(1990) 25-28

Pulmonary Pharmacology 3 Q Longman Group UK Ltd

1990

0952-0600/90/0003--0025/$10 .00 PULMONARY PHARMACOLOGY

Pulmonary Clearance of Atrial Natriuretic Peptides N . A . Numan*, M . N . Gillespie, R . J . Altieret University of Kentucky College of Pharmacy, Lexington, Kentucky, USA

SUMMARY. Pulmonary clearance and metabolism of atrial natriuretic peptides were studied in isolated perfused rat lungs utilizing high performance liquid chromatography and radioimmunoassay analyses . The disappearance rate of atrial peptides from the perfusion medium followed pseudo-first order kinetics and was associated with formation of lower molecular weight products. Pretreatment of the lungs with MK-521, an angiotensin converting enzyme inhibitor, decreased pulmonary clearance values for both atrial natriuretic peptide and atriopeptin III but did not affect clearance of atriopeptin II. Aminopeptidase inhibition also decreased the rate of atrial natriuretic peptide clearance, however carboxypeptidase inhibition did not affect atrial natriuretic peptide, atriopeptin III or atriopeptin II clearance . The results suggest that the lung through uptake and metabolic processes participates in the clearance and enzymatic metabolism of the atrial natriuretic peptides .

INTRODUCTION

MATERIALS AND METHODS Isolated perfused rat lungs

The atria of mammalian hearts contain endocrine-like secretory granules which store bioactive proteins referred to as atrial natriuretic peptides. These peptides exhibit diuretic/natriuretic and vasorelaxant activities .' Upon release from the right atrium, atrial peptides must traverse the lungs before entering the systemic circulation . The lung is a highly metabolic organ that plays an important role in the metabolism of a variety of neurohumoral substances .' Pretreatment of the lungs with various peptidase inhibitors alters their vasorelaxant activities,6 suggesting that atrial peptides may be metabolized by peptidases in the pulmonary vascular bed . In addition, atrial natriuretic peptide (ANP) and atriopeptin III (AP III) are cleared from perfusion medium by the lungs . 7 Other investigators have studied the pulmonary uptake of atrial peptides either by bioassay where changes in vascular relaxant activity of the pulmonary perfusates were determined or by measuring disappearance of radiolabeled ANP from the perfusion medium . 9 The current study was undertaken to determine the pulmonary clearance and metabolism of atrial peptides in isolated lungs by measuring the loss of peptides from the perfusate and the appearance of lower molecular weight metabolites using a radioimmunoassay or HPLC separation and detection by ultraviolet spectroscopy .

Rat lungs were isolated and perfused as described previously .6 Male Sprague-Dawley rats (200-250 g) were anesthetized with sodium pentobarbital (50 mg/kg, i .p .) . The trachea was cannulated and positive pressure ventilation (peak inspiratory pressure 9 cm H2 0 ; expiratory pressure of 2 cm H20) with warmed (37°C), humidified room air was initiated using a Harvard respirator . The thoracic cavity was opened by a midline sternotomy and animals were heparinized by intracardiac injection with 400 IU of heparin . The main pulmonary artery and the left ventricle were cannulated. The heart and lungs were removed en bloc and mounted in a water jacketed (37°C), humidified chamber. Ventilation was then changed from room air to a mixture of 95% room air-5% CO 2 and the rate set at 65 breaths/min . Single pass perfusion with 20 ml of prewarmed modified Krebs solution (composition in mM : NaCl, 118 .2 ; KCl, 4 .7 ; CaC1 2 , 2 .5 ; MgSO4 , 1 .2 ; KH2PO4, 1 .2 ; NaHCO3 , 26 .2 ; dextrose, 11 .1 ; no BSA included as it interfered with HPLC analysis) at constant flow (10 ml/min) was instituted with a ColePartner pump to wash residual blood from the lungs . The perfusion of the preparation was then switched to a recirculating system and perfused for a 10-15 min stabilization period . Atrial peptide (0 .1-0 .2 mg) was added to 20 ml of perfusate and recirculated through the system excluding the lungs for a 2 min equilibration period . The preparation was then reconnected and perfusion of the lungs with atrial peptide was initiated . Status of the lungs, particularly with regard to development of edema, was assessed by monitoring pulmonary artery perfusion pressure from a side-arm

* Present address : Division of Pharmacology and Toxicology, College of Pharmacy, University of Baghdad, Iraq . t To whom correspondence should be addressed . Present address : University of Colorado, School of Pharmacy, Campus Box 297, Boulder, CO 80309-0297 USA . 25



26 Pulmonary Pharmacology

off the inflow cannula . A rapid rise in perfusion pressure indicated edema and the lungs were discarded . Aliquots were taken from the reservoir at predetermined time points during the perfusion . The peptide content in an accurate volume (0 .1 ml) of perfusate was determined by reverse-phase high performance liquid chromatography (HPLC) on a 4 .6 X 250 mm ODS column eluted at I ml/min with an ionpairing mobile phase (24% acetonitrile in phosphate buffer pH 3, containing 0 .01 M triethylamine and 0 .1 % trifluoracetic acid) . Peptide products eluted from the column were detected by UV absorption (205 nM) . Retention times and peak heights were compared with those produced by analysis of known concentrations of atrial peptides. The concentrations of atrial peptides in lung perfusates were determined from standard curves of peptide concentration vs peak height . When used, peptidase inhibitors were added to the perfusate reservoir and perfused through the lungs for 20 min before atrial peptides were administered . Similar procedures were used when lower concentrations (1 ,ug/ml) of ANP or AP III were perfused through the lungs . Aliquots of perfusate were collected at predetermined time points and separated by HPLC . Because the concentrations of the peptides were too low to be detected by ultraviolet spectroscopy, fractions from the HPLC effluent were collected at previously determined retention times for ANP and AP III and the peptide content was analysed by radioimmunoassay (RIA kit 9103, Peninsula Labs) (ANP and AP III fractions were not collected separately when AP III was perfused through the lungs) . These experiments were restricted to a 5 min perfusion and collection period to limit the number of samples to be processed by HPLC separation and RIA analysis .

RESULTS The reverse phase HPLC analysis system provided a sensitive and accurate method to measure the concentrations of atrial peptides in perfusate samples . Figure 1 illustrates a typical chromatogram of the peptides used in the current study . Atrial peptides disappeared from the perfusion medium in a curvilinear, time-dependent manner suggesting a first-order process . Atrial natriuretic peptide (ANP) was metabolized to AP III and AP I (Fig . 2) . Similarly, AP III was metabolized to AP II and AP I and AP II was metabolized to AP I . Inhibition of carboxypeptidase N by 1 mM guanidinoethylmercapto-succinic acid (GEMSA)10 had no effect on ANP, AP III or AP II metabolism nor did it affect the rate of disappearance of these peptides from the perfusion medium . Pretreatment with the

APII

ANP

P

APIII

1 .-A 2 min.

INJECTION

Fig. 1-Typical HPLC chromatograms of atrial natriuretic peptide (ANP), atriopeptin I, II and III (API, AP II and AP III) .

MATERIALS The following drugs, chemicals and peptides were used : rat atrial natriuretic peptide (ANP), atriopeptins I, II and III (AP I, AP II and AP III) (Calbiochem or Peninsula Laboratories) ; bestatin (Sigma) ; MK-521 (Merck) ; guanidinoethylmercapto-succinic acid (GEMSA) (Calbiochem) .

5 .0

• E lL O

ANP 1API o AP III

4 .0

°'

z cn 3 .0 0 W N Q 0 Ir F-

Data analysis All data are expressed as concentration of peptide per ml of perfusate vs time of perfusion . Clearance of atrial peptides from the perfusion medium by the lungs was calculated according to the equation Clearance (ml/min) = Dose (µg) AUCO-• oo (,ug/ml x min) where dose= total amount of atrial peptide added to reservoir and AUC = area under the perfusate concentration vs time curve .

z w 2 .0 u.J a. CJ J z Q

a

1 .0 0-, 1 0

5

10

15

,

20

25

TIME (min)

Fig. 2-Disappearance of ANP during perfusion through isolated rat lungs and conversion to AP III and AP I (n =4) .



Pulmonary Clearance of Atrial Natriuretic Peptides

angiotensin converting enzyme inhibitor, MK-521 11 (10 pM), prevented formation of AP I from both ANP and AP III . MK-521 also decreased pulmonary clearance of ANP and AP III (Table 1) but it did not affect pulmonary clearance or metabolism of AP II . In separate experiments, lungs were perfused with lower concentrations of atrial peptides (1 pg/ml) . Because the concentrations of atrial peptides in perfusates were below the limits of detection using ultraviolet spectroscopy, peptide concentrations were estimated using radioimmunoassay . Since the RIA antibody reacts with ANP and AP III, they were the only peptides utilized in these studies . ANP disappeared from the perfusion medium in a curvilinear, time-dependent manner with formation of AP III (Fig . 3) . MK-521 (10,W) inhibited clearance of ANP and led to accumulation of AP III . Aminopeptidase inhibition by bestatin (10 µM) inhibited clearance of 400 *ANP oAPlll E g 300 _ z 0

c zz 0 200 Z O

Table l . Clearance values for atrial peptides perfused through isolated rat lungs in the absence and in the presence of peptidase inhibitors . Atrial peptide

ANP ANP+MK-521 (10µM) ANP+Bestatin (10µM) AP III APIll+ MK-521(10µM) AP II

Clearance (ml/min) • 5-10 µg/mlb 1 µg/mlb 3 .34 1 .52 n.d ., 0.88 0.69 0.89

10 .95 0 .78 0 .09 0 .73 0 .21 n .d .

a

Clearance values calculated from mean curve of peptide concentration vs (n = 2 to 5) . Values for individual experiments did not differ by greater than 15% . bConcentration of atrial peptide in perfusion medium . °n.d .-not determined

ANP and AP III but did not affect formation of AP III from ANP . MK-521 (10,W) inhibited disappearance of AP III from the perfusion medium (Fig . 4). Effects of peptidase inhibition on pulmonary clearance values are summarized in Table 1 .

\\ DISCUSSION I I

F

a-,--i

a E

27

Q

ff

100

a

a 0 0

I

3 TIME (min)

Fig . 3-Uptake and metabolism of ANP by isolated perfused rat lungs determined by RIA analysis (n = 4) . Points shown before zero time indicate amount of ANP and AP III present in perfusate before adding exogenous ANP . Similar concentrations were obtained at the end of the experiment (points shown after 5 min mark) when fresh physiological salt solution was recirculated through lungs for 15 min . 2000

• AP III

o APIII+MK - 521

1050

E z

az r

z „ 1000

0 aW 00 950

S

5

TIME (min.)

Fig. 4-Effect of MK-521 (10,W) treatment on pulmonary uptake and metabolism of AP III determined by RIA analysis (n = 3) .

The disappearance rate of atrial peptides from the systemic circulation is rapid with half-lives ranging from 16 .8 sec to 2 .5 min, 12-16 indicating that their disappearance is likely the result of binding to receptors on cells or due to breakdown of the peptides in various organs and tissues . Tang et al .12 found the relative degradation rates of AP III in various organs to be kidney > liver > lung>plasma> heart and that smaller peptide fragments were formed in these tissues . Other investigators" reported that hepatectomy and nephrectomy did not cause major prolongation of disappearance rates for ANP, Arg-Arg-AP III and AP III, suggesting that these two organs may not be the primary sites involved in removal of these peptides from the circulation . The lung, a highly metabolic organ that takes up and metabolizes numerous substances from the blood, may be expected to participate in the removal and/or degradation of atrial peptides from plasma . Weselcouch et al .e reported that approximately 25% of atrial peptide vasodilator activity was lost when a crude atrial extract was perfused through isolated guinea-pig lungs and that the vasorelaxant activity of AP II was decreased 21 % upon passage through the lungs . From results of the current study which showed that atrial peptides were cleared from the perfusate and metabolized upon passage through the pulmonary circulation, the decrease in vasorelaxant activity of atrial peptides observed by Weselcouch et al . 8 probably resulted from uptake and/or metabolism of atrial peptides by the pulmonary vasculature . Turrin and Gillis9 studied removal of 125 1-ANP by rabbit lungs and observed that pulmonary removal of this peptide (66 .9% upon single passage through the

28

Pulmonary Pharmacology

lungs) was a saturable and reversible process that did not appear to involve binding to angiotensin converting enzyme present on pulmonary vascular endothelial cells . In the current study, MK-521, an inhibitor of angiotensin converting enzyme, decreased clearance of ANP and AP III, suggesting that converting enzyme in the pulmonary vasculature may contribute to the clearance of ANP and AP III by the lungs . The difference between these results and those of Turrin and Gillis 9 may be due to labeling of ANP with iodine which may cause a loss in binding affinity,' -e.g. to converting enzyme . Alternatively, an enzyme other than angiotensin converting enzyme that is sensitive to inhibition by MK-521 may degrade ANP and AP III . Another difference between these studies is the considerably greater uptake observed by Turrin and Gillis 9 compared to the clearance of ANP observed in our study . The discrepancy is likely due to the concentrations of ANP used in the two studies . With radiolabeled ANP, a very low concentration (6 .5 pmoles) of the peptide was perfused through the lungs, whereas in the current study a considerably higher concentration (30-60 nmoles) was used to permit detection of metabolic products by ultraviolet absorbance . It is probable that sites of removal were being saturated, thereby causing an apparently lower clearance rate of ANP and other atrial peptides . To investigate this possibility, a separate study was conducted utilizing lower concentrations of ANP and AP III . The results indicated that saturation of removal sites at high concentrations of atrial peptides was likely for ANP because the clearance rate was greater when a lower concentration of this peptide was perfused through the lungs (Table 1) . Similar results were not observed for AP III, perhaps because ANP (converted to AP III) and/or AP III secreted from the heart or lungs was measured, thereby interfering with determination of the true clearance rate of AP III . From a prior study, which showed that pulmonary vasodilator properties of atrial peptides were enhanced by peptidase inhibitors 6 and, the current study on the clearance and metabolism of atrial peptides by the lung, a tentative degradation pathway for atrial peptides upon passage through the pulmonary I

DES-SER - AP III 1,2

DES-SER -AP III

Fig. 5-Tentative pathway for degradation of atrial peptides in the pulmonary circulation . APase = aminopeptidase ; CPase = dipeptidyl or other carboxypeptidase associated with metabolism of AP III or AP II .

vascular bed may be proposed as shown in Figure 5 . To summarize, atrial peptides are partially cleared by the lungs where they can be converted to lower molecular weight atrial peptides (and perhaps to smaller fragments) by peptidases in the pulmonary vasculature, suggesting that the lungs participate in the removal and degradation of circulating atrial peptides, especially those secreted from the right heart . Acknowledgements The authors wish to thank Melinda J . McIntyre for preparation of the illustrations and the University of Kentucky Medical Center Research Fund for support of this work . References 1 . DeBold A J . Atrial natriuretic factor : a hormone produced by the heart. Science 1985 ; 230 : 767-770. 2 . Atlas S A, Laragh J H . Atrial natriuretic peptide : a new factor in hormonal control of blood pressure and electrolyte homeostasis. Ann Rev Med 1986 ; 37 : 397-414. 3 . Palluk R, Gaida W, Hoefke W . Atrial natriuretic factor . Life Sci 1985 ; 36 : 1415-1425 . 4 . Thibault G, Garcia R, Gutkowksa J, Genest J, Cantin M . Atrial natriuretic factor . A newly discovered hormone with significant clinical implications. Drugs 1986 ;31 :369-375 . 5 . Gillis C N . Pharmacological aspects of metabolic processes in the pulmonary microcirculation . Ann Rev Pharmacol Toxicol 1986 ; 26 : 183-200 . 6 . Numan N A, Gillespie M N, Altiere R J . Pulm Pharmacol, submitted for publication . 7 . Numan N A, Gillespie M N, Altiere R J . Metabolism of atrial natriuretic peptides by isolated perfused rat lungs . Fed Proc 1986 ; 45 : 907 . 8 . Weselcouch E 0, Humphrey W R, Aiken J W . Effects of pulmonary and renal circulations on activity of atrial natriuretic factor . Am J Physiol 1985 ; 249 : R595-R602 . 9 . Turrin M, Gillis C N . Removal of atrial natriuretic peptide by perfused rabbit lungs in situ . Biochem Biophys Res Commun 1986 ; 140 : 868-873 . 10 . Plummer T H, Ryan T J . A potent mercapto bi-product analogue inhibitor for human carboxypeptidase N . Biochem Biophys Res Commun 1981 ; 98 : 448-454 . 11 . Patchett A A, Harris E, Tristram E W et al . A new class of angiotensin-converting enzyme inhibitors . Nature 1980 ; 288 : 280-283 . 12 . Tang J, Webber R J, Chang D, Chang J K, Kiang J, Wei E T . Depressor and natriuretic activities of several atrial peptides . Regul Peptides 1984 ; 9 : 53-59 . 13 . Yandle T G, Richards A M, Nicholls M G, Cuneo R, Espiner E A, Livesey J H . Metabolic clearance rate and plasma half life of alpha-human atrial natriuretic peptide in man . Life Sci 1986 ; 38 : 1827-1833 . 14 . Luft F C, Lang R E, Aronoff G R, Ruskoaho H, Toth M, Ganten D, Sterzel R B, Unger T . Atriopeptin III kinetics and pharmacodynamics in normal and anephric rats . J Pharmacol Exp Ther 1986 ; 236 : 416-418 . 15 . Murthy K K, Thibault G, Schiffrin E L, Garcia R, Chartier L, Gutkowska J, Genest J, Cantin M . Disappearance of atrial natriuretic factor from circulation in the rat. Peptides 1986 ; 7 : 241-246 . 16 . Katsube N, Schwartz D, Needleman P . Atriopeptin turnover : quantitative relationship between in vivo changes in plasma levels and atrial content . J Pharmacol Exp Ther 1986 ; 239 : 474-479. Date received : 25 December 1988 Date revised : 5 July 1989 Date accepted : 15 August 1989