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J Appl Physiol 88: 827-834, 2000;
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Vol. 88, Issue 3, 827-834, March 2000

Endothelium-dependent relaxation differs in porcine pulmonary arteries from the left and right caudal lobes

Lynelle R. Johnson1, John R. Dodam1,2, and M. Harold Laughlin1

Departments of 1 Veterinary Biomedical Sciences and 2 Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that pulmonary arteries (PA) from identical branch orders within left and right caudal lung lobes would exhibit similar vasomotor responses. Arterial rings from caudal lung lobes of female swine were examined in vitro. Vascular smooth muscle contraction to KCl and norepinephrine did not differ. Vascular relaxation to endothelium-dependent (bradykinin, acetylcholine, A-23187) and -independent (sodium nitroprusside, zero-calcium Krebs solution) vasodilators was assessed. Right PA exhibited less maximal relaxation to acetylcholine (50%) than did left PA (69%; P < 0.001). Maximal relaxation to sodium nitroprusside did not differ, although right PA had a lower drug concentration resulting in half-maximal relaxation (6.26 × 10-8 M) than did left PA (9.57 × 10-8 M; P < 0.05). Nitric oxide synthase inhibition with an arginine analog (Nomega -nitro-L-arginine methyl ester) depressed acetylcholine-induced relaxation but the left vs. right difference persisted. Indomethacin enhanced relaxation to acetylcholine and abolished the difference between left and right. We conclude that endothelium-dependent vasorelaxation is less in porcine right than in left PA because of greater release of one or more constricting prostanoids in arteries from the right caudal lobe.

pulmonary circulation; arachidonic acid metabolites; porcine; endothelial cell function; blood flow distribution


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DETERMINANTS OF PULMONARY blood flow distribution include hydrostatic pressure, gravitational effects, and regional differences in vasomotor function, although in recent years the importance of gravity has been questioned (2, 3). Perfusion studies using canine, porcine, and equine models describe a relatively minor role for gravitational effects on pulmonary blood flow distribution at rest, during positional changes, during exercise, and during increased gravitational force (1-4, 7). The heterogeneous geometry of pulmonary arterial branching and differences in vascular distensibility may play an important role in structure-function properties that affect blood flow distribution. Importantly, in the horse, blood flow distribution appears to be influenced by regional variations in endothelial cell function, because arteries in a dorsocaudal position display enhanced endothelium-dependent vasorelaxation in comparison to arteries from a ventral location within the same lung lobe (8).

Preliminary studies on pulmonary vasoreactivity in a porcine model of exercise training suggested that a majority of both trained and sedentary subjects exhibited lateralizing differences in endothelium-dependent vasorelaxation. However, because the hemodynamic conditions in the left and right pulmonary arteries are similar, arterial sizes are similar, and vascular compliance is likely equivalent, one would expect that the vasomotor responses of these two arteries and reliance on vasoactive mediators such as nitric oxide (NO) and prostacyclin for relaxation would also be comparable. The purpose of this study was to test the null hypothesis that endothelium-dependent vasomotor responses of left and right pulmonary arteries are similar.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Design

This analysis emerged from an ongoing study to test the hypothesis that vasomotor responsiveness of pulmonary arteries is altered by exercise training. The experimental design required examination of a number of pulmonary artery rings from each animal. We initially assumed that vasomotor responsiveness of pulmonary arteries from identical branch orders within the left and right caudal lung lobes would be similar. We conducted a preliminary study to test this hypothesis and discovered that acetylcholine and bradykinin administration produced greater vasorelaxation in left pulmonary arteries than in pulmonary arteries from the right side. We therefore designed the study reported herein to test the hypothesis that vasomotor responsiveness of pulmonary arteries from identical branch orders within the left and right caudal lung lobes are similar. Second, because our preliminary results indicated that responses to two vasoactive compounds that are believed to be endothelium dependent were different between left and right pulmonary arteries, we tested the hypothesis that this difference was endothelium dependent. Endothelium dependency was determined by comparing responses of intact pulmonary arteries with those of arteries with the endothelium removed (denuded). We used an arginine analog to determine whether the proposed difference between endothelium-dependent vasorelaxation on the right and left sides was mediated by the NO synthase (NOS) pathway and indomethacin to determine whether the proposed difference between endothelium-dependent vasorelaxation on the right and left sides was mediated by the cyclooxygenase pathway. Finally, to determine whether different responses resulted from receptor-specific mechanisms, we compared vasorelaxation responses produced by the calcium ionophore A-23187.

Isolation and Preparation of Pulmonary Arteries

Female, adult swine weighing 25-45 kg were used in all experiments. Pigs were sedated with ketamine (25 mg/kg im) and xylazine (2 mg/kg im), anesthetized with pentobarbital (30 mg/kg iv), and administered heparin (2,000 U/kg iv). The heart was removed by transection of the pulmonary artery and ascending aorta, and lungs were immediately placed in ice-cold Krebs solution containing (in mM) 131.5 NaCl, 5.0 KCl, 1.2 MgCl2 · 6H2O, 2.5 CaCl2 · 2H2O, 1.2 NaH2PO4 · H2O, 11.2 glucose, and 20.8 NaHCO3 for vessel isolation.

Pulmonary arteries (2- to 3-mm OD) were located by their position medial to the bronchi and were taken from a precise, reproducible location within the pulmonary circulation. Lobar pulmonary arteries to the left and right caudal lung lobes were identified, and the first ventrally oriented branch of each lobar artery was carefully isolated from associated bronchial and lymphoid tissue. Vessels were dissected in ice-cold Krebs solution, and adventitia was gently removed from the vessel surface. Adjacent segments of pulmonary arteries were cut into four ring segments, each 2-4 mm in length. Internal and outer diameter and ring length were recorded in millimeters for each vascular ring, and measurements were used to calculate vessel area.

Pulmonary artery rings from left and right caudal lobes were mounted in pairs on two stainless steel wires. One wire was connected to a force transducer (ETH-200/400 Series, CB Science, Dover, NH) to measure developed tension. The other wire was attached to a micrometer microdrive, allowing stretch of the vessel by known increments. Vessel rings were stretched to a passive tension of 1.0 g for 1 h in individual 20-ml baths containing Krebs solution equilibrated at 37°C and continuously bubbled with 95% O2-5% CO2. Krebs solution contained propranolol (3 × 10-6 M) to oppose beta 2-adrenergic receptor-mediated vasorelaxation. Isometric tension (in g) was continuously recorded on a polygraph connected to a computer acquisition system (MacLab, CB Sciences, Milford, MA). Individual length-tension curves were generated for each artery through repeated exposure to 30 mM KCl at increasing vessel diameter. Vessels were progressively stretched by 10% of the outer diameter for successive tension determinations. The optimal length for a given artery (Lmax) was defined as the length at which contractile force evoked by KCl failed to increase by >10% of the previous measurement. Vessels were stabilized at Lmax for 30 min before experimentation.

Contractile and Relaxation Responses

Maximum contraction to 80 mM KCl was determined as a reference point in all arterial rings. Arteries were washed to resting tension, and then a concentration-response curve was constructed for norepinephrine (NE; 10-8 to 10-4 M, in half-log doses) by cumulative addition of stock solutions to vessel baths. In a separate group of pigs, concentration-response relationship to KCl was generated by using osmotically balanced solutions containing increasing concentrations of KCl (10-100 mM) as replacement for NaCl. KCl solutions were made fresh weekly in modified Krebs buffer. Response to contractile agonists was expressed as the developed change in tension (in g) from resting tension. A concentration-response curve was constructed by plotting developed tension against millimolar K+ concentration or log NE concentration, and the concentration for each agonist that resulted in half-maximal contraction (EC50) was determined for arteries from the left and right caudal lung lobe.

Arteries were washed to resting tension then precontracted with the predetermined EC50 of NE (5.75 × 10-7 M) and stabilized for 20 min. Preliminary experiments indicated that NE resulted in stable contraction for >150 min in porcine pulmonary arteries. The presence of endothelium was confirmed by observing >60% relaxation to a single dose of bradykinin (10-6 M) after NE precontraction. Arterial rings that did not exhibit relaxation to bradykinin were discarded.

Pulmonary artery rings from each pig were studied under separate conditions; one pair of left and right rings was placed into a single treatment group throughout the experiment. Conditions were defined as control (untreated) vessels, vessels denuded of endothelium, vessels treated with indomethacin (10-5 M) to block cyclooxygenase activity, vessels treated with 300 µM Nomega -nitro-L-arginine methyl ester (L-NAME) for inhibition of NOS, and vessels receiving combined treatment with indomethacin (10-5 M) and L-NAME (300 µM). Endothelial denudation was achieved by gently rubbing the internal surface of each ring with the edge of stainless steel forceps. Vessels were reconstricted with 5.75 × 10-7 M NE, and response to 10-6 M bradykinin was reassessed. Denudation was considered successful when <5% relaxation was observed after reexposure to 10-6 M bradykinin. Denudation was confirmed by histological examination. Response to 10-6 M bradykinin was reevaluated in the presence of pharmacological inhibition of NOS or cyclooxygenase to determine the contribution of NO and prostaglandin metabolites to single-dose bradykinin-induced relaxation.

Basal Release of Factors

The effects of pharmacological inhibitors on NE-induced developed tension and resting tension were determined, and responses in left and right pulmonary arteries were compared in the presence and absence of inhibitors. In one group of pigs, vessels were precontracted with 5.75 × 10-7 M NE (EC50), and tension was stabilized for 20 min. L-NAME or indomethacin was then added to the appropriate vessel bath, and tension was restabilized for 20 min. The percent change in tension induced by initial exposure to pharmacological antagonists was calculated by dividing developed tension after addition of inhibitor (in g) by the tension developed in response to 5.75 × 10-7 M NE (in g) alone and multiplying by 100. In another group of pigs, pharmacological inhibitors were added to the vessel bath before NE was applied to assess the effect of L-NAME or indomethacin on resting tension. Response was calculated by subtracting resting tension (in g) from tension developed after addition of inhibitors (in g).

Role of Endothelial-Derived Mediators in Vasoactive Responses

After the second bradykinin response, arterial rings were washed to resting tension, and then 5.75 × 10-7 M NE and inhibitors were added to appropriate vessel baths. Endothelium-dependent relaxation was determined through cumulative addition of ACh (10-10 to 10-4 M, in half-log doses) stock solutions to the vessel bath (Fig. 1). Percent relaxation was determined as percent reduction in tension from precontracted developed tension. After the concentration-response curve, arteries were washed to resting tension, and then 5.75 × 10-7 M NE and appropriate inhibitors were added to each vessel bath. Tension was stabilized for 20 min, and relaxation to sodium nitroprusside (SNP; 10-10 to 10-4 M, in half-log doses) was determined to assess endothelium-independent relaxation. Maximal relaxation was identified through incubation in zero-calcium Krebs solution containing 2 mM EGTA.


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  		Fig. 1.   Experimental traces from a representative experiment showing ACh-induced relaxation from norepinephrine (NE)-induced developed tension (in g). Left: left pulmonary arteries. Right: right pulmonary arteries. Channels display responses in control vessels (1 and 2), in presence of 300 µM Nomega -nitro-L-arginine methyl ester (L-NAME) (3 and 4), in presence of 10-5 M indomethacin (5 and 6), and in presence of combined inhibition with L-NAME and indomethacin (7 and 8).

In a separate group of pigs, pulmonary arteries from the left and right caudal lobe were contracted with 5.75 × 10-7 M NE, and endothelium-dependent relaxation to half-log doses of the calcium ionophore A-23187 (10-8 to 10-6 M) was measured to assess receptor-independent relaxation.

Control experiments were performed in the presence of 300 µM Nomega -nitro-D-arginine methyl ester (D-NAME), the inactive isomer of L-NAME, to confirm that inhibition of NOS was responsible for the observed effects. In some pigs, control experiments were performed in the presence of 0.07% ethanol to confirm that responses noted were related to pharmacological inhibition of cyclooxygenase by indomethacin.

Solutions

All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted. Krebs solution was made fresh daily, equilibrated at 37°C, bubbled with 95% O2-5% CO2 for 20 min, and adjusted to pH 7.4 before use. Stock solutions of bradykinin (Bachem California, Torrance, CA), ACh, and NE dissolved in distilled, deionized water were made in a single batch and stored at -20°C until use. Dilutions used in experiments were made fresh daily using Krebs solution as a diluent. NE was kept covered and on ice throughout the experiment to prevent oxidative degradation. SNP was made fresh daily in Krebs solution and was kept in the dark immediately before addition to the bath. L-NAME or D-NAME was dissolved in distilled, deionized water and stored as a stock solution of 300 mM at -20°C. Indomethacin was prepared in 70% ethanol as a stock solution of 10-2 M and stored at -20°C before use. A stock solution 10-3 M A-23187 was prepared in 100% ethanol and diluted with Krebs solution before use.

Data Analysis

Data are presented as means ± SE. Measurements from left and right vessels were handled separately. Contractile tension was calculated by subtracting resting tension from tension measured after each addition of drug. Maximal responses were compared by using the Student's t-test. A concentration-response curve was constructed by plotting KCl concentration or log of NE concentration vs. developed tension. The EC50 was determined from the standard or semilog curve with a linear regression computer program (Basica IC50).

Relaxation was expressed as percent reduction from precontracted tension induced by 5.75 × 10-7 M norepinephrine (EC50) at each dose added. IC50 was defined as the drug concentration resulting in half-maximal relaxation. These values were generated by a linear regression computer program (Basica IC50). Cumulative concentration-response curves for ACh and SNP were analyzed by using repeated-measures analysis of variance (SuperANOVA, Abacus Concepts). Comparisons were made between responses from left and right pulmonary arteries for ACh and SNP concentration-response curves in the presence and absence of indomethacin, L-NAME, combined inhibition, and endothelial denudation. When indicated by a significant F-test, planned contrasts were performed to detect differences between individual means. IC50 concentrations for left and right vessels were compared by using the Student's t-test. For all analyses, significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of Pulmonary Arteries

Vessel rings from the left and right caudal lung lobe exhibited similar passive characteristics, physical dimensions, and percent stretch required to reach Lmax (Table 1). Passive tension necessary to attain Lmax did not differ between left and right arteries.

                              
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  		Table 1.   Physical characteristics of pulmonary arterial rings from the left and right caudal lung lobes

Contraction

Response to 80 mM KCl did not differ between vessel rings from the left (2.77 ± 0.13 g) and right (3.01 ± 0.20 g) lung lobe (n = 13; P = 0.33). Addition of increasing concentrations of NE produced dose-dependent increases in developed tension in arteries from the left and right caudal lung lobe. The concentration-response relationship to NE did not differ between arteries from left and right caudal lung lobes, and EC50 values were similar for arteries from the left (5.22 ± 0.72 × 10-7 M) and right (4.66 ± 0.42 × 10-7 M) lobes (P = 0.52; Fig. 2). Tension developed in response to NE at its EC50 concentration did not differ between left (1.26 ± 0.13 g) and right (1.56 ± 0.21 g) arteries (P = 0.24). The concentration-response relationship to isosmotic KCl solutions and EC50 values did not differ between arteries from the left and right caudal lobe (n = 5; P = 0.30; data not shown).


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  		Fig. 2.   Vasoconstrictor responses to increasing doses (log M) of NE in pulmonary arteries from the left and right caudal lung lobe of the pig. Developed tension was calculated by subtracting resting tension from tension resulting at each successive dose of vasoactive drug. Values are means ± SE; n = 5 pigs. Brackets denote concentration. No significant differences were found between left and right arteries.

Endothelium-Dependent Relaxation

Endothelium-mediated relaxation to 10-6 M bradykinin was significantly greater in arteries from the left caudal lung lobe (90.9 ± 1.4%) than in arteries from the right (82.3 ± 1.4%; n = 12; P < 0.0005; Fig. 3A). Cumulative addition of increasing concentrations of ACh resulted in dose-dependent reduction in tension from NE-induced contraction (Fig. 3B). Arteries from the left caudal lung lobe showed greater concentration-related relaxation to ACh, suggesting enhanced endothelium-mediated control of relaxation (P < 0.05). Sensitivity to ACh did not differ between arteries from the left and right caudal lobe as indicated by similar IC50 values. In separate experiments, A-23187 also appeared to produce greater endothelium-dependent relaxation in arteries from the left (85.6 ± 3.9%) vs. the right (76.7 ± 1.7%) caudal lobe (n = 5; P = 0.05; Fig. 3C).




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  		Fig. 3.   Relaxation responses to endothelium-dependent relaxing agents. Percent relaxation was calculated as percent reduction in tension from NE-induced tension. Values are means ± SE. A: vasorelaxation to a single dose of bradykinin (BK; 10-6 M); n = 14 pigs. * Arteries from the left side had significantly greater relaxation than did arteries from the right caudal lobe (P < 0.0005). B: concentration-response relationship to ACh (10-10 to 10-4 M) in intact (solid symbols; n = 14) and denuded (open symbols, n = 8) arteries. * Arteries from the left side had significantly greater relaxation than did arteries from the right caudal lobe (P < 0.05). C: concentration-response relationship to receptor-independent vasodilator A-23187 (10-8 to 10-6 M) in left and right pulmonary arteries; n = 5 pigs. * Arteries from the left side had significantly greater relaxation than arteries from the right caudal lobe (P = 0.05).

Endothelium-Independent Relaxation

Arteries from the left and right caudal lung lobe exhibited similar responses to SNP. However, left pulmonary arteries exhibited slightly less sensitivity in response to endothelium-independent stimulation with SNP as indicated by a greater IC50 (9.6 ± 1.4 × 10-8 M on the left vs. 6.3 ± 0.6 × 10-8 M on the right; n = 14; P < 0.05; Fig. 4). Arteries relaxed equally after incubation in zero-calcium Krebs solution.


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  		Fig. 4.   Relaxation response to endothelium-independent relaxation with sodium nitroprusside (SNP). Values are means ± SE; n = 14 pigs. Percent relaxation was calculated as percent reduction in tension from NE-induced tension. Concentration-response relationship to SNP (10-10 to 10-4 M) did not differ between arteries from the left and right caudal lung lobe; however, arteries from the right side had greater sensitivity to SNP as indicated by a lower drug concentration resulting in half-maximal relaxation (P < 0.05).

Basal Release of Factors

In control vessels, NE-induced tension did not differ in arteries from left and right caudal lung lobes (P = 0.24). In vessels where L-NAME was added after NE contraction, developed tension was significantly and equally increased above NE-induced tension alone (P < 0.05) in left (154%) and right (163%) arteries. However, after L-NAME, NE-induced tension was significantly greater in arteries from the right side (1.98 ± 0.11 g) than in arteries from the left side (1.58 ± 0.11 g; P < 0.05). Exposure to indomethacin after NE contraction resulted in a significant reduction in NE-induced tension in arteries from left (P = 0.05) but not in arteries from the right side. As was true for L-NAME, indomethacin treatment resulted in less precontracted tension in arteries from the left side (1.06 ± 0.09 g) than in arteries from the right side (1.43 ± 0.12 g; P < 0.05). Combined treatment with L-NAME and indomethacin resulted in a significant increase in tension from control levels in both left and right arteries (P < 0.05), and there was no difference in developed tension between arteries from the left (1.95 ± 0.18 g) and right (1.86 ± 0.20 g) side. Left and right arteries denuded of endothelium did not differ in contracted tension.

Role of Endothelial-Derived Mediators in Vasoactive Responses

Response to bradykinin. In the presence of L-NAME, relaxation to bradykinin was suppressed in both left and right arteries; however, relaxation was still greater in arteries from the left (50.3 ± 2.7%) than in arteries from the right (41.1 ± 2.2%; n = 10; P < 0.05). When indomethacin was present, relaxation was equivalent in arteries from the left and right caudal lung lobe (n = 12; P = 0.31). With combined inhibition of NOS and cyclooxygenase, relaxation to bradykinin was suppressed in both left and right arteries to equivalent levels (P = 0.31).

Response to ACh. Arteries denuded of endothelium relaxed <10% to ACh and responses did not differ between left and right pulmonary arteries (Fig. 3B). These results indicate that the response to ACh and the left vs. right difference are endothelium dependent.

L-NAME inhibited ACh-mediated relaxation in both left and right vessels; however, the concentration-response relationship remained significantly greater in left arteries (P < 0.05; Fig. 5). In arteries from the left lobe, maximal relaxation was reduced to 47.1% in the presence of L-NAME, whereas in arteries from the right, maximal relaxation was reduced to 35.5%. Sensitivity to ACh was not different between left and right arteries. Relaxation was not suppressed by the presence of D-NAME.


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  		Fig. 5.   Relaxation response to ACh (10-10 to 10-4 M) in control vessels (solid symbols) and in presence of inhibition of nitric oxide synthase with 300 µM L-NAME (open symbols) Values are means ± SE. Percent relaxation was calculated as percent reduction in tension from NE-induced tension. * Arteries from the left (n = 11 pigs) had significantly greater relaxation in response to ACh than did arteries from the right (n = 12 pigs) caudal lung lobe in each group (P < 0.05).

In the presence of indomethacin, ACh-mediated relaxation was significantly enhanced in arteries from the right caudal lung lobe (P < 0.05), but relaxation was not significantly changed in arteries from the left (Fig. 6). Dose-response curves no longer differed between arteries from the left and right caudal lobe after indomethacin treatment. Relaxation was not enhanced by the addition of an equivalent volume of 70% ethanol, the diluent used in formulating indomethacin, to the vessel bath.


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  		Fig. 6.   Relaxation response to ACh (10-10 to 10-4 M) in control vessels (solid symbols) and in presence of 10-5 M indomethacin (Indo; open symbols). Values are means ± SE; n = 14 pigs. Percent relaxation was calculated as percent reduction in tension from NE-induced tension. Indo abolished difference between arteries from the left and right caudal lung lobe.

Response to SNP. With denudation, endothelium-independent relaxation to SNP was enhanced in both left (127.8 ± 12.1%) and right arteries (113.2 ± 7.2%), and denudation abolished the difference between left and right responses (Fig. 7). Maximal endothelium-independent relaxation was also enhanced compared with control levels in the presence of either NOS or cyclooxygenase inhibition, and differences between left and right arteries were abolished; arteries from the left caudal lung lobe displayed increased sensitivity to SNP compared with control when L-NAME or indomethacin was present in the vessel bath (Table 2).


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  		Fig. 7.   Relaxation response to endothelium-independent relaxation with SNP (10-10 to 10-4 M) in control (solid symbols; n = 14 pigs) and denuded (open symbols; n = 8 pigs) arteries. Values are means ± SE. Percent relaxation was calculated as percent reduction in tension from NE-induced tension. Denudation abolished difference in sensitivity to SNP noted between left and right control vessels.


                              
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  		Table 2.   Relaxation response of left- and right-sided pulmonary arteries to sodium nitroprusside


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that pulmonary arteries taken from identical branch orders within the left and right caudal lung lobe of the pig would exhibit similar vasoactive responses to contractile and relaxant agonists. In our opinion, this hypothesis was reasonable because the arteries are exposed to the same perfusion pressure, partial pressure of oxygen, and gravitational effects, since the arteries are located at the same relative position on each side of the lung. Use of similar-sized arteries removed the possibility of diameter-dependent differences in relaxation properties (11). Results indicate that vasocontractile and vasorelaxation responses mediated by vascular smooth muscle (endothelium-independent responses) are similar in left and right pulmonary arteries, consistent with our hypothesis. Importantly, our results reveal significant differences in endothelium-dependent vasorelaxation between pulmonary arteries from the left and right caudal lung lobe. These regional variations in endothelial cell function may impact distribution of pulmonary blood flow.

Contractile Responses

Maximal contractile response to activation of voltage-gated calcium channels with 80 mM KCl and the concentration-response relationship to isosmotic KCl concentrations did not differ between arteries from the left and right caudal lung lobe. This would suggest that there are no lateralizing differences in populations of vascular smooth muscle cells at this level. The concentration-response relationship to stimulation with NE and contraction to EC50 levels of NE also did not differ, suggesting that beta -adrenergic receptor number and distribution are similar between arteries from the left and right caudal lung lobe. Thus our results indicate that vascular smooth muscle responses to various contractile agonists do not differ in arteries from the left and right caudal lung lobe.

Relaxation Responses

Our data reveal that the pulmonary artery from the left caudal lobe exhibits greater endothelium-dependent vasorelaxation than does the pulmonary artery from the right. Diminished relaxation to ACh in arteries from the right appeared to be endothelium dependent because response to the endothelium-independent vasodilator SNP was actually augmented in arteries from the right side. Also, arteries denuded of endothelium exhibited equivalent responses to both endothelium-dependent and -independent relaxing agents. To our knowledge, this represents the first description of differences in vasorelaxation in arteries from the right caudal lung lobe compared with the left and indicates that a lateralizing variation in responses is present within the pulmonary circulation of the pig.

Endothelium-dependent relaxation to two different receptor-mediated agonists and a receptor-independent agent was significantly less in arteries from the right caudal lung lobe than in arteries from the left lobe (Fig. 3). ACh and bradykinin both act through binding of hormone to endothelial cell receptors with activation of G-protein-mediated cell signaling cascades to increase intracellular calcium. NO and/or prostacyclin released in response to these agonists increase cyclic nucleotides within vascular smooth muscle cells to initiate relaxation. The diminished response to ACh in arteries from the right caudal lung lobe could be related to decreased receptors on endothelial cells, decreased activation or efficiency of the signaling cascade, or altered production of endothelium-derived mediators from arteries on different sides. If diminished relaxation in right pulmonary arteries was related to decreased receptor number or density on endothelial cells of arteries from the right caudal lung lobe, on the basis of our results, both muscarinic and bradykinin receptors would have to be affected. This mechanism appears unlikely because A-23187, a receptor-independent agonist of endothelium-mediated relaxation, also appeared to produce less relaxation in arteries from the right, supporting an intrinsic difference in endothelial cells. Vasomotor responses to ACh in the presence of indomethacin were enhanced in arteries from the right caudal lung lobe and not affected in arteries from the left (Fig. 6). These results suggest that the difference in endothelium-dependent relaxation results from greater production of a prostanoid constrictor in right pulmonary arteries. Because there was no evidence of differences in basal release between left and right pulmonary arteries, our results indicate that this proposed prostanoid constrictor is released during stimulation with bradykinin, ACh, or A-23187.

Although this is the first report of left- and right- sided differences in pulmonary vasorelaxant responses, others have detected varying endothelium-dependent relaxation within the pulmonary vasculature on the basis of vessel location. Pelletier et al. (8) reported significant differences in vascular responses of arteries taken from a caudodorsal vs. ventral position within the equine lung, with dorsal arteries exhibiting greater endothelium-mediated relaxation than ventrally positioned arteries (8). Inhibition of NOS totally abolished endothelium-dependent relaxation of both dorsal and ventral pulmonary arteries in the horse, suggesting that differential release of NO was responsible for the observed difference between arteries taken from different planes within the lung. In the present study, differential release of NO did not appear to explain the right-to-left differences because after L-NAME treatment, endothelium-dependent relaxation in arteries from the left caudal lung lobe was still greater than in right pulmonary arteries. Thus, although NO plays a role in ACh-mediated relaxation in both left and right pulmonary arteries in the pig, it is unlikely that differences in NOS expression or activity are responsible for increased relaxation in arteries from the left side.

Endothelial cells may also release a variety of vasoactive prostaglandin products through metabolism of membrane phospholipids. Arachidonic acid is metabolized through at least three pathways, resulting in production of both vasodilators and vasoconstrictors. The cyclooxygenase pathway results in generation of prostacyclin, a vasodilator produced primarily by the endothelium, and constrictor prostaglandins and thromboxane A2. The lipoxygenase pathway produces leukotrienes and hydroxyeicosatrienoic acids, whereas the epoxygenase pathway produces epoxyeicosatrienoic acids. Revtyak et al. (9) found significant differences in metabolism of arachidonic acid in endothelial cell cultures derived from separate conduit or microvascular beds. Our experiments indicate that inhibition of the cyclooxygenase pathway with indomethacin abolishes the difference in endothelium-dependent relaxation between left and right arteries. If right pulmonary arteries were continually producing a prostanoid constrictor, indomethacin would have an effect on NE-induced contraction, which was not seen here. Enhanced smooth muscle responsiveness to a prostanoid constrictor is not a likely explanation for differences noted here given the equivalent responses of left and right pulmonary arteries to SNP in the presence of indomethacin. Thus our results indicate that endothelial cells of pulmonary arteries from the right caudal lobe release more prostanoid constrictor only during stimulation with ACh, bradykinin, and A-23187.

Kovitz et al. (5) reported endothelium-dependent differences in response to hypoxia for different-sized pulmonary arteries isolated from porcine lungs. Large proximal pulmonary arteries displayed transient contraction to moderate hypoxia whereas relaxation predominated in small arteries. The response in distal arteries appeared to be mediated by both NO and prostacyclin. In contrast to this difference between distinct sizes of pulmonary arteries, our results in the present study and those of Pelletier et al. (8) confirm that, in the normal lung, location of the artery within the pulmonary circulation can play a key role in defining endothelial function.

The diminished relaxation in pulmonary arteries on the right might serve as a mechanism to limit blood flow into the right lung and facilitate flow into the left lung. Asymmetrical left vs. right pulmonary perfusion was documented in miniature swine in vivo through the use of radiolabeled microspheres (10) but was thought to be related to the larger size of the right lung lobe. The right lung of the pig is composed of four lobes (tracheobronchial, middle, caudal, and accessory) and makes up ~56% of total lung wet weight, whereas the left lung is composed of two lobes (cranial and caudal). Although the left and right caudal lung lobes are approximately equal in wet weight, the right caudal lobe is 36% of total lung weight compared with 31% for the left caudal lobe. Pelletier et al. (8) proposed that enhanced endothelium-dependent relaxation in caudodorsal arteries plays a role in preferential distribution of blood flow to dorsal lung regions in the horse. In rats, the right lung is larger and lobar lung blood flow per minute is greater to the right lung than to the left; however, blood flow per gram of tissue is equal in left and right lungs (6). In a similar manner, right pulmonary arteries of the pig might produce a vasoconstrictor to optimize distribution of blood flow among lobes.

In conclusion, we hypothesized that arteries taken from the same branch order and same relative location within left and right caudal lung lobes would exhibit similar vasomotor responses. Although vascular smooth muscle responsiveness was similar, our results revealed decreased endothelium-dependent relaxation in pulmonary arteries from the right side. Because indomethacin increased endothelium-dependent relaxation of pulmonary arteries from the right and abolished left vs. right differences, we conclude that the differences result from increased production of one or more prostanoid vasoconstrictors by the endothelium of right pulmonary arteries.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-03856 and HL-52490.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: L. R. Johnson, Dept. of Veterinary Biomedical Sciences, E102 Veterinary Medicine Bldg., Columbia, MO 65211 (E-mail: JohnsonLR{at}missouri.edu).

Received 1 July 1999; accepted in final form 1 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 88(3):827-834
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