similar in Norm Mec (9 arteries from 5 piglets) and Norm Indo (8 arteries from 5 piglets) groups. For example, alkalosis-induced relaxation was 40.9 ± 9.8 and 44.4 ± 11.5%, respectively. Therefore, data from both groups were combined into a single Cyclo-inhibited group (Norm Cyclo). Only 10⁵ M Mec was used to inhibit Cyclo activity in arteries from Hyp piglets (Hyp Cyclo).

Preliminary experiments also showed that both 10⁶ and 10⁵ M glibenclamide (Glib) blocked relaxation due to the ATP-dependent K⁺ (K_ATP)-channel opener cromakalim (data not shown). Because previous studies (7, 32) used this concentration range of Glib, Norm Glib arteries were treated with 5 × 10⁶ M Glib to evaluate the contribution of K_ATP channels to alkalosis-induced relaxation in this study. Previous studies (5, 20) have shown that iberiotoxin (IbTX) in concentrations ranging from 10⁹ to 10⁷ M inhibited Ca²⁺-dependent K⁺ (K_Ca)-channel activation. We measured responses to alkalosis in arteries after adding 10⁹ (n = 2), 10⁸ (n = 5), 3 × 10⁸ (n = 3), and 10⁷ (n = 4) M IbTX to the bath. Alkalosis caused a 40.6 ± 16.5% relaxation in arteries treated with 10⁹ M IbTX vs. 37.4 ± 8.8, 36.4 ± 12, and 35.7 ± 12.8%, respectively, in those treated with the three higher concentrations. Although there were no significant differences between the four concentrations used, only arteries treated with >10⁹ M IbTX, to minimize the possibility that insufficient inhibitor was present to block the K_Ca channels, were included when the effects of alkalosis on the Norm IbTX group were studied. K_V-channel inhibition has been described with a range of 10³ to 10² M 4-aminopyridine (4-AP) in studies of pulmonary arterial smooth muscle cells and pulmonary vessels (29, 37, 43, 44). However, high concentrations of 4-AP may result in depolarization due to non-K_V-dependent mechanisms (19). Therefore, we measured the responses to alkalosis in arteries from both Norm and Hyp piglets after the addition of either 10³ or 10² M 4-AP to the bath. In addition, the effects of depolarization on alkalosis-induced relaxation were also assessed in arteries from Norm piglets treated with the Na⁺-K⁺-ATPase inhibitor ouabain (Ouab; 10³ M) and arteries precontracted with 40 mM KCl rather than with U-46619.

Preparation of drugs and solutions. The MKS consisted of (in mM) 120 NaCl, 4.7 KCl, 1.7 NaH₂PO₄, 0.72 MgSO₄ · 7H₂O, 2.5 CaCl₂ · 2H₂O, 20 NaHCO₃, and 10 glucose. KCl challenges were achieved by substituting MKS with 80 mM NaCl and 40 mM KCl, thus maintaining isosmolar conditions. This solution was also used in those arteries in which responses to alkalosis were measured after precontraction with 40 mM KCl rather than with U-46619. ACh, BK, IbTX, sodium nitroprusside, Ouab (all from Sigma, St. Louis, MO), and Mec (BIOMOL Research Laboratories, Plymouth Meeting, PA) were prepared in normal saline. The stock solutions of Indo (Sigma) and U-46619 (BIOMOL) were prepared in ethanol and then added to normal saline. Glib, ODQ, and cromakalim (all from Sigma) were prepared in dimethyl sulfoxide. L-NNA (Sigma) was dissolved in normal saline with 12 N HCl. 4-AP (Sigma) was also dissolved in saline, and HCl was added to titrate the pH back to 7.40. Neither addition of the different drugs nor their vehicles in the volumes (5-100 µl) used in this study altered the pH of the MKS. All drug concentrations are expressed as the final molar concentrations in the organ bath.

Data analysis. All data are expressed as means ± SE. The number of piglets and arteries studied under each condition are indicated in Figs. 1-9 and Tables 1-3. Absolute tension and changes in tension in response to the various interventions are expressed in milligrams per milligram of vessel weight. The percentage of alkalosis-induced relaxation [calculated as (decrease in tension during alkalosis divided by increase in tension in response to U-46619 challenge 2) × 100] was also compared among groups to facilitate comparisons among groups with differing absolute tensions during U-46619 challenge 2. All groups were compared by one- or two-way ANOVA and Tukey's or the least significant difference test as appropriate. Differences were considered significant at P < 0.05.

	RESULTS

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Alkalosis-induced relaxation in arteries from Norm piglets. There were no differences in piglet or artery weight, artery diameter, initial resting tension, tension after the third challenge with 40 mM KCl, or tension after U-46619 challenge 1 to the bath. Table 3 shows the mean preparation characteristics for all Norm and Hyp piglets. Figure 1 illustrates the effects of endothelium-derived modulators on the absolute tension measurements made under the four study conditions. The tension measured under resting conditions and after the U-46619 challenge 2 to the bath was significantly higher in Norm Endo arteries compared with Norm Con arteries (Fig. 1). U-46619 challenge 2 also caused a greater increase in tension in arteries treated with a nitric oxide synthase inhibitor (Norm L-NNA) or a guanylate cyclase inhibitor (Norm ODQ) but not in arteries treated with Cyclo inhibitors (Norm Cyclo; Fig. 1). In Norm Con and Norm Cyclo arteries, tension decreased significantly in response to alkalosis, then returned to the previous U-46619 challenge 2 level when normocarbia was restored. In contrast, tension did not change in response to alkalosis in Norm Endo, Norm L-NNA, or Norm ODQ arteries (Fig. 1). To facilitate comparison of the effects of alkalosis among groups with differing absolute resting and U-46619 challenge 2-induced tensions, the percent decrease in the U-46619 challenge 2 response during alkalosis was also measured and is illustrated as percent alkalosis-induced relaxation (Fig. 2). The percent alkalosis-induced relaxation was similar in Norm Cyclo and Norm Con arteries but was significantly blunted in Norm Endo, Norm L-NNA, and Norm ODQ arteries.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   In normoxic (Norm) piglets, tension measured under resting conditions was significantly higher () in endothelium-negative (Endo) and nitric oxide synthase-inhibited [with N-monomethyl-L-arginine (L-NNA)] arteries compared with control (Con) arteries. Tension measured after 2nd submaximal contraction induced by U-46619 (U-46619 challenge 2) was significantly higher (**) in Norm Endo, Norm L-NNA, and Norm guanylate cyclase-inhibited (with ODQ) arteries compared with Norm Con and Norm cyclooxygenase (Cyclo)-inhibited arteries. Alkalosis caused a significant decrease in tension in Norm Con and Norm Cyclo arteries (*) but not in Norm Endo, Norm L-NNA, or Norm ODQ arteries. Nos. in parentheses, no. of arteries, piglets.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   In Norm piglets, alkalosis-induced relaxation, calculated as percent decrease in U-46619-induced tension during alkalosis, was significantly blunted in Norm Endo, Norm L-NNA, and Norm ODQ arteries (). Nos. in parentheses, no. of arteries, piglets.

The effects of K⁺-channel inhibition on absolute tension are shown in Fig. 3. U-46619 challenge 2 caused a significant increase in tension in all groups. Tension measurements in K_ATP channel-inhibited (Norm Glib) and K_Ca channel-inhibited (Norm IbTX) arteries did not differ from Norm Con arteries (Fig. 3A). In contrast, comparison of tension measurements made in K_V channel-inhibited (Norm 4-AP 10³ M and Norm 4-AP 10² M) and Norm Con arteries approached significance (ANOVA interaction term, P = 0.07; Fig. 3B). However, only arteries treated with 10² M 4-AP had a blunted response to alkalosis when the percent alkalosis-induced relaxation was compared in all groups (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A: in Norm piglets, tension measured under resting conditions and after U-46619 challenge 2 did not differ between ATP-dependent K+ or Ca2+-dependent K+ channel-inhibited [with glibenclamide (Glib) or iberiotoxin (IbTX), respectively] and Norm Con arteries. Alkalosis caused a significant decrease in tension in all groups (*). B: there was no difference in tension measured under resting conditions or after U-46619 challenge 2 among Norm Con and voltage-dependent K+ channel-inhibited [with 103 and 102 M 4-aminopyridine (4-AP)] arteries. Alkalosis-induced relaxation was significant in Norm Con and Norm 4-AP 103 M arteries. Nos. in parentheses, no. of arteries, piglets.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   In Norm piglets, alkalosis-induced relaxation, calculated as percent decrease in U-46619-induced tension during alkalosis, was significantly blunted only in 4-AP 102 arteries (). Nos. in parentheses, no. of arteries, piglets.

Figure 5 illustrates the effects of alkalosis in Norm 4-AP 10² M arteries, arteries treated with the Na⁺-K⁺-ATPase inhibitor Ouab (Norm Ouab), and those precontracted with 40 mM KCl (Norm KCl) rather than with U-46619 challenge 2. Absolute tension was significantly higher under all experimental conditions in Norm 4-AP 10² M arteries compared with Norm Ouab or Norm KCl arteries (Fig. 5A). However, alkalosis failed to reduce tension in any of these groups, and the percent alkalosis-induced relaxation was significantly blunted in all groups compared with the Norm Con group (Fig. 5B).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   A: in Norm piglets, tension was significantly higher in Norm 4-AP 102 M compared with Norm ouabain (Ouab)-inhibited or Norm KCl-contracted arteries, but alkalosis had no effect on absolute tension in any group. B: percent relaxation was significantly decreased in all groups compared with Norm Con group (). Nos. in parentheses, no. of arteries, piglets.

Alkalosis-induced relaxation in arteries from Hyp piglets. There were no differences in piglet or artery weight, artery diameter, initial resting tension, tension after 40 mM KCl, or tension in response to U-46619 challenge 1 among the 55 arteries from the 7 Hyp piglets. Table 3 shows the mean preparation characteristics for all Norm and Hyp piglets. Although the Hyp piglets weighed significantly less than the Norm piglets, both the weight and diameter of the arteries were the same as those from Norm piglets (Table 3). The increase in tension in response to both the final challenge with 40 mM KCl and the U-46619 challenge 1 was significantly higher in arteries from Hyp compared with Norm piglets (Fig. 6A) even though a significantly higher concentration of U-46619 was used in the Norm piglets (8.7 × 10⁸ ± 1.1 × 10⁸ and 2.5 × 10⁸ ± 0.1 × 10⁸ M, respectively; P < 0.001). Moreover, BK and ACh caused significantly less relaxation in arteries from Hyp than from Norm piglets (Fig. 6B).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   A: change in tension in response to 3rd challenge with 40 mM KCl or 1st submaximal contraction induced by U-46619 (U-46619 challenge 1; #1) was significantly higher in Hyp Con compared with Norm Con arteries (*). Nos. in parentheses, no. of arteries, piglets. B: percent relaxation in response to bradykinin (BK) and ACh was significantly less in Hyp Con compared with Norm Con arteries ().

Resting tension measured before U-46619 challenge 2 was similar in Norm Con compared with Hyp Con arteries (Fig. 7A). However, tension measured during U-46619 challenge 2 was significantly higher in Hyp Con arteries (Fig. 7A) even though the concentration of U-46619 challenge 2 was significantly higher in Norm Con than in Hyp Con arteries (1.1 × 10⁷ ± 2.0 × 10⁸ and 3.9 × 10⁸ ± 0.2 × 10⁸ M U-46619, respectively). Alkalosis caused a significant decrease in tension in both groups (Fig. 7A). However, the percent alkalosis-induced relaxation was significantly blunted in Norm Con compared with Hyp Con arteries (Fig. 7B).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   A: tension measured under resting conditions did not differ between Norm Con and Hyp Con arteries, but tension was significantly greater after U-46619 challenge 2 to Hyp Con arteries (**). Alkalosis caused a significant decrease in tension in both groups (*). B: however, percent relaxation in response to alkalosis was significantly lower in Hyp Con compared with Norm Con arteries (). Nos. in parentheses, no. of arteries, piglets.

In contrast to arteries from Norm piglets (Fig. 1B), neither resting tension nor tension measured after U-46619 challenge 2 differed between Hyp Con and Hyp L-NNA arteries from Hyp piglets (Fig. 8A). However, as in Norm piglets, alkalosis had no effect on tension in Hyp L-NNA arteries and the effects of alkalosis were similar in Hyp Con and Hyp Cyclo arteries (Figs. 8A and 9). Moreover, alkalosis-induced relaxation was significantly inhibited in Hyp 4-AP 10² M arteries (Figs. 8B and 9). However, the effects of 10³ M 4-AP did approach significance in the Hyp 4-AP 10³ M arteries (Figs. 8B and 9).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   A: in Hyp piglets, resting tension and tension after U-46619 challenge 2 did not differ between Hyp Con, Hyp L-NNA, and Hyp Cyclo arteries. However, alkalosis reduced tension in Hyp Con and Hyp Cyclo arteries (*) but not in Hyp L-NNA arteries. Mec, meclofenamate. B: tension was higher in Hyp 4-AP 102 M arteries (**), and alkalosis had no effect in that group. Differences between Hyp 4-AP 103 M and Hyp Con arteries approached but did not reach significance (* P = 0.07). Nos. in parentheses, no. of arteries, piglets.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   In Hyp piglets, alkalosis-induced relaxation was significantly attenuated in Hyp L-NNA and Hyp 4-AP 102 M arteries (). Nos. in parentheses, no. of arteries, piglets.

	DISCUSSION

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This study identifies the relative contribution of endothelium-derived modulators and K⁺-channel activation to alkalosis-induced relaxation in isolated, 0.5-mm-diameter pulmonary arteries from Norm piglets. In addition, the magnitude and mediators of alkalosis-induced vasodilation were determined for the first time in an animal model of preexistent pulmonary hypertension. Endothelium-derived nitric oxide appeared to be the predominant mediator of alkalosis-induced relaxation in arteries from both Norm piglets and piglets with pulmonary hypertension due to 3 days of chronic hypoxia. However, alkalosis-induced relaxation was blunted in arteries from chronically Hyp piglets. This was likely due to decreased endothelium-derived nitric oxide activity because endothelium-derived relaxation to ACh and BK was also diminished in the arteries from chronically Hyp piglets. Although alkalosis-induced relaxation was inhibited by 10² M 4-AP in arteries from both Norm and Hyp piglets, this was likely due to a nonspecific effect of 4-AP rather than to K_V-channel inhibition.

Mediators of alkalosis-induced relaxation in arteries from Norm piglets. Our finding that alkalosis-induced relaxation was blunted in arteries without functional endothelium suggests that the response is endothelium dependent (Figs. 1 and 2). Moreover, the significant effects of nitric oxide synthase and guanylate cyclase inhibition, but lack of effect of Cyclo inhibition, suggest that the nitric oxide-cGMP pathway mediates alkalosis-induced vasodilation in pulmonary arteries from Norm piglets. How alkalosis enhances nitric oxide activity is not known, but because alkalosis increases cytosolic Ca²⁺ in several cell types (15, 40), it may also increase endothelial cytosolic Ca²⁺, a potent stimulus for endothelial nitric oxide synthesis (23). Increased cytosolic Ca²⁺ can also stimulate endothelial prostacyclin synthesis. However, Cyclo inhibition had no effect on alkalosis-induced relaxation in this study, suggesting that prostacyclin played little role in alkalosis-induced relaxation in pulmonary arteries from Norm piglets.

Our data are consistent with those obtained in bovine aortic endothelial cells showing that alkalosis caused increased release of a nonprostanoid endothelium-derived relaxing factor (24) and with preliminary data presented by Aschner and Smith (2) suggesting that nitric oxide mediated alkalosis-induced relaxation of piglet pulmonary arteries. In addition, Indo failed to block alkalosis-induced vasodilation in previous studies (12, 25) of intact lambs. In contrast, however, nitric oxide did not seem to be involved in alkalosis-induced vasodilation of adult rabbit lungs (42) or intact lambs (12) and prostacyclin did appear to mediate the vasodilator response to alkalosis in mature rats (41) and intact piglets (18). Thus the contribution of different endothelium-derived modulators to alkalosis-induced vasodilation may vary with species and/or age. This conclusion is supported by studies showing that basal nitric oxide and prostacyclin activities differed between species (4) and that both nitric oxide and dilator prostaglandin activities changed with age (16, 30).

Several lines of evidence led us to investigate the contribution of K⁺ channels to alkalosis-induced vascular relaxation in piglet pulmonary arteries. In recent patch-clamp studies of canine (1) and rat (39) pulmonary arterial smooth muscle cells, alkalosis promoted K_V-channel activation, and in newborn piglets, K_ATP channels appeared to contribute to basal pulmonary vasomotor tone (32). In addition, alkalosis increases vascular smooth muscle cytosolic Ca²⁺ concentration (40), a potent stimulus for K_Ca-channel activation. However, neither IbTX at concentrations sufficient to inhibit K_Ca-channel activation (5, 20) nor Glib at concentrations sufficient to inhibit K_ATP-channel activation (7, 32) altered the response to alkalosis (Figs. 3A and 4). 4-AP at 10³ M, a concentration thought to inhibit K_V channels (29, 37), also failed to significantly alter the response to alkalosis (Figs. 3B and 4). In contrast, 10² M 4-AP did significantly blunt the response to alkalosis. Even though this may indicate that K_V-channel activation contributed to alkalosis-induced relaxation, 10² M 4-AP has several nonspecific effects, including inhibition of Ca²⁺-ATPase and Na⁺-K⁺-ATPase (19). Our observation that alkalosis failed to induce relaxation in arteries treated with Ouab or precontracted with KCl (Fig. 5) provides indirect support for the hypothesis that 10² M 4-AP blunted alkalosis-induced relaxation by causing sustained vascular smooth muscle depolarization rather than K_V-channel inhibition. Further studies on isolated vessels and intact lungs from piglets and other species are needed to determine whether the K_V-channel activation described in patch-clamp studies (1, 39) plays a physiologically significant role in alkalosis-induced vasodilation.

Effects of chronic hypoxia on piglet pulmonary arteries. The finding that pulmonary arteries from piglets exposed to 3 days of 12% O₂ exhibited greater reactivity to both KCl and U-46619 challenge 1 than did arteries of a similar size and generation from Norm piglets (Fig. 6A) is consistent with previous observations by Fike and Kaplowitz (10) that Hyp pulmonary vascular reactivity was enhanced after 3 days of chronic hypoxia. The enhanced reactivity to KCl and U-46619 together with our finding that the responses to the endothelium-dependent dilators BK and ACh were blunted and abolished, respectively, in arteries from Hyp piglets (Fig. 6B) suggests that nitric oxide activity was decreased in pulmonary arteries from piglets exposed to 3 days of chronic hypoxia.

Magnitude and mediators of alkalosis-induced relaxation after chronic hypoxia. Alkalosis-induced relaxation was significantly blunted in arteries from Hyp piglets studied under Con conditions (Fig. 7). This was also likely a consequence of decreased nitric oxide activity. However, nitric oxide synthase inhibition further reduced alkalosis-induced relaxation from 25 to <1% (Figs. 8A and 9), suggesting that the vasodilator response was mediated by residual nitric oxide activity. As with the arteries from Norm piglets, 10² M 4-AP also attenuated the response to alkalosis in arteries from Hyp piglets (Figs. 8B and 9). However, unlike the arteries from Norm piglets, the effects of 10³ M 4-AP approached significance (Figs. 8B and 9), raising the possibility that K_V channels might play a role in the arteries from Hyp piglets.

Whether alkalosis-induced vasodilation would be further reduced or abolished in a model of more severe pulmonary hypertension remains to be determined. A previous study by Fike and Kaplowitz (11) demonstrating a greater decrease in nitric oxide synthase after 10 days of chronic hypoxia suggested that nitric oxide would contribute less to alkalosis-induced relaxation in piglet pulmonary arteries as pulmonary hypertension due to chronic hypoxia worsened. However, other vasodilator modulators such as epoxygenase metabolites of arachidonic acid (31) may contribute to alkalosis-induced vasodilation in more severe pulmonary hypertension.

Summary. Our data suggest that hypocarbic alkalosis-induced vascular relaxation is mediated predominantly by nitric oxide in isolated pulmonary arteries from newborn piglets and that this response is attenuated after 3 days of chronic hypoxia. Although this calls into question the efficacy of alkalosis therapy in the treatment of neonatal and pediatric pulmonary hypertension, several issues need yet to be addressed. For example, the contribution of both endothelium-derived modulators and K⁺-channel activation to alkalosis-induced vasodilation must be examined in isolated lungs and/or intact animals to evaluate their roles in a more physiological preparation. In addition, interspecies variability in the consequences of more prolonged hypoxia and other hypertensive stimuli on alkalosis-induced vasodilation must be evaluated. Finally, understanding how alkalosis leads to increased synthesis of nitric oxide or other modulators of vasomotor tone may lead to a more effective treatment strategy for infants and children with severe pulmonary hypertension.