Development of intrinsic tone in isolated pulmonary arterioles

Qiang Liu and J. T. Sylvester

Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In isolated porcine pulmonary arterioles with endothelium, intraluminal diameter measured at a transmural pressure of 20 mmHg decreased spontaneously from 233 ± 11 to 171 ± 12 µm in 135 min. This intrinsic constriction was not prevented by indomethacin, tetraethylammonium, or superoxide dismutase. Indomethacin plus NG-nitro-L-arginine methyl ester caused initial constriction and BQ-123 or BQ-123 plus BQ-788 caused initial dilation, but these treatments did not prevent subsequent progressive constriction. In pulmonary arterioles with endothelium exposed to calcium-free conditions and pulmonary arterioles without endothelium, the intraluminal diameter measured at a transmural pressure of 20 mmHg was constant at 239 ± 16 and 174 ± 7 µm, respectively. Thus the spontaneous development of tone in isolated pulmonary arterioles required extracellular calcium and resulted from 1) time-independent smooth muscle contraction caused by mechanisms intrinsic to smooth muscle and 2) time-dependent contraction caused by decreasing activity of endothelium-derived relaxing factors other than nitric oxide, vasodilator prostaglandins, and hyperpolarizing factors acting on calcium-dependent potassium channels or increasing activity of endothelium-derived contracting factors other than endothelin-1, vasoconstrictor prostaglandins, and superoxide anions. Further investigation is indicated to identify these unknown mechanisms and determine their role in pulmonary vasoreactivity.

endothelium-derived relaxing factor; endothelium-derived contracting factor; nitric oxide; prostaglandins; endothelin-1; superoxide anions; extracellular calcium concentration; pig


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VASOMOTOR RESPONSES having the greatest impact on pulmonary vascular resistance and ventilation-perfusion matching are thought to occur in small, distal "resistance" pulmonary arteries rather than in large, proximal "conduit" arteries; therefore, isolated small pulmonary arteries are being used with increasing frequency to study the mechanisms of pulmonary vasoreactivity, and the characteristics of these vessels are becoming better appreciated (14, 22, 23, 27, 28). Recent studies (24, 25, 27) in isolated cat and pig pulmonary arteries 200-600 µm in diameter revealed that these resistance vessels developed intrinsic tone, a phenomenon that is not typical of larger conduit pulmonary arteries. Because baseline tone and the manner in which it is generated can exert profound effects on vasomotor responses (1, 3, 20, 33), the mechanisms by which isolated small pulmonary arteries develop intrinsic tone could be important determinants of vasoreactivity in this tissue.

In general, development of intrinsic tone could involve mechanisms intrinsic to vascular smooth muscle, endothelium, or both. For example, tone could develop because a progressive decrease in the activity of endothelium-derived relaxing factors (EDRFs) unmasked a contraction generated by mechanisms intrinsic to smooth muscle. Alternatively, a progressive increase in the activity of endothelium-derived contracting factors (EDCFs) could cause otherwise quiescent smooth muscle to contract. In isolated systemic arteries where intrinsic tone develops routinely and has long been used as a criterion of preparation acceptability (19, 21, 34), only a few studies have been reported, and these yielded inconsistent results. In coronary arteries, Rubanyi (32) found that development of intrinsic tone depended on the endothelium and elevation of transmural pressure (Ptm), whereas Cohen et al. (6) found that intrinsic tone was attenuated by the endothelium. In cerebral arterioles, intrinsic tone was decreased by reductions in Ptm and increased by inhibitors of nitric oxide (NO) synthase (NOS) (19, 34). To our knowledge, the only experiments investigating the mechanisms of intrinsic tone in small pulmonary arteries were those of Madden et al. (27), who demonstrated a requirement for extracellular calcium.

We performed the present study to determine the magnitude and mechanisms of intrinsic tone in porcine pulmonary arteries < 300 µm in diameter. Our results demonstrate that intrinsic constriction in these vessels was time dependent and quantitatively significant, achieving ~35% of maximum possible vasoconstriction at 135 min under control conditions and ~70% of maximum vasoconstriction when modulation by NO and vasodilator prostaglandins was blocked. In addition, our results confirm the dependence of this response on extracellular calcium and suggest that the initial spontaneous tone was generated by mechanisms intrinsic to smooth muscle and modulated by endothelium-derived NO and endothelin (ET)-1, whereas subsequent progressive tone development was due to altered activity of endothelium-derived vasoactive factors other than NO, prostaglandins, hyperpolarizing factors acting on calcium-dependent potassium channels, ET-1, or superoxide anions (O-2·). Further investigation is warranted to identify these unknown mechanisms and determine their role in pulmonary vasoreactivity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of isolated pulmonary arterioles. All protocols and procedures were approved by the Animal Care and Use Committee in accordance with National Institutes of Health guidelines. Male pigs (20-25 kg) were anesthetized with ketamine (30 mg/kg im) followed by pentobarbital sodium (12.5 mg/kg iv). After exsanguination via the femoral arteries, the lungs were rapidly removed and placed in ice-cold Krebs-Ringer bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose. Under a dissecting microscope, pulmonary arterioles ~100-150 µm in diameter and 1 mm long were isolated from seventh-generation branches of intrapulmonary arteries. The vessels were placed in a microvascular chamber, cannulated at one end with a glass micropipette, and secured with 12-0 nylon monofilament suture. Krebs-Ringer bicarbonate solution was infused slowly through the pipette until the arteriole was completely filled. The other end of the vessel was then cannulated with a second micropipette prefilled with Krebs-Ringer bicarbonate solution. Both cannulas were connected to a reservoir that could be raised or lowered to control Ptm. Ptm was measured with a pressure transducer positioned at the level of the vessel lumen. In the microvascular chamber, the vessels were superfused with Krebs-Ringer bicarbonate solution (total volume 100 ml) gassed with 16% O2-5% CO2-balance N2 (pH 7.35-7.45), maintained at 37°C, and recirculated except as noted in Experimental protocols. The chamber was placed on the stage of an inverted microscope (Nikon TMS-F) connected to a video camera (Panasonic CCTV camera). The vascular image was projected onto a video monitor, and the intraluminal diameter (ID) was continuously measured with a video dimension analyzer (Living Systems Instrumentation, Burlington, VT). The ID and Ptm were recorded with a recorder (Gould, Cleveland, OH). In some vessels, endothelial cells were disrupted by gently rubbing the intraluminal surface with a steel wire (70 µm in diameter). These vessels were then perfused with 2 ml of air bubbles followed by 2 ml of Krebs-Ringer bicarbonate solution (perfusion pressure < 5 mmHg) before being mounted in the microvascular chamber.

Experimental protocols. Initially, isolated pulmonary arterioles were allowed to equilibrate in the microvascular chamber for 20 min at a Ptm of 5 mmHg. Ptm was then increased to 20 mmHg in 5-mmHg steps at 5- to 7-min intervals and thereafter held constant. Measurements of the ID at Ptm of 20 mmHg (ID20) began 5 min after the Ptm was increased to 20 mmHg (time 0) and continued until the end of the experiment. This Ptm was chosen because it approximated the normal mean pulmonary arterial pressure in the pig (37). To assess viability, the vessels were exposed to KCl (60 mM) at 20 min and the thromboxane A2 agonist U-46619 (10-8 M) followed by ACh (10-6 M) at 50 min. In each case, the agonists were washed out of the microvascular chamber by 20 min of nonrecirculating superfusion with fresh Krebs-Ringer bicarbonate solution after the responses had stabilized (5-10 min). Thus, during the first 75 min of the experiment, the superfusate was recirculated for 35 min and not recirculated for 40 min. In most arterioles, KCl was administered again at the end of the experiment. In some untreated control arterioles with (Endo+; n = 6) and without (Endo-; n = 6) endothelium, KCl and U-46619-ACh were administered only at the end of the experiment. Control Endo+ (n = 19) and Endo- (n = 8) arterioles, which received no other treatment, were considered acceptable if 1) the contractile responses (decreased ID20) to KCl and U-46619 were >= 30% of the immediately preceding control ID20 and 2) the dilator responses (increased ID20) to ACh were >= 50 (Endo+ vessels) or <= 0% (Endo- vessels) of the U-46619 response.

Experimental Endo+ arterioles were continuously exposed to one of the following treatments from the time of mounting in the microvascular chamber: 1) Ca2+-free Krebs-Ringer bicarbonate solution containing EGTA (10-3 M; n = 6); 2) indomethacin (10-5 M; n = 6); 3) indomethacin (10-5 M) plus NG-nitro-L-arginine methyl ester (L-NAME; 3 × 10-5 M; n = 6); 4) indomethacin (10-5 M) plus L-NAME (3 × 10-5 M) + L-arginine (10-4 M; n = 6); 5) L-arginine (10-4 M; n = 5); 6) tetraethylammonium chloride (TEA; 5 × 10-3 M; n = 4); 7) charybdotoxin (CTX; 10-7 M; n = 4); 8) BQ-123 (3 × 10-6 M; n = 6); 9) BQ-788 (3 × 10-6 M; n = 6); 10) BQ-123 (3 × 10-6 M) plus BQ-788 (3 × 10-6 M; n = 6); and 11) superoxide dismutase (SOD; 150 U/ml; n = 5), where n is the number of animals. We also determined the effects of L-NAME (3 × 10-5 M) administered 75 min into the experiment to Endo+ (n = 9) and Endo- (n = 5) arterioles pretreated with indomethacin (10-5 M). Acceptance criteria on viability testing with KCl and U-46619-ACh for the experimental arterioles were the same as for the control arterioles except that 1) the responses to KCl and U-46619-ACh were not required in Endo+ arterioles exposed to calcium-free conditions and 2) the dilator responses to ACh in Endo+ arterioles exposed to indomethacin plus L-NAME had to be <= 0% of the U-46619 response.

In additional experiments, we measured the contractile responses to progressively increasing superfusate concentrations of ET-1 (10-11 to 10-8 M) in untreated control Endo+ arterioles (n = 6) and Endo+ arterioles treated with BQ-123 (3 × 10-6 M; n = 4), BQ-788 (3 × 10-6 M; n = 5), or both BQ-123 and BQ-788 (n = 9). After each administration of ET-1, sufficient time was allowed for the vessels to achieve a stable ID20. These experiments were performed either at the end of the time-course experiments described above or in separate studies.

Drugs. ACh, indomethacin, L-NAME, L-arginine, TEA, BQ-788, and bovine copper-zinc SOD were purchased from Sigma (St. Louis, MO); CTX was from from Peptides International (Louisville, KY); BQ-123 and ET-1 were from American Peptide (Sunnyvale, CA); and U-46619 was from Cayman Chemical (Ann Arbor, MI). Stock solutions of ACh, L-NAME, L-arginine, TEA, CTX, SOD, and ET-1 were prepared each day in deionized water and stored at 4°C until used. Indomethacin was dissolved each day in an aqueous solution of NaHCO3 (2.35 × 10-2 M). BQ-123 and BQ-788 were dissolved in absolute ethanol, stored at -60°C, and, on the day of the experiment, diluted as necessary with deionized water. Concentrations are expressed as final molar concentrations in the superfusate.

Data analysis. Student's t-test or one- or two-factor analyses of variance were used for statistical analysis as appropriate. When significant F-ratios were obtained with analysis of variance, Dunnett's test was used to compare individual means with a control value, and least significant differences were used to compare means at a specific time or level of treatment. P values < 0.05 were assumed to indicate significance. Values are means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Table 1 shows the average responses to KCl and U-46619-ACh administered at the beginning of the experiment to test vessel viability. KCl (60 mM) elicited vigorous contractile responses in Endo+ and Endo- arterioles but not in Endo+ arterioles exposed to calcium-free conditions. During exposure to U-46619, ACh (10-6 M) caused vasodilation in all groups except the Endo- arterioles and Endo+ arterioles exposed to L-NAME or calcium-free conditions. When viability testing was delayed until the end of the experiment, KCl and U-46619 caused contraction in Endo+ and Endo- arterioles, but ACh reversed the U-46619-induced contraction only in Endo+ arterioles (data not shown).

                              
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Table 1.   Changes in pulmonary arteriolar ID20 in response to KCl, U-46619, and ACh at beginning of experiment

As shown in Fig. 1, ID20 in Endo+ arterioles decreased progressively and spontaneously from 233 ± 11 µm at time 0 to 171 ± 12 µm at the end of the experiment (135 min). This intrinsic contraction did not occur in arterioles exposed to calcium-free conditions in which ID20 remained at its baseline value of 238 ± 10 µm. If this value represented the maximum (fully relaxed) ID20 in these vessels and the minimum (fully constricted) ID20 value was ~35 µm (Table 1), intrinsic vasoconstriction achieved ~35% of maximum possible vasoconstriction at 135 min.


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Fig. 1.   Time course of intraluminal diameter at transmural pressure of 20 mmHg (ID20) in isolated pulmonary arterioles with (E+; n = 19) and without (E-; n = 8) endothelium. Arterioles exposed to Ca2+-free conditions (n = 6) were superfused with Ca2+-free physiological salt solution containing 10-3 M EGTA. Data are means ± SE.

Although ID20 at time 0 was the same in untreated Endo+ arterioles and Endo+ arterioles exposed to calcium-free conditions, it was markedly diminished in Endo- vessels (169 ± 21 µm). This contraction of Endo- arterioles, which began immediately after endothelial denudation under the dissecting microscope, remained constant throughout the experiment. As a result, ID20 was the same in Endo- and Endo+ vessels at 135 min. Similar results were obtained in Endo+ and Endo- arterioles in which viability testing with KCl and U-46619-ACh was delayed until the end of the experiment (Fig. 2).


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Fig. 2.   Time course of ID20 in isolated E+ and E- pulmonary arterioles (n = 6/group) in which viability testing with KCl and U-46619-ACh was delayed until end of experiment. Data are means ± SE.

Indomethacin (10-5 M), an inhibitor of cyclooxygenase (9, 38), did not alter the constrictor responses to KCl or U-46619, the dilator responses to ACh, or the development of intrinsic tone (Table 1, Fig. 3). Treatment with both indomethacin and L-NAME (3 × 10-5 M), an inhibitor of NOS (2, 11), abolished the vasodilator response to ACh (Table 1) and decreased ID20 at time 0 but did not prevent further intrinsic vasoconstriction (Fig. 3). In these vessels, ID20 decreased from 194 ± 17 µm at 0 min to 99 ± 21 µm at 135 min. When 10-4 M L-arginine, a substrate for NOS (11), was included as pretreatment with indomethacin and L-NAME, vasodilator responses to ACh were restored (Table 1) and intrinsic constriction was markedly attenuated (Fig. 4A). In Endo+ arterioles treated with L-arginine alone, ACh caused vasodilation (Table 1) and development of intrinsic tone was abolished (Fig. 4B).


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Fig. 3.   Effects of indomethacin (Indo; 10-5 M; n = 6) and Indo plus NG-nitro-L-arginine methyl ester (L-NAME; 3 × 10-5 M; n = 6) on intrinsic constriction in isolated porcine E+ pulmonary arterioles. Data are means ± SE.



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Fig. 4.   A: reversal of effects of Indo (10-5 M) plus L-NAME (3 × 10-5 M) on intrinsic constriction in isolated porcine E+ pulmonary arterioles by L-arginine (L-arg; 10-4 M; n = 6). B: effects of L-arg (10-4 M; n = 5) on intrinsic constriction in E+ pulmonary arterioles. Data are means ± SE.

TEA, a nonspecific potassium-channel antagonist (8, 17), did not alter intrinsic vasoconstriction (Fig. 5A). CTX, a specific inhibitor of calcium-dependent potassium channels (8, 17), did not alter ID20 at 0 min, but it enhanced intrinsic tone at 0-20 min and inhibited intrinsic tone at 70-135 min (Fig. 5A). Neither agent altered the vasoconstrictor response to KCl at the beginning of the experiment (Table 1); however, CTX had abolished this response by the end of the experiment (Fig. 5B).


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Fig. 5.   Effects of tetraethylammonium (TEA; 5 × 10-3 M; n = 4) and charybdotoxin (CTX; 10-7 M; n = 4) on intrinsic vasoconstriction (A) and vasoconstrictor (B) responses to KCl (60 mM) at end of experiment in isolated porcine E+ pulmonary arterioles. Delta ID20, change in ID20. Data are means ± SE.

Figure 6A directly compares Endo+ arterioles pretreated with indomethacin plus L-NAME with Endo- arterioles. Development of intrinsic tone was greater in Endo+ arterioles treated with indomethacin plus L-NAME. Figure 6B shows the effects of delayed administration of L-NAME (75 min) in Endo+ and Endo- arterioles pretreated with indomethacin. L-NAME caused contraction in Endo+ arterioles but had no effect in Endo- arterioles.


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Fig. 6.   A: comparison of intrinsic constriction in isolated porcine E- pulmonary arterioles and E+ pulmonary arterioles treated with Indo (10-5 M) plus L-NAME (3 × 10-5 M). B: effects of L-NAME (3 × 10-5 M) administered at 75 min (arrow) to E+ (n = 9) and E- (n = 5) pulmonary arterioles pretreated with Indo (10-5 M). Data are means ± SE.

As shown in Fig. 7, ET-1 (10-11 to 10-8 M) caused a concentration-dependent contraction in Endo+ arterioles, which was blocked by the ETA-receptor antagonist BQ-123 (3 × 10-6 M) but not by the ETB-receptor antagonist BQ-788 (3 × 10-6 M). In arterioles treated with both BQ-123 and BQ-788, high concentrations of ET-1 caused a slight vasoconstriction, but this contractile response was still markedly reduced compared with that in control Endo+ arterioles.


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Fig. 7.   Effects of BQ-123 (3 × 10-6 M; n = 4), BQ-788 (3 × 10-6 M; n = 5), and BQ-123 (3 × 10-6 M) plus BQ-788 (3 × 10-6 M; n = 9) on constriction induced by endothelin-1 (ET-1) in isolated porcine E+ pulmonary arterioles. C, control. Data are means ± SE.

Figure 8 shows the effects of ET-1-receptor antagonists on the time course of ID20. BQ-123 (3 × 10-6 M) increased ID20 at 0 min (262 ± 23 µm); however, it did not alter subsequent intrinsic vasoconstriction (ID20 = 225 ± 28 µm at 135 min). BQ-788 (3 × 10-6 M) did not alter ID20 at 0 min (256 ± 15 µm) but increased subsequent intrinsic vasoconstriction (ID20 = 134 ± 21 µm at 135 min). Treatment with BQ-123 and BQ-788 together increased both ID20 at time 0 (284 ± 15 µm) and subsequent constriction (ID20 = 178 ± 14 µm at 135 min).


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Fig. 8.   Effects of BQ-123 (3 × 10-6 M; n = 6), BQ-788 (3 × 10-6 M; n = 6), and BQ-123 (3 × 10-6 M) plus BQ-788 (3 × 10-6 M; n = 6) on intrinsic constriction in isolated porcine E+ pulmonary arterioles. Data are means ± SE.

SOD (150 U/ml) did not affect the time course of ID20 (Fig. 9).


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Fig. 9.   Effects of superoxide dismutase (SOD; 150 U/ml) on intrinsic constriction in isolated porcine E+ pulmonary arterioles (n = 5). Data are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Control Endo+ pulmonary arterioles constricted in response to KCl and U-46619 and dilated in response to ACh after precontraction with U-46619 (Table 1). Arterioles subjected to endothelial denudation maintained vigorous constrictor responses to KCl and U-46619 but did not exhibit dilator responses to ACh. These results indicate that contractile function and receptor-linked signal transduction were intact in both Endo+ and Endo- arterioles, whereas endothelial function was intact in only Endo+ arterioles.

As shown in Fig. 1, Endo+ arterioles exhibited progressive constriction, with ID20 decreasing from 233 ± 11.0 µm at 0 min to 170 ± 12.0 µm at 135 min. Most of this constriction occurred during the first 75 min of the experiment. Because superfusate was not recirculated for 40 of these 75 min, the decrease in ID20 is unlikely to have been caused by a time-dependent change in superfusate composition, such as increasing osmolality due to evaporation. Rather, it must have resulted from factors or mechanisms intrinsic to the vessel.

The time course of ID20 in Endo+ arterioles differed markedly from that in Endo- arterioles, which were already constricted at 0 min (ID20 = 169 ± 21.4 µm) and did not exhibit a significant change in ID20 thereafter. Constriction of Endo- arterioles at time 0 is more likely to have resulted from endothelium removal than vascular smooth muscle damage due to the denudation procedure because ID20 decreased rapidly in response to KCl and U-46619 and returned promptly to baseline on washout of these agonists, indicating normal smooth muscle function. Furthermore, the differences between Endo+ and Endo- arterioles were present when viability testing was delayed until the end of the experiment (Fig. 2), indicating that they were not the result of transient exposures to KCl, U-46619, or ACh. We conclude that the progressive development of tone seen in control Endo+ pulmonary arterioles was the product of two influences: 1) smooth muscle contraction already established at time 0 by mechanisms intrinsic to smooth muscle and 2) endothelial antagonism of smooth muscle contraction, which was present at time 0 and progressively diminished thereafter.

In Endo+ arterioles exposed to calcium-free conditions, vasoconstrictor responses to KCl were absent (Table 1). This finding is consistent with a previous work (5) that indicated that KCl causes smooth muscle contraction primarily via membrane depolarization and calcium influx through voltage-dependent calcium channels. In contrast, vasoconstriction induced by U-46619, although diminished, was still present in these arterioles (Table 1). This finding is consistent with previous results (7) that suggested that vasoconstrictor responses to U-46619 utilize calcium released from intracellular stores. As shown in Fig. 1, Endo+ arterioles exposed to calcium-free conditions remained dilated throughout the experiment (ID20 = 240 ± 16.1 µm), confirming previous studies (13, 15, 27) in both pulmonary and systemic arteries. These results indicate that the smooth muscle or endothelial mechanisms responsible for intrinsic constriction in Endo+ pulmonary arterioles required extracellular calcium.

Studies of the mechanisms responsible for intrinsic tone in systemic arteries have not been conclusive. Some (6, 30) suggested that constriction was caused by mechanisms intrinsic to smooth muscle, whereas others (19, 31) concluded that endothelial mechanisms played a major role. Because short periods of decreased Ptm reduced intrinsic tone in cerebral arteries (34), the mechanisms responsible for intrinsic constriction could be similar to those mediating myogenic responses (29). For example, depolarization and calcium influx in smooth muscle due to stretch-induced activation of ion channels (12, 29) could explain the constriction we observed at time 0 in Endo- arterioles. Alternatively, stretch could act on endothelium, altering the release of EDRFs such as NO and vasodilator prostaglandins or EDCFs such as ET, vasoconstrictor prostaglandins, and superoxide anions (12, 18, 29, 32). To evaluate the roles of EDRFs and EDCFs, we determined the effects of 1) indomethacin, an inhibitor of cyclooxygenase, which catalyzes production of prostaglandins from arachidonic acid; 2) L-NAME, an inhibitor of NOS, which catalyzes production of NO from L-arginine; 3) TEA and CTX, antagonists of calcium-dependent potassium channels, which may mediate the vasodilating effects of endothelium-derived hyperpolarizing factors (EDHFs); 4) BQ-123 and BQ-788, antagonists of ETA and ETB receptors, respectively; and 5) SOD, which catabolizes superoxide anions.

As shown in Fig. 3, indomethacin did not alter development of intrinsic tone in pulmonary arterioles. This cyclooxygenase inhibitor has been shown to block prostaglandin production and contractile responses to arachidonic acid in systemic vascular tissue at concentrations less than or equal to that used in the present study (10-5 M) (9, 35, 38). Furthermore, indomethacin did not alter either the contractile responses to KCl and U-46619 or the dilator responses to ACh (Table 1), indicating the absence of nonspecific effects. We conclude that prostaglandins did not mediate intrinsic constriction in isolated pulmonary arterioles.

To evaluate the role of the EDRF NO, we determined the effects of L-NAME, an inhibitor of NOS (2, 11). As shown in Table 1, ACh caused a marked dilation in pulmonary arterioles treated with indomethacin but not in arterioles treated with indomethacin plus L-NAME. ACh-induced vasodilation was restored when L-arginine was added to indomethacin plus L-NAME. Vasoconstrictor responses to KCl and U-46619 were present in all three groups. These results indicate that L-NAME inhibited NO synthesis and did not impair contractile function.

The effects of L-NAME on the time course of ID20 are shown in Fig. 3. At 0 min, Endo+ arterioles treated with L-NAME plus indomethacin were markedly constricted compared with both untreated and indomethacin-treated Endo+ arterioles; however, L-NAME did not prevent the subsequent progressive decline in ID20. These results indicate that although NO modulated vasomotor tone at time 0, a decrease in NO-mediated relaxation did not cause the progressive vasoconstriction that occurred from 0 to 135 min.

The addition of L-arginine (10-4 M) to indomethacin plus L-NAME (Fig. 4A) or treatment with L-arginine alone (Fig. 4B) abolished intrinsic tone. These results suggest that NO production in these vessels was limited by L-arginine concentration and that enhancement of NO production could overcome the mechanisms responsible for intrinsic constriction.

EDHF is an unknown vasodilator that may act by opening calcium-dependent potassium channels in smooth muscle (8); however, 5 mM TEA, which blocks these channels and the effects of EDHF (8, 17), did not alter intrinsic vasoconstriction in pulmonary arterioles (Fig. 5A). CTX, a more specific antagonist of calcium-dependent potassium channels (8, 17), did inhibit intrinsic constriction (Fig. 5A), but unlike TEA, it also abolished the vasoconstrictor responses to KCl at the end of the experiment (Fig. 5B). These results suggest that the effects of CTX were due to nonspecific toxicity. Thus our data provide no evidence that intrinsic vasoconstriction was caused by decreasing activity of an EDHF acting on calcium-dependent potassium channels.

As illustrated in Fig. 6A, intrinsic constriction was more severe in Endo+ arterioles treated with L-NAME plus indomethacin than in untreated Endo- arterioles. This difference could be due to smooth muscle vasodilator mechanisms in Endo- arterioles or unopposed EDCF activity in Endo+ arterioles treated with L-NAME plus indomethacin. To assess the role of smooth muscle type II NOS, which may have been induced by cytokines or other factors (36, 39), we administered L-NAME at 75 min to Endo- and Endo+ arterioles pretreated with indomethacin (Fig. 6B). L-NAME had no effect in Endo- arterioles but caused a marked constriction in Endo+ arterioles. These results indicate that NOS was absent or inactive in smooth muscle.

It seems more likely that the differences shown in Fig. 6A were due to increasing EDCF activity. The magnitude of tone generated by these contractile factors was impressive. Assuming again that arterioles exposed to calcium-free conditions were maximally relaxed (Fig. 1) and that fully constricted arterioles had an ID20 of ~35 µm (Table 1), intrinsic vasoconstriction in the absence of modulation by NO and vasodilator prostaglandins (Fig. 6A) achieved ~70% of the maximum potential constriction at 135 min. The EDCFs involved could be ET or superoxide anions (18, 26) but not vasoconstrictor prostaglandins. To evaluate these possibilities, we studied arterioles exposed to ET antagonists or SOD.

The ETA-receptor antagonist BQ-123 abolished the constrictor responses to ET-1, whereas the ETB-receptor antagonist BQ-788 (3 × 10-6 M) had no effect (Fig. 7). When these antagonists were administered together, contraction to ET-1 was still inhibited, but the vessels were more contracted than after BQ-123 alone. These results indicate that ET-1-induced constriction of porcine pulmonary arterioles was mediated by ETA rather than ETB receptors. In contrast, previous results (16) in rat intrapulmonary arteries suggested that ET-1-induced vasoconstriction was mediated by both ETA and ETB receptors. This difference may be due to species-dependent differences in receptor density (10). Indeed, in the presence of BQ-123, ET-1 caused vasodilation in pig pulmonary arterioles (Fig. 7), suggesting that activation of ETB receptors mediated vasodilation, perhaps by increasing release of NO from the endothelium as previously suggested (16).

BQ-123 alone or in combination with BQ-788 increased ID20 at 0 min, suggesting that ETA receptor-mediated constriction contributed to the initial vasomotor tone; however, neither BQ-123 nor BQ-788 prevented subsequent progressive constriction (Fig. 8), indicating that this response was not due to activation of ETA or ETB receptors. Indeed, BQ-788 accelerated the decline in ID20, suggesting that ETB receptors exerted a vasodilatory influence that modulated progressive constriction.

At concentrations of 100-200 U/ml, SOD eliminated or markedly reduced release of superoxide anions from vascular tissues (4, 40). In preliminary studies of isolated porcine coronary arterioles exposed to anoxia-reoxygenation, we found that SOD (150 U/ml) caused a 70% decrease in superoxide anion release as assessed by lucigenin-enhanced chemilumenescence (data not shown). However, this concentration of SOD had no effect on the time course of ID20 in pulmonary arterioles (Fig. 9), indicating that intrinsic tone was not due to superoxide anions. These results do not eliminate the possibility that intrinsic constriction was mediated by H2O2, the product of SOD-induced catabolism of superoxide anions; however, in this case, SOD would be expected to enhance intrinsic constriction, which did not occur.

The development of intrinsic tone in pig pulmonary arterioles was similar to that described by Madden et al. (27) in cannulated cat pulmonary arterioles 200-600 µm in diameter. The ID measured at a Ptm of 10 mmHg decreased spontaneously by 23.9 ± 4.4% over 60-90 min. In contrast, Leach et al. (23) were unable to demonstrate intrinsic tone in rat pulmonary arterioles of similar size. In this study, resting isometric tension, adjusted to be equivalent to a Ptm of 30 mmHg, was not altered by exposure to calcium-free conditions or several vasodilator agents. This disparity may be due to differences in species, preparation, experimental conditions, or other factors. For example, it is likely that rat arterioles 200 µm in diameter were obtained from more proximal loci in the pulmonary vasculature than in pig or cat arterioles of the same diameter. Endothelium-dependent vasodilation was reduced or absent in rat pulmonary arterioles (23), whereas Endo+ arterioles that did not dilate at least 50% in response to 10-6 M ACh were excluded in our study. The higher resting Ptm and the isometric conditions used in the rat arterioles may have activated mechanisms not operative at lower constant levels of Ptm. Such considerations also lead to important questions about the role of intrinsic pulmonary arteriolar tone in the intact animal. Further investigation will be necessary to obtain answers.

In summary, our results demonstrate that development of intrinsic tone in isolated pulmonary arterioles was dependent on time and extracellular calcium concentration. Initially, tone was generated by mechanisms intrinsic to vascular smooth muscle and modulated by endothelium-derived NO and ET-1 but not by prostaglandins; however, subsequent progressive constriction could not be explained by these mechanisms. Rather, our results indicate that progressive development of intrinsic tone was caused by 1) decreased activity of an EDRF other than NO, vasodilator prostaglandins, and EDHFs acting via calcium-dependent potassium channels or 2) increased activity of an EDCF other than vasoconstrictor prostaglandins, ET-1, or superoxide anions. Additional research is indicated to determine the identity of the unknown mechanisms responsible for intrinsic tone, their role in pulmonary vasoreactivity, and whether they are unique to pulmonary vessels.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-51912 and a research grant from the American Lung Association (to Q. Liu).


    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: Q. Liu, Div. of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: qliu{at}welchlink.welch.jhu.edu).

Received 19 November 1998; accepted in final form 8 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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