COMBINED CLINICAL AND BASIC SCIENCE SEMINAR Selected and edited by Richard T. Silver, M.D. and Alexander Department
of Medicine,
The New York Hospital-Cornell
Medical Center,
G. Bearn. M.D.,
New York, bIew York
Is the Lung a Para-Endocrine Organ?
Modera tot HENRY 0. HEINEMANN, M.D. New York, New York Lecturers: JAMES W. RYAN, M.D., D.Phil. UNA S. RYAN, Ph.D. Miami, Florida
From the Department of Medicine, The New York Hospital-Cornell Medical Center, the Papanicolaou Cancer Research Institute and the Department of Medicine, University of Miami. This work was supported by grants from the U.S. Public Health Service (HL 15691, HL 16407, HL 19764 and Contract HR3-30 15) and from the John A. Hartford Foundation, Inc., the Council for Tobacco Research U.S.A., Inc., and by an Established Investigatorship Award to Dr. Una S. Ryan from the American Heart Association and with funds contributed by the Heart Association of Palm Beach County, Florida. Requests for reprints should be addressed to Dr. James W. Ryan, Department of Medicine, University of Miami School of Medicine, Biscayne Annex, P.O. Box 52-0875, Miami, Florida 33152.
H. 0. Heinemann: It has become apparent that the lung, although primarily concerned with gas exchange, has other functions which are unrelated to ventilation. These functions are carried out by individual cell populations residing within this organ. The endothelium lining the vessels of the pulmonary vasculature represents one category of cells involved in some of these nonventilatory functions of the lung. This metabolically active cell population characteristically affects substances circulating in blood. This is facilitated by the immense surface area of the capillary bed, well suited for the dual function of gas exchange and interaction with blood constituents. The concept that cells lining the capillaries have functions other than to serve as partitions between the fluid and gas phase has many physiologic implications, some of which will be reviewed here. Evidence for the role of the lung in modifying circulating vasoactive blood constituents can be found in the literature dating back to the first quarter of this century. At that time, Starling and Verney [l] were engaged in their classic experiments on the formation of urine. They included an isolated lung in the system to prevent vasoconstriction in the isolated perfused kidneys. Page [2] later demonstrated that extracts of lung tissue could inactivate a “serum vasoconstrictor substance” which was eventually identified as serotonin. We now know that serotonin is taken up by the endothelial cells of the pulmonary vasculature, that the uptake is coupied to ion transport and that this amine is effectively converted to 5-hydroxyindolacetic acid (5HIAA) [3-71. The concept which emerges from such observations is that the lung may play a pivotal role in reconstituting the blood prior to its entry into the systemic circulation, not only in regard to the oxygen and carbon dioxide content but also in relation to vasoactive substances [8,9]. We are fortunate to have with us here Dr. James W. Ryan and Dr. Una S. Ryan, who have contributed so much to our understanding of the structure and function of the endothelial lining of the pulmonary vasculature. J. W. Ryan and U. S. Ryan: The lungs conduct activities which bear similarities to functions of endocrine glands: The lungs are capable of processing selectively hormones, prohormones and other excitatory substances as they pass through the pulmonary circulation-some are inactivated or otherwise eliminated from the blood, a second group is allowed free passage and yet a third group is activated; the active products of which pass into the systemic arterial circulation (for review,
October 1977
The American Journal of Medicine
Volume 63
595
COMBINED
CLINICAL AND BASIC SCIENCE
SEMINAR
see
[9]). In addition, recent evidence indicates that the lungs are capable of synthesizing prostaglandin-related substances (in particular prostaglandin GP (PGG2) and the thromboxanes) which are released into the systemic arterial circulation [ 10,111. Thus, as the lungs convert venous blood to arterial blood in terms of exchanging carbon dioxide for oxygen, they also regulate the entry of certain hormonal substances into the systemic arterial circulation. Through the processing of hormones and prohormones, it would appear that the product of specific reactions of the lungs may well influence specific actions of tissues and organs at a distance. In this sense the term, “endocrine,” conveys an understanding of the activity; however, it is only in terms of synthesis and release of prostaglandin-related substances that the word roots “internal secretion” may apply. The processing of hormones and other excitatory substances by the lung covers a broad range of types of compounds, including steroids, biogenic amines, prostaglandins, polypeptide hormones and adenine nucleotides. The range of substances processed would appear to bespeak little specificity. However, the lungs distinguish within chemical groups of hormones; e.g., norepinephrine is taken up by the lungs but its methylated homolog, epinephrine, is not. Similarly, prostaglandins of the E and F series are metabolized as they pass through the pulmonary circulation, but prostaglandins of the A series pass intact through the lungs [12-141. Angiotensins II and III are not metabolized during passage through the lungs [ 151, but their higher homolog and precursor, angiotensin I, is degraded to smaller polypeptides, among them being angiotensin II and des-Asp’-angiotensin I, a possible precursor of angiotensin Ill (des-Asp’-angiotensin II) [16-181. Furthermore, bradykinin is quantitatively metabolized as it passes through the lungs, but its higher homologs are degraded at slower rates. Polistes kinin, an 18 amino acid residue peptide having bradykinin as its C-terminal nonapeptide moiety, is not metabolized at all [ 19,201. The apparent selectivity of the lungs in processing these substances is all the more remarkable in that the lungs once disrupted (for example by homogenization) can be shown to contain an abundance of enzymes which are capable of inactivating all the aforementioned substances [ 191. No selectivity is evident. Thus, it would appear that within intact lungs, enzymes are so partitioned that some have access to circulating hormones whereas others do not. As one might suspect, this partitioning of enzymes among different cells of the lungs and among different organelles of a given cell type is a major determinant of the fate of hormones and of the ultimate disposition of the metabolic products. For the remainder of this presentation, we will dis-
596
October
1977
The American
Journal
of Medlclne
Volume
cuss primarily the processing of two vasoactive polypeptide hormones: angiotensin I and bradykinin. The immediate metabolic fates of these two polypeptides are known in some detail, and the cellular and molecular mechanisms by which these hormones are processed have been defined by several independent technics. Furthermore, these two compounds are of interest because they have opposite or, in some respects, reciprocal biologic activities. In a physiologic sense, they have opposite fates. Specifically, bradykinin, a potent hypotensive substance, is completely inactivated during passage through the lungs. In contrast, angiotensin I is activated by being converted to its lower homolog, angiotensin II, the most potent hypertensive substance known. The ability of the lungs to eliminate a hormone which lowers blood pressure while forming a hormone which raises blood pressure suggests that the lungs may possibly play a role in blood pressure homeostasis. Furthermore, it now appears that a single lung enzyme (angiotensin converting enzyme, kininase II) may, in large part, account for the inactivation of bradykinin and for the conversion of angiotensin I to angiotensin II. Thus, there is the distinct possibility that an understanding of a role of the lungs in blood pressure homeostasis might be obtained at the cellular and molecular levels. Our interest in the role of the lungs in the processing of polypeptide hormones arose from efforts to understand three observations made by Vane and colleagues at the Royal College of Surgeons, London, in 1967. Ferreira and Vane [21] noted that bradykinin does not survive a single passage through the pulmonary circulation. In contrast, Hodge, Ng and Vane [15a] confirmed a previous observation by Goffinet and Mulrow [22] which indicated that angiotensin II is not cleared or inactivated during passage through the lungs. On the other hand, Ng and Vane [23,24] observed that the venous effluent of lungs perfused with angiotensin I contained more angiotensin-like activity than could be explained in terms of the amount of angiotensin I used for perfusion. Ng and Vane postulated that the lungs convert angiotensin I to its active lower homolog, angiotensin II; a hypothesis that was subsequently shown to be largely correct [ 16,251. In 1967 we began our efforts to confirm these observations and to examine the enzymic and cellular mechanisms by which bradykinin and angiotensin I might be processed. Our first experiments provided virtually all the essential observations on which the remainder of the work has been based. We confirmed, using (‘4C)Pro2-bradykinin, that bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) does, in fact, disappear as it passes through the lungs. However, we found that its disappearance is not accounted for by cellular uptake or by transfer to extravascular spaces. Indeed, all the radioactivity pumped into the pulmonary
63
IS THE LUNG A PARA-ENDDCRINE DRGAN?
artery could be recovered without delay in the venous effluent of the lungs. However, none of the radioactivity remained in the form of bradykinin. The radioactivity coming out in the venous effluent was found to occur in two forms; namely, the dipeptide, Pro-Pro, and the tetrapeptide, Arg-Pro-Pro-Gly [26]. We then repeated these experiments using (3H)Phes-bradykinin [ 19,271. When this compound was perfused through the pulmonary vascular bed, all the radioactivity was found to emerge in the form of the dipeptide, Phe-Arg. The latter metabolite proved to be one of the keys to the discovery that bradykinin and angiotensin I are metabolized by the same lung enzyme [28a]. Efforts to examine for the immediate metabolic fates of angiotensin II and angiotensin I (H-Asp-Arg-ValTyr-Ile-His-Pro-Phe-His-Leu-OH) in the pulmonary circulation were begun when radioactive analogs of these two compounds became available commercially. Using (14C)lle5-angiotensin II, we were able to confirm and to extend the previous findings of Mulrow and colleagues [22] and Hodge et al. [15a]. None of the radioactivity was taken up by the lungs; essentially all emerged in the venous effluent still in the form of angiotensin II [28b]. In contrast, we found that (14C)Leu’o-angiotensin I was completely metabolized as it passed through the lungs. The major radioactive metabolite was the dipeptide, His-Leu [ 161. Using ( 14C)Leu10-angiotensin I, it was not possible to detect the formation of angiotensin II by following changes in radioactivity. The formation of angiotensin II was established unequivocally when we used (14C)Phes-angiotensin I as the starting material [25,29]. Although our data showed that the lungs had converted angiotensin I to angiotensin II, we were surprised to find that most of the angiotensin-like biologic activity emerging from the lungs was neither angiotensin I nor angiotensin II. Most of the biologic activity, possibly due to a prostaglandin-related substance, appeared to originate in the lungs themselves [ 161. Although blood itself contains enzymes capable of degrading bradykinin and of converting angiotensin I to angiotensin II, we found, as had Vane’s group, that the degradation of these compounds in the lung was much faster than could be accounted for by enzyme activity in blood. In fact, blood is not required at all, since the reactions are as brisk in lungs made free of blood and perfused with artificial salt solutions. The possibility was considered that the lungs might secrete peptide hydrolase enzymes into the vascular space: but enzymes of the requisite activity and specificity were not found in the effluent of lungs perfused with these salt solutions. Taking each of these observations into account, we postulated that bradykinin is inactivated and that angiotensin I is activated by enzymes on or very close to the innermost surface of the cells lining the blood vessels;
namely, on the luminal surface of pulmonary endothelial cells [ 16,19,25-27,291. Considering the narrow caliber and the vast surface area of the lung capillaries along with the apparent high efficiency of metabolic reactions, we proposed that the degradations of bradykinin and angiotensin I would occur most efficiently in the capillary bed. The hypothesis implies some specificity of function of endothelial cells, yet these cells were thought to do little more than provide part of the physical barrier between blood and air. Indeed, until the lungs were examined by electron microscopy, it was not known that lung capillaries were lined by cells; rather it was thought that air and blood were separated by a membrane (Figure 1). Even when seen by electron microscopy, endothelial cells are among the least impressive in terms of structural complexity and morphologic features of all the various cell types of the lungs (Figure 2). Thus, one could well ask where within these cells could enzymes capable of degrading bradykinin and angiotensin I be accommodated? On the other hand, our hypothesis had some testable features. If the enzymes which metabolize circulating bradykinin and angiotensin I exist on the luminal surface of endothelial cells, then the plasma membrane fraction of lung should account fully for the inactivation of bradykinin and the conversion of angiotensin I to angiotensin II. We found this to be the case [30,31]. In addition, isolated monolayers of pulmonary endothelium should be capable of conducting the relevant reactions. Using monolayers of endothelial cells derived from the main-stem pulmonary artery, we found that ( 14C)Phe8angiotensin I was converted to (14C)Phea-angiotensin II and that (14C)Pro2s3-bradykinin was degraded to yield ( 14C)Pro-Pro [32,33]. More recently, we have found that pulmonary endothelial cells in culture are capable of metabolizing (1251)Tyra-bradykinin to yield (1251)Tyr-Arg, the product expected from the reaction of substrate with angiotensin-converting enzyme [34]. The latter studies have used endothelial cells collectecl from the mainstem pulmonary artery and from pulmonary vein. The enzymic activity is detectable in primary cultures and in later passages (X0 passages). In efforts to determine the cellular and subcellular sites of enzymes capable of metabolizing bradykinin and angiotensin I, it was important to look for ultrastructural specializations that could account for the requisite specificity as well as explain the speed and efficiency of the metabolic processes and make provision for the return of metabolic products to the circulation. Many aspects of the structure and organization of the pulmonary endothelium are well known. First, it is of the continuous type, cells being linked by tight junctions, and second, the endothelium as it occurs in pulmonary capillaries has a high density of caveolae intracellulares
October 1977
The American Journal of Medicine
Volume 63
597
Figure 1. Light micrograph of 0.5 p section of rat lung which had been perfused free of blood. The air spaces (A) are surrounded by capillary loops (C). The alveolar-capillary unit is extremely thin (e.g., at arrows) and its cellular nature cannot be resolved in the light microscope. An electron micrograph of a capillary is shown in Figure 2. Original magnification X425, reduced by 24 per cent.
(pinocytotic vesicles), a large proportion of which open to the vascular lumen (Figures 2 and 3). As has been pointed out recently, the small vessels of the pulmonary circulation differ in some respects from those of the bronchial circulation. Pietra et al. [35] have presented evidence that arterioles of the bronchial circulation contain fibrillar material and are capable of contracting. Our experience in studies of the endothelium of the small pulmonary vessels indicates that fibrillar material is not a prominent feature. In contrast, the endothelium of the main-stem pulmonary artery, at least in the dog, rat and cow, contains abundant fibrillar material, a point possibly consistent with the previous findings of others that the large vessels of the pulmonary circulation show a much greater response to the vasoconstrictor actions of angiotensin II than do the small vessels [36]. For these reasons, our first studies were directed toward the fine structure of endothelial caveolae (pinocytotic vesicles) as they occur in pulmonary capillaries. From the beginning, these structures were of particular interest to us because of their position in direct communication with circulating blood and because of the enormous surface area which they represent in the capillary beds. Those caveolae “open” to the
598
October 1977
The American Journal of Medicine
capillary lumen are covered by a delicate diaphragm composed of a single lamella (Figure 3). We believed that the diaphragm might contribute to a microenvironment favoring the observed metabolic reactions. The caveolae are clearly involved in the metabolism of adenine nucleotides. Adenine nucleotides provide the opportunity of studying metabolism by cytochemical technics. As their phosphate ester bonds are hydrolyzed, the phosphate can be trapped at or near the site of the relevent esterase enzyme as insoluble, electron-dense, lead phosphate. When blood-free lungs are perfused with Sadenosine monophosphate and lead nitrate, the electron-dense material, presumably lead phosphate, is virtually restricted to those caveolae directly facing the vascular lumen (Figure 4) [30,37]. Unfortunately, there are no cytochemical technics for the localization of the peptidase enzymes responsible for the metabolism of angiotensin I and the kinins. Therefore, until recently the data implicating caveolae as the site of these enzymes were inferential. The kinetics of disappearance of angiotensin I and bradykinin are virtually identical to the kinetics of disappearance of the adenine nucleotides, a fact which suggests that the location of enzymes affecting their metabolism is similar [ 19,25,27,29].
Volume 83
IS THE LUNG A PARA-.ENDOCRINE ORGAN?
Figure 2. Electron micrograph of a rat pulmonary capillary, The alveolar capillary unit is composed of type 1 alveolar epithelium (Ep) and endothelium (En) and their basement membranes. Except in the region of the nucleus (NJ,the endothelial cell is extremely thin and contains few organelles. The luminal surface area of the endothelial cell is increased by endothelial projections (arrowhead) and large numbers of caveolae (arrows). Caveolae are shown at higher magnification in Figure 3. Original magnification X 15,000, reduced by 24 per cent. Figure 3. Higher magnification of luminal surface of capillary endothelial cell showing caveolae. The luminal stoma is covered by a delicate diaphragm (arrows). Original magnification X 17 1,000, reduced by 24 per cent.
Recently, we have been able to gain direct evidence on the subcellular localization of an enzyme capable of degrading bradykinin and of converting angiotensin
I to angiotensin II. In 1972, Dorer et al. [38] succeeded in purifying, to homogeneity, angiotensin-converting enzyme from pig lung. Having the pure enzyme available, they were able to confirm the previous inferential evidence that the enzyme is also capable of degrading bradykinin to yield substances like those produced by
intact lungs [28]. The enzyme acts as a dipeptidyl carboxypeptidase having little or no :specificity. However, our recent studies indicate that the enzyme has remarkable selectivity of action [ 18,39,40]. Indeed, the selectivity of the enzyme in hydrolyzing bradykinin and its higher homologs parallels the selectivity of metabolism of the kinins by intact lungs [20,39]. Curiously, bradykinin is a better substrate for angiotensin-converting enzyme than is angiotensin I.
October 1977
The American Journal of Medlclne
Volume 63
599
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Figure
4. This figure illustrates the cytochemica) localization of S’nucleotidase. The endothelial cell is from a rat lung which had been perfused with 5’-AMP and lead nitrate. At the site of hydrolysis of the phosphate ester bond, insoluble electron-dense lead phosphate is precipitated. The reaction product, indicating sites of 5’-nucleotidase activity, is specifically localized in those caveolae facing the vascular lumen (arrows). Original magnification X 120,000, reduced by 24 per cent, Reprinted from Smith, Ryan [ 571. Figure 5. Micrograph illustrating the morphology of pig pulmonary endothelium in primary culture. Cells were obtained by perfusion of the vascular bed of the lungs with 0.2 per cent collagenase. The cells grow as a monolayer with a characteristic cobblestone appearance when viewed in the light microscope. The plastic culture flask in which the cells were grown, fixed and embedded is indicated by an asterisk. The field shown contains features characteristic of endothelial cells in situ and in culture, e.g., caveolae (arrows). Golgi apparatus (G), rough endoplasmic reticulum (ER). Original magnification X40,500, reduced by 24 per cent.
600
October 1977
The American Journal of Medicine
Volume 63
IS THE LUNG A PARA-ENDOCRINE
ORGAN?
Endothelial cell from pig lung tissue which had been incubated with antibodies to angiotensin-converting enzyme coupled to microperoxidase (B-M/7. The peroxidase moiety was then reacted with 3,3’-diaminobenzidine and t&OS. The reaction product is localized along the luminal plasma membrane of the endothelial cell (arrows). The section was not stained. Original magnification X92,000. reduced by 24 per cent.
Figure 6.
Figure 7. Endothelial cell in culture derived from pig aorta. The cells were incubated as described in Figure 6. The reaction product, indicating sites of angiotensin converting enzyme, is localized along the plasma membrane and caveolae (arrows). The section was not stained. Original magnification X 116,000, reduced by 24 per cent. The availability
of pure enzyme
made it possible
to
prepare monospecific antibodies to the protein. The antibodies have been used in direct immunocytochemistry studies. In the direct technic, anti-(pig lung angiotensin-converting enzyme) was coupled to 1 l-MP (microperoxidase, a heme-undecapeptide of cytochrome c) or 8-MP (a heme-octapeptide of cytochrome c). Both prefixed blocks of lung and fixed or unfixed endothelial cells in culture (Figure 5) (pulmonary and aortic) were exposed to the antibody-peroxidase conjugate. Examination of intact lung tissue showed that angiotensin-converting enzyme is distributed along the Iuminal surface of virtually all endothelial cells and is especially prominent at the level of the capillaries and other small vessels [41-431. Both luminal caveolae and areas of undifferentiated plasma membrane were reactive (Figure 6). Endothelial cells in culture (both pulmonary and aortic) also were reactive with the specific antibody-peroxidase conjugate (Figure 7) and antibody-fluorophor conjugates. Similarly, the cells reacted with the specific antibody and then, with species specific anti-IgG-fluorophor conjugate, showed specific fluorescence. Thus, it appears that angiotensin-converting enzyme (kininase II) is situated in such a position
that the circulating vasoactive polypeptides, angiotensin I and bradykinin, can be metabolized and the metabolic products can be released into the systemic arterial circulation without cellular uptake or secretion of soluble enzymes into the blood. Recently, Caldwell et al. [44] have gained evidence by immunofluorescence studies which indicates that angiotensin-converting enzyme occurs in association with the vascular bed of a large number of tissues (e.g., liver, pancreas and adrenal gland) and that it may also be prominent in the epithelium of the renal proximal tubules. Previous studies have shown that angiotensin-converting enzyme is not unique to the pulmonary vascular bed [45,46]. Small amounts occur in plasma and larger amounts occur in kidney, testes and other tissues. There are, however, two unique features of lung which strongly favor the hypothesis that the ‘lung forms angiotensin II for distribution to distant organs: first, the lungs are apparently unique in being unable to degrade angiotensin II to inactive products. This rnay also be true for angiotensin Ill [ 15b]. Second, the venous effluent of the lungs empties directly into the systemic arterial circulation. Thus, their metabolic products can be delivered to all target tissues, including (via the bronchial circulation) the lungs themselves.
1977
The American Journal of Medicine
Volume 63
601
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
When one considers the strategic location of the lungs within the circulation and some of the characteristics of their normal function, it seems evident that the lungs have a number of ways by which they can vary the amounts of angiotensin II entering the systemic arterial circulation. The lungs receive the entire cardiac output. This may vary from 5 to 7 liters/min at rest to 40 liters/min during strenuous exercise. Not all capillaries of the lungs are open at any given time. In view of the fact that angiotensin-converting enzyme is located on the luminal surface of pulmonary endothelial cells, vascular surface area, caliber of vessels, rates of flow, distribution of flow and atrioventricular (A-V) shunting are likely to be important determinants of the quantities of angiotensin II and bradykinin allowed to enter the systemic circulation. In this presentation, we have described the ability of lungs to conduct a number of extremely fast reactions which both eliminate a series of blood pressure lowering substances and produce one or several compounds which have the capability to raise blood pressure. One substance, angiotensin II, is capable of raising the blood pressure level by increasing peripheral resistance and also by increasing the rate at which aldosterone is secreted. It now appears likely that the lungs can also form
angiotensin Ill, an aldosterone secretogogue that is even more potent than angiotensin II [9,10]. However, as shown by Laragh and colleagues [47], it may be that the renin-angiotensin system is important to the maintenance or sustained elevation of systemic arterial blood pressure levels only when plasma volume is low or when the plasma renin level is extremely high, circumstances that occur as a result of treatment with diuretics and that may also occur as a result of strenuous exercise. At present, there are few empiric data bearing on the physiologic or pathologic implications of the ability of the lungs to inactivate bradykinin and to form angiotensin II. Nonetheless, it appears reasonable to ask the question: What are the consequences when the ability of the lungs to eliminate bradykinin and to form angiotensin II is compromised? For example, during treatment with specific inhibitors of angiotensin-converting enzyme [47], or during operations requiring complete cardiopulmonary bypass, or following a pulmonary embolus, or in diseases such as emphysema in which the microcirculation of the lungs is reduced. At this point, one can well appreciate why the title of our presentation ends with a question mark: Is the lung a para-endocrine organ?
REFERENCES
2. 3.
4.
5.
6.
8. 9. 10.
11.
12.
602
Starling EH, Verney EB: The excretion of urine as studied on the isolated kidney. Proc Roy Sot (Lond), Ser B 97: 321, 1924-25. Page IH: Serotonin (5-hydroxytryptamine); the last four years. Physiol Rev 38: 277, 1958. Gaddum JH, Hebb CO, Silver A, et al.: 5hydroxytryptamine: pharmacological action and destruction in perfused lungs. Q J Exp Physiol 38: 255, 1953. Gruby LA, Rowlands C, Varley BQ, et al.: The fate of 5-hydroxytryptamine in the lungs. Br J Surg 58: 525, 1971. Strum JM, Junod AF: Radioautographic demonstration of 5hydroxytryptamine-3H uptake by pulmonary endothelial cells. J Cell Biol 54: 456, 1972. Junod AF: Uptake, metabolism and efflux of 14C-5-hydroxytryptamine in isolated perfused rat lungs. J Pharmacol Exp Therap 183: 341. 1972. Crawford N: The lung and serotonin metabolism: a study of the pulmonary arterio-venous levels of plasma free and platelet-bound serotonin in man. Clin Chim Acta 12: 264, 1965. Fishman AP: Dietary pulmonary hypertension. Circ Res 35: 657, 1974. Vane JR: The release and fate of vaso-active hormones in the circulation. Br J Pharmacol 35: 209, 1969. Hamberg M, Samuelsson B: Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung. Biochem Biophys Res Commun 61: 942, 1974. Hamberg M, Svensson J, Samuelsson B: Thromboxanes. A new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA 72: 2994, 1975. Ferreira SH, Vane JR: Prostaglandins. Their disappearance and release into the circulation. Nature (Lond) 216: 868, 1967.
October 1977
The American Journal of Medicine
13.
Piper PJ, Vane JR, Wyllie HJ: Inactivation of prostaglandins by the lungs. Nature (Lond) 225: 600, 1970. 14. Ryan JW, Niemeyer RS, Ryan U: Metabolism of prostaglandin F,,, in the pulmonary circulation. Prostaglandins 10: 101, 1975. 15. (a) Hodge RL, Ng KKF, Vane JR: Disappearance of angiotensin from the circulation of the dog. Nature (Lond) 215: 138, 1967. (b) Chiu AT, Ryan JW: Unpublished data. 16. Ryan JW, Stewart JM, Leary WP, et al.: Metabolism of angiotensin I in the pulmonary circulation. Biochem J 120: 221, 1970. 17. Tsai BS, Peach fvlJ, Khosla MC, et al.: Synthesis and evaluation of (Des-Asp’) angiotensin I as a precursor for (Des-Asp’)angiotensin II (“angiotensin Ill”). J Med Chem 18: 1180, 1975. 18. Chiu AT, Ryan JW, Stewart JM, et al.: Formation of angiotensin Ill by angiotensin-converting enzyme. Biochem J 155: 189, 1976. 19. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in rat lung. Adv Exp Med Biol 8: 263, 1970. 20. Roblero J, Ryan JW, Stewart JM: Assay of kinins by their effects on blood pressure. Res Commun Chem Pathol Pharmacol 6: 207, 1973. 21. Ferreira SH, Vane JR: The disappearance of bradykinin and eledoisin in the circulation and vascular beds of the cat. Br J Pharmacol 30: 417, 1967. 22. Goffinet JA, Mulrow PJ: Estimation of angiotensin clearance by an in vivo assay. Clin Res 11: 408, 1963. 23. Ng KKF, Vane JR: The conversion of angiotensin I to angiotensin Il. Nature (London) 216: 762, 1967. 24. Ng KKF, Vane JR: Fate of angiotensin I in the circulation. Nature (Land) 218: 144, 1968. 25. Ryan JW, Niemeyer RS, Goodwin DW, et al.: Metabolism of (8-L-(14C)phenylalanine)-angiotensin I in the pulmonary
Volume 63
IS THE LUNG A PARA-ENDOCRINE ORGAN?
26. 27. 28.
29. 30.
31.
32.
33.
34.
35.
36. 37. 38.
circulation. Biochem J 125: 921, 1971. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in the pulmonary circulation. Biochem J 110: 795, 1968. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in rat lung. Pharmacol Res Commun 1: 192, 1969. (a) Dorer FE, Kahn JR, Lentz KE, et al.: Hydrolysis of bradykinin byangiotensin-convertingenzyme.CircRes34: 824.1974. (b) Leary WP, Ryan JW: Unpublished data. Ryan JW, Smith U. Niemeyer RS: Angiotensin I. Metabolism by plasma membrane of lung. Science 176: 64, 1972. Ryan JW, Smith U: Metabolism of adenosine-5’-monophosphate during circulation through the lungs. Trans Assoc Am Physicians 84: 297. 1971. Ryan JW. Smith U: A rapid, simple method for isolating pinocytotic vesicles and plasma membrane of lung. Biochim Biophys Acta 249: 177, 1971. Ryan JW, Smith U: The metabolism of angiotensin I by endothelial cells. Protides of the Biological Fluids (Peeters H, ed), Oxford, England, Pergamon Press, 1973, p 379. Smith U, Ryan JW: Electron microscopy of endothelial cells collected on cellulose acetate paper. Tissue Cell 5: 333, 1973. Chiu AT, Ryan JW. Ryan US, et al.: A sensitive radioachemical assay for angiotensin-converting enzyme (kininase II). Biochem J 149: 297, 1975. Pietra GG, Szidon JP, Leventhal MM, et al.: Histamine and interstitial pulmonary edema in the dog. Circ Res 29: 323, 1971. De Bono E, Lee G de J, Mottram FR, et al.: The action of angiotensin in man. Clin Sci 25: 123, 1963. Smith U, Ryan JW: Pinocytotic vesicles of the pulmonary endothelial cell. Chest 59: 12S, 1971. Dorer FE, Kahn JR, Lentz KE, et al.: Purification and properties
39.
40.
41.
42.
43.
44.
45.
46.
47.
October 1977
of angiotensin-converting enzyme frolm hog lung. Circ Res 31: 356, 1972. Dorer FE, Ryan JW, Stewart JM: Hydrolysis of bradykinin and its higher homologues by angiotensin-converting enzyme. Biochem J 141: 915. 1974. Dorer FE, Kahn JR, Lentz KE, et al.: Forrnation of angiotensin II from tetradecapeptide renin substrate by angiotensinconverting enzyme. Biochem Pharmacol 24: 1137, 1975. Ryan JW, Ryan US, Schultz DR, et al.: Subcellular localization of pulmonary angiotensin converting enzyme (kininase II). Biochem J 146: 497, 1975. Ryan JW, Day AR, Ryan US, et al.: Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-heme-octapeptide conjugates. Tissue Cell 8: 111, 1976. Ryan US, Ryan JW, Whitaker C, et al.: Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence. Tissue Cell 8: 125, 1976. Caldwell PRB, Seegal BC, Hsu KC, et al.: Angiotensin-converting enzyme. Vascular endothelial localization. Science 191: 1050, 1976. Aiken JW, Vane JR: Inhibition of converting enzyme of the renin-angiotensin system in kidneys and hindlegs of dogs. Circ Res 30: 263, 1972. Cushman DW, Cheung HS: Concentrations of angiotensinconverting enzyme in tissues of the rat. Biochim Biophys Acta 250: 261, 1971. Gavras H, Brunner HR. Laragh JH, et al.: An angiotensin converting enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients. N Engl J Med 291: 817, 1974.
The American Journal of Medlclne
Volume 63
603
of Medicine,
The New York Hospital-Cornell
Medical Center,
G. Bearn. M.D.,
New York, bIew York
Is the Lung a Para-Endocrine Organ?
Modera tot HENRY 0. HEINEMANN, M.D. New York, New York Lecturers: JAMES W. RYAN, M.D., D.Phil. UNA S. RYAN, Ph.D. Miami, Florida
From the Department of Medicine, The New York Hospital-Cornell Medical Center, the Papanicolaou Cancer Research Institute and the Department of Medicine, University of Miami. This work was supported by grants from the U.S. Public Health Service (HL 15691, HL 16407, HL 19764 and Contract HR3-30 15) and from the John A. Hartford Foundation, Inc., the Council for Tobacco Research U.S.A., Inc., and by an Established Investigatorship Award to Dr. Una S. Ryan from the American Heart Association and with funds contributed by the Heart Association of Palm Beach County, Florida. Requests for reprints should be addressed to Dr. James W. Ryan, Department of Medicine, University of Miami School of Medicine, Biscayne Annex, P.O. Box 52-0875, Miami, Florida 33152.
H. 0. Heinemann: It has become apparent that the lung, although primarily concerned with gas exchange, has other functions which are unrelated to ventilation. These functions are carried out by individual cell populations residing within this organ. The endothelium lining the vessels of the pulmonary vasculature represents one category of cells involved in some of these nonventilatory functions of the lung. This metabolically active cell population characteristically affects substances circulating in blood. This is facilitated by the immense surface area of the capillary bed, well suited for the dual function of gas exchange and interaction with blood constituents. The concept that cells lining the capillaries have functions other than to serve as partitions between the fluid and gas phase has many physiologic implications, some of which will be reviewed here. Evidence for the role of the lung in modifying circulating vasoactive blood constituents can be found in the literature dating back to the first quarter of this century. At that time, Starling and Verney [l] were engaged in their classic experiments on the formation of urine. They included an isolated lung in the system to prevent vasoconstriction in the isolated perfused kidneys. Page [2] later demonstrated that extracts of lung tissue could inactivate a “serum vasoconstrictor substance” which was eventually identified as serotonin. We now know that serotonin is taken up by the endothelial cells of the pulmonary vasculature, that the uptake is coupied to ion transport and that this amine is effectively converted to 5-hydroxyindolacetic acid (5HIAA) [3-71. The concept which emerges from such observations is that the lung may play a pivotal role in reconstituting the blood prior to its entry into the systemic circulation, not only in regard to the oxygen and carbon dioxide content but also in relation to vasoactive substances [8,9]. We are fortunate to have with us here Dr. James W. Ryan and Dr. Una S. Ryan, who have contributed so much to our understanding of the structure and function of the endothelial lining of the pulmonary vasculature. J. W. Ryan and U. S. Ryan: The lungs conduct activities which bear similarities to functions of endocrine glands: The lungs are capable of processing selectively hormones, prohormones and other excitatory substances as they pass through the pulmonary circulation-some are inactivated or otherwise eliminated from the blood, a second group is allowed free passage and yet a third group is activated; the active products of which pass into the systemic arterial circulation (for review,
October 1977
The American Journal of Medicine
Volume 63
595
COMBINED
CLINICAL AND BASIC SCIENCE
SEMINAR
see
[9]). In addition, recent evidence indicates that the lungs are capable of synthesizing prostaglandin-related substances (in particular prostaglandin GP (PGG2) and the thromboxanes) which are released into the systemic arterial circulation [ 10,111. Thus, as the lungs convert venous blood to arterial blood in terms of exchanging carbon dioxide for oxygen, they also regulate the entry of certain hormonal substances into the systemic arterial circulation. Through the processing of hormones and prohormones, it would appear that the product of specific reactions of the lungs may well influence specific actions of tissues and organs at a distance. In this sense the term, “endocrine,” conveys an understanding of the activity; however, it is only in terms of synthesis and release of prostaglandin-related substances that the word roots “internal secretion” may apply. The processing of hormones and other excitatory substances by the lung covers a broad range of types of compounds, including steroids, biogenic amines, prostaglandins, polypeptide hormones and adenine nucleotides. The range of substances processed would appear to bespeak little specificity. However, the lungs distinguish within chemical groups of hormones; e.g., norepinephrine is taken up by the lungs but its methylated homolog, epinephrine, is not. Similarly, prostaglandins of the E and F series are metabolized as they pass through the pulmonary circulation, but prostaglandins of the A series pass intact through the lungs [12-141. Angiotensins II and III are not metabolized during passage through the lungs [ 151, but their higher homolog and precursor, angiotensin I, is degraded to smaller polypeptides, among them being angiotensin II and des-Asp’-angiotensin I, a possible precursor of angiotensin Ill (des-Asp’-angiotensin II) [16-181. Furthermore, bradykinin is quantitatively metabolized as it passes through the lungs, but its higher homologs are degraded at slower rates. Polistes kinin, an 18 amino acid residue peptide having bradykinin as its C-terminal nonapeptide moiety, is not metabolized at all [ 19,201. The apparent selectivity of the lungs in processing these substances is all the more remarkable in that the lungs once disrupted (for example by homogenization) can be shown to contain an abundance of enzymes which are capable of inactivating all the aforementioned substances [ 191. No selectivity is evident. Thus, it would appear that within intact lungs, enzymes are so partitioned that some have access to circulating hormones whereas others do not. As one might suspect, this partitioning of enzymes among different cells of the lungs and among different organelles of a given cell type is a major determinant of the fate of hormones and of the ultimate disposition of the metabolic products. For the remainder of this presentation, we will dis-
596
October
1977
The American
Journal
of Medlclne
Volume
cuss primarily the processing of two vasoactive polypeptide hormones: angiotensin I and bradykinin. The immediate metabolic fates of these two polypeptides are known in some detail, and the cellular and molecular mechanisms by which these hormones are processed have been defined by several independent technics. Furthermore, these two compounds are of interest because they have opposite or, in some respects, reciprocal biologic activities. In a physiologic sense, they have opposite fates. Specifically, bradykinin, a potent hypotensive substance, is completely inactivated during passage through the lungs. In contrast, angiotensin I is activated by being converted to its lower homolog, angiotensin II, the most potent hypertensive substance known. The ability of the lungs to eliminate a hormone which lowers blood pressure while forming a hormone which raises blood pressure suggests that the lungs may possibly play a role in blood pressure homeostasis. Furthermore, it now appears that a single lung enzyme (angiotensin converting enzyme, kininase II) may, in large part, account for the inactivation of bradykinin and for the conversion of angiotensin I to angiotensin II. Thus, there is the distinct possibility that an understanding of a role of the lungs in blood pressure homeostasis might be obtained at the cellular and molecular levels. Our interest in the role of the lungs in the processing of polypeptide hormones arose from efforts to understand three observations made by Vane and colleagues at the Royal College of Surgeons, London, in 1967. Ferreira and Vane [21] noted that bradykinin does not survive a single passage through the pulmonary circulation. In contrast, Hodge, Ng and Vane [15a] confirmed a previous observation by Goffinet and Mulrow [22] which indicated that angiotensin II is not cleared or inactivated during passage through the lungs. On the other hand, Ng and Vane [23,24] observed that the venous effluent of lungs perfused with angiotensin I contained more angiotensin-like activity than could be explained in terms of the amount of angiotensin I used for perfusion. Ng and Vane postulated that the lungs convert angiotensin I to its active lower homolog, angiotensin II; a hypothesis that was subsequently shown to be largely correct [ 16,251. In 1967 we began our efforts to confirm these observations and to examine the enzymic and cellular mechanisms by which bradykinin and angiotensin I might be processed. Our first experiments provided virtually all the essential observations on which the remainder of the work has been based. We confirmed, using (‘4C)Pro2-bradykinin, that bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) does, in fact, disappear as it passes through the lungs. However, we found that its disappearance is not accounted for by cellular uptake or by transfer to extravascular spaces. Indeed, all the radioactivity pumped into the pulmonary
63
IS THE LUNG A PARA-ENDDCRINE DRGAN?
artery could be recovered without delay in the venous effluent of the lungs. However, none of the radioactivity remained in the form of bradykinin. The radioactivity coming out in the venous effluent was found to occur in two forms; namely, the dipeptide, Pro-Pro, and the tetrapeptide, Arg-Pro-Pro-Gly [26]. We then repeated these experiments using (3H)Phes-bradykinin [ 19,271. When this compound was perfused through the pulmonary vascular bed, all the radioactivity was found to emerge in the form of the dipeptide, Phe-Arg. The latter metabolite proved to be one of the keys to the discovery that bradykinin and angiotensin I are metabolized by the same lung enzyme [28a]. Efforts to examine for the immediate metabolic fates of angiotensin II and angiotensin I (H-Asp-Arg-ValTyr-Ile-His-Pro-Phe-His-Leu-OH) in the pulmonary circulation were begun when radioactive analogs of these two compounds became available commercially. Using (14C)lle5-angiotensin II, we were able to confirm and to extend the previous findings of Mulrow and colleagues [22] and Hodge et al. [15a]. None of the radioactivity was taken up by the lungs; essentially all emerged in the venous effluent still in the form of angiotensin II [28b]. In contrast, we found that (14C)Leu’o-angiotensin I was completely metabolized as it passed through the lungs. The major radioactive metabolite was the dipeptide, His-Leu [ 161. Using ( 14C)Leu10-angiotensin I, it was not possible to detect the formation of angiotensin II by following changes in radioactivity. The formation of angiotensin II was established unequivocally when we used (14C)Phes-angiotensin I as the starting material [25,29]. Although our data showed that the lungs had converted angiotensin I to angiotensin II, we were surprised to find that most of the angiotensin-like biologic activity emerging from the lungs was neither angiotensin I nor angiotensin II. Most of the biologic activity, possibly due to a prostaglandin-related substance, appeared to originate in the lungs themselves [ 161. Although blood itself contains enzymes capable of degrading bradykinin and of converting angiotensin I to angiotensin II, we found, as had Vane’s group, that the degradation of these compounds in the lung was much faster than could be accounted for by enzyme activity in blood. In fact, blood is not required at all, since the reactions are as brisk in lungs made free of blood and perfused with artificial salt solutions. The possibility was considered that the lungs might secrete peptide hydrolase enzymes into the vascular space: but enzymes of the requisite activity and specificity were not found in the effluent of lungs perfused with these salt solutions. Taking each of these observations into account, we postulated that bradykinin is inactivated and that angiotensin I is activated by enzymes on or very close to the innermost surface of the cells lining the blood vessels;
namely, on the luminal surface of pulmonary endothelial cells [ 16,19,25-27,291. Considering the narrow caliber and the vast surface area of the lung capillaries along with the apparent high efficiency of metabolic reactions, we proposed that the degradations of bradykinin and angiotensin I would occur most efficiently in the capillary bed. The hypothesis implies some specificity of function of endothelial cells, yet these cells were thought to do little more than provide part of the physical barrier between blood and air. Indeed, until the lungs were examined by electron microscopy, it was not known that lung capillaries were lined by cells; rather it was thought that air and blood were separated by a membrane (Figure 1). Even when seen by electron microscopy, endothelial cells are among the least impressive in terms of structural complexity and morphologic features of all the various cell types of the lungs (Figure 2). Thus, one could well ask where within these cells could enzymes capable of degrading bradykinin and angiotensin I be accommodated? On the other hand, our hypothesis had some testable features. If the enzymes which metabolize circulating bradykinin and angiotensin I exist on the luminal surface of endothelial cells, then the plasma membrane fraction of lung should account fully for the inactivation of bradykinin and the conversion of angiotensin I to angiotensin II. We found this to be the case [30,31]. In addition, isolated monolayers of pulmonary endothelium should be capable of conducting the relevant reactions. Using monolayers of endothelial cells derived from the main-stem pulmonary artery, we found that ( 14C)Phe8angiotensin I was converted to (14C)Phea-angiotensin II and that (14C)Pro2s3-bradykinin was degraded to yield ( 14C)Pro-Pro [32,33]. More recently, we have found that pulmonary endothelial cells in culture are capable of metabolizing (1251)Tyra-bradykinin to yield (1251)Tyr-Arg, the product expected from the reaction of substrate with angiotensin-converting enzyme [34]. The latter studies have used endothelial cells collectecl from the mainstem pulmonary artery and from pulmonary vein. The enzymic activity is detectable in primary cultures and in later passages (X0 passages). In efforts to determine the cellular and subcellular sites of enzymes capable of metabolizing bradykinin and angiotensin I, it was important to look for ultrastructural specializations that could account for the requisite specificity as well as explain the speed and efficiency of the metabolic processes and make provision for the return of metabolic products to the circulation. Many aspects of the structure and organization of the pulmonary endothelium are well known. First, it is of the continuous type, cells being linked by tight junctions, and second, the endothelium as it occurs in pulmonary capillaries has a high density of caveolae intracellulares
October 1977
The American Journal of Medicine
Volume 63
597
Figure 1. Light micrograph of 0.5 p section of rat lung which had been perfused free of blood. The air spaces (A) are surrounded by capillary loops (C). The alveolar-capillary unit is extremely thin (e.g., at arrows) and its cellular nature cannot be resolved in the light microscope. An electron micrograph of a capillary is shown in Figure 2. Original magnification X425, reduced by 24 per cent.
(pinocytotic vesicles), a large proportion of which open to the vascular lumen (Figures 2 and 3). As has been pointed out recently, the small vessels of the pulmonary circulation differ in some respects from those of the bronchial circulation. Pietra et al. [35] have presented evidence that arterioles of the bronchial circulation contain fibrillar material and are capable of contracting. Our experience in studies of the endothelium of the small pulmonary vessels indicates that fibrillar material is not a prominent feature. In contrast, the endothelium of the main-stem pulmonary artery, at least in the dog, rat and cow, contains abundant fibrillar material, a point possibly consistent with the previous findings of others that the large vessels of the pulmonary circulation show a much greater response to the vasoconstrictor actions of angiotensin II than do the small vessels [36]. For these reasons, our first studies were directed toward the fine structure of endothelial caveolae (pinocytotic vesicles) as they occur in pulmonary capillaries. From the beginning, these structures were of particular interest to us because of their position in direct communication with circulating blood and because of the enormous surface area which they represent in the capillary beds. Those caveolae “open” to the
598
October 1977
The American Journal of Medicine
capillary lumen are covered by a delicate diaphragm composed of a single lamella (Figure 3). We believed that the diaphragm might contribute to a microenvironment favoring the observed metabolic reactions. The caveolae are clearly involved in the metabolism of adenine nucleotides. Adenine nucleotides provide the opportunity of studying metabolism by cytochemical technics. As their phosphate ester bonds are hydrolyzed, the phosphate can be trapped at or near the site of the relevent esterase enzyme as insoluble, electron-dense, lead phosphate. When blood-free lungs are perfused with Sadenosine monophosphate and lead nitrate, the electron-dense material, presumably lead phosphate, is virtually restricted to those caveolae directly facing the vascular lumen (Figure 4) [30,37]. Unfortunately, there are no cytochemical technics for the localization of the peptidase enzymes responsible for the metabolism of angiotensin I and the kinins. Therefore, until recently the data implicating caveolae as the site of these enzymes were inferential. The kinetics of disappearance of angiotensin I and bradykinin are virtually identical to the kinetics of disappearance of the adenine nucleotides, a fact which suggests that the location of enzymes affecting their metabolism is similar [ 19,25,27,29].
Volume 83
IS THE LUNG A PARA-.ENDOCRINE ORGAN?
Figure 2. Electron micrograph of a rat pulmonary capillary, The alveolar capillary unit is composed of type 1 alveolar epithelium (Ep) and endothelium (En) and their basement membranes. Except in the region of the nucleus (NJ,the endothelial cell is extremely thin and contains few organelles. The luminal surface area of the endothelial cell is increased by endothelial projections (arrowhead) and large numbers of caveolae (arrows). Caveolae are shown at higher magnification in Figure 3. Original magnification X 15,000, reduced by 24 per cent. Figure 3. Higher magnification of luminal surface of capillary endothelial cell showing caveolae. The luminal stoma is covered by a delicate diaphragm (arrows). Original magnification X 17 1,000, reduced by 24 per cent.
Recently, we have been able to gain direct evidence on the subcellular localization of an enzyme capable of degrading bradykinin and of converting angiotensin
I to angiotensin II. In 1972, Dorer et al. [38] succeeded in purifying, to homogeneity, angiotensin-converting enzyme from pig lung. Having the pure enzyme available, they were able to confirm the previous inferential evidence that the enzyme is also capable of degrading bradykinin to yield substances like those produced by
intact lungs [28]. The enzyme acts as a dipeptidyl carboxypeptidase having little or no :specificity. However, our recent studies indicate that the enzyme has remarkable selectivity of action [ 18,39,40]. Indeed, the selectivity of the enzyme in hydrolyzing bradykinin and its higher homologs parallels the selectivity of metabolism of the kinins by intact lungs [20,39]. Curiously, bradykinin is a better substrate for angiotensin-converting enzyme than is angiotensin I.
October 1977
The American Journal of Medlclne
Volume 63
599
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Figure
4. This figure illustrates the cytochemica) localization of S’nucleotidase. The endothelial cell is from a rat lung which had been perfused with 5’-AMP and lead nitrate. At the site of hydrolysis of the phosphate ester bond, insoluble electron-dense lead phosphate is precipitated. The reaction product, indicating sites of 5’-nucleotidase activity, is specifically localized in those caveolae facing the vascular lumen (arrows). Original magnification X 120,000, reduced by 24 per cent, Reprinted from Smith, Ryan [ 571. Figure 5. Micrograph illustrating the morphology of pig pulmonary endothelium in primary culture. Cells were obtained by perfusion of the vascular bed of the lungs with 0.2 per cent collagenase. The cells grow as a monolayer with a characteristic cobblestone appearance when viewed in the light microscope. The plastic culture flask in which the cells were grown, fixed and embedded is indicated by an asterisk. The field shown contains features characteristic of endothelial cells in situ and in culture, e.g., caveolae (arrows). Golgi apparatus (G), rough endoplasmic reticulum (ER). Original magnification X40,500, reduced by 24 per cent.
600
October 1977
The American Journal of Medicine
Volume 63
IS THE LUNG A PARA-ENDOCRINE
ORGAN?
Endothelial cell from pig lung tissue which had been incubated with antibodies to angiotensin-converting enzyme coupled to microperoxidase (B-M/7. The peroxidase moiety was then reacted with 3,3’-diaminobenzidine and t&OS. The reaction product is localized along the luminal plasma membrane of the endothelial cell (arrows). The section was not stained. Original magnification X92,000. reduced by 24 per cent.
Figure 6.
Figure 7. Endothelial cell in culture derived from pig aorta. The cells were incubated as described in Figure 6. The reaction product, indicating sites of angiotensin converting enzyme, is localized along the plasma membrane and caveolae (arrows). The section was not stained. Original magnification X 116,000, reduced by 24 per cent. The availability
of pure enzyme
made it possible
to
prepare monospecific antibodies to the protein. The antibodies have been used in direct immunocytochemistry studies. In the direct technic, anti-(pig lung angiotensin-converting enzyme) was coupled to 1 l-MP (microperoxidase, a heme-undecapeptide of cytochrome c) or 8-MP (a heme-octapeptide of cytochrome c). Both prefixed blocks of lung and fixed or unfixed endothelial cells in culture (Figure 5) (pulmonary and aortic) were exposed to the antibody-peroxidase conjugate. Examination of intact lung tissue showed that angiotensin-converting enzyme is distributed along the Iuminal surface of virtually all endothelial cells and is especially prominent at the level of the capillaries and other small vessels [41-431. Both luminal caveolae and areas of undifferentiated plasma membrane were reactive (Figure 6). Endothelial cells in culture (both pulmonary and aortic) also were reactive with the specific antibody-peroxidase conjugate (Figure 7) and antibody-fluorophor conjugates. Similarly, the cells reacted with the specific antibody and then, with species specific anti-IgG-fluorophor conjugate, showed specific fluorescence. Thus, it appears that angiotensin-converting enzyme (kininase II) is situated in such a position
that the circulating vasoactive polypeptides, angiotensin I and bradykinin, can be metabolized and the metabolic products can be released into the systemic arterial circulation without cellular uptake or secretion of soluble enzymes into the blood. Recently, Caldwell et al. [44] have gained evidence by immunofluorescence studies which indicates that angiotensin-converting enzyme occurs in association with the vascular bed of a large number of tissues (e.g., liver, pancreas and adrenal gland) and that it may also be prominent in the epithelium of the renal proximal tubules. Previous studies have shown that angiotensin-converting enzyme is not unique to the pulmonary vascular bed [45,46]. Small amounts occur in plasma and larger amounts occur in kidney, testes and other tissues. There are, however, two unique features of lung which strongly favor the hypothesis that the ‘lung forms angiotensin II for distribution to distant organs: first, the lungs are apparently unique in being unable to degrade angiotensin II to inactive products. This rnay also be true for angiotensin Ill [ 15b]. Second, the venous effluent of the lungs empties directly into the systemic arterial circulation. Thus, their metabolic products can be delivered to all target tissues, including (via the bronchial circulation) the lungs themselves.
1977
The American Journal of Medicine
Volume 63
601
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
When one considers the strategic location of the lungs within the circulation and some of the characteristics of their normal function, it seems evident that the lungs have a number of ways by which they can vary the amounts of angiotensin II entering the systemic arterial circulation. The lungs receive the entire cardiac output. This may vary from 5 to 7 liters/min at rest to 40 liters/min during strenuous exercise. Not all capillaries of the lungs are open at any given time. In view of the fact that angiotensin-converting enzyme is located on the luminal surface of pulmonary endothelial cells, vascular surface area, caliber of vessels, rates of flow, distribution of flow and atrioventricular (A-V) shunting are likely to be important determinants of the quantities of angiotensin II and bradykinin allowed to enter the systemic circulation. In this presentation, we have described the ability of lungs to conduct a number of extremely fast reactions which both eliminate a series of blood pressure lowering substances and produce one or several compounds which have the capability to raise blood pressure. One substance, angiotensin II, is capable of raising the blood pressure level by increasing peripheral resistance and also by increasing the rate at which aldosterone is secreted. It now appears likely that the lungs can also form
angiotensin Ill, an aldosterone secretogogue that is even more potent than angiotensin II [9,10]. However, as shown by Laragh and colleagues [47], it may be that the renin-angiotensin system is important to the maintenance or sustained elevation of systemic arterial blood pressure levels only when plasma volume is low or when the plasma renin level is extremely high, circumstances that occur as a result of treatment with diuretics and that may also occur as a result of strenuous exercise. At present, there are few empiric data bearing on the physiologic or pathologic implications of the ability of the lungs to inactivate bradykinin and to form angiotensin II. Nonetheless, it appears reasonable to ask the question: What are the consequences when the ability of the lungs to eliminate bradykinin and to form angiotensin II is compromised? For example, during treatment with specific inhibitors of angiotensin-converting enzyme [47], or during operations requiring complete cardiopulmonary bypass, or following a pulmonary embolus, or in diseases such as emphysema in which the microcirculation of the lungs is reduced. At this point, one can well appreciate why the title of our presentation ends with a question mark: Is the lung a para-endocrine organ?
REFERENCES
2. 3.
4.
5.
6.
8. 9. 10.
11.
12.
602
Starling EH, Verney EB: The excretion of urine as studied on the isolated kidney. Proc Roy Sot (Lond), Ser B 97: 321, 1924-25. Page IH: Serotonin (5-hydroxytryptamine); the last four years. Physiol Rev 38: 277, 1958. Gaddum JH, Hebb CO, Silver A, et al.: 5hydroxytryptamine: pharmacological action and destruction in perfused lungs. Q J Exp Physiol 38: 255, 1953. Gruby LA, Rowlands C, Varley BQ, et al.: The fate of 5-hydroxytryptamine in the lungs. Br J Surg 58: 525, 1971. Strum JM, Junod AF: Radioautographic demonstration of 5hydroxytryptamine-3H uptake by pulmonary endothelial cells. J Cell Biol 54: 456, 1972. Junod AF: Uptake, metabolism and efflux of 14C-5-hydroxytryptamine in isolated perfused rat lungs. J Pharmacol Exp Therap 183: 341. 1972. Crawford N: The lung and serotonin metabolism: a study of the pulmonary arterio-venous levels of plasma free and platelet-bound serotonin in man. Clin Chim Acta 12: 264, 1965. Fishman AP: Dietary pulmonary hypertension. Circ Res 35: 657, 1974. Vane JR: The release and fate of vaso-active hormones in the circulation. Br J Pharmacol 35: 209, 1969. Hamberg M, Samuelsson B: Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung. Biochem Biophys Res Commun 61: 942, 1974. Hamberg M, Svensson J, Samuelsson B: Thromboxanes. A new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA 72: 2994, 1975. Ferreira SH, Vane JR: Prostaglandins. Their disappearance and release into the circulation. Nature (Lond) 216: 868, 1967.
October 1977
The American Journal of Medicine
13.
Piper PJ, Vane JR, Wyllie HJ: Inactivation of prostaglandins by the lungs. Nature (Lond) 225: 600, 1970. 14. Ryan JW, Niemeyer RS, Ryan U: Metabolism of prostaglandin F,,, in the pulmonary circulation. Prostaglandins 10: 101, 1975. 15. (a) Hodge RL, Ng KKF, Vane JR: Disappearance of angiotensin from the circulation of the dog. Nature (Lond) 215: 138, 1967. (b) Chiu AT, Ryan JW: Unpublished data. 16. Ryan JW, Stewart JM, Leary WP, et al.: Metabolism of angiotensin I in the pulmonary circulation. Biochem J 120: 221, 1970. 17. Tsai BS, Peach fvlJ, Khosla MC, et al.: Synthesis and evaluation of (Des-Asp’) angiotensin I as a precursor for (Des-Asp’)angiotensin II (“angiotensin Ill”). J Med Chem 18: 1180, 1975. 18. Chiu AT, Ryan JW, Stewart JM, et al.: Formation of angiotensin Ill by angiotensin-converting enzyme. Biochem J 155: 189, 1976. 19. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in rat lung. Adv Exp Med Biol 8: 263, 1970. 20. Roblero J, Ryan JW, Stewart JM: Assay of kinins by their effects on blood pressure. Res Commun Chem Pathol Pharmacol 6: 207, 1973. 21. Ferreira SH, Vane JR: The disappearance of bradykinin and eledoisin in the circulation and vascular beds of the cat. Br J Pharmacol 30: 417, 1967. 22. Goffinet JA, Mulrow PJ: Estimation of angiotensin clearance by an in vivo assay. Clin Res 11: 408, 1963. 23. Ng KKF, Vane JR: The conversion of angiotensin I to angiotensin Il. Nature (London) 216: 762, 1967. 24. Ng KKF, Vane JR: Fate of angiotensin I in the circulation. Nature (Land) 218: 144, 1968. 25. Ryan JW, Niemeyer RS, Goodwin DW, et al.: Metabolism of (8-L-(14C)phenylalanine)-angiotensin I in the pulmonary
Volume 63
IS THE LUNG A PARA-ENDOCRINE ORGAN?
26. 27. 28.
29. 30.
31.
32.
33.
34.
35.
36. 37. 38.
circulation. Biochem J 125: 921, 1971. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in the pulmonary circulation. Biochem J 110: 795, 1968. Ryan JW, Roblero J, Stewart JM: Inactivation of bradykinin in rat lung. Pharmacol Res Commun 1: 192, 1969. (a) Dorer FE, Kahn JR, Lentz KE, et al.: Hydrolysis of bradykinin byangiotensin-convertingenzyme.CircRes34: 824.1974. (b) Leary WP, Ryan JW: Unpublished data. Ryan JW, Smith U. Niemeyer RS: Angiotensin I. Metabolism by plasma membrane of lung. Science 176: 64, 1972. Ryan JW, Smith U: Metabolism of adenosine-5’-monophosphate during circulation through the lungs. Trans Assoc Am Physicians 84: 297. 1971. Ryan JW. Smith U: A rapid, simple method for isolating pinocytotic vesicles and plasma membrane of lung. Biochim Biophys Acta 249: 177, 1971. Ryan JW, Smith U: The metabolism of angiotensin I by endothelial cells. Protides of the Biological Fluids (Peeters H, ed), Oxford, England, Pergamon Press, 1973, p 379. Smith U, Ryan JW: Electron microscopy of endothelial cells collected on cellulose acetate paper. Tissue Cell 5: 333, 1973. Chiu AT, Ryan JW. Ryan US, et al.: A sensitive radioachemical assay for angiotensin-converting enzyme (kininase II). Biochem J 149: 297, 1975. Pietra GG, Szidon JP, Leventhal MM, et al.: Histamine and interstitial pulmonary edema in the dog. Circ Res 29: 323, 1971. De Bono E, Lee G de J, Mottram FR, et al.: The action of angiotensin in man. Clin Sci 25: 123, 1963. Smith U, Ryan JW: Pinocytotic vesicles of the pulmonary endothelial cell. Chest 59: 12S, 1971. Dorer FE, Kahn JR, Lentz KE, et al.: Purification and properties
39.
40.
41.
42.
43.
44.
45.
46.
47.
October 1977
of angiotensin-converting enzyme frolm hog lung. Circ Res 31: 356, 1972. Dorer FE, Ryan JW, Stewart JM: Hydrolysis of bradykinin and its higher homologues by angiotensin-converting enzyme. Biochem J 141: 915. 1974. Dorer FE, Kahn JR, Lentz KE, et al.: Forrnation of angiotensin II from tetradecapeptide renin substrate by angiotensinconverting enzyme. Biochem Pharmacol 24: 1137, 1975. Ryan JW, Ryan US, Schultz DR, et al.: Subcellular localization of pulmonary angiotensin converting enzyme (kininase II). Biochem J 146: 497, 1975. Ryan JW, Day AR, Ryan US, et al.: Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-heme-octapeptide conjugates. Tissue Cell 8: 111, 1976. Ryan US, Ryan JW, Whitaker C, et al.: Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence. Tissue Cell 8: 125, 1976. Caldwell PRB, Seegal BC, Hsu KC, et al.: Angiotensin-converting enzyme. Vascular endothelial localization. Science 191: 1050, 1976. Aiken JW, Vane JR: Inhibition of converting enzyme of the renin-angiotensin system in kidneys and hindlegs of dogs. Circ Res 30: 263, 1972. Cushman DW, Cheung HS: Concentrations of angiotensinconverting enzyme in tissues of the rat. Biochim Biophys Acta 250: 261, 1971. Gavras H, Brunner HR. Laragh JH, et al.: An angiotensin converting enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients. N Engl J Med 291: 817, 1974.
The American Journal of Medlclne
Volume 63
603
