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(Circulation. 1996;94:1578-1584.)
© 1996 American Heart Association, Inc.

Articles

Human Pulmonary Circulation Is an Important Site for Both Clearance and Production of Endothelin-1

Jocelyn Dupuis, MD; Duncan J. Stewart, MD; Peter Cernacek, MD; Gilbert Gosselin, MD

the Department of Medicine, Montreal Heart Institute, St. Michael's Hospital, Toronto, and the Royal Victoria Hospital, Montreal, Canada.

Correspondence to Jocelyn Dupuis, MD, Montreal Heart Institute, 5000 Belanger St, Montreal, Quebec H1T 1C8, Canada.

	Abstract

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Background Animal studies suggest a major role of the pulmonary circulation in the clearance of circulating endothelin-1 (ET-1). The contribution of the human pulmonary circulation to plasma ET-1 clearance, however, has never been quantified. The absence of an AV gradient in plasma ET-1 has previously been interpreted as evidence that the lungs do not have a role in modulating circulating ET-1 levels. This study was designed to quantify and discern between pulmonary ET-1 clearance and production in humans.

Methods and Results We studied 13 subjects by combining the multiple indicator-dilution technique with the measurement of immunoreactive ET-1 (irET-1). All patients had normal left ventricular ejection fractions (61±7%, mean±SD) and baseline hemodynamics. Mean pulmonary ET-1 extraction was 47±7%. The ET-1 extracted does not return to circulation and can be characterized by a sequestration rate constant: K_seq=0.048±0.019 s^-1. There was no significant difference between irET-1 levels from the pulmonary artery and aorta (0.61±0.29 and 0.68±0.33 pg/mL, respectively; P=.22); the normal lung consequently produces an amount of ET-1 that is quantitatively similar to the amount that has been extracted.

Conclusions The human lung is an important site for both clearance and production of ET-1. There is a normal physiological balance of ET-1 across the pulmonary circulation, which explains the absence of difference in AV ET-1 levels despite a 47±7% clearance. Reduced pulmonary clearance or increased production of this peptide may contribute to the increase in circulating levels found in various cardiovascular conditions.

Key Words: endothelin • circulation • metabolism • endothelium

	Introduction

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Endothelin-1 (ET-1) is a 21–amino-acid peptide with potent vasoconstrictive and proliferative actions on the vascular smooth muscle and other cells.¹ The vascular endothelium produces ET-1 in a preferential paracrine fashion with a smaller clinically measurable luminal secretion.² Numerous animal studies suggest that the lungs are a major site for ET-1 clearance^{3 4 5 6 7 8 9} and production^{10 11} and that they may modulate circulating ET-1 levels. The role of the human pulmonary circulation in the clearance and production of circulating ET-1, however, has never been established. In the normal human, there is no significant difference between immunoreactive ET-1 (irET-1) levels across the pulmonary circulation^{12 13} ; a simple analysis of this observation would be that the human lung is metabolically inactive toward ET-1 and does not modify circulating ET-1 levels. A more complex "black box" hypothesis would be that there is a metabolic balance for ET-1 across the human pulmonary circulation; the human lung may be an important site for ET-1 clearance but with a simultaneous equal release into circulation that explains the observed absence of an AV gradient in ET-1 levels. Such a balance is present in dogs with a mean pulmonary ET-1 clearance of 31±8% and a simultaneous quantitatively similar release³ (there also is no AV ET-1 gradient across dog lungs).

Circulating ET-1 levels are increased in conditions of primary or secondary pulmonary hypertension^{14 15 16 17} and may be a marker or mediator of disease.¹⁸ In heart failure, the ET-1 levels are linearly related to the severity of pulmonary hypertension.^{19 20} Some patients with pulmonary hypertension also have a positive ET-1 gradient across the lungs^{14 15 21} ; the immunoreactive levels in the systemic circulation are slightly higher than in the mixed venous blood. The human pulmonary circulation may thus modulate circulating ET-1 by maintaining a normal physiological balance and contribute to the pathological hyperendothelinemia by an increased production, a reduced clearance, or a combination of both.

The importance of the human pulmonary circulation in the clearance or production of ET-1, however, is still unknown. We designed a set of experiments to quantify and discern between pulmonary clearance and production of ET-1 in humans without pulmonary hypertension. Combination of the multiple indicator-dilution technique and simultaneous measurements of irET-1 levels across the lungs enables dissection of the black-box elements and was used for this purpose.

	Methods

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Study protocol and the consent form were designed in accordance with the research and ethics committees of the Montreal Heart Institute. Thirteen subjects were recruited among patients awaiting elective diagnostic coronary angiograms for diagnosed or suspected stable angina pectoris. Criteria for exclusion were history of heart failure, previous myocardial infarction, pulmonary hypertension, diabetes, and renal failure with a creatinine level 200 mmol/L.

The study protocol was carried out before the coronary angiogram and left ventriculogram. The right femoral artery and vein were punctured by use of the Seldinger technique, and introducers were inserted. A pigtail catheter was positioned about 2 cm above the aortic valve, and a multipurpose catheter was positioned in the right ventricular outflow tract just below the pulmonary valve. Companion aortic and mixed venous blood samples were simultaneously obtained in EDTA-containing tubes for determination of irET-1 levels. A 2-cm extension with a three-way stopcock was added to the venous catheter, and a 2-mL bolus mixture for the indicator-dilution study was introduced into the catheters (the bolus consequently was completely contained within the injection system). The arterial catheter was then connected to a Masterflex Roller pump that was connected to a circular fraction collector. The dilution experiment was carried out by rapid flushing of the bolus with 10 mL of the patient's and with simultaneous collection of aortic blood in 50 consecutive tubes (0.6 seconds per tube) at 200 mL/min. The angiographic procedure was then performed as scheduled. A 3-mL blood sample was taken from the injection lines to determine the amount of activity retained in the injection system.

Injection Mixture Preparation
The preparation was carried out under sterile conditions. Human albumin (25%) was added to 3 mL Evans blue dye to obtain a final bolus concentration of 4% of albumin. The blue dye binds tightly to albumin, so this procedure results in labeling of albumin, the concentration of which can be determined later spectrophotometrically. ¹²⁵I-labeled ET-1 (10 µCi; Du Pont–New England Nuclear; specific activity, 2200 Ci/mmol) was then added to the mixture. The final volume was 3.5 mL per experiment: 2 mL was taken to be used as the injection mixture, and the remaining 1.5 mL was kept for the preparation of dilution curve standards.

Sample Processing
All collected tubes, 1/10- and 1/100-diluted standards, and injection line samples were treated identically. Blood (200 µL) was pipetted from each tube and assayed in a gamma counter to determine ¹²⁵I activity. The remaining samples were centrifuged at 3000 rpm for 10 minutes, and 100 µL plasma was pipetted and added to 1.0 mL of 0.9% NaCl in a spectrophotometer cuvette. Evans blue dye absorbance was determined as the difference between the absorbance at 620 and 740 nm.

Indicator-Dilution Curve Construction and Analysis
The activity of tracer ET-1 and tracer albumin for each tube was corrected for background activity. The exact quantity of tracer injected was determined by subtracting the activity retained in the injection system from the amount contained in 2 mL injection mixture. For each sample, the fractional recovery of tracer ET-1 and albumin could then be determined. A plot of the fractional recoveries per 1 mL blood as a function of time was then constructed to obtain the indicator-dilution curve for each tracer. The albumin was used as a plasmatic vascular reference tracer; it remains in the vascular space within a pulmonary transit time. A plasmatic tracer that is cleared from the pulmonary circulation will exhibit an outflow profile that deviates from that of albumin; its fractional recovery per 1 mL blood at each time point will be lower than that of albumin. This technique has been used extensively to quantify microcirculatory events in the pulmonary circulation in vivo. It helped characterization of the pulmonary hydrolysis of ACE substrates,²² clearance of vasoactive amines like serotonin and norepinephrine,²³ and more recently pulmonary clearance of ET-1 in dogs.³

The pulmonary blood flow was computed with the following formula:

where q_o is the total amount of Evans blue dye–labeled albumin injected. The integral in the denominator represents the area under the extrapolated fractional recovery versus time curve for tracer albumin. The downslope of indicator-dilution curves is characteristically semiexponential, so its terminal portion can easily be linearly extrapolated on a logarithmic scale to remove any parasite recirculation of the tracers.

The pulmonary mean transit time (MTT) for each tracer is then calculated as

where C is the tracer outflow fractional recovery and _cath is the mean transit time of the collection system. This computed transit time includes the pulmonary large vessels and microcirculatory transit time plus the transit time of the left chambers of the heart.

Instantaneous tracer ET-1 extraction (EXT[t]) for each time point is calculated with the following:

Cumulative tracer ET-1 extraction for each curve is obtained by

where the numerator and denominator are the areas under the extrapolated fractional recovery versus time curve for tracer ET-1 and albumin, respectively. Mean tracer ET-1 extraction represents the difference between the area of these two curves.

From the outflow profile relationship of the two tracers, we can also estimate the sequestration rate constant for pulmonary ET-1 clearance (K_seq). We have previously discussed the significance of K_seq in terms of ordinary capillary modeling.³ It represents the product of capillary permeability to endothelin and the surface-to-volume ratio of the capillaries.

t_o, the common large-vessel transit time, cannot be determined in our preparation.

In the present experiment, in the absence of correction for catheter distortion and large-vessel transit time, K_seq represents a lower-bound estimate: it will be slightly smaller than the actual value. K_seq can be obtained by linear regression of log ratio plots for ET-1 and albumin (Fig 2).

View larger version (11K): [in this window] [in a new window]   Figure 2. Natural logarithm of the ratio of the fractional recovery for albumin and tracer endothelin-1 (ET-1) as a function of time (from patient 6; see Fig 1). This plot depicts a linear increase over the entire primary dilution curve. A sequestration rate constant, Kseq, characterizing the unidirectional pulmonary removal of circulating ET-1, can be estimated from the slope of this relationship. See text for details.

irET-1 Measurements
Measurements were done according to a previously described technique.¹⁴ Before the bolus injection, paired samples of 10 mL blood were simultaneously collected from the aorta and the right ventricular outflow tract in EDTA-containing tubes and centrifuged at 1800g for 20 minutes. Plasma samples were then extracted with SepPak C₁₈ cartridges (Waters) that had been activated with methanol, 8 mol/L urea, and water. Endothelin was eluted with 100% methanol, yielding a recovery of 75±3.3% (mean±SD). Samples and standards (ET-1; Peninsula Laboratories) were reconstituted in assay buffer and incubated for 24 hours with rabbit anti–ET-1 serum (Peninsula Laboratories) at 4°C. The addition of 4000 counts per minute of ¹²⁵I-ET-1 (Amersham) was followed by a second 24-hour incubation. Bound and free radioligands were separated by use of the second antibody method. The bound radioactivity data were evaluated after logit-log transformation.

The antibody exhibited a cross-reactivity of 10% for human big endothelin and 5% with endothelin-3 but no cross-reactivity with unrelated peptides (atrial natriuretic factor[1-28], brain natriuretic peptide, vasopressin, and angiotensins I and II). The limit of detection, defined as the least amount of irET-1 distinguishable from zero at a 95% confidence level, was 0.12 pg per tube. The intra-assay and interassay coefficients of variation were 9% and 12%, respectively, at the midpoint of the standard curve. Heparin in concentrations exceeding those achieved in the present experiments did not interfere with the assay. High-performance liquid chromatography of the plasma extract showed a dominant peak of irET-1 coeluting with the synthetic ET-1.

Endothelin Clearance and Production
Pulmonary ET-1 clearance was computed as follows:

where F_p is pulmonary plasma flow [pulmonary blood flow multiplied by (1-hematocrit)], MV_ET-1 represents mixed venous irET-1 level, and EXT is the mean cumulative ET-1 extraction.

Pulmonary ET-1 production was computed as follows:

where net pulmonary ET-1 balance is

Consequently, if aortic ET-1 levels are identical to MV_ET-1 levels, net ET-1 balance is 0 pg/min, and ET-1 clearance is equal to ET-1 production.

Statistical Analysis
All values are reported as mean±SD. When applicable, linear regression was used to detect correlations between different parameters. Two-tailed t tests were used to compare aortic and mixed venous ET-1 levels and pulmonary clearance and production of ET-1. Values were considered significantly different if P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
All patients had normal systemic and pulmonary hemodynamic parameters (Table 1). Mean left ventricular ejection fraction was 61±8%, and all patients had normal left ventricular angiograms.

View this table: [in this window] [in a new window]   Table 1. Demographic and Hemodynamic Parameters of the Study Patients Undergoing Elective Diagnostic Coronary Angiography

Fig 1 shows a typical set of indicator-dilution curves obtained from patient 6 in linear (top) and logarithmic ordinate formats (bottom). Tracer albumin and ET-1 outflow profiles progressively diverge, the outflow fractional recovery of ET-1 becoming a progressively smaller proportion relative to that of tracer albumin; this is particularly evident on the logarithmic plot. Tracer recirculation is mathematically removed by linear extrapolation of the semilogarithmic downslope of the curves as shown in Fig 1. The difference between the areas of the extrapolated curves of albumin and ET-1 represents cumulative tracer ET-1 extraction during a single pulmonary transit time (52% for the patient shown in Fig 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Typical set of indicator-dilution curves obtained from patient 6. The fractional recovery per 1 mL blood for each tracer is plotted as a function of time in linear (top) and logarithmic (bottom) ordinate formats. Fractional recovery for tracer endothelin-1 (ET-1) rapidly diverges from the albumin, becoming a progressively smaller fraction with time. Linear extrapolation of the semilogarithmic downslope enables the mathematical removal of recirculating tracer. The difference between the areas of the extrapolated curves represents the cumulative circulating ET-1 extraction during a single pulmonary transit time. In this experiment, the extraction was 52%. The arrow on the abscissa represents the transit time of the collection system.

Indicator-dilution curve–derived parameters are assembled in Table 2. The mean pulmonary transit time for albumin was 11.9±1.4 seconds; that for tracer ET-1 was slightly lower at 11.4±1.4 seconds (P<.001). The central blood volume (composed primarily of pulmonary blood) was 1008±169 mL. Cardiac output and cardiac index were within normal limits: mean cardiac output, 5.16±1.01 L/min; mean cardiac index, 2.82±0.52 L·min^-1·m^-2. There was a substantial cumulative tracer ET-1 extraction in every patient ranging from 34.9% to 60.7% (mean, 47.4±7.1%). There was no correlation between ET-1 extraction and cardiac index (r=-.33, P=.27) or mean pulmonary artery pressure (r=-.164, P=.59). For each patient, instantaneous ET-1 extraction (the extraction at each time point of the dilution curves) increases linearly (not shown). By plotting the logarithmic ratio of the outflow profile of albumin over endothelin, we can gain additional insight into the microcirculatory behavior or of circulating ET-1. This ratio (Fig 2; patient 6) increases linearly over the entire primary dilution curve. This typical microcirculatory behavior of substances modified or inactivated by the pulmonary circulation has previously been well described and studied but is confirmed here for the first time in the human pulmonary circulation. This linear increase in extraction with time reflects the heterogeneity of the pulmonary capillary transit time distribution.²⁴ Early in time, capillaries with shorter transit times contribute to the outflow profile. Later in time, capillaries with progressively increasing transit times contribute to an increase in ET-1 extraction. This resting heterogeneity of the pulmonary circulation has already been well described for ET-1 and for serotonin in dogs.^{3 24} The major difference for a tracer like serotonin is an apparent leveling off and plateau of the log ratio plot caused by return (or reentry) of tracer serotonin into circulation after it has been extracted. In this instance, the linear increase in the log ratio plot is maintained over the entire primary dilution curve, suggesting that ET-1 is extracted by the pulmonary circulation following a unidirectional process without any return of the extracted tracer to circulation.

View this table: [in this window] [in a new window]   Table 2. Pulmonary Clearance and Production of Circulating ET-1 in Humans From Indicator-Dilution Tracer ET-1 Extraction and Immunoreactive ET-1 Measurements Across the Pulmonary Circulation

Mean aortic irET-1 levels were not significantly different from mean pulmonary artery values: 0.68±33 and 0.61±0.29 pg/mL, respectively; P=.22 (Table 2). The absence of a significant AV difference in irET-1 across the pulmonary circulation in the presence of 47% mean cumulative tracer ET-1 extraction therefore supports a production of ET-1 quantitatively similar to the extracted amount. For each experiment, the indicator-dilution data were combined with irET-1 levels to determine net ET-1 clearance and production by the lungs. Mean ET-1 clearance is 884±458 pg/min, whereas production is slightly but nonsignificantly higher at 1101±682 pg/min (P=.15; Table 2). Fig 3 gives the correlation between aortic and pulmonary irET-1 plasma values. There is a close linear relationship between the two following the regression equation,

(r=.85, P<.001). Despite a notable variability in individual irET-1 measurements, the regression line does not systematically deviate from a line of identity.

View larger version (15K): [in this window] [in a new window]   Figure 3. Plasma immunoreactive (ir) endothelin-1 levels measured simultaneously in the pulmonary arteries and aortas of patients undergoing diagnostic coronary angiograms. The solid line is a regression through the data points. The dotted line, representing a perfect correlation, is included for comparison.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have shown that the human lungs are an important site for both clearance and production of circulating ET-1. Within a single pulmonary transit time, 47±7% of ET-1 is extracted from the pulmonary circulation, with a simultaneous quantitatively similar release explaining the absence of an AV difference in irET-1 levels across the pulmonary vascular bed.

Previous studies have interpreted the lack of an ET-1 gradient across the human pulmonary circulation as evidence for the absence of extraction or release of this peptide by the lungs.^{12 13} The present analysis, however, clearly indicates that such conclusions must be made with caution when the experiments are not designed to simultaneously quantify and discern between clearance and production. In this study, the measurement of tracer ET-1 extraction combined with the simultaneous measurements of irET-1 levels enabled such an analysis. Such a methodology was previously validated in anesthetized dogs³ : the dog lungs removed 31±8% of tracer ET-1 with an equal production also explaining the absence in ET-1 gradient across the pulmonary vascular bed. The important role of the pulmonary circulation in the removal of circulating ET-1 has been established in various species: in rats, 82% of injected ET-1 is found in the lungs, with a smaller proportion in the kidneys.⁵ With the use of various techniques, it has been demonstrated that the lungs of dogs, rats, rabbits, and guinea pigs could extract 40% to 70% of circulating ET-1, depending on the species and methodology used.^{3 5 6 7 8 9} This accumulating fundamental data strongly suggested a major role for the pulmonary circulation in the removal of circulating ET-1. This study confirms this important role in humans. A more detailed analysis of the indicator-dilution outflow profile for tracer ET-1 in relation to the albumin tracer supports the concept of heterogeneity of pulmonary capillary transit time distribution previously shown in animals.^{3 24} The outflow profile also indicates that ET-1 is removed unidirectionally following a single rate constant without any return of the extracted tracer to circulation in a single transit time. A simple model describing these events was applied to the data, and a sequestration rate constant for ET-1 was estimated: K_seq=0.048±0.019 s^-1. This value is slightly lower than the value we obtained in dogs with the same technique: K_seq=0.056±0.016 s^-1.³ In rats, injected ET-1 has a half-life of <7 minutes, and no ET-1 degradation products can be found in the circulation up to 60 minutes after injection.⁶ The retained ET-1 is found mostly in the lungs, associated with membranes and intracellular organelles.⁷ These observations, together with the present results, suggest that the extracted ET-1 is sequestrated by the vascular endothelium and does not return to circulation. Receptor-mediated endocytosis may mediate this extraction; the ET_B receptor is present in the vascular endothelium, and the specific ET_B antagonist BQ788 can reduce removal of ET-1 from the circulation.²⁵

In normal subjects and patients with coronary artery disease, Stewart et al¹⁴ found slightly but significantly lower irET-1 levels in the aorta than in the pulmonary artery with a ratio that was significantly lower than unity (0.54±0.64 for coronary patients, P<.02). This led the authors to conclude that there may be net ET-1 clearance across the normal pulmonary circulation. In the same study, pulmonary hypertension patients had a ratio equal to (secondary hypertension) or greater than (primary hypertension) unity, suggesting a reduced clearance with possibly a net ET-1 production in pulmonary hypertensive conditions. Three other studies (including this one), however, found no significant AV difference in ET-1 levels across the human pulmonary circulation.^{12 13} It is possible that the different sampling site in the study of Stewart et al¹⁴ may have contributed to the lower AV ratio; some arterial samples were taken from the radial artery, and some venous samples were taken from the antecubital vein rather than from a mixed venous site. The intrinsic limitations of the irET-1 assay need to be considered in the overall analysis of these data. The intra-assay variability itself can explain many of the individual differences in paired aortic and pulmonary samples; mean values must therefore more adequately reflect true differences. In the present study, there was no significant difference between mean aortic and pulmonary artery irET-1 values.

The absence of an irET-1 gradient across the lungs, in the presence of simultaneous tracer ET-1 extraction (47±7%), suggests that the lung produces an amount of ET-1 similar to the amount that has been extracted. The human lung expresses ET-1 mRNA,¹¹ and this expression is increased in patients with pulmonary hypertension.²⁶ The pulmonary production of ET-1 therefore significantly contributes to circulating ET-1 levels, which explains the normal physiological ET-1 balance across the lungs.

Primary and secondary pulmonary hypertensions are associated with increased circulating ET-1 levels.^{14 15 16 17 19 20 21} In congestive heart failure, there is a direct correlation between pulmonary artery pressure and circulating ET-1 levels.^{19 20} In these patients, plasma ET-1 levels increase significantly from the pulmonary artery to the capillary wedge region, with a consequent net pulmonary ET-1 production that is also proportional to pulmonary vascular resistance.²¹ It is still debated whether circulating ET-1 is a simple marker or mediator of disease. ET-1 infused at pathophysiological concentrations exerts significant hemodynamic effects and may thus contribute to the counterregulatory mechanisms of chronic congestive heart failure.^{27 28} The present study brings new insight into the mechanisms of increased circulating ET-1 levels in humans. The lung may contribute to this process through a reduced clearance, an increase in production, or a combination of both. An increase in ET-1 production in pulmonary hypertension has been shown in humans.²⁶ In the monocrotaline hypertensive rat, a reduced expression of the ET_B receptor has been established.²⁹ In normal dogs, the nonspecific ET_A and ET_B antagonist bosentan acutely increase circulating ET-1 levels up to 30-fold.³⁰ Downregulation or reduced sensitivity of the ET_B receptor may therefore contribute to increases in circulating ET-1 levels.

The human lung is an important metabolic organ. It produces, modifies, and inactivates various circulating amines and peptides. We must now add ET-1 to the list of its substrates and products. Close to 50% of circulating ET-1 is cleared in a single passage, with a simultaneous equal production that explains the normal physiological ET-1 balance across the human lungs. Future studies are needed to verify whether pharmacological or pathological alteration of this balance may modify circulating ET-1 levels. These new concepts should help in the clinical interpretation of increased circulating ET-1 values.

	Acknowledgments

 
This work is supported by the Medical Research Council of Canada, the Fonds de la recherche en sante du Quebec, and the Canadian Heart and Stroke Foundation. We would like to thank Nathalie Ruel and Dominique Blais for their expert technical assistance, the staff of the invasive hemodynamic laboratory for their constant collaboration, and Claire Bertrand-St-Hilaire for typing the manuscript.

Received December 27, 1995; revision received April 16, 1996; accepted April 23, 1996.

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