Pergamon Press
Life Sciences, Vol . 24, pp . 1419-1424 Printed in the U .S .A .
ANGIOTENSIN I CONVERTING ENZYME (KININASE II) IN ISOLATED RETINAL MICROYESSELS Patrick E . Ward; Tess A . Stewart, Katy J . Hammon, Rolland C . Reynolds and Rajko P . Igic Departments of Pharmacology and Pathology University of Texas Health Science Center Dallas, Texas 75235, U .S .A . (Received in final form March 1,
1979)
Summary Swine retinae were homogenized and fractions enriched in retinal microvasculature were prepared by techniques of selective sieving and centrifugation . The identity and purity of the preparations were investigated by phase contrast and electron microscopy . Angiotensin I converting enzyme (kininase II) was concentrated in the retinal microvessels . Metabolism of angiotensins and kinins in localized sites of the vasculature may contribute to local regulation of blood flow . Angiotensin I converting enzyme (EC 3 .4 .15 .1) is a carboxy-terminal peptidyl dipeptidase which catalyzes the release of His-Leu from the decapeptide angiotensin I to form angiotensin II and also inactivates kinins (1 ,2 ) . The metabolism of kinins and angiotensins can have significant effects on systemic circulation . Angiotensin I converting enzyme (kininase II) has been localized in a variety of vascular beds (2-8) and in epithelial cells of the kidney and intestine (9-11) . Igic et al . (12) have found converting enzyme in crude homogenates of the retina~F~e retina is one of the few tissue beds from which pure, metabolically active microvessels approaching capillary size can be isolated (13) . In the present study we investigated the distribution of converting enzyme in purified microvessels of the retina . The presence of converting enzyme in retinal vessels may be indicative of a functional role of kinins and angiotensins in control of retinal blood flow . Methods Animals : Eyes were obtained at a local slaughter house frrom freshly slang tare swine . Retinal vessels were prepared within two hrs . of death . Microvessels : As described by Meezan et al . (13), swine eyes were bisecte amend-the retinae dissected and placed~n~arle's balanced salt solution buffered with 28 mM HEPES, pH 7 .4 at 4oC . After mincing, the retinae were homogenized in 25% (w/v) of the above buffer with 10 up and down strokes *Established Investigator - American Heart Association 0024-3205/79/151419-0502 .00/0 Copyright (c) 1979 Pergamon Press Ltd
142 0
Converting Enzyme in the Retina
Vol . 24, No . 15, 1979
of a hand-held glass-Teflon homogenizer (Thomas, size B) . The homogenate was poured over an 85 micron nylon sieve and the sieve washed extensively with buffer . The material remaining on the sieve was washed into a test tube and centrifuged (1000 x g, 2 min) and the pellet resuspended . After microscopic examination, the preparation was disrupted by sonication or freezing and thawing . En me Ass ~: Angiotensin I converting enzyme (kininase II) was assayed by incu at ng ractions with 1 mM hippurylglycylglycine in 0 .1 M Tris, pH 7 .4, containing 0 .1 M-NaCI at 37oC (10, 14) . Converting enzyme activity was calcu lated as the amount of hippurylglycylglycine hydrolysed that could be inhibited by 0 .1 mM of the specific inhibitor, SQ 20881 . The amount of diglycine released was assayed in a Beckman 121 amino acid analyzer . Kininase was assayed by incubating samples with bradykinin in 0 .1 M Tris, pH 7 .4, 0 .2 M NaCI at 37oC then following the inactivation of the peptide on the isolated rat uterus . Protein was determined by the Lowry method with bovine serum albunin as standard (15) . Electron Microsco : Swine retinal vessels were pelleted and fixed in 2% glutara e yde in l00 mM sodium phosphate buffer (pH 7 .4) for two hrs . The pellets were washed and stored overnight in 200 mM sucrose with 100 mM sodium phosphate . The pellets were post-fixed in 2% osmium tetroxide in 100 mM sodium phosphate . After rinsing with distilled water, dehydration with graded ethanol solutions and treatment with propylene oxide, the pellets were infiltrated with and embedded in Epon 812 which was subsequently polymerized at 60oC for 24 hrs . Sections were stained with urar~yl acetate and lead citrate prior to observation and photography . Materials Bradykinin and the converting enzyme substrate (hippurylglycylglycine) were obtained from Schwarz-Mann (Orangeburg, NY) . The converting enzyme inhibitors SQ 20881 and SQ 14225 were obtained from Dr . Z . Horovitz of Squibb, Inc . (Princeton, NJ) . The sieve used in vessel isolation was from Nitex Nylon monofilament bolting cloth b Tetko (Houston, TX) . Earle's salt solution was from Gibco (Grand Island, NY~ . Results Microvessels : Retinal microvessels were prepared from approximately 100 eyes in three experiments . The purified preparations, examined by phase contrast microscopy, were devoid of non-vascular contamination other than trapped blood cells (Figure 1) . Most of the vessels measured 10-20 microns in diameter with some as much as 100 microns in width . Frequently dichotomous branching at acute angles was apparent as was the normal pyramidal shape distribution pattern of small vessels and interconnecting collateral branches . Electron microscopy revealed that the structural integ rity of the isolated vessels was maintained by the intact basal lamina surrounding them . No perivascular inters ti ti al cells were attached to the blood vessels although fragIn general, the endothelial ments of cellular debri were occasionally seen . cells, their intracellular organelles, and intercellular junctions were intact . Endothelial damage, characterized by loss of ribosomes, edema and swelling, was minimal . In spaces between layers of the basal lamina, electron dense intramural pericytes (16,17) and occasional bundles of connective tissue were seen (Figure 2) . In summary, morphologic examination revealed intact capillaries and some
Vol . 24, No . 15,
1979
Converting Enzyme in the Retina
1421
FIG . 1 Phase contrast micrograph of isolated swine retinal blood vessels (X 400) .
FIG . 2 Electron micrograph of isolated swine retinal blood vessels (X 18,500) . Structural details include : red blood cell (RBC), lumen (L), endothelial cell (E), mitochondria (M), basal lamina (BL) and interstitial pericytes (P) .
142 2
Converting Enzyme in the Retina
Vol . 24, No . 15, 1979
pre- and post-capillary vessels . The endothelial cells and intramural pericytes of the isolated vessels were relatively undamaged by the isolation procedure . The specific activity of angiotensin I converting enzyme (kininase II) in the purified vessel preparation was approximately twenty times higher than in the homogenate and recovery of total activity was near 40% (Table I) . This concentration of activity was not due to the presence of blood cells (which had no detectible activity) or to serum, which had low levels of converting enzyme (0 .03 mol/hr/mg) . The vascular converting enzyme wa~ also susceptible to inhibition by SQ 14225 . Concentrations as low as 10 - M produced 100% inhibition .
TABLE I Angiotensin Converting Enzyme Activity of Swine Retinal Blood Vessels Converting Enzyme }unol /hr/mg
Relative Specific Activity
Recovery %
Retinal Homogenate
0 .010 + 0 .001
1
100
Retinal Vessels
0 .210 + 0 .026
21
37
~erum
0 .031 + 0 .006
3 .1
---
Retinal blood vessels were prepared as described by Meezan et al . (13) . All values are means + S .E .M . of three experiments . Relative specific activity is (mean s ecificactivity in the sample)/(mean specific activity in the hornogenate~ .
The isolated vasculature also inactivated bradykinin at a rate of 4 .8 micromole per hour per mg protein . This ~Cininase activity was almost completely inhibited (>90%) by SQ 20881 (10 - M) . Thus most of the kininase activity could be ascribed to the angiotensin I converting enzyme . Discussion The results of the present study demonstrate that angiotensin I converting enzyme (kininase II) is present in the microvessels of the retina . The level of converting enzyme (kininase II) in the microvessels is more than five times higher (units/mg protein) than that seen in blood . Such a local concentration of enzyme may indicate a significant role for angiotensin II and/or Indeed, Legant et _al . (18) have kinin in local control of retinal blood flow . reported data which suggest that in hypoxic tissues local]y generated bradykinin will acc~snulatel and angiotensin II levels decrease) due to inhibition of systemic vascular converting enzyme (kininase II) . Such a mechanism may provide for a graded increase in blood flow to hypoxic tissues . If the presence of a kinin and angiotensin metabolizing enzyme in specific vascular beds is related to local control of blood flow, administration of converting enzyme inhibitors to hypertensive patients may alter local blood
Vol . 24, No . 15, 1979
Converting Enzyme in the Retina
1423
flow disproportionately to changes in systemic blood pressure . Oral therapy with the converting enzyme inhibitor SQ 14225 may be used widely to treat sane forms of hypertension (19) . Converting en~,yme is present in a variety of vascular beds including the lung (1,2,8), liver, adrenal cortex, pancreas (5,9) and brain (6) . Although changes in blood flow in such tissue beds would be impossible to monitor routinely in patients receiving the converting en~yrne inhibitor, alterations in retinal blood flow could be more easily observed . It might be of interest to monitor possible changes in the state of the retinal vasculature in patients receiving converting enzyme inhibitor canpared to patients receiving other types of antihypertensive medication . Arty significant changes may be relevant not only to the pathophysiology of the eye but may also be indicative of changes occurring in other vascular beds . Acknowledgements We are grateful for the skilled assistance of Ms . Martha Ann Sheridan and for the helpful advice and criticisms of Dr . E. G. Erdôs . This work was supported by the following grants : American Heart - Texas Affiliate, N .I .H . HL 16320, and Institutional Grant 5-S07-RR-07175 . References 1. 2.
E.G . ERDOS, Am . J . Med . 60, 749-759 (1976) . Y.S . BAKHLE, ngi~otén Win , (Eds . I .H . Page and F .M . Bumpus) pp . 41-80, Springer-Verlag, Heidelberg (1974) . 3. K.K .F . NG and J .R . VANE, Nature London , 218, 114-150 (1968) . 4. Y .S . BAKHLE, A.M . REYNARD D .R . V NE, Nature (London) 222, 956-959 (1969) . 5. P .R .B . CALDWELL, B.C . SEEGAL, K.C . HSU, M. DAS and R .L . SOFFER, Science 191, 1050-1051 (7976) . 6 . F~ORLOWSKI and E. WILK, ed . Proc . _37, 602 (1978) . 7 . A.R . JOHNSON and E .G . ERD S, J. Clin Invest . 59, 684-695 (1977) . 8. J .W . RYAN and U .S . RYAN, Card ovas . Me . , 531-538 (1978) . 9 . H .J . WIGGER and S.A STALCUP, Lab . Invest. 38, 581-585 (1978) . 10 . P .E . WARD, E.G . ERDBS, C .D . GE~tEY,~F~DOR~EN and R.C . REYNOLDS, Biochem . J.157, 643-650 (1976) . 11 . ~.~WARD, R.J . KLAUSER and E.G . ERDO , Pharmacolo 1st 20, 260 (1978) . 12 . R . IGIC, C .J .G . ROBINSON and E.G . ERD~S, entral ctions of An iotensin and Related Hormones (Eds . J . P . Buckley an C . M. Ferrario p. 23 Pergamon Press, New York (1977) . 13 . E . MEEZAN, K. BRENDEL and E .C . CARLSON, Nature 251, 65-67 (1974) . 14 . H .Y .T . YANG, E .G . ERDÔS and Y. LEVIN, J .~rmaco . Exp. Ther . 177, 291 (1971) . 15 . O.H . LOWRY, N .J . ROSEBROUGH, A.L . FARR and R.J . RANDALL, J. Biol . Chem . 193, 265-275 (1951) . 16 . P:R. AGRAWAL, Orient . Arch . 0 thal . 3, 23-26 (1965) . 17 . T. KUWABARA an D .G . CO N, Arch . _0~thal . 492, 69-74 (1963) . 18 . P.M . LEGANT, S.A . STALCUP, J~CiFSET,-R .~OTNONES and R .B . MILLENS, Fed . Proc . 37, 602 (1978) . 19 . Ham, H .R . BRUNWER, G .A . TURINI, G.R . KERSHAW, C .P . TIFFT, S . CUTTE- . LOD, I . GAVRAS, R .A . VUKOVICH and D.N . McKINSTRY, N. Eng . J . Med. 298, 991-995 (1978) .
Life Sciences, Vol . 24, pp . 1419-1424 Printed in the U .S .A .
ANGIOTENSIN I CONVERTING ENZYME (KININASE II) IN ISOLATED RETINAL MICROYESSELS Patrick E . Ward; Tess A . Stewart, Katy J . Hammon, Rolland C . Reynolds and Rajko P . Igic Departments of Pharmacology and Pathology University of Texas Health Science Center Dallas, Texas 75235, U .S .A . (Received in final form March 1,
1979)
Summary Swine retinae were homogenized and fractions enriched in retinal microvasculature were prepared by techniques of selective sieving and centrifugation . The identity and purity of the preparations were investigated by phase contrast and electron microscopy . Angiotensin I converting enzyme (kininase II) was concentrated in the retinal microvessels . Metabolism of angiotensins and kinins in localized sites of the vasculature may contribute to local regulation of blood flow . Angiotensin I converting enzyme (EC 3 .4 .15 .1) is a carboxy-terminal peptidyl dipeptidase which catalyzes the release of His-Leu from the decapeptide angiotensin I to form angiotensin II and also inactivates kinins (1 ,2 ) . The metabolism of kinins and angiotensins can have significant effects on systemic circulation . Angiotensin I converting enzyme (kininase II) has been localized in a variety of vascular beds (2-8) and in epithelial cells of the kidney and intestine (9-11) . Igic et al . (12) have found converting enzyme in crude homogenates of the retina~F~e retina is one of the few tissue beds from which pure, metabolically active microvessels approaching capillary size can be isolated (13) . In the present study we investigated the distribution of converting enzyme in purified microvessels of the retina . The presence of converting enzyme in retinal vessels may be indicative of a functional role of kinins and angiotensins in control of retinal blood flow . Methods Animals : Eyes were obtained at a local slaughter house frrom freshly slang tare swine . Retinal vessels were prepared within two hrs . of death . Microvessels : As described by Meezan et al . (13), swine eyes were bisecte amend-the retinae dissected and placed~n~arle's balanced salt solution buffered with 28 mM HEPES, pH 7 .4 at 4oC . After mincing, the retinae were homogenized in 25% (w/v) of the above buffer with 10 up and down strokes *Established Investigator - American Heart Association 0024-3205/79/151419-0502 .00/0 Copyright (c) 1979 Pergamon Press Ltd
142 0
Converting Enzyme in the Retina
Vol . 24, No . 15, 1979
of a hand-held glass-Teflon homogenizer (Thomas, size B) . The homogenate was poured over an 85 micron nylon sieve and the sieve washed extensively with buffer . The material remaining on the sieve was washed into a test tube and centrifuged (1000 x g, 2 min) and the pellet resuspended . After microscopic examination, the preparation was disrupted by sonication or freezing and thawing . En me Ass ~: Angiotensin I converting enzyme (kininase II) was assayed by incu at ng ractions with 1 mM hippurylglycylglycine in 0 .1 M Tris, pH 7 .4, containing 0 .1 M-NaCI at 37oC (10, 14) . Converting enzyme activity was calcu lated as the amount of hippurylglycylglycine hydrolysed that could be inhibited by 0 .1 mM of the specific inhibitor, SQ 20881 . The amount of diglycine released was assayed in a Beckman 121 amino acid analyzer . Kininase was assayed by incubating samples with bradykinin in 0 .1 M Tris, pH 7 .4, 0 .2 M NaCI at 37oC then following the inactivation of the peptide on the isolated rat uterus . Protein was determined by the Lowry method with bovine serum albunin as standard (15) . Electron Microsco : Swine retinal vessels were pelleted and fixed in 2% glutara e yde in l00 mM sodium phosphate buffer (pH 7 .4) for two hrs . The pellets were washed and stored overnight in 200 mM sucrose with 100 mM sodium phosphate . The pellets were post-fixed in 2% osmium tetroxide in 100 mM sodium phosphate . After rinsing with distilled water, dehydration with graded ethanol solutions and treatment with propylene oxide, the pellets were infiltrated with and embedded in Epon 812 which was subsequently polymerized at 60oC for 24 hrs . Sections were stained with urar~yl acetate and lead citrate prior to observation and photography . Materials Bradykinin and the converting enzyme substrate (hippurylglycylglycine) were obtained from Schwarz-Mann (Orangeburg, NY) . The converting enzyme inhibitors SQ 20881 and SQ 14225 were obtained from Dr . Z . Horovitz of Squibb, Inc . (Princeton, NJ) . The sieve used in vessel isolation was from Nitex Nylon monofilament bolting cloth b Tetko (Houston, TX) . Earle's salt solution was from Gibco (Grand Island, NY~ . Results Microvessels : Retinal microvessels were prepared from approximately 100 eyes in three experiments . The purified preparations, examined by phase contrast microscopy, were devoid of non-vascular contamination other than trapped blood cells (Figure 1) . Most of the vessels measured 10-20 microns in diameter with some as much as 100 microns in width . Frequently dichotomous branching at acute angles was apparent as was the normal pyramidal shape distribution pattern of small vessels and interconnecting collateral branches . Electron microscopy revealed that the structural integ rity of the isolated vessels was maintained by the intact basal lamina surrounding them . No perivascular inters ti ti al cells were attached to the blood vessels although fragIn general, the endothelial ments of cellular debri were occasionally seen . cells, their intracellular organelles, and intercellular junctions were intact . Endothelial damage, characterized by loss of ribosomes, edema and swelling, was minimal . In spaces between layers of the basal lamina, electron dense intramural pericytes (16,17) and occasional bundles of connective tissue were seen (Figure 2) . In summary, morphologic examination revealed intact capillaries and some
Vol . 24, No . 15,
1979
Converting Enzyme in the Retina
1421
FIG . 1 Phase contrast micrograph of isolated swine retinal blood vessels (X 400) .
FIG . 2 Electron micrograph of isolated swine retinal blood vessels (X 18,500) . Structural details include : red blood cell (RBC), lumen (L), endothelial cell (E), mitochondria (M), basal lamina (BL) and interstitial pericytes (P) .
142 2
Converting Enzyme in the Retina
Vol . 24, No . 15, 1979
pre- and post-capillary vessels . The endothelial cells and intramural pericytes of the isolated vessels were relatively undamaged by the isolation procedure . The specific activity of angiotensin I converting enzyme (kininase II) in the purified vessel preparation was approximately twenty times higher than in the homogenate and recovery of total activity was near 40% (Table I) . This concentration of activity was not due to the presence of blood cells (which had no detectible activity) or to serum, which had low levels of converting enzyme (0 .03 mol/hr/mg) . The vascular converting enzyme wa~ also susceptible to inhibition by SQ 14225 . Concentrations as low as 10 - M produced 100% inhibition .
TABLE I Angiotensin Converting Enzyme Activity of Swine Retinal Blood Vessels Converting Enzyme }unol /hr/mg
Relative Specific Activity
Recovery %
Retinal Homogenate
0 .010 + 0 .001
1
100
Retinal Vessels
0 .210 + 0 .026
21
37
~erum
0 .031 + 0 .006
3 .1
---
Retinal blood vessels were prepared as described by Meezan et al . (13) . All values are means + S .E .M . of three experiments . Relative specific activity is (mean s ecificactivity in the sample)/(mean specific activity in the hornogenate~ .
The isolated vasculature also inactivated bradykinin at a rate of 4 .8 micromole per hour per mg protein . This ~Cininase activity was almost completely inhibited (>90%) by SQ 20881 (10 - M) . Thus most of the kininase activity could be ascribed to the angiotensin I converting enzyme . Discussion The results of the present study demonstrate that angiotensin I converting enzyme (kininase II) is present in the microvessels of the retina . The level of converting enzyme (kininase II) in the microvessels is more than five times higher (units/mg protein) than that seen in blood . Such a local concentration of enzyme may indicate a significant role for angiotensin II and/or Indeed, Legant et _al . (18) have kinin in local control of retinal blood flow . reported data which suggest that in hypoxic tissues local]y generated bradykinin will acc~snulatel and angiotensin II levels decrease) due to inhibition of systemic vascular converting enzyme (kininase II) . Such a mechanism may provide for a graded increase in blood flow to hypoxic tissues . If the presence of a kinin and angiotensin metabolizing enzyme in specific vascular beds is related to local control of blood flow, administration of converting enzyme inhibitors to hypertensive patients may alter local blood
Vol . 24, No . 15, 1979
Converting Enzyme in the Retina
1423
flow disproportionately to changes in systemic blood pressure . Oral therapy with the converting enzyme inhibitor SQ 14225 may be used widely to treat sane forms of hypertension (19) . Converting en~,yme is present in a variety of vascular beds including the lung (1,2,8), liver, adrenal cortex, pancreas (5,9) and brain (6) . Although changes in blood flow in such tissue beds would be impossible to monitor routinely in patients receiving the converting en~yrne inhibitor, alterations in retinal blood flow could be more easily observed . It might be of interest to monitor possible changes in the state of the retinal vasculature in patients receiving converting enzyme inhibitor canpared to patients receiving other types of antihypertensive medication . Arty significant changes may be relevant not only to the pathophysiology of the eye but may also be indicative of changes occurring in other vascular beds . Acknowledgements We are grateful for the skilled assistance of Ms . Martha Ann Sheridan and for the helpful advice and criticisms of Dr . E. G. Erdôs . This work was supported by the following grants : American Heart - Texas Affiliate, N .I .H . HL 16320, and Institutional Grant 5-S07-RR-07175 . References 1. 2.
E.G . ERDOS, Am . J . Med . 60, 749-759 (1976) . Y.S . BAKHLE, ngi~otén Win , (Eds . I .H . Page and F .M . Bumpus) pp . 41-80, Springer-Verlag, Heidelberg (1974) . 3. K.K .F . NG and J .R . VANE, Nature London , 218, 114-150 (1968) . 4. Y .S . BAKHLE, A.M . REYNARD D .R . V NE, Nature (London) 222, 956-959 (1969) . 5. P .R .B . CALDWELL, B.C . SEEGAL, K.C . HSU, M. DAS and R .L . SOFFER, Science 191, 1050-1051 (7976) . 6 . F~ORLOWSKI and E. WILK, ed . Proc . _37, 602 (1978) . 7 . A.R . JOHNSON and E .G . ERD S, J. Clin Invest . 59, 684-695 (1977) . 8. J .W . RYAN and U .S . RYAN, Card ovas . Me . , 531-538 (1978) . 9 . H .J . WIGGER and S.A STALCUP, Lab . Invest. 38, 581-585 (1978) . 10 . P .E . WARD, E.G . ERDBS, C .D . GE~tEY,~F~DOR~EN and R.C . REYNOLDS, Biochem . J.157, 643-650 (1976) . 11 . ~.~WARD, R.J . KLAUSER and E.G . ERDO , Pharmacolo 1st 20, 260 (1978) . 12 . R . IGIC, C .J .G . ROBINSON and E.G . ERD~S, entral ctions of An iotensin and Related Hormones (Eds . J . P . Buckley an C . M. Ferrario p. 23 Pergamon Press, New York (1977) . 13 . E . MEEZAN, K. BRENDEL and E .C . CARLSON, Nature 251, 65-67 (1974) . 14 . H .Y .T . YANG, E .G . ERDÔS and Y. LEVIN, J .~rmaco . Exp. Ther . 177, 291 (1971) . 15 . O.H . LOWRY, N .J . ROSEBROUGH, A.L . FARR and R.J . RANDALL, J. Biol . Chem . 193, 265-275 (1951) . 16 . P:R. AGRAWAL, Orient . Arch . 0 thal . 3, 23-26 (1965) . 17 . T. KUWABARA an D .G . CO N, Arch . _0~thal . 492, 69-74 (1963) . 18 . P.M . LEGANT, S.A . STALCUP, J~CiFSET,-R .~OTNONES and R .B . MILLENS, Fed . Proc . 37, 602 (1978) . 19 . Ham, H .R . BRUNWER, G .A . TURINI, G.R . KERSHAW, C .P . TIFFT, S . CUTTE- . LOD, I . GAVRAS, R .A . VUKOVICH and D.N . McKINSTRY, N. Eng . J . Med. 298, 991-995 (1978) .
