Journal of Applied Physiology
Vol. 81, No. 5, pp. 2013-2019, November 1996
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Developmental changes in endothelium-dependent relaxation of pulmonary arteries: role of EDNO and prostanoids

Denise C. O'Donnell, Mary L. Tod, and John B. Gordon

Departments of Pediatrics, Medicine, and Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

O'Donnell, Denise C., Mary L. Tod, and John B. Gordon. Developmental changes in endothelium-dependent relaxation of pulmonary arteries: role of EDNO and prostanoids. J. Appl. Physiol. 81(5): 2013-2019, 1996.We hypothesized that maturational changes in both prostaglandin and endothelium-derived nitric oxide (EDNO) activity contribute to developmental changes in endothelium-dependent relaxation of newborn pulmonary arteries. Responses to endothelium-dependent vasodilators acetylcholine, bradykinin, and calcium ionophore A-23187 were determined in phenylephrine-constricted third- and fourth-generation (1- to 2-mm-diameter) pulmonary artery rings from 2-day (2d)- and 1-mo (1m)-old lambs under control conditions (Con), after inhibition of EDNO synthesis with N-nitro-L-arginine (L-NNA), after inhibition of prostanoid synthesis with meclofenamate (Mec), or both modulators with both inhibitors. Endothelium-independent responses to sodium nitroprusside (SNP) were also measured in Con rings. Endothelium-dependent relaxation was greater in 2d than 1m Con rings, particularly at high concentrations when an increase in tension occurred in 1m rings. L-NNA attenuated endothelium-dependent relaxation more in 2d rings, and SNP caused greater relaxation in 2d rings. However, Mec abolished all age-related differences by attenuating relaxation in 2d rings and constriction in 1m rings. These data suggest that developmental changes in endothelium-dependent responses of ovine pulmonary artery rings reflect both a decrease in EDNO activity and maturational differences in the relative influence of dilator and constrictor prostanoids.

endothelium-derived relaxing factor; thromboxane; acetylcholine; bradykinin; calcium ionophore; sodium nitroprusside; endothelium-derived nitric oxide

INTRODUCTION

ENDOTHELIUM-DERIVED NITRIC OXIDE (EDNO) is an important modulator of increased pulmonary vasomotor tone in mature animals (3, 16, 18) and during the perinatal period (1). However, developmental changes in the synthesis of and /or response to EDNO in the pulmonary circulation remain unclear, for both age-dependent increases and decreases in EDNO activity have been demonstrated. For example, inhibition of EDNO synthesis enhanced hypoxic pulmonary vascular resistance (PVR) more in isolated lungs of 1-mo (1m)- than 2-day-old lambs (2d) (12), and endothelium-dependent relaxation increased with age in isolated pulmonary arteries from sheep (2) and piglets (32), suggesting that the modulating effects of EDNO increased with age. In contrast, inhibition of EDNO synthesis accentuated thromboxane-induced vasoconstriction more in isolated lungs of 1d compared with 7d piglets (22). Moreover, endothelium-dependent vasodilation was greater in lungs of 2d than 1m lambs (11, 12) and in isolated pulmonary arteries from 3-10d compared with 1m piglets (19), suggesting that pulmonary vascular synthesis of and /or response to EDNO decreased during the newborn period.

Several factors may have led to the discordance between these studies. For example, synthesis of and /or response to EDNO may vary between species or preparations (3, 27). In addition, differences in oxygen tension (16, 25, 26) or vasomotor tone (11, 15) may alter synthesis of or response to EDNO, thus leading to apparently conflicting developmental changes. Finally, endothelium-dependent vasodilators may also induce hyperpolarization (5, 6, 20) or stimulate dilator prostaglandin (PG) synthesis (7, 8, 17), and the modulating effects of dilator PG appear to decrease with age (10, 13, 24). In this study, we sought to identify the relative contributions of EDNO and dilator PG to developmental changes in endothelium-dependent relaxation in newborn pulmonary arteries. Dose-response relationships to both receptor-dependent [acetylcholine (ACh) and bradykinin (BK)] and -independent (A-23187) endothelium-dependent vasodilators were measured in third- and fourth-generation (1- to 2-mm-diameter) pulmonary artery rings from 2d and 1m lambs under control (Con) conditions and after inhibition of EDNO and/or PG synthesis. Responses to the endothelium-independent vasodilator sodium nitroprusside (SNP) were also measured.

METHODS

Isolated pulmonary artery rings were prepared from 2d (n = 14) and 1m (n = 19) lambs as previously described by Tseng et al. (29) in a study of mature dog pulmonary arteries. Briefly, lambs were anesthetized with ketamine (50 mg/ kg im), and after cannulation of the femoral artery and vein, they were heparinized (5,000 U iv) and exsanguinated. The heart and lungs were removed en bloc through a midline sternotomy, and the lungs were immersed in cold physiological saline and stored at 4°C until use. As in the previous studies by Tseng et al. (29), there were no differences in responses of rings studied on the day of death or the subsequent 2 days (data not shown). On the day of study, third- and fourth-generation (1- to 2-mm-ID) pulmonary arteries were dissected from parenchymal tissue, and adherent adventitia was carefully removed. Vessels were cut into 1- to 2-mm-length rings and placed between two parallel stainless steel hooks in 10-ml organ baths. The top hook was suspended from an isometric force transducer (model 52-9529, Harvard Apparatus, South Natick, MA), and the bottom hook was attached to the organ chamber. Rings were bathed in modified Krebs solution (MKS; see below) and bubbled with 95% O₂-5% CO₂ at 37.5°C. The transducer was mounted on a micromanipulator (Stoelting model 55020, Chicago, IL) so that resting tension could be adjusted as desired, and vessel tension was constantly recorded on a flatbed recorder (Linseis model L6514-4, Princeton Junction, NJ).

In preliminary studies, length-tension curves were generated in response to 40 mM KCl, and the optimal resting tensions were identified at 625-750 mg for 2d and 750 mg for 1m rings, respectively (data not shown). Phenylephrine (PE) dose-response relationships were also determined, and the concentration of PE resulting in an 80% maximal contraction (EC₈₀) was identified as 10⁵ M for both age groups (data not shown). Endothelium-dependent responses were determined in a similar manner in all rings. Briefly, rings were allowed to equilibrate in MKS at optimal resting tension for 60-90 min. After stability was achieved, contractility was assessed by challenging the rings three times with 40 mM K⁺. Eight and one-half percent of the rings from 2d lambs and 6.2% of the rings from 1m lambs increased tension by <100 or <200 mg, respectively, in response to the third K⁺ challenge and were excluded from further study. After fresh MKS had been added to the organ chamber, the EC₈₀ concentration of PE was added and is termed the PE₁ response. After a stable PE₁ response had been achieved, 10⁷ M BK was added to the bath to verify endothelial integrity (9, 31). From 2d lungs, 6.9% of the rings and 25.2% of the rings from 1m lungs that had <20% relaxation to BK were excluded from further study of endothelium-dependent vasodilators; however, some of these rings were included in the endothelium-independent protocol described below. After BK challenge, rings were allowed to stabilize for 15 min at optimal resting tension in fresh MKS buffer under Con conditions or after addition of the nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine (L-NNA; 10³ M), the cyclooxygenase inhibitor Mec (2 × 10⁶ M), or both inhibitors (Both) to the bath. The EC₈₀ concentration of PE was then again added to the bath, and the second PE response, termed PE₂, was measured. Rings that exhibited an increase in tension of <125 mg were excluded from further study. Cumulative dose responses to one of three endothelium-dependent vasodilators [ACh (3 × 10⁹ to 10⁴ M), BK (3 × 10¹⁰ to 10⁶ M) or calcium ionophore A-23187 (10⁹ to 10⁵ M)] were then determined under Con, L-NNA, Mec, and Both conditions. Each ring was subjected to only one dilator-study condition combination. Cumulative dose responses to SNP (10⁹ to 10⁴ M) were evaluated in a separate group of vessels. At the end of each experiment, rings were blotted dry and weighed.

The MKS was composed of (in mM) 118 NaCl, 4.7 KCl, 1.2 NaH₂PO₄, 1.2 MgSO₄ ^· 7H₂O, 1.8 CaCl₂ ^· 2H₂O, 20 NaHCO₃, and 10 glucose. The K⁺ challenges were accomplished by altering the MKS so that it contained 83 mM NaCl and 40 mM KCl, thus maintaining an isosmolar solution. ACh, BK, PE, SNP (Sigma Chemical, St. Louis, MO) and Mec (Biomol Research Laboratories, Plymouth Meeting, PA) were prepared in physiological saline. A-23187 (Biomol Research Laboratories) was dissolved in dimethyl sulfoxide. L-NNA (Sigma Chemical) was dissolved in physiological saline titrated with 12 N hydrochloric acid. All drug concentrations are expressed as final molar concentrations in the organ bath. The various vasodilators and inhibitors were added in <0.1-ml aliquots to 10-ml baths; thus the volume of these drugs was minimal relative to total volume and had no effect on the chemical composition or pH of the MKS. Moreover, addition of vehicle (saline or dimethyl sulfoxide) had no effect on ring tension in the concentrations used.

All data are expressed as means ± SE. The increase in tension in response to the third K⁺ challenge and both PE₁ and PE₂ was measured in all rings and normalized to individual ring weight. As well as exclusion of rings that did not demonstrate a sufficient absolute increase in tension (see above), rings were excluded from further statistical analysis if the increase in tension was <40 mg tension/mg wt in response to the third K⁺ challenge or <30 mg tension/mg wt in response to the PE₂ challenge at either age. The percentage of relaxation of each ring in response to different vasodilators was calculated as the decrease in tension relative to the PE₂ response. Changes in tension for study conditions and ages were compared by analysis of variance and least significant difference test (LSD) with results considered significant at P < 0.05.

RESULTS

After normalization for weight, the increase in tension measured in response to the third K⁺ challenge was similar at both ages (Fig. 1, top). However, the increase in tension in response to PE₁ was significantly greater in 1m than in 2d rings (Fig. 1, top). At both ages, the increase in tension in response to PE₂ was similar to the response to PE₁ in Con rings, but L-NNA, MEC, and Both all resulted in a significant increase in the ratio of PE₂ to PE₁ (Fig. 1, bottom).

In Con rings, all concentrations of ACh over 3 × 10⁶ M caused a greater decrease in tension in 2d compared with 1m rings (Fig. 2A; LSD = 17.2%). Maximal relaxation of 2d Con rings was 44.2 ± 13.6% of the PE₂ contraction and occurred at 10⁵ M ACh. Inhibition of EDNO and/or PG synthesis with Mec, L-NNA, or Both significantly blunted the responses to ACh in 2d rings (Fig. 2, B-D). In contrast, maximal relaxation (12.2 ± 4.6% of the PE₂ contraction) of 1m Con rings did not differ statistically from 0% (Fig. 2A), and none of the inhibitors altered this response (Fig. 2, B-D). The age-dependent difference in response to ACh seen in Con rings (Fig. 2A) was abolished by the addition of L-NNA and/or Mec to the organ bath (Fig. 2, B-D).

The dose-response relationship to BK also differed significantly between 2d and 1m Con rings. Relaxation was greater in younger rings at all BK concentrations 10⁷ M (Fig. 3A; LSD = 15.6%). Maximal relaxation was 62.8 ± 14.8% of the PE₂ contraction and occurred at 3 × 10⁷ M BK in the 2d Con rings. Whereas maximal relaxation was only 38.4 ± 6.8% of the PE₂ response in 1m rings, it occurred at a lower concentration of BK (10⁷ M). Higher concentrations of BK resulted in a progressively smaller decrease in tension in the 1m rings such that at 10⁶ M BK no decrease occurred. In 2d rings, L-NNA caused a marked attenuation, Mec caused a smaller but significant decrease, and Both resulted in complete abolition of any response to BK (Fig. 3, B-D). In 1m rings, L-NNA reduced the maximal decrease in tension achieved but did not alter the increase in tension seen at higher concentrations of BK (Fig. 3B). In contrast, Mec had no effect on maximal relaxation but markedly reduced the increase in tension seen at high concentrations of BK (Fig. 3C). The addition of Both resulted in a total abolition of any response to BK (Fig. 3D). Comparisons between the two ages show that after L-NNA the decrease in tension in response to BK was greater in 1m than in 2d rings. However, there was no difference between the two ages after Mec or Both (Fig. 3, B-D).

The dose-response relationship to A-23187 also differed significantly between Con 2d and 1m rings (Fig. 4A; LSD = 15.1%). However, this was due to a shift in the dose-response curve rather than to a difference in maximal relaxation. In 2d rings, maximal relaxation was 35.5 ± 9.2% of the PE₂ response and occurred at 10⁵ M A-23187, the highest concentration used. Maximal relaxation in 1m Con rings (34.6 ± 4.9% of the PE₂ response) did not differ from that of 2d rings, but it occurred at a lower concentration of A-23187 (3 × 10⁷ M). As with BK, higher concentrations of A-23187 caused a return of tension toward the PE₂ baseline in 1m rings. In 2d rings, L-NNA significantly attenuated the response to A-23187, but Mec had little effect (Fig. 4, B and C). The combination of Mec and L-NNA further attenuated the response to A-23187 in 2d rings (Fig. 4D), but a significant decrease in tension persisted at high concentrations. In 1m rings, L-NNA reduced the maximal dilator response to A-23187 seen in Con rings, and Mec markedly attenuated the increase in tension seen at concentrations >3 × 10⁷ M A-23187 (Fig. 4, B and C). As in the 2d rings, Both markedly attenuated the response to A-23187 in 1m rings, but a significant decrease in tension still occurred at 3 × 10⁶ M A-23187 (Fig. 4D). Responses to A-23187 were similar at both ages in L-NNA, Mec, and Both rings.

5

7

Endothelium-independent relaxation to SNP was significantly greater in 2d than in 1m rings (Fig. 5, LSD = 12.4%). Not only was maximal relaxation greater in the younger rings (140.1 ± 12.4 vs. 100.4 ± 6.3%) but also the dose-response curve was shifted to the left in the 2d rings, suggesting that they were both more sensitive and more responsive to SNP.

DISCUSSION

Previous studies have described both increases and decreases in endothelium-dependent relaxation during newborn development. For example, several endothelium-dependent vasodilators caused greater relaxation in pulmonary artery rings from 1m compared with fetal lambs (2) and 10d or 30d compared with 3d piglets (32). In contrast, ACh caused greater relaxation of pulmonary artery rings from 3-10d than 3- to 8-wk- or 6-9m piglets (19), and ACh-induced pulmonary vasodilation was greater in hypoxic lungs from 2d than 1d lambs (11). At first glance, our data appeared more consistent with the latter studies, for under Con conditions the receptor-dependent vasodilators, ACh and BK, both caused markedly greater relaxation of third- and fourth- generation pulmonary artery rings from 2d compared with 1m lambs (Figs. 2 A and 3A). Furthermore, tension was lower in 2d rings exposed to high concentrations (10⁵ M) of the receptor-independent vasodilator, A-23187 (Fig. 4 A). However, the percent decrease in PE-induced tension varied among the different vasodilators at both ages (Figs. 2, 3, 4), suggesting that multiple mediators of relaxation may be involved. Furthermore, maximal relaxation to A-23187 and BK occurred at lower concentrations in 1m compared with 2d rings (Figs. 3A and 4 A), suggesting that pulmonary arteries from older lambs were more sensitive to one or more of these mediators. The effects of L-NNA, Mec, and Both on responses of 2d and 1m rings (Figs. 2, 3, 4) suggest that maturational changes in both EDNO and prostanoid activity stimulated by ACh, BK, and A-23187 contributed to the developmental differences in endothelium-dependent responses seen in this study.

It has long been argued that because NOS inhibition attenuated or blocked endothelium-dependent responses, then developmental differences in endothelium-dependent relaxation during the newborn period reflect changes in synthesis of and/or responsiveness to EDNO (2, 11, 19, 22, 32). In this study, we found that the NOS inhibitor L-NNA markedly attenuated both ACh- and BK-induced relaxation and significantly reduced the response to A-23187 in 2d rings (Figs. 2B, 3B, 4B). Thus EDNO probably contributed to the endothelium-dependent responses of the younger rings. In contrast, ACh caused little relaxation of Con 1m rings, and L-NNA had no effect on this response (Fig. 2, A and B). In addition, L-NNA caused only a modest decrease in BK- and A-23187-induced relaxation in 1m rings (Figs. 3B and 4B). Thus these endothelium-dependent vasodilators appeared to stimulate greater EDNO activity in 2d than in 1m rings. It is of note, however, that L-NNA increased PE-induced tension to the same extent at both ages (Fig. 1B). A similar developmental difference between basal and stimulated EDNO activity was also described in isolated lamb lungs (12) and may contribute to the discordance between previous studies showing an increase or decrease in EDNO activity during development.

Whether the apparent developmental decrease in EDNO activity seen in response to endothelium-dependent vasodilators in our study reflects increased endothelial synthesis of or vascular smooth muscle responsiveness to EDNO in younger rings is uncertain, for neither nitric oxide (NO) synthesis nor vascular responses to authentic NO were directly measured. However, SNP caused a greater decrease in tension in 2d rings, and its dose-response curve was shifted to the left when compared with 1m rings (Fig. 5). Although these findings differ from some studies of pulmonary arterial rings from developing piglets and lambs (2, 19, 32), they are similar to the developmental decrease in SNP-induced vasodilation seen in isolated lamb (11) and piglet (22) lungs. Although these data suggest that pulmonary arterial responsiveness to EDNO decreases with age, such a conclusion must be cautiously drawn, for SNP may cause vasodilation through several mechanisms in addition to a NO-induced increase in guanylate cyclase activity (28, 30), and developmental changes in response to SNP may not fully parallel changes in response to EDNO (22, 32).

Maturational changes in synthesis of and /or responsiveness to EDNO were not the only factors contributing to the developmental changes in endothelium-dependent responses seen in this study. The cyclooxygenase inhibitor Mec attenuated the relaxation of 2d rings to ACh and BK (Figs. 2C and 3C). Furthermore, although Mec alone had little effect on the response to A-23187 in 2d rings, the combination of Mec and L-NNA caused a greater reduction in tension than did L-NNA alone. These data are consistent with other studies suggesting that dilator PG contributes to endothelium-dependent relaxation (4, 7, 8, 17, 27). For example, indomethacin reduced ACh-induced relaxation in vascular rings from newborn and juvenile lambs (27), and ACh-induced prostacyclin synthesis attenuated the vasoconstrictor responses to hypoxia and angiotensin II in isolated rat lungs (8). Our observation that Mec failed to attenuate endothelium-dependent relaxation in 1m rings (Figs. 2C, 3C, 4C) suggests that dilator PG activity was greater in 2d than in 1m rings. This was consistent with previous studies by Gordon et al. (10, 13) showing that dilator PG attenuated hypoxic pulmonary vasoconstriction more in lungs from <4d compared with 1m lambs.

Although Mec had little effect on endothelium-dependent relaxation in the 1m rings, it did block the increase in tension seen at high concentrations of all three endothelium-dependent vasodilators (Figs. 2, 3, 4). This finding was consistent with previous studies of mature animals (4, 7, 17) in which endothelium-dependent vasodilators appeared to stimulate synthesis of a constrictor prostanoid such as thromboxane. Indeed, after Mec the 1m rings exhibited significant relaxation in response to maximal concentrations of BK and A-23187, and all age-dependent differences in response to the endothelium-dependent vasodilators were abolished (Figs. 2C, 3C, 4C). Thus our data suggest that both dilator and constrictor prostanoids play a role in the developmental changes in effects of endothelium-dependent vasodilators.

Several other factors may influence developmental changes in endothelium-dependent relaxation. For example, maturational differences in endothelial cell- receptor density or function may have played a role in our study, for BK caused significantly greater relaxation than did ACh at either age (Figs. 2 and 3). In addition, we found that the response to A-23187 was not completely blocked by Both inhibitors at either age (Fig. 4D), suggesting that other mediators may contribute to the response. Several studies have shown that endothelium-dependent vasodilators may cause vascular smooth muscle hyperpolarization (6, 20, 21), but few studies have addressed developmental changes in this response (22, 23). The higher baseline tone typical of younger newborn lungs has also been implicated in their greater endothelium-dependent vasodilation (11). However, this was not a factor in our present study, for the PE-induced tension was greater in 1m than in 2d rings (Fig. 1, top). Similarly, although differences in O₂ tension may have led to differences in EDNO activity, all rings were tested in 95% O₂ in our study, obviating this factor from consideration. Finally, differences in EDNO activity between large and small mesenteric and pulmonary arteries (3, 14) and newborn pulmonary veins and arteries (12, 27) may contribute to the previously reported differences in endothelium-dependent responses of whole lungs and isolated conducting arterial rings.

In conclusion, our findings suggest that although EDNO activity of ovine pulmonary arterial rings appeared to decrease between 2d and 1m, developmental changes in the influence of both constrictor and dilator prostanoids played a major role in the developmental changes in endothelium-dependent relaxation. These findings may, in part, account for the apparent discordance in developmental changes in EDNO activity seen in previous studies. However, other factors, such as differences in basal and stimulated EDNO activity, developmental differences in synthesis of and response to EDNO between arteries and veins, and the potential role of endothelium-dependent hyperpolarization during development, require further investigation.

ACKNOWLEDGEMENTS

This study was supported by a research grant from the American Lung Association (to J. B. Gordon). M. L. Tod was supported by a National Heart, Lung, and Blood Institute FIRST Award (HL-43304) and holds an American Heart Association Established Investigator Award. J. B. Gordon was an Edward Livingston Trudeau Scholar of the American Lung Association.

FOOTNOTES

Address for reprint requests: J. B. Gordon, Children's Hospital of Wisconsin, Pediatric Critical Care Section, Rm. 647, MS 681, 9000 W. Wisconsin Ave., PO Box 1997, Milwaukee, WI 53201.

Received 11 May 1995; accepted in final form 14 June 1996.

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