Angiotensin-Converting Enzyme Activity in the Isolated Perfused Guinea Pig Lung
L. MOR, A. BOMZON, R. FRENKEL, AND M. B. H. YOUDIM
We have developed a pressure-dependent isolated lung perfusion system that can be used for the determination of pulmonary enzyme activity and kinetics under physiologic conditions. This development was done using two different and artificial radiolabeled substrates, glycine-I-hippuryl-L-histidyl-L-leucine phenyl-4(n)-hippuryl-glycyl-glycine, for the pulmonary enzyme, angiotensin-convetting enzyme. With this system, we assessed the effects of different perfusate types upon the stability of the perfusion as well as the independent effects of pressure, flow, and substrate concentration on the activity of this enzyme. We concluded that this system enables the operator to determine the kinetics and activities of pulmonary enzymes independently of the effects of pressure and flow in a perfusion system that is stable for at least 3 h under physiologic conditions. Key Words:
Angiotensin-converting
enzyme
INTRODUCTION
(ACE) is a carboxypeptidase that is located on cells and converts angiotensin I to II, as well as inactivating bradykinin (Ryan, 1982). Determination of kinetics of this enzyme in the lung in vivo is difficult because its activity is influenced by vascular parameters in the lung, which include the rate of blood flow, the diameter of the blood vessels, and the surface area of the endothelial lining (Oparil et al., 1982). In addition, the efficiency of gas exchange and the quality of inhaled air have also been shown to affect pulmonary ACE activity (Stalcup et al., 1981). Angiotensin-converting enzyme activity in the lung has been researched by many investigators using angiotensin I as the substrate (Bakhle et al., 1969; lgic et al., 1972; Ryan et al., 1972; Fanburg and Glazier, 1973; Roth et al., 1979; Wallace et al., 1980; Leuenberger et al., 1980; Szidon et al., 1980). Because the product, angiotensin II, is vasoactive and may change the hemodynamic parameters in the lung, several nonvasoactive synthetic substrates have been developed (Ryan, 1983; Ryan et al., 1983; Howell et al., 1984; Ashton et al., 1985). These synthetic substrates are specific for ACE and have a broad spectrum of affinity to the enzyme. Moreover, they yield hydrolytic products that can be easily separated from the parent compound and Angiotensin-converting
the luminal
enzyme
surface of pulmonary
endothelial
From the Rappaport Institute for Medical Research, Research Center for Work Safety and Human Engineering, S. Neamann Institute for Advanced Studies in Science & Technology; and Department of Pharmacology, Faculty of Medicine, Technion-Israel Institute for Technology, Haifa, Israel. Address reprint requests to: Dr. Arieh Bomzon, Rappaport institute for Medical Research, TechnionIsrael Institute for Technology, P.O. Box 9697, Haifa 31096, Israel. Received October 1988; revised and accepted July, 1989. 141 Journalof
Pharmacological
Methods
8 1990 Elsevier Science Publishing
23, 141-153 (1990) Co., Inc., 655 Avenue of the Americas,
New York, NY 10010
142
1. Mor et al.
readily measured radiometrically (Ryan, 1984; Ryan et al., 1977; Ryan et al., 1978; Ryan et al., 1980). Because synthetic substrates have different kinetic parameters and their metabolisms may differ in response to changes in pulmonary perfusion, we decided to examine the effect of altering pulmonary blood flow upon the apparent reaction rates of two synthetic ACE substrates viz. glycine-l-l4 C-hippuryl-L-histidyl-L-leucine (HHL) and phenyl- 4(n)-3H-hippuryl-glycyl-glycine (HGG). In order to do so, we have developed an isolated perfused guinea pig lung preparation in which the lungs can be perfused for at least 3 h under physiologic conditions while the hydrostatic pressure and flow rate of the perfusion system can be manipulated and controlled. MATERIALS AND METHODS Isolated Perfused Guinea Pig Lung System The perfusion system is schematically described in Figure 1. It consisted of a 1-L reservoir of the perfusing solution that was pumped into the lungs using a peristaltic pump (Watson-Marlow MHRE 200, United Kingdom), through a heat exchanger to the inflow reservoir immediately proximal to the perfused lung. The perfusate consisted of a Krebs’-Ringer bicarbonate buffer of the following composition (in mM): NaCl 118, KCI 4.7, MgS04 1.16, NaH,P04 1.27, CaC12 2.45, NaHC03 2.62, glucose 11 .I, to which was added 10 units/mL heparin, 45 g/L bovine albumin (Fraction V, Sigma Chemical Co., St. Louis, Missouri), and guinea pig autologous blood to give a final hematocrit of 2-5%. The basis for the use of such a perfusate is described in the results section where data on the effect of various additives on the stability of the perfusion are presented. From the inflow reservoir, the fluid passed through a catheter inserted into the pulmonary artery catheter to perfuse the lungs that were suspended in a heated chamber. The inflow reservoir had a second entry port through which the radioactive substrates could be added by a syringe pump and mixed with the inflowing perfusing medium. The height of the reservoir above the perfused lungs was adjustable in order to control the hy drostatic pressure in the pulmonary artery. The perfusate outflow was collected from a pulmonary vein catheter that passed through an outlet port of the heated lung chamber. In a single pass experiment, this perfusate was collected whereas in a recirculation experiment, it was returned to the main reservoir. In addition to the outlet to the lung, the inflow reservoir had an additional outlet port through which the overflow perfusate could be returned to the main reservoir via the oxygenator. All the reservoirs had double walls between which heated water was circulated to maintain the temperature at 37°C. The water temperature in the heating bath was controlled by a thermoregulator in the inflow reservoir. The perfusate in the main reservoir was continuously stirred and aerated using a gas mixture of oxygen, carbon dioxide, and nitrogen obtained by mixing air, carbogen (95% oxygen:5% carbon dioxide) and 100% carbon dioxide using gas flow meters (Manostat, New York). The pH of the inflowing perfusate was monitored continuously by a pH electrode in the main reservoir and maintained at 7.4 f 0.05 by controlling the carbon dioxide concentration in the gas mixture. Gas tensions in the inflow and outflow perfusates
Angiotensin-Converting
Enzyme Activity
FIGURE 1. Schematic representation of the pressure-dependent isolated perfusion system highlighting the important features described in the text (see Materials and Methods section). 1, main reservoir; 2, peristaltic pump; 3, heat exchanger; 4, inflow reservoir; 5, thermoregulator; 6, lungs; 7, lung chamber; 8, oxygenator; 9, arterial line pressure transducer; 10, recorder; 11, ventilator; 12, pH meter; 13, gas flow meter; 14, heating bath; 15, syringe pump; 16, humidifier; 17, tracheal pressure transducer.
were determined regularly using a blood gas analyzer (Instrumentation Laboratory System 1302, Milan, Italy). The pulmonary artery pressure was continuously recorded during the perfusion on a Brush-Gould 2200s pen recorder and was varied between 0 and 25 mmHg by adjusting the height of the inflow reservoir. The flow rate of perfusate into the inflow reservoir was 2.73 f 0.07 mUmin/mmHg/kg; this value was determined in a series of experiments that are described in the results section. The lungs were ventilated with room air throughout the experiment by a constant volume ventilator (Harvard Apparatus, Series 683, Rodent Respirator) at a rate of 60 breath/min with the tidal volume set at 6-7 mUkg body weight. The ventilated air was warmed and humidified by passing it through a 37°C water bath before its entry into the lung. In addition, a positive end expiratory pressure of 23 mmHg was maintained during the perfusion. The ventilation pressure was measured continuously using a pressure transducer whose output was recorded on the pen recorder.
143
144 1. Mor et al.
Surgical Procedure For ail the experiments, male guinea pigs weighing between 250-700 g were used. Under sodium phenobarbitone anesthesia (60-75 mg/kg), the animal was intubated via a tracheotomy and through the tracheal cannula, the animal was ventilated using the constant volume ventilator. After opening the chest, the heart and pulmonary artery were exposed. A loose ligature was placed around the pulmonary artery close to its exit from the right ventricle. A second ligature was then placed around the pulmonary vein and left atrium. Heparin (800 units/kg) was injected into the right ventricle and about 1 min later, the right ventricle was incised. Through this incision, a catheter (i.d. 2.5 mm, o.d. 3 mm) was placed into the pulmonary artery and anchored by tying the preplaced ligature. The perfusate was flowing through the catheter at the time of its placement into the pulmonary artery. Within seconds, the lungs became “white” due to the flushing of the flood from the lungs by the perfusate. During the next 5 min, the blood was flushed from the vascular system through the incision in the right ventricle and collected for subsequent addition to the perfusate. After this period of “whole body perfusion,” the left ventricle was incised and a second catheter (i.d. 2.5 mm, o.d. 3 mm) was passed into the pulmonary vein and anchored by the prepositioned ligature. The lungs were then dissected free from the rib cage except the section with the spinal column and suspended by the cannulated trachea within the perfusion apparatus. Perfusion and ventilation of the lungs was continuous during the placement of the pulmonary vein catheter and the transfer to the perfusion apparatus. Upon completion of the experiment, the lung was completely dissected free from the nonpulmonary tissue and weighed (wet weight). After an overnight drying at IlO”, the lung was reweighed (dry weight). Measurement
of Pulmonary ACE Activity
Two substrates were used to determine the pulmonary activity of ACE-_[phenyl4(n)-3H]-hippuryl-glycl-glycine (HGC) and [glycine-l-14C]hippuryl-histidyl-l-leucine (HHL) that were purchased from Amersham International, United Kingdom). Either 0.1 mmol HCG or 1.67 mmol HHL were infused at a rate of 0.28 mUmin into the inflow reservoir. The outflow was collected into preweighed test tubes containing 1 mol HCI:l mol NaCl (1:lO) for fixed times at known time intervals or as a single fraction. The HCl:NaCl was added to prevent any further conversion. The test tubes were then reweighed and the volume contained therein calculated. From each sample, duplicate aliquots of 0.1 mL and 0.5 mL were removed. The O.-l-mL aliquots were used to measure the total amount of radioactivity. To the 0.5-mL aliquots, 0.5mL ethyl acetate was added, stirred for 5 set and then spun in an Eppendorf centrifuge for 3 min. The upper phase containing the metabolic product of the conversion was separated from the resultant two phase solution and the radioactivity determined in two O.l-mL duplicate samples. If the total outflow was collected as a single large volume, duplicate O.l-mL and 0.5mL aliquots of the sample were removed from this volume and added to test tubes containing a O.l-mL Imol HCI:lmol NaCI. The samples were prepared for the measurement of ACE activity in the same manner as described previously.
Angiotensin-Converting
Enzyme Activity
The radioactivity in the samples was measured using a scintillation cocktail of lumax:xylene (30:70) in a RackBeta Model 1211 (LKB Wallac) scintillation counter. The radioactivity was expressed in dpm using the external ratio standard method. The actual product dpm in each sample was calculated according to Stewart et al., (1981) that takes into account the partitioning of the product and substrate into the two layers. The activity of the enzyme was calculated as a percentage of the product radioactivity from the total radioactivity and expressed as percentage conversion of the substrate. Statistical Analysis of the Data The statistical significance was determined using the Student’s t test for pai red variables. All the data is presented as mean -t standard error of the mean. RESULTS Stability of the Perfusion These experiments were undertaken with the explicit aim of extending the duration of the perfusion by delaying the onset of edema. Table 1 summarizes the effect of adding varying amounts of dextran and albumin and red blood cells to the Krebs’-Ringer bicarbonate buffer. It can be seen the onset of edema could be delayed for at least 3 h with the addition of red blood cells and albumin. Further support for this conclusion was obtained by the nonsignificant difference in the TABLE 1 Effect of Various Additives to the Basic Krebs’-Ringer Bicarbonate Buffer Perfusion Medium on the Duration of the Perfusion and the Visual Onset of Edema in the Pressure-Dependent Isolated Perfused Guinea Pig Lung
ADDITIVES None
4% Dextran
RBC 4% Dextran + RBC 4% Dextran + 3% Albumin 2% Albumin + RBC
Abbreviations:
EXP # 1 2 3 4 5 5 1 2 2 1 1 1 1 1 1 2 3 4 ND, not determined;
DURATION
OF PERFUSION 15 15 70 90 50 90 30 60 220 240 50 130 70 180 130 150 120 170
PRESENCE OF EDEMA
FLOWRATE (h4U~lN)
yes yes yes yes
7 7 2 2.5 IO 7 7 3 3 2 2.6 1 4 1.8 2.5 12 17 8
min min min min min min min min min min min min min min min min min min
RBC, red blood cells.
no yes no no yes no no yes no yes no no no no
PRESSURE (MMHG) ND ND 10 IO IO IO ND IO IO IO IO IO 10 IO IO 15 17
a
145
146
1. Mor et al.
7.600I
7.700~-
..’
L
7.600-I*
7.500-. 7.400
ii
7.500
--
7.200
J
-
*
P
1
P
P
3
p
P
40 I
1004
:
25.000
15.0000 Time
(minutes)
FIGURE 2. The mean arterial and venous “blood” pH and gas tensions obtained during the perfusion of the lung. The first sample, time 0, was taken before the catheterizationof the lung and the remaining sampleswere obtained every 15 min. 0 = artery; 0 = vein
Angiotensin-Converting
Enzyme Activity
wet:dry weight ratios between the lungs perfused for at least 3 h with such a perfusate (17.93 + 1.85) and washed lungs i.e., washed for 3 min only with the same perfusate (18.18 + 1.94). The changes in the arterial and venous perfusate pH, pCOz, pOZ, and bicarbonate levels with time obtained from lungs perfused with the buffer, albumin, and red blood cells is described in Figure 2. It can be seen that over a 2 h period of lung perfusion, the mean values and the differences were stable. For all subsequent experiments, albumin and red blood cells were added and the specific amounts have been described in the Materials and Methods section. Pressure-Flow
Relationship
The flow rate of the lung perfusion is directly related to the hydrostatis pressure in the pulmonary artery catheter. In vivo, the pulmonary blood flow rate is related to the body weight. In this series of experiments (11 perfusion experiments conducted in eight guinea pig lungs), the flow rates were normalized to the body weight and the results are shown in Figures 3A and 36. In Figure 3A, the 11 regression lines are presented showing a linear relationship between the normalized blood flow rate and hydrostatic pressure in the pulmonary artery catheter or pulmonary artery
FLOW RATE ml/min/kg BW 100
....-A
90 -
&I70 6050 40 30-
2010 0
81 I
0
I
I
10
I 20
0 30
PA PRESSURE mmHg FIGURE 3. Pressure-flow relationship in the isolated perfused guinea pig lung. The individual flow rates have been normalized to the body weight. A: the ll.individual regression lines obtained from 8 guinea pig lung perfusion experiments; B: the common constrained linear regression line with its 95% confidence limits of the mean.
147
148
1. Mor et
al.
pressure. From Figure 3B in which the common constrained regression line and the 95% confidence limits are shown, the slope of the line gave a value of 2.73 + 0.07 mL/min/mmHg/kg, and this constant was utilized in the determination of the flow rate of the perfusate into the inflow reservoir for each individual lung perfusion experiment. Because the height of the inflow reservoir could be changed, it thus enabled the operator to perfuse the lungs at different pulmonary artery pressures and flow rates independent of flow rate of the peristaltic pump. Accuracy
of the Technique
for Measuring
ACE Activity
In these experiments, the pulmonary artery pressure was maintained at 10 mmHg. The two substrates were infused for 1 m into the inflow reservoir and the outflow collected every 15 set as separate fractions. Figures 4A and 48 show the results of atypical experiment using HGG and HHL, respectively as the substrates. As expected the total radioactivity increased and decreased with time. However, the percentage conversion remained constant over the collection period. In order to check the reproducibility of the measurement in the same lung, each substrate was infused five times at 20-min intervals for 1 min and the outflow collected for 3 min at 15set intervals. Between the individual infusions of the substrates, the perfusate was allowed to recirculate. Because radioactivity could accumulate in the perfusate because of each previous injections, a sample for measurement of the total radioactivity and percentage conversion was taken before each infusion. These values served as the blank for each subsequent infusion. The results of the reproducibility experiments are summarized in Table 2.
a.
b. 6000
60 40 20 _I-
7
1
Time (min)
4 l
Substrate-DPM
6
6 10 12 14 16 Sample No.
0 % Conversion FIGURE 4. The results of a typical experiment demonstrating the reproducibility of the experiments when more than one determination of enzyme activity is made in the same lung preparation. The upper figure shows the total amount of radioactivity in a sample obtained from inflowing perfusate immediately before the injection of the substrate, whereas the lower figure shows the activity of the enzyme from a sample of the outflow perfusate when collected as a single fraction. The duration of the infusion was 5 min and the outflow collected every 15 set (see text for details).
Angiotensin-Converting
Enzyme Activity
TABLE2. Coefficient of Variation of Pulmonary ACE Activity Measured Within Fractions of the Same Injection of Either Substrate (HGC or HHL); Within Samples Injected Consecutively in the Same Lung Perfusion; and Within Different Lung Perfusion Experiments HGC
HIA.
10.6 (n = IO) 20.0 (n = IO) 48.4 (n = 5)
10.9 (n = 11) 11.1 (n = 10) 46.1 (n = 7)
MODE OF COMPARISON
Within fractions Within samples Within experiments
Substrate concentration for experiments involving HGG was 0.002 nM and for HHL, it was O.OlmM. For all the experiments the perfusion pressure was 10 mmHg.
Effect of Pulmonary Blood Flow Rate on ACE Activity In these experiments, the effect of pulmonary blood flow rate on ACE activity was determined by changing the perfusion flow rate by altering the hydrostatic pressure in the pulmonary artery. This was done by increasing the height of the inflow reservoir above the lungs to give a pressure range of O-25 mmHg in increments of 5 mmHg. A 15-min equilibration period between pressure changes was
50
1
R 40
0” N
30
0
_.__‘_+ _._.-.-.-.-+.- .-.-._._,_._._,_._.
0 N 0
0.03
0.05
1 /FLOW
0.12
0.09
mln
kgBW
/
ml
----.’ -I-.-
predicted upper 95%
-.-.-
lower
95%
0.15
*.**.’ predicted -.-.- upper 95% -‘-‘lowlw 95% oI+’
I
+ 0
0.03
1 /FLOW
0.00
min
0.09
kgEW
/
ml
0.12
0.15
FIGURE 5. The relationship between perfusion rate and conversion in the isolated perfused lung showing the common linear regression line and its 95% confidence limits for each substrate. A (upper), HGG; B (lower), HHL.
149
150
1. Mor et al,
allowed to elapse before infusion of either of the two substrates. In these experiments, each substrate was infused for 15 set and collected as a single fraction, usually between 20-50 mL depending upon the flow rate. Aliquots were removed for the counting of total radioactivity and determination of ACE activity. Between each injection, the outflow was allowed to recirculate and the appropriate blanks were taken before each injection. Figures 5A and 58 describe the relationship between the pulmonary activity of ACE and the reciprocal of the flow rate for HHL and HGG, respectively. It can be seen that variation in percentage conversion at the same flow rate can be as high as 40%. Furthermore, it would appear that conversion is not always dependent upon flow rate because it can decrease, increase, or not change whatsoever.
DISCUSSION The system developed and described in this study incorporates all of the features that other investigators considered important for the perfusion of the isolated guinea pig lung (Rosenbloom and Bass, 1970; Watkins and Rannels, 1979; Smith and Bend, 1981; Olson and Rankin, 1983). Irrespective of the feature, the primary objective of their incorporation into this technique was to eliminate any pulmonary damage induced by the surgical isolation of the lung because such damage can hasten the onset of pulmonary edema and hence shorten the viable duration of the perfused lung. The combination of a physiologic perfusing medium, pulmonary ventilation, and the minimilization of surgical and ischemic damage increased the longevity of the perfusion by at least l-2 h to at least 3 h and enabled us to undertake numerous repetitive experiments with reproducible results. In this study, the development of pulmonary edema was assessed visually and using the wet:dry weight ratio. Based upon the representative data that has been presented, the addition of red blood cells and albumin to the perfusing medium improved the longevity of the perfusion because edema was not observed up to 3 h of perfusion. The reasons for this enhancement are obvious because their addition to the perfusate improves oxygen delivery and buffering capacity as well as contributing significantly to the maintenance of the pulmonary oncotic and osmotic pressures. Although dextran is widely used as an additive to overcome the problem of edema formation in organ perfusion, our results indicate that it may not be an appropriate additive when experiments involving lung perfusion longer than 2 h are undertaken. Watkins and Rannels (1979) emphasized the importance of pulmonary ventilation during perfusion of the lung because it influences the rate of onset of edema. Our experiences indicate that the maintenance of a positive end expiratory pressure during ventilation with warm, humidified air also contributed the enhanced integrity of the perfusion. Another advantage of this perfusion system is that it is pressure-dependent as opposed to being flow-dependent. This dependence upon pressure ensures the integrity of the whole pulmonary vasculature system with flow rates approximating those obtained for cardiac output. The flow rates obtained in our system are in
Angiotensin-Converting
Enzyme Activity
agreement with pulmonary pressure-flow relationships reported in the in situ dog lung perfusion reported by Pitt et al., (1982). In this regard, one cannot emphasize the importance of using catheters that will enable high perfusion rates to be obtained. We found the best results could be obtained using catheters of an internal diameter of 2.5 mm. If larger catheters were used, a problem of placement into the pulmonary artery occurred and often resulted in ischemic damage to the lung because of the interruption of flow to the lung. The maintenance of physiologic conditions and the minimal handling of the lungs during the surgical procedures involved in the isolation of the lungs from the cardiovascular system are also important contributors to the stability of the preparation. Pulmonary damage induced by handling, surgery, and transient ischemia promote the early onset of edema. Many of these factors can be eliminated by leaving the dorsal rib cage intact during the perfusion and maintaining flow and ventilation during the placement of the appropriate pulmonary artery and vein catheters. For example, the rib cage serves as a protective casing while the lungs are being transferred to the perfusion chamber. ACE Activity The activity of enzymes is normally assayed in vitro under conditions in which the reaction obeys zero-order enzyme kinetics i.e., at substrate concentration higher than its K, value (Howell et al, 1984). The in vivo determination of enzyme activity is difficult because the reaction obeys first order enzyme kinetics i.e., the substrate concentration is always lower than the K,. In the case of ACE, the circulating plasma concentration is 0.1 nM (Niarchos et al, 19791, whereas its K, is 33 PM (Chiu et al, 1976). In this study, the ACE activity was measured in the range of the first-order enzyme kinetics with both synthetic substrates. The resultant concentrations of HGC and HHL in the inflowing perfusate after their infusion into the inflow reservoir results in concentrations below their respective K, values of 3.49 mM and 0.103 mM (Ryan et al, 1980). Figures 3A and 36 demonstrate that the activity obeys the first-order assumption because conversion was constant despite changes in the perfusate substrate concentration. This suggests that the pulmonary activity of ACE is not dependent upon substrate concentration that is in accordance with the conclusions obtained by Ryan (1982). Because the concentration of the substrate is below its K,, one could anticipate considerable within and between experiment variation. In fact, the variation within experiments was small; this contrasts with the large variation found between experiments. The small within experiment variation has a distinct advantage because it would allow the collection of the outflow as single fraction and enable more than one determination of activity per lung to be undertaken. On the other hand, the large between experiment variation due to varying inflow concentrations of substrate undoubtedly makes the comparison between individual experiments difficult. It should also be noted that the pulmonary activity of ACE is dependent upon the pulmonary surface area being perfused (Fanburg and Glazier, 1973; Stalcup et al., 1981). In a flow-dependent perfusion system, in which more pulmonary capillaries would be perfused at the high pressure, the ACE activity should increase. However,
151
152
L. Mor et al.
the ACE activity could also be reduced by virtue of a reduction in the capillary transit time due to the increased flow rates and a consequent reduction in the time needed for the conversion. In contrast, in the pressure-dependent perfusion system, the flow rate and perfusion pressure can be independently manipulated and their individual effects upon ACE activity can be evaluated. Such an evaluation is not possible in a flow-dependent perfusion system because these two variables cannot be independently manipulated. In conclusion, the pressure-dependent isolated lung perfusion system for the determination of pulmonary ACE activity has been evaluated. Such a system enables an independent assessment of the effects of pressure, flow, and substrate concentration upon the activity of this enzyme in a perfusion system that is stable for at least 3 h under physiologic conditions. The authors wish to acknowledge the technical assistance of Bilha Pinhassi and to thank the Committee for Research and Prevention in Occupational Safety and Health, Ministry of Labour and Social Affairs, Jerusalem, Israel and the S. Neamann Institute for Advanced Studies in Science and Technology, Technion, Haifa, Israel for the financial support in this project.
REFERENCES Ashton JH, Pitt BR, Gillis CN (1985) Apparent kinetics of angiotensin converting enzyme: hydrolysis of [‘HI benzoyl-phenylalanyl-proline in isolated perfused lung. / Pharmacol Exp Ther 232:602-607. Bakhle YS, Reynard AM, Vane JR (1960) Metabolism of the angiotensins in isolated perfused tissues. Nature 222 :956-960. Chiu AT, Ryan JW, Stewart JM, Dorer FE (1976) Formation of angiotensin III by angiotensin converting enzyme. Biochem J 155:189-194. Fanburg BL, Glazier JB (1973) Conversion of angiotensin 1 to angiotensin 2 in isolated perfused dog lung. / Appl Physiol35:325-331. Howell RE, Moalli R, Gillis CN (1984) Analysis of rabbit pulmonary angiotensin converting enzyme kinetics in vivo. / Pharmacol Exp Ther 228:154-160. lgic R, Erdos EC, Yeh HSJ, Sorrells K, Nakajima T (1972) Angiotensin I converting enzyme of the lung. Cir Res 30, 31: supplement ll:51-61. Leuenberger PJ, Stalcup SA, Creenbaum LM, Mellins RB, Torino GM (1980) Angiotensin I conversion and vascular reactivity in pathophysiological states in dogs. 1 Appl Physiol48:308-312. Niarchos AP, Roberts AJ, Laragh JH (1979) Effects of converting enzyme inhibitors (SQ20,881) on the pulmonary circulation in man. Am J Med67:785791.
Olson EB, Rankin J (1983) Isolated, perfused fetal rabbit lungs: Preparation and flow relationships. Lung 161:87-98. Oparil S, Winternitz S, Gould V, Baerwald M, Szidon P (1982) Effect of hypoxia on the conversion of angiotensin I to II in the isolated perfused rat lung. Biochem Pharmacol31:1375-1379. Pitt BR, Hammond CL, Gillis CN (1982) Comparison of pulmonary and extrapulmonary extraction of biogenic amines. I Appl Physiol52:1545-1551. Rosenbloom PM, Bass AD (1970) A lung perfusion preparation for the study of drug metabolism. 1 Appl Physiol29:138-144. Roth RA, Wallace KB, Alper RH, Bailie MD (1979) Effect of paraquat treatment of rats on disposition of 5-hydroxytryptamine and angiotensin I by perfused lung. Biochem Pharmacol 28:23492355. Ryan JW (1982) Processing of endogenous polypeptides by the lungs. Ann Rev Physiol44:241255. Ryan JW (1983) Assay of peptidase and protease enzymes in vivo. Biochem Pharmacol32:2127-2137. Ryan JW (1984) Angiotensin I converting enzyme (Kininase II). In Methods of Enzymatic Analysis. Ed., Bergmeyer, 3rd ed., vol. V. Weinheim: Verlag Chemie Gmbh, pp. 20-34. Ryan JW, Smith U, Niemeyer RS (1972) Angiotensin
Angiotensin-Converting I: Metabolism by plasma membrane of lung. Science 17664-66. Ryan JW, Chung A, Ammons C, Carlton ML (1977) A simple radioassay for angiotensin-converting enzyme. Biochem / 167:501-504. Ryan JW, Chung A, Martin LC, Ryan US (1978) New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture. Tissue Cell 10:555-562. Ryan JW, Chung A, Ryan US (1980) Angiotensinconverting enzyme: I. New strategies for assay. Environ Health Perspect 35:165-170. Ryan JW, Berryer P, Chung A (1983) Assay of angiotensin converting enzyme in vivo. Adv Exp Med Biol (Part B) 156:805-812. Smith BR, Bend R (1981) Lung perfusion techniques for xenobiotic metabolism and toxicity studies. Methods Enzymol77:105-120.
Enzyme Activity
Stalcup SA, Leuenberger PI, Lipset JS, Osman MM, Cerreta JM, Mellins RB, Turin0 GM (1981) Impaired angiotensin conversion and bradykinin clearance in experimental canine pulmonary emphysema. 1 C/in Invest 67:201-209. Stewart TA, Weare JA, Erdos EC (7981) Human peptidy1 dipeptidase (converting enzyme, kininase II). Methods Enzyme/80:450-460. Szidon P, Bauey N, Oparil S (1980) Effect of acute hypoxia on the pulmonary conversion of angiotensin I to angiotensin II in dogs. Circ Res 46:221-226. Wallace KB, Roth RA, Hook JB, Bailie MD (1980) Age-related differences in angiotensin I metabolism by isolated perfused rat lungs. Am\ Physiol 238:R395-R399. Watkins CA, Rannels DE (1979) In situ perfusion of rat lungs: stability and effects of oxygen tension. / Appl Physiol47:325-329.
153
L. MOR, A. BOMZON, R. FRENKEL, AND M. B. H. YOUDIM
We have developed a pressure-dependent isolated lung perfusion system that can be used for the determination of pulmonary enzyme activity and kinetics under physiologic conditions. This development was done using two different and artificial radiolabeled substrates, glycine-I-hippuryl-L-histidyl-L-leucine phenyl-4(n)-hippuryl-glycyl-glycine, for the pulmonary enzyme, angiotensin-convetting enzyme. With this system, we assessed the effects of different perfusate types upon the stability of the perfusion as well as the independent effects of pressure, flow, and substrate concentration on the activity of this enzyme. We concluded that this system enables the operator to determine the kinetics and activities of pulmonary enzymes independently of the effects of pressure and flow in a perfusion system that is stable for at least 3 h under physiologic conditions. Key Words:
Angiotensin-converting
enzyme
INTRODUCTION
(ACE) is a carboxypeptidase that is located on cells and converts angiotensin I to II, as well as inactivating bradykinin (Ryan, 1982). Determination of kinetics of this enzyme in the lung in vivo is difficult because its activity is influenced by vascular parameters in the lung, which include the rate of blood flow, the diameter of the blood vessels, and the surface area of the endothelial lining (Oparil et al., 1982). In addition, the efficiency of gas exchange and the quality of inhaled air have also been shown to affect pulmonary ACE activity (Stalcup et al., 1981). Angiotensin-converting enzyme activity in the lung has been researched by many investigators using angiotensin I as the substrate (Bakhle et al., 1969; lgic et al., 1972; Ryan et al., 1972; Fanburg and Glazier, 1973; Roth et al., 1979; Wallace et al., 1980; Leuenberger et al., 1980; Szidon et al., 1980). Because the product, angiotensin II, is vasoactive and may change the hemodynamic parameters in the lung, several nonvasoactive synthetic substrates have been developed (Ryan, 1983; Ryan et al., 1983; Howell et al., 1984; Ashton et al., 1985). These synthetic substrates are specific for ACE and have a broad spectrum of affinity to the enzyme. Moreover, they yield hydrolytic products that can be easily separated from the parent compound and Angiotensin-converting
the luminal
enzyme
surface of pulmonary
endothelial
From the Rappaport Institute for Medical Research, Research Center for Work Safety and Human Engineering, S. Neamann Institute for Advanced Studies in Science & Technology; and Department of Pharmacology, Faculty of Medicine, Technion-Israel Institute for Technology, Haifa, Israel. Address reprint requests to: Dr. Arieh Bomzon, Rappaport institute for Medical Research, TechnionIsrael Institute for Technology, P.O. Box 9697, Haifa 31096, Israel. Received October 1988; revised and accepted July, 1989. 141 Journalof
Pharmacological
Methods
8 1990 Elsevier Science Publishing
23, 141-153 (1990) Co., Inc., 655 Avenue of the Americas,
New York, NY 10010
142
1. Mor et al.
readily measured radiometrically (Ryan, 1984; Ryan et al., 1977; Ryan et al., 1978; Ryan et al., 1980). Because synthetic substrates have different kinetic parameters and their metabolisms may differ in response to changes in pulmonary perfusion, we decided to examine the effect of altering pulmonary blood flow upon the apparent reaction rates of two synthetic ACE substrates viz. glycine-l-l4 C-hippuryl-L-histidyl-L-leucine (HHL) and phenyl- 4(n)-3H-hippuryl-glycyl-glycine (HGG). In order to do so, we have developed an isolated perfused guinea pig lung preparation in which the lungs can be perfused for at least 3 h under physiologic conditions while the hydrostatic pressure and flow rate of the perfusion system can be manipulated and controlled. MATERIALS AND METHODS Isolated Perfused Guinea Pig Lung System The perfusion system is schematically described in Figure 1. It consisted of a 1-L reservoir of the perfusing solution that was pumped into the lungs using a peristaltic pump (Watson-Marlow MHRE 200, United Kingdom), through a heat exchanger to the inflow reservoir immediately proximal to the perfused lung. The perfusate consisted of a Krebs’-Ringer bicarbonate buffer of the following composition (in mM): NaCl 118, KCI 4.7, MgS04 1.16, NaH,P04 1.27, CaC12 2.45, NaHC03 2.62, glucose 11 .I, to which was added 10 units/mL heparin, 45 g/L bovine albumin (Fraction V, Sigma Chemical Co., St. Louis, Missouri), and guinea pig autologous blood to give a final hematocrit of 2-5%. The basis for the use of such a perfusate is described in the results section where data on the effect of various additives on the stability of the perfusion are presented. From the inflow reservoir, the fluid passed through a catheter inserted into the pulmonary artery catheter to perfuse the lungs that were suspended in a heated chamber. The inflow reservoir had a second entry port through which the radioactive substrates could be added by a syringe pump and mixed with the inflowing perfusing medium. The height of the reservoir above the perfused lungs was adjustable in order to control the hy drostatic pressure in the pulmonary artery. The perfusate outflow was collected from a pulmonary vein catheter that passed through an outlet port of the heated lung chamber. In a single pass experiment, this perfusate was collected whereas in a recirculation experiment, it was returned to the main reservoir. In addition to the outlet to the lung, the inflow reservoir had an additional outlet port through which the overflow perfusate could be returned to the main reservoir via the oxygenator. All the reservoirs had double walls between which heated water was circulated to maintain the temperature at 37°C. The water temperature in the heating bath was controlled by a thermoregulator in the inflow reservoir. The perfusate in the main reservoir was continuously stirred and aerated using a gas mixture of oxygen, carbon dioxide, and nitrogen obtained by mixing air, carbogen (95% oxygen:5% carbon dioxide) and 100% carbon dioxide using gas flow meters (Manostat, New York). The pH of the inflowing perfusate was monitored continuously by a pH electrode in the main reservoir and maintained at 7.4 f 0.05 by controlling the carbon dioxide concentration in the gas mixture. Gas tensions in the inflow and outflow perfusates
Angiotensin-Converting
Enzyme Activity
FIGURE 1. Schematic representation of the pressure-dependent isolated perfusion system highlighting the important features described in the text (see Materials and Methods section). 1, main reservoir; 2, peristaltic pump; 3, heat exchanger; 4, inflow reservoir; 5, thermoregulator; 6, lungs; 7, lung chamber; 8, oxygenator; 9, arterial line pressure transducer; 10, recorder; 11, ventilator; 12, pH meter; 13, gas flow meter; 14, heating bath; 15, syringe pump; 16, humidifier; 17, tracheal pressure transducer.
were determined regularly using a blood gas analyzer (Instrumentation Laboratory System 1302, Milan, Italy). The pulmonary artery pressure was continuously recorded during the perfusion on a Brush-Gould 2200s pen recorder and was varied between 0 and 25 mmHg by adjusting the height of the inflow reservoir. The flow rate of perfusate into the inflow reservoir was 2.73 f 0.07 mUmin/mmHg/kg; this value was determined in a series of experiments that are described in the results section. The lungs were ventilated with room air throughout the experiment by a constant volume ventilator (Harvard Apparatus, Series 683, Rodent Respirator) at a rate of 60 breath/min with the tidal volume set at 6-7 mUkg body weight. The ventilated air was warmed and humidified by passing it through a 37°C water bath before its entry into the lung. In addition, a positive end expiratory pressure of 23 mmHg was maintained during the perfusion. The ventilation pressure was measured continuously using a pressure transducer whose output was recorded on the pen recorder.
143
144 1. Mor et al.
Surgical Procedure For ail the experiments, male guinea pigs weighing between 250-700 g were used. Under sodium phenobarbitone anesthesia (60-75 mg/kg), the animal was intubated via a tracheotomy and through the tracheal cannula, the animal was ventilated using the constant volume ventilator. After opening the chest, the heart and pulmonary artery were exposed. A loose ligature was placed around the pulmonary artery close to its exit from the right ventricle. A second ligature was then placed around the pulmonary vein and left atrium. Heparin (800 units/kg) was injected into the right ventricle and about 1 min later, the right ventricle was incised. Through this incision, a catheter (i.d. 2.5 mm, o.d. 3 mm) was placed into the pulmonary artery and anchored by tying the preplaced ligature. The perfusate was flowing through the catheter at the time of its placement into the pulmonary artery. Within seconds, the lungs became “white” due to the flushing of the flood from the lungs by the perfusate. During the next 5 min, the blood was flushed from the vascular system through the incision in the right ventricle and collected for subsequent addition to the perfusate. After this period of “whole body perfusion,” the left ventricle was incised and a second catheter (i.d. 2.5 mm, o.d. 3 mm) was passed into the pulmonary vein and anchored by the prepositioned ligature. The lungs were then dissected free from the rib cage except the section with the spinal column and suspended by the cannulated trachea within the perfusion apparatus. Perfusion and ventilation of the lungs was continuous during the placement of the pulmonary vein catheter and the transfer to the perfusion apparatus. Upon completion of the experiment, the lung was completely dissected free from the nonpulmonary tissue and weighed (wet weight). After an overnight drying at IlO”, the lung was reweighed (dry weight). Measurement
of Pulmonary ACE Activity
Two substrates were used to determine the pulmonary activity of ACE-_[phenyl4(n)-3H]-hippuryl-glycl-glycine (HGC) and [glycine-l-14C]hippuryl-histidyl-l-leucine (HHL) that were purchased from Amersham International, United Kingdom). Either 0.1 mmol HCG or 1.67 mmol HHL were infused at a rate of 0.28 mUmin into the inflow reservoir. The outflow was collected into preweighed test tubes containing 1 mol HCI:l mol NaCl (1:lO) for fixed times at known time intervals or as a single fraction. The HCl:NaCl was added to prevent any further conversion. The test tubes were then reweighed and the volume contained therein calculated. From each sample, duplicate aliquots of 0.1 mL and 0.5 mL were removed. The O.-l-mL aliquots were used to measure the total amount of radioactivity. To the 0.5-mL aliquots, 0.5mL ethyl acetate was added, stirred for 5 set and then spun in an Eppendorf centrifuge for 3 min. The upper phase containing the metabolic product of the conversion was separated from the resultant two phase solution and the radioactivity determined in two O.l-mL duplicate samples. If the total outflow was collected as a single large volume, duplicate O.l-mL and 0.5mL aliquots of the sample were removed from this volume and added to test tubes containing a O.l-mL Imol HCI:lmol NaCI. The samples were prepared for the measurement of ACE activity in the same manner as described previously.
Angiotensin-Converting
Enzyme Activity
The radioactivity in the samples was measured using a scintillation cocktail of lumax:xylene (30:70) in a RackBeta Model 1211 (LKB Wallac) scintillation counter. The radioactivity was expressed in dpm using the external ratio standard method. The actual product dpm in each sample was calculated according to Stewart et al., (1981) that takes into account the partitioning of the product and substrate into the two layers. The activity of the enzyme was calculated as a percentage of the product radioactivity from the total radioactivity and expressed as percentage conversion of the substrate. Statistical Analysis of the Data The statistical significance was determined using the Student’s t test for pai red variables. All the data is presented as mean -t standard error of the mean. RESULTS Stability of the Perfusion These experiments were undertaken with the explicit aim of extending the duration of the perfusion by delaying the onset of edema. Table 1 summarizes the effect of adding varying amounts of dextran and albumin and red blood cells to the Krebs’-Ringer bicarbonate buffer. It can be seen the onset of edema could be delayed for at least 3 h with the addition of red blood cells and albumin. Further support for this conclusion was obtained by the nonsignificant difference in the TABLE 1 Effect of Various Additives to the Basic Krebs’-Ringer Bicarbonate Buffer Perfusion Medium on the Duration of the Perfusion and the Visual Onset of Edema in the Pressure-Dependent Isolated Perfused Guinea Pig Lung
ADDITIVES None
4% Dextran
RBC 4% Dextran + RBC 4% Dextran + 3% Albumin 2% Albumin + RBC
Abbreviations:
EXP # 1 2 3 4 5 5 1 2 2 1 1 1 1 1 1 2 3 4 ND, not determined;
DURATION
OF PERFUSION 15 15 70 90 50 90 30 60 220 240 50 130 70 180 130 150 120 170
PRESENCE OF EDEMA
FLOWRATE (h4U~lN)
yes yes yes yes
7 7 2 2.5 IO 7 7 3 3 2 2.6 1 4 1.8 2.5 12 17 8
min min min min min min min min min min min min min min min min min min
RBC, red blood cells.
no yes no no yes no no yes no yes no no no no
PRESSURE (MMHG) ND ND 10 IO IO IO ND IO IO IO IO IO 10 IO IO 15 17
a
145
146
1. Mor et al.
7.600I
7.700~-
..’
L
7.600-I*
7.500-. 7.400
ii
7.500
--
7.200
J
-
*
P
1
P
P
3
p
P
40 I
1004
:
25.000
15.0000 Time
(minutes)
FIGURE 2. The mean arterial and venous “blood” pH and gas tensions obtained during the perfusion of the lung. The first sample, time 0, was taken before the catheterizationof the lung and the remaining sampleswere obtained every 15 min. 0 = artery; 0 = vein
Angiotensin-Converting
Enzyme Activity
wet:dry weight ratios between the lungs perfused for at least 3 h with such a perfusate (17.93 + 1.85) and washed lungs i.e., washed for 3 min only with the same perfusate (18.18 + 1.94). The changes in the arterial and venous perfusate pH, pCOz, pOZ, and bicarbonate levels with time obtained from lungs perfused with the buffer, albumin, and red blood cells is described in Figure 2. It can be seen that over a 2 h period of lung perfusion, the mean values and the differences were stable. For all subsequent experiments, albumin and red blood cells were added and the specific amounts have been described in the Materials and Methods section. Pressure-Flow
Relationship
The flow rate of the lung perfusion is directly related to the hydrostatis pressure in the pulmonary artery catheter. In vivo, the pulmonary blood flow rate is related to the body weight. In this series of experiments (11 perfusion experiments conducted in eight guinea pig lungs), the flow rates were normalized to the body weight and the results are shown in Figures 3A and 36. In Figure 3A, the 11 regression lines are presented showing a linear relationship between the normalized blood flow rate and hydrostatic pressure in the pulmonary artery catheter or pulmonary artery
FLOW RATE ml/min/kg BW 100
....-A
90 -
&I70 6050 40 30-
2010 0
81 I
0
I
I
10
I 20
0 30
PA PRESSURE mmHg FIGURE 3. Pressure-flow relationship in the isolated perfused guinea pig lung. The individual flow rates have been normalized to the body weight. A: the ll.individual regression lines obtained from 8 guinea pig lung perfusion experiments; B: the common constrained linear regression line with its 95% confidence limits of the mean.
147
148
1. Mor et
al.
pressure. From Figure 3B in which the common constrained regression line and the 95% confidence limits are shown, the slope of the line gave a value of 2.73 + 0.07 mL/min/mmHg/kg, and this constant was utilized in the determination of the flow rate of the perfusate into the inflow reservoir for each individual lung perfusion experiment. Because the height of the inflow reservoir could be changed, it thus enabled the operator to perfuse the lungs at different pulmonary artery pressures and flow rates independent of flow rate of the peristaltic pump. Accuracy
of the Technique
for Measuring
ACE Activity
In these experiments, the pulmonary artery pressure was maintained at 10 mmHg. The two substrates were infused for 1 m into the inflow reservoir and the outflow collected every 15 set as separate fractions. Figures 4A and 48 show the results of atypical experiment using HGG and HHL, respectively as the substrates. As expected the total radioactivity increased and decreased with time. However, the percentage conversion remained constant over the collection period. In order to check the reproducibility of the measurement in the same lung, each substrate was infused five times at 20-min intervals for 1 min and the outflow collected for 3 min at 15set intervals. Between the individual infusions of the substrates, the perfusate was allowed to recirculate. Because radioactivity could accumulate in the perfusate because of each previous injections, a sample for measurement of the total radioactivity and percentage conversion was taken before each infusion. These values served as the blank for each subsequent infusion. The results of the reproducibility experiments are summarized in Table 2.
a.
b. 6000
60 40 20 _I-
7
1
Time (min)
4 l
Substrate-DPM
6
6 10 12 14 16 Sample No.
0 % Conversion FIGURE 4. The results of a typical experiment demonstrating the reproducibility of the experiments when more than one determination of enzyme activity is made in the same lung preparation. The upper figure shows the total amount of radioactivity in a sample obtained from inflowing perfusate immediately before the injection of the substrate, whereas the lower figure shows the activity of the enzyme from a sample of the outflow perfusate when collected as a single fraction. The duration of the infusion was 5 min and the outflow collected every 15 set (see text for details).
Angiotensin-Converting
Enzyme Activity
TABLE2. Coefficient of Variation of Pulmonary ACE Activity Measured Within Fractions of the Same Injection of Either Substrate (HGC or HHL); Within Samples Injected Consecutively in the Same Lung Perfusion; and Within Different Lung Perfusion Experiments HGC
HIA.
10.6 (n = IO) 20.0 (n = IO) 48.4 (n = 5)
10.9 (n = 11) 11.1 (n = 10) 46.1 (n = 7)
MODE OF COMPARISON
Within fractions Within samples Within experiments
Substrate concentration for experiments involving HGG was 0.002 nM and for HHL, it was O.OlmM. For all the experiments the perfusion pressure was 10 mmHg.
Effect of Pulmonary Blood Flow Rate on ACE Activity In these experiments, the effect of pulmonary blood flow rate on ACE activity was determined by changing the perfusion flow rate by altering the hydrostatic pressure in the pulmonary artery. This was done by increasing the height of the inflow reservoir above the lungs to give a pressure range of O-25 mmHg in increments of 5 mmHg. A 15-min equilibration period between pressure changes was
50
1
R 40
0” N
30
0
_.__‘_+ _._.-.-.-.-+.- .-.-._._,_._._,_._.
0 N 0
0.03
0.05
1 /FLOW
0.12
0.09
mln
kgBW
/
ml
----.’ -I-.-
predicted upper 95%
-.-.-
lower
95%
0.15
*.**.’ predicted -.-.- upper 95% -‘-‘lowlw 95% oI+’
I
+ 0
0.03
1 /FLOW
0.00
min
0.09
kgEW
/
ml
0.12
0.15
FIGURE 5. The relationship between perfusion rate and conversion in the isolated perfused lung showing the common linear regression line and its 95% confidence limits for each substrate. A (upper), HGG; B (lower), HHL.
149
150
1. Mor et al,
allowed to elapse before infusion of either of the two substrates. In these experiments, each substrate was infused for 15 set and collected as a single fraction, usually between 20-50 mL depending upon the flow rate. Aliquots were removed for the counting of total radioactivity and determination of ACE activity. Between each injection, the outflow was allowed to recirculate and the appropriate blanks were taken before each injection. Figures 5A and 58 describe the relationship between the pulmonary activity of ACE and the reciprocal of the flow rate for HHL and HGG, respectively. It can be seen that variation in percentage conversion at the same flow rate can be as high as 40%. Furthermore, it would appear that conversion is not always dependent upon flow rate because it can decrease, increase, or not change whatsoever.
DISCUSSION The system developed and described in this study incorporates all of the features that other investigators considered important for the perfusion of the isolated guinea pig lung (Rosenbloom and Bass, 1970; Watkins and Rannels, 1979; Smith and Bend, 1981; Olson and Rankin, 1983). Irrespective of the feature, the primary objective of their incorporation into this technique was to eliminate any pulmonary damage induced by the surgical isolation of the lung because such damage can hasten the onset of pulmonary edema and hence shorten the viable duration of the perfused lung. The combination of a physiologic perfusing medium, pulmonary ventilation, and the minimilization of surgical and ischemic damage increased the longevity of the perfusion by at least l-2 h to at least 3 h and enabled us to undertake numerous repetitive experiments with reproducible results. In this study, the development of pulmonary edema was assessed visually and using the wet:dry weight ratio. Based upon the representative data that has been presented, the addition of red blood cells and albumin to the perfusing medium improved the longevity of the perfusion because edema was not observed up to 3 h of perfusion. The reasons for this enhancement are obvious because their addition to the perfusate improves oxygen delivery and buffering capacity as well as contributing significantly to the maintenance of the pulmonary oncotic and osmotic pressures. Although dextran is widely used as an additive to overcome the problem of edema formation in organ perfusion, our results indicate that it may not be an appropriate additive when experiments involving lung perfusion longer than 2 h are undertaken. Watkins and Rannels (1979) emphasized the importance of pulmonary ventilation during perfusion of the lung because it influences the rate of onset of edema. Our experiences indicate that the maintenance of a positive end expiratory pressure during ventilation with warm, humidified air also contributed the enhanced integrity of the perfusion. Another advantage of this perfusion system is that it is pressure-dependent as opposed to being flow-dependent. This dependence upon pressure ensures the integrity of the whole pulmonary vasculature system with flow rates approximating those obtained for cardiac output. The flow rates obtained in our system are in
Angiotensin-Converting
Enzyme Activity
agreement with pulmonary pressure-flow relationships reported in the in situ dog lung perfusion reported by Pitt et al., (1982). In this regard, one cannot emphasize the importance of using catheters that will enable high perfusion rates to be obtained. We found the best results could be obtained using catheters of an internal diameter of 2.5 mm. If larger catheters were used, a problem of placement into the pulmonary artery occurred and often resulted in ischemic damage to the lung because of the interruption of flow to the lung. The maintenance of physiologic conditions and the minimal handling of the lungs during the surgical procedures involved in the isolation of the lungs from the cardiovascular system are also important contributors to the stability of the preparation. Pulmonary damage induced by handling, surgery, and transient ischemia promote the early onset of edema. Many of these factors can be eliminated by leaving the dorsal rib cage intact during the perfusion and maintaining flow and ventilation during the placement of the appropriate pulmonary artery and vein catheters. For example, the rib cage serves as a protective casing while the lungs are being transferred to the perfusion chamber. ACE Activity The activity of enzymes is normally assayed in vitro under conditions in which the reaction obeys zero-order enzyme kinetics i.e., at substrate concentration higher than its K, value (Howell et al, 1984). The in vivo determination of enzyme activity is difficult because the reaction obeys first order enzyme kinetics i.e., the substrate concentration is always lower than the K,. In the case of ACE, the circulating plasma concentration is 0.1 nM (Niarchos et al, 19791, whereas its K, is 33 PM (Chiu et al, 1976). In this study, the ACE activity was measured in the range of the first-order enzyme kinetics with both synthetic substrates. The resultant concentrations of HGC and HHL in the inflowing perfusate after their infusion into the inflow reservoir results in concentrations below their respective K, values of 3.49 mM and 0.103 mM (Ryan et al, 1980). Figures 3A and 36 demonstrate that the activity obeys the first-order assumption because conversion was constant despite changes in the perfusate substrate concentration. This suggests that the pulmonary activity of ACE is not dependent upon substrate concentration that is in accordance with the conclusions obtained by Ryan (1982). Because the concentration of the substrate is below its K,, one could anticipate considerable within and between experiment variation. In fact, the variation within experiments was small; this contrasts with the large variation found between experiments. The small within experiment variation has a distinct advantage because it would allow the collection of the outflow as single fraction and enable more than one determination of activity per lung to be undertaken. On the other hand, the large between experiment variation due to varying inflow concentrations of substrate undoubtedly makes the comparison between individual experiments difficult. It should also be noted that the pulmonary activity of ACE is dependent upon the pulmonary surface area being perfused (Fanburg and Glazier, 1973; Stalcup et al., 1981). In a flow-dependent perfusion system, in which more pulmonary capillaries would be perfused at the high pressure, the ACE activity should increase. However,
151
152
L. Mor et al.
the ACE activity could also be reduced by virtue of a reduction in the capillary transit time due to the increased flow rates and a consequent reduction in the time needed for the conversion. In contrast, in the pressure-dependent perfusion system, the flow rate and perfusion pressure can be independently manipulated and their individual effects upon ACE activity can be evaluated. Such an evaluation is not possible in a flow-dependent perfusion system because these two variables cannot be independently manipulated. In conclusion, the pressure-dependent isolated lung perfusion system for the determination of pulmonary ACE activity has been evaluated. Such a system enables an independent assessment of the effects of pressure, flow, and substrate concentration upon the activity of this enzyme in a perfusion system that is stable for at least 3 h under physiologic conditions. The authors wish to acknowledge the technical assistance of Bilha Pinhassi and to thank the Committee for Research and Prevention in Occupational Safety and Health, Ministry of Labour and Social Affairs, Jerusalem, Israel and the S. Neamann Institute for Advanced Studies in Science and Technology, Technion, Haifa, Israel for the financial support in this project.
REFERENCES Ashton JH, Pitt BR, Gillis CN (1985) Apparent kinetics of angiotensin converting enzyme: hydrolysis of [‘HI benzoyl-phenylalanyl-proline in isolated perfused lung. / Pharmacol Exp Ther 232:602-607. Bakhle YS, Reynard AM, Vane JR (1960) Metabolism of the angiotensins in isolated perfused tissues. Nature 222 :956-960. Chiu AT, Ryan JW, Stewart JM, Dorer FE (1976) Formation of angiotensin III by angiotensin converting enzyme. Biochem J 155:189-194. Fanburg BL, Glazier JB (1973) Conversion of angiotensin 1 to angiotensin 2 in isolated perfused dog lung. / Appl Physiol35:325-331. Howell RE, Moalli R, Gillis CN (1984) Analysis of rabbit pulmonary angiotensin converting enzyme kinetics in vivo. / Pharmacol Exp Ther 228:154-160. lgic R, Erdos EC, Yeh HSJ, Sorrells K, Nakajima T (1972) Angiotensin I converting enzyme of the lung. Cir Res 30, 31: supplement ll:51-61. Leuenberger PJ, Stalcup SA, Creenbaum LM, Mellins RB, Torino GM (1980) Angiotensin I conversion and vascular reactivity in pathophysiological states in dogs. 1 Appl Physiol48:308-312. Niarchos AP, Roberts AJ, Laragh JH (1979) Effects of converting enzyme inhibitors (SQ20,881) on the pulmonary circulation in man. Am J Med67:785791.
Olson EB, Rankin J (1983) Isolated, perfused fetal rabbit lungs: Preparation and flow relationships. Lung 161:87-98. Oparil S, Winternitz S, Gould V, Baerwald M, Szidon P (1982) Effect of hypoxia on the conversion of angiotensin I to II in the isolated perfused rat lung. Biochem Pharmacol31:1375-1379. Pitt BR, Hammond CL, Gillis CN (1982) Comparison of pulmonary and extrapulmonary extraction of biogenic amines. I Appl Physiol52:1545-1551. Rosenbloom PM, Bass AD (1970) A lung perfusion preparation for the study of drug metabolism. 1 Appl Physiol29:138-144. Roth RA, Wallace KB, Alper RH, Bailie MD (1979) Effect of paraquat treatment of rats on disposition of 5-hydroxytryptamine and angiotensin I by perfused lung. Biochem Pharmacol 28:23492355. Ryan JW (1982) Processing of endogenous polypeptides by the lungs. Ann Rev Physiol44:241255. Ryan JW (1983) Assay of peptidase and protease enzymes in vivo. Biochem Pharmacol32:2127-2137. Ryan JW (1984) Angiotensin I converting enzyme (Kininase II). In Methods of Enzymatic Analysis. Ed., Bergmeyer, 3rd ed., vol. V. Weinheim: Verlag Chemie Gmbh, pp. 20-34. Ryan JW, Smith U, Niemeyer RS (1972) Angiotensin
Angiotensin-Converting I: Metabolism by plasma membrane of lung. Science 17664-66. Ryan JW, Chung A, Ammons C, Carlton ML (1977) A simple radioassay for angiotensin-converting enzyme. Biochem / 167:501-504. Ryan JW, Chung A, Martin LC, Ryan US (1978) New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture. Tissue Cell 10:555-562. Ryan JW, Chung A, Ryan US (1980) Angiotensinconverting enzyme: I. New strategies for assay. Environ Health Perspect 35:165-170. Ryan JW, Berryer P, Chung A (1983) Assay of angiotensin converting enzyme in vivo. Adv Exp Med Biol (Part B) 156:805-812. Smith BR, Bend R (1981) Lung perfusion techniques for xenobiotic metabolism and toxicity studies. Methods Enzymol77:105-120.
Enzyme Activity
Stalcup SA, Leuenberger PI, Lipset JS, Osman MM, Cerreta JM, Mellins RB, Turin0 GM (1981) Impaired angiotensin conversion and bradykinin clearance in experimental canine pulmonary emphysema. 1 C/in Invest 67:201-209. Stewart TA, Weare JA, Erdos EC (7981) Human peptidy1 dipeptidase (converting enzyme, kininase II). Methods Enzyme/80:450-460. Szidon P, Bauey N, Oparil S (1980) Effect of acute hypoxia on the pulmonary conversion of angiotensin I to angiotensin II in dogs. Circ Res 46:221-226. Wallace KB, Roth RA, Hook JB, Bailie MD (1980) Age-related differences in angiotensin I metabolism by isolated perfused rat lungs. Am\ Physiol 238:R395-R399. Watkins CA, Rannels DE (1979) In situ perfusion of rat lungs: stability and effects of oxygen tension. / Appl Physiol47:325-329.
153
