Exp. Eye Res. (1992) 55, 563-568
Local Generation and Action of Angiotensin II in Dog Iris Sphincter Muscle TOMIO OKAMURA*,YAN
W A N G AND N O B O R U T O D A
Department of Pharmacology, Shiga University of Medical Sciences, Seta, Ohtsu 520-21. Japan (Received Chicago 2 October 1991 and accepted in revised form 20 January 1992) Existence of the renin-angiotensin system was pharmacologically investigated in the dog isolated iris sphincter muscle. The sphincter muscle contracted in response to tetradecapeptide, a synthetic renin substrate, angiotensin (ANG) I and ANG II dose-dependently. The contractions induced by these peptides were suppressed by treatment with saralasin, indomethacin and aspirin. Contractile responses to tetradecapeptide and ANG I were also reduced by KRI-1314, a renin inhibitor, and captopril, respectively. ANG II stimulated the release of prostaglandin (PG) F~ from the sphincter muscle. Angiotensinconverting enzyme activity was measurable in the sphincter muscle. Miosis was observed by intracameral injection of ANG I and ANG II into the anterior chamber. These results strongly suggest that angiotensin generating enzymes function in the sphincter muscle and ANG It produced by these enzymes contracts the sphincter muscle via the formation of PG (s), possibly PG F2~. Key words: renin; angiotensin I; angiotensin II; tetradecapeptide; angiotensin-converting enzyme; prostaglandin F2~; miosis; sphincter muscle contraction. 1. Introduction
2. Materials and Methods
Angiotensin (ANG) II, a bioactive product of the renin-angiotensin system, is known as a blood-borne peptide regulating blood pressure, local blood flow and water-electrolyte balance. Recent studies have demonstrated the local formation of ANG II in various tissues such as brain (Printz et al., 1982), vascular wall (Okamura et al., 1990), adrenal (Nakamaru et al., 1985) and reproductive organs (Pandy, Misono and Inagami, 1984), suggesting the important roles of the peptide in functioning of these organs and tissues. Locally produced ANG II has been suggested to regulate ocular functions. Administration of ANG II through the lingual artery or into the vitrous body in vivo (Eakins, 1964) and through the ophthalmic artery in vitro (Macri, 1965) lowers the intraocular pressure. The peptide contracts isolated dog sphincter pupillae via a synthesis of cyclooxygenase products by acting on specific ANG II receptors (Lu et al., 1988). Although the renin activity in the aqueous humor (Ikemoto and Yamamoto, 1978) and the angiotensinconverting enzyme (ACE) activity in retina (Ferraridries et al., 1988 ; Igic and Kojovic, 1980), choroid (Igic and Kojovic, 1980), ciliary body (Igic and Kojovic, 1980), aqueous humor (Weinreb et al., 1985; Vita et al., 1981) and tear (Vita et al., 1981) have been detected, functional significance of these enzymes has not been elucidated in the ocular tissues. The present study was thus undertaken to compare the actions of ANG II and its precursors on dog sphincter muscles in vitro and in vivo, in order to clarify the local generation and action of ANG II in the ocular tissues.
Preparation of Isolated Sphincter Pupillae
*For correspondence at: Department of Pharmacology, Shiga University of Medical Sciences, Seta, Ohtsu 520-21. Japan. 0014-4835/92/100563 + 06 $08.00/0
Mongrel dogs of both sexes, weighing 8-15 kg, were anesthetized with intravenous injections of sodium pentobarbital (30 mg kg -1) and killed by bleeding from the common carotid arteries. Eyes were rapidly removed. One strip of the iris sphincter was isolated from each eye. The strip was fixed vertically between hooks in the muscle bath of 20 ml capacity containing the modified Ringer-Locke solution, which was maintained at 37+_0-3°C and aerated with a mixture of 95% 02 and 5% CO2. The hook anchoring the upper end of the strip was connected to the lever of a forcedisplacement transducer (Nihonkohden Kogyo Co., Tokyo, ]apan). The resting tension was adjusted to 50 rag. Constituents of the solution were as follows (raM): NaC1, 120; KC1, 5.4; NaHCO 3, 25.0; CaC12, 2.2; MgCI~, 1.0; and dextrose, 5"6. The pH of the solution was 7.3-7"4. Before the start of experiments, the strips were allowed to equilibrate for 6 0 - 9 0 rain in control media, during which time the solutions were replaced every 10-15 rain.
Tension Recording Isometric contractions were recorded on an inkwriting oscillograph (Nihonkohden Kogyo Co.). Test drugs were added directly to the bathing media in cumulative concentrations (acetylcholine) or in single concentrations (tetradecapeptide, ANG I and ANG II) because of tachyphylaxis by cumulative applications. The contractile response to 30 mM K÷ was first obtained and the preparations were washed three times with control media and equilibrated for 4 0 -
© 1992 Academic Press Limited
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50 rain. Then, the concentration-contraction curve for acetylcholine was obtained. Contractions induced by 10 -4 M acetylcholine were taken as a standard for comparison on the contractile responses to the peptides. To determine the concentration-response relationship for ANG I or II, the response to 10-VM ANG II was obtained twice and then the response to one of the concentrations (5 x 10 -9 to 2 x 10 -6 M) of ANG II was obtained. Preparations were treated for 20 rain with pharmacological inhibitors, before the response to the agonist was obtained.
Measurement of Angiotensin-converting Enzgme (ACE) Activity Tissue ACE activity was determined by using the modified method of Cushman and Cheung (1970) as previously described (Okamura et al., 1986). Briefly, flesh tissues (dog iris sphincters, mesenteric arteries with and without the endothelium and lungs, and monkey lungs; wet weight 3 0 - 5 0 0 mg) were homogenized with ten volumes of homogenization buffer. This solution consisted of 20 mM Tris-HC1 buffer (pH 8-3), 5 mM magnesium acetate, 30 mM KC1, 0-25 M sucrose and 0'5% Nonidet P-40 (Okamura et al., 1986). The homogenized sample was stored overnight at 4°C and then centrifuged for 20 rain at 2 0 0 0 0 g and 4°C. The supernatant was then incubated in the presence and absence of captopril, an ACE inhibitor, at 37°C for 30 min with hippuryl-L-histidylr-leucine (5 raM) in lOmM potassium phosphate buffer, pH 8.3, containing 800 m ~ or 600 mM sodium chloride for dog or monkey tissues, respectively (Miyazaki et al., 1984). The enzyme reaction was stopped by the addition of 3 % metaphosphate. The reaction mixture then was centrifuged at 12 000 g for 10 min and the supernatant was applied to a reversed phase column and eluted at 38°C with lOmM KH~PQ:methanol (1:1, pH3"O) at a rate of 0.7 ml rain -1. Hippurate was detected by ultraviolet absorbance at 228 nm. The activity of ACE was expressed as specific activity (mUmg -1 protein) using tissue protein concentration values determined by the modified method of Bradford (1976).
Measurement of PGF2~ Two sphincter muscle strips obtained from eyes of the same dogs were used for the paired analysis of PGF~ release in the bathing media. After 20 rain preincubation at 37°C, one of the strips was treated with 10 7 M ANG II and the other with vehicle. The preparation was incubated for 15 rain, before adding indomethacin (10 -~ M) to stop the further production of PGF~. The bathing medium was then quickly collected by aspiration and stored frozen at below - 6 0 ° C until the assay. Samples were concentrated ten times by SEP-PAK C18 column (Waters, MA, U.S.A.) prior to the assay. PGF2~ was measured by
radioimmunoassay using a commercial kit (PGF~[3H] assay system, Amersham, U.K.).
Miotic Response In Vivo Mongrel dogs were anesthetized with intravenous administration of sodium pentobarbital as described above. Before topical applications of testing drugs, both eyes received a couple of drops of Mydrin P~ (0'5% tropicamide and 0"5% phenylephrine hydrochloride) in order to obtain sufficient dilatation of iris sphincter muscles. After stabilization, a small part of cornea was incised with a razor blade. All drugs (100 #1) were diluted with saline and injected into the anterior chamber through the incision site of either eye and saline was injected into the other eye, as a control. Whenever the drug was injected, 100 #1 of aqueous humor was taken just before the injection. Time-dependent changes of the pupil sizes were photographed. Miotic responses were quantitatively analysed by measuring the pupil size which was calculated as a ratio of' white-to-white' a n d ' black-toblack' diameters.
Statistics and Drugs Results shown in the text, table and figures are expressed as mean+s.E.M. Statistical analyses were made using the Student's paired and unpaired t-test and Tukey's method after one-way analysis of variance. Drugs used were PGF~ (Ono Pharmaceutical Co., Osaka, Japan), indomethacin and tetradecapeptide (Sigma, St Louis, MO, U.S.A.), angiotensin I, angiotensin I[, [Sar 1, AlaS]angiotensin II (saralasin) and hippuryl-L-histydyl-L-leucine (Peptide Institute Inc., Minoh, Japan), acetylsalicylic acid (Nacalai Tesque, Kyoto, Japan) acetylcholine chloride (Daiichi Pharmaceutical Co., Tokyo, Japan), captopril (Sankyo Pharmaceutical Co., Tokyo), KRI-1314 (Kissei Pharmaceutical Co., Nagano, Japan), Mydrin P® (0"5% tropicamide and 0.5 % phenylephrine, Santen Pharmaceutical Co., Osaka) and sodium pentobarbital (Abbott Lab., North Chicago, IL, U.S.A.).
3. Results Responses to ANG I and ANG II The addition of ANG I (2 x 10 -s to 2 x 10 -6 M) and ANG II (5 x 10 -9 to 10 -6 M) caused a concentrationrelated contraction in the dog isolated sphincter muscle (Fig. 1). Mean values of the median effective concentration (EC60) of ANG I and ANG II were ( 3 " 6 7 _ 1 - 0 0 ) × 1 0 - 7 M ( n = 7 ) and (3.96+1.08) x 10-8M (n = 7), respectively. Maximum contractions caused by these peptides were obtained at 10 -6 M and the magnitude of the contractions were almost the same. The contractile response to 5 x lO-TM ANG I was almost abolished by treatment with lO-VM
RENIN-ANGIOTENSIN
SYSTEM
IN IRIS
565
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Control
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Soralasin Captopril Indomefhocin KRI 1514
FIG. 3. Modification by 10 .7 M saralasin (n = 7), 10 -s M captopril (n = 6), 10-6M indomethacin (n = 7) and 5 x 10-5M KPd-1314 (n = 11) of the tetradecapeptide (5 x 10 -7 M)-induced contraction in iris sphincter strips. Contractions induced by tetradecapeptide in control preparations (n = 23) were taken as 100%; the m e a n absolute values was 30.0 _+4.9 mg. * Significantly different from controls at P < 0"01. Vertical hines indicate S.E.M.
8
o
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Concenfrcflion of pepfide (M)
FIG. 1. Concentration-response curves for angiotensin [ (C), n = 7) and angiotensin II ( 0 , n = 7) in the dog iris sphincter muscle. Contractions induced by 10-4M acetylcholine were taken as 100 %; the m e a n absolute value was 141"2 +- 13"0 mg (n = 14). Vertical lines indicate S.E.M.
v
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o
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Concentration of KRI-1314(M)
o Control Soralasin Captopril Indomethacin Aspirin FIG. 2. Modification by 10 -7 M saralasin (n = 11), 10 5 M captopril (n = 10), 10 -6 M indomethacin (n = 8) and 5 x 10-SM aspirin (n = 10) of angiotensin I (5X10-TM) induced contraction in iris sphincter strips. Contractions induced by angiotensin I in control preparations (n = 10) were taken as 100%; the m e a n absolute value was 54.6 +_9.6 mg. * Significantly different from controls at P < 0-001. Vertical lines indicate S.E.M.
s a r a l a s i n , ]O-SM captopril, 1 0 - 6 M i n d o m e t h a c i n or 5 x 10 -5 M a s p i r i n (Fig. 2). The i n h i b i t i o n w a s reversed by r e p e a t e d r i n s i n g of t h e p r e p a r a t i o n w i t h fresh m e d i a , T r e a t m e n t w i t h e a c h a n t a g o n i s t did n o t s i g n i f i c a n t l y alter t h e c o n t r a c t i o n i n d u c e d b y 30 mM K + (n = 3 ) .
Fro. 4. Effects of KRI-1314 on the angiotensin lI (O, n = 5) and tetradecapeptide-induced contractions (©, n = 5) in iris sphincter strips. Contractions induced by angiotensin II and tetradecapeptide in the absence of KRI1314 were taken as 100%; the m e a n absolute values were 65'7+-_14"0 mg and 26.1+-,4.2 mg, respectively. *Significantly different from controls at P < 0'01. Vertical lines indicate S.E.M.
Responses to Tetradecapeptide T e t r a d e c a p e p t i d e (5 x 1 0 _7 M), a s y n t h e t i c r e n i n substrate, contracted the sphincter muscle preparat i o n s ; m e a n v a l u e s were ] 2 . 9 + 1 ' 5 % of t h e c o n t r a c t i o n i n d u c e d by 1 0 - 4 M a c e t y l c h o l i n e . The c o n tractile r e s p o n s e w a s a l m o s t a b o l i s h e d by t r e a t m e n t w i t h i n d o m e t h a c i n a n d m a r k e d l y suppressed b y s a r a l a s i n , captopril or 5 x 1 0 -5 M K R I - 1 3 1 4 (Fig. 3). K R I - 1 3 1 4 ( 1 0 - 7 - 1 0 -5 M) i n h i b i t e d t h e t e t r a d e c a p e p -
566
T. O K A M U R A ET AL. I00
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FIG. 5. Concentration-inhibition curves for captopril on the ACE activities in (A) monkey lung (C), n = 3), dog lung (O, n = 3) and dog mesenteric artery (&, n = 3) and in (B) dog iris sphincter muscle (&, n = 3). ACE activities in monkey lung, dog lung, dog mesenteric artery and iris sphincter without captopril were taken as 100%: the mean absolute values were 109"4+ 16'8, 27.6_+ 3"1, 4.9 + 1-2 and 0"5 _+0'1 mU mg-~ protein, respectively. tide-induced contraction in a concentration-related manner, but did not significantly affect the contractile response to ANG II at the concentrations used (Fig. 4).
Release of PGF2~ by Angiotensin II Paired comparisons were made of the amount of PGF~ released in the bathing media in which sphincter muscle preparations from the same dogs were treated
I00
Tissue ACE Activity The ACE activity in dog and monkey tissues measured is summarized in Table I. The enzyme activity in dog sphincter muscle preparations was low but significantly detectable; the value was similar to that in mesenteric arteries without the endothelium and approximately 1/70 of that in the lung. Captopril (10-9-10 -7 M) dose-dependently inhibited the ACE activity in dog sphincter muscles as well as the activities in the monkey lung, dog lung and mesenteric artery (Fig. 5). Mean values of the median inhibitory concentration (IC~0) of captopril were [8.1_+2.3], [9.7 1.6], [12.0+_2.6] and [14.4+_3"8]x10 -gM in dog sphincter muscles (n -= 3), monkey lung (n = 3), dog mesenteric arteries (n = 3) and dog lung (n = 3), respectively; the differences in these values are statistically not significant.
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_ .........i f ' ~
TABLE I
Angiotensin-converting enzyme (ACE) activities in various tissues Animal Dog
Monkey
Tissue Iris sphincter Mesenteric EM (+) artery EM ( - ) Lung Lung
EM, Endothelium.
n
ACE activity (mU mg -1 protein)
8 6 3 14 3
0.4 + 0"1 4'7+ 1"5 0"5__+0'2 28.6 + 2'6 109'4 + 16.8
0
~
I I0
i
I (( I (.~ J 20 40 60
Time (rain) FIG. 6. Typical time-dependent changes in pupil size induced by injection of saline (O), 5 x 10 -5 M angiotensin I (A) and 5 x 10 5M angiotensin II ( I ) into the anterior chamber of anesthetized dogs. Pupil size was expressed as a ratio of'white-to-white' diameter (A) and 'black-to-black' diameter (B) from the photographs. Eyes were topically treated with 0"5 % tropicamide and 0-5 % phenylephrine. One-hundred microliters of the aqueous humor was drawn before the drug injection (100 #1).
RENIN-ANGIOTENSIN SYSTEM IN IRIS
with either ANG II (10 -v M) or its vehicle. The amount of PGF.a~ released from control preparations averaged 1 5 9 + 3 5 pg mg -1 wet tissue weight (n = 5). Treatment with ANG II significantly increased the release by 18_+6% (n = 5, P < 0.025). Miosis Induced by ANG I and ANG II
Miotic responses were studied by the direct injection of a drug into the anterior chamber. The ratio of 'black-to-black' diameter and 'white-to-white' diameter was in a range between 0"59-0-77 ( 0 . 6 6 + 0"02, n = 10) under control conditions. ANG I or ANG II at 5 x 10 -~ M caused a rapid, marked miosis. The typical time-courses are illustrated in Fig. 6. Maximal responses to the peptides were obtained at 1 0 - 2 0 min and gradually recovered to the control level until 60 min after injection. Mean ratios of the maximal response to ANG II and ANG I were 0"20_+0.01 ( 2 6 ' 7 + 0 " 9 % of control, n = 3 ) and 0"27+0"02 (31.7 + 4" 1%, n = 3), respectively. The peak response to ANG II was obtained at 10 rain after injection, which tended to be earlier than that to ANG I. The injection of saline did not significantly alter the ratio for at least 60 rain. 4. Discussion The synthetic renin substrate and ANG I contracted the dog isolated sphincter muscle in a concentrationrelated manner. The contractions by both peptides appear to be mediated by activation of the ANG II receptor, as the responses to these ANG precursors were almost abolished by treatment with saralasin and significantly suppressed by captopril (for tetradecapeptide and ANG I) and KRI-1314 (for tetradecapeptide). Treatment with cyclooxygenase inhibitors such as indomethacin or aspirin also almost abolished the contractions by tetradecapeptide, ANG I (present study) and ANG II (Lu et al., 1988), but did not alter the PGF2~-induced contraction (Lu et al., 1988). These findings suggest that the contractions induced by ANG II and its precursors via the ANG II receptor are mediated by PGs, possibly synthesized by cyclooxygenase from arachidonic acid and demonstrated in the vasculature (McGiff et al., 1970; Toda and Miyazaki, 1981; Toda, Ayajiki and Okamura, 1990) and the cultured vascular smooth muscle cells (Alexander and Gimbrone, 1976). in the present study, ANG II was found to stimulate the release of PGF2~ from isolated sphincter muscle. PGF~ is one of the most potent constrictor PGs in the sphincter muscle (Lu et al., 1988), therefore, the PG may be responsible for the contraction caused by ANG I and tetradecapeptide. ANG I appears to be converted to ANG II mainly by ACE located in the sphincter muscle, since the contractile response to ANG I was almost abolished by captopril. The ACE activity was chemically detected in the homogenate of the sphincter muscle. The activity
567
was similar to that in the arteries without the endothelium and the affinity of captopril to sphincter muscle ACE was comparable with that to vascular or lung ACE. ACE activity has also been detected in the vascular smooth muscle layer (Velletri and Bean, 1982). Tetradecapeptide seems to be converted to ANG II via ANG I by catalyses of renin and ACE, since the contractions by tetradecapeptide were markedly attenuated by treatment with captopril or KR[-1314, a specific renin inhibitor (Miyazaki et al., 1989), in concentrations in which the ANG II-induced contraction was not attenuated. Tonin in rat tissues (Thibault and Genest, 1981), unlike renin, converts tetradecapeptide directly to A N G / [ ; however, this is not the case in dog sphincter muscle. ANG I caused miosis in vivo; the potency was comparable with that of ANG IL Since ACE activity is present not only in the sphincter muscle (present study) but also in the choroid, ciliary body (Igic and Kojovic, 1980) and aqueous humor (Weinreb et al., 1985; Vita et al., 1981), ANG I seems to be converted rapidly and effectively to ANG II in the anterior chamber and then constrict the sphincter muscle. Irnmunohistochemical evidence suggests the involvement of ACE located in the ciliary process internal epithelium in aqueous humor secretion, but not in reabsorption (Laliberte et al., 1988). Further study is underway to determine whether or not ANG II generated intracamerally is involved in the regulation of the volume of aqueous humor. The present study revealed that the contractile responses of iris sphincter to tetradecapeptide and ANG I were suppressed by inhibitors of renin and ACE and abolished by saralasin, it seems likely that the renin-angiotensin system exists in the dog iris sphincter muscle. We hypothesize that ANG II synthesized locally from its substrates plays a physiological role in the regulation of the muscle tone by generating the constrictor PGs, possibly PGF2~.
Acknowledgements We thank Drs Kenji Sasamoto and Wei Lu for their excellent technical assistance.
References Alexander, R. W. and Gimbrone, M. A. (1976). Stimulation of prostaglandin E synthesis in cultured human umbilical vein smooth muscle ceils. Proc. Natl. Acad. Sci. U.S.A. 73, 1617-20. Bradford, M. M. (1976). A rapid and sensitive method for the quantiation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-54. Cushman, D.W. and Cheung, F. S. (1970). Spectrophotometric assay and properties of the angiotensin-convcrting enzyme of rabbit lung. Biochem. Biophys. Res. Commun. 20, 1637-48. Eakins, K.E. (1964). Effects of angiotensin on intraocular pressure. Nature 202, 813-14. Ferrari-diles, G., Ryan, J. W., Rockwood, E.J., Davis, E. B.
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and Anderson, D.R. (1988). Angiotensin-converting enzyme in bovine, feline and human ocular tissues. Invest. OphthalmoI. Vis. Sci. 29, 876-81. Igic, R. and Kojovic, V. (1980). Angiotensin I converting enzyme (kininase II) in ocular tissues. Exp. Eye Res. 30, 299-303. Ikemoto, F. and Yamamoto, K. (1978). Renin-angiotensin system in the aqueous humor of rabbits, dogs and monkeys. Exp. Eye Res. 27, 723-5. Laliberte, M. F., Laliberte, F., Alhenc-Gelas, F. and Chevillard, C. (1988). Immunohistochemistry of angiotensin Pconverting enzyme in rat eye structures involved in aqueous humor regulation. Lab. Invest. 59, 263-70. Lu, W., Okamura, T., Bian, K., Inatomi, A. and Toda, N. (1988). Prostaglandins involved in contractions by angiotensin II and bradykinin of isolated dog sphincter pupillae. Br. J. Pharmacol. 95, 544-50. Macri, F. ]. (1965). The action of angiotensin on intraocular pressure. Arch. Ophthalmol. 73, 528-39. McGiff, J. C., Crowshaw, K., Terragno, N.A. and Lonigro, A. J. (1970). Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II. Circ. Res. 26 & 27 (Suppl. I) I121-30. Miyazaki, M., Etoh, Y., Iizuka, K. and Toda, N. (1989). An orally active renin inhibitor: cyclohexylnorstatinecontaining dipeptide (KRI-1314). J. Hypertens. 7 (Suppl. 2). S25-7. Miyazaki, M., Okunishi, H., Nishimura, K. and Toda, N. (1984). Vascular angiotensin-converting enzyme activity in man and other species. Clin. Sci. 66, 39-45. Nakamaru, M., Misono, K. S., Naruse, M., Workman, R. ]. and Inagami, T. (1985). A role for the adrenal renin-angiotensin system in the regulation of potassium-stimulated aldosterone production. Endocrinol. 117, 1772-8. Okamura, T., Miyazaki, M , Inagami, T. and Toda, N. (1986). Vascular renin-angiotensin system in two-
T. OKAMURA ET AL.
kidney, one clip hypertensive rats. Hypertension 8, 560-5. Okamura, T., Okunishi, H., Ayajiki, K. and Toda, N. (1990). Conversion of angiotensin I to angiotensin II in dog isolated renal artery: role of two different angiotensin II-generating enzymes. ]. Cardiovasc. PharmacoI. 15, 353-9. Pandy, K. N., Misono, K, S. and Inagami, T. (1984). Evidence for intracellular formation of angiotensins: coexistence of renin and angiotensin-converting enzyme in Leydig cells of rat testis. Biochem. Biophys. Res. Commun. 122, 1337-43. Printz, M.P., Ganten, D., Unger, T. and Phillips, M.I. (1982). Minireview. The brain renin-angiotensin system 1982. In Experimental Brain Research Supplementura 4: The Renin Angiotensin System in the Brain. (Eds Ganten, D., Printz, M., Phillips, M.I. and Scholkens, B. A.). Pp. 3-52. Springer-Verlag: Berlin. Thibault, G. and Genest, J. (1981). Tonin, an esteroprotease from rat submaxillary glands. Biochem. Biophys. Acta 660, 23-9. Toda, M., Ayajiki, K. and Okamura, T. (1990). Modifications by endogenous prostaglandins of angiotensin II-induced contractions in dog and monkey cerebral and ruesenteric arteries. ]. Pharmacol. Exp. Ther. 252, 374-9. Toda, N. and Miyazaki, M. (1981). Angiotensin-induced relaxation in isolated dog renal and cerebral arteries. Am. ]. Physiol. 240, H247-54. Velletri, P. and Bean, B. L. (1982). The effects of captopril on rat aortic angiotensin-converting enzyme. J. Cardiovasc. Pharmacol. 4, 315-25. Vita, ]. B., Anderson, ]. A., Hulem, C. D. and Leopold, I. H. (1981). Angiotensin-converting enzyme activity in ocular fluids. Invest. Ophthalmol. Vis. Sci. 20, 255-7. Weinreb, R. N., Sandman, R., Ryder, M. I. and Friberg, T. R. (1985). Angiotensin-converting enzyme activity in human aqueous humor. Arch. Ophthalmol. 103, 34-6.
Local Generation and Action of Angiotensin II in Dog Iris Sphincter Muscle TOMIO OKAMURA*,YAN
W A N G AND N O B O R U T O D A
Department of Pharmacology, Shiga University of Medical Sciences, Seta, Ohtsu 520-21. Japan (Received Chicago 2 October 1991 and accepted in revised form 20 January 1992) Existence of the renin-angiotensin system was pharmacologically investigated in the dog isolated iris sphincter muscle. The sphincter muscle contracted in response to tetradecapeptide, a synthetic renin substrate, angiotensin (ANG) I and ANG II dose-dependently. The contractions induced by these peptides were suppressed by treatment with saralasin, indomethacin and aspirin. Contractile responses to tetradecapeptide and ANG I were also reduced by KRI-1314, a renin inhibitor, and captopril, respectively. ANG II stimulated the release of prostaglandin (PG) F~ from the sphincter muscle. Angiotensinconverting enzyme activity was measurable in the sphincter muscle. Miosis was observed by intracameral injection of ANG I and ANG II into the anterior chamber. These results strongly suggest that angiotensin generating enzymes function in the sphincter muscle and ANG It produced by these enzymes contracts the sphincter muscle via the formation of PG (s), possibly PG F2~. Key words: renin; angiotensin I; angiotensin II; tetradecapeptide; angiotensin-converting enzyme; prostaglandin F2~; miosis; sphincter muscle contraction. 1. Introduction
2. Materials and Methods
Angiotensin (ANG) II, a bioactive product of the renin-angiotensin system, is known as a blood-borne peptide regulating blood pressure, local blood flow and water-electrolyte balance. Recent studies have demonstrated the local formation of ANG II in various tissues such as brain (Printz et al., 1982), vascular wall (Okamura et al., 1990), adrenal (Nakamaru et al., 1985) and reproductive organs (Pandy, Misono and Inagami, 1984), suggesting the important roles of the peptide in functioning of these organs and tissues. Locally produced ANG II has been suggested to regulate ocular functions. Administration of ANG II through the lingual artery or into the vitrous body in vivo (Eakins, 1964) and through the ophthalmic artery in vitro (Macri, 1965) lowers the intraocular pressure. The peptide contracts isolated dog sphincter pupillae via a synthesis of cyclooxygenase products by acting on specific ANG II receptors (Lu et al., 1988). Although the renin activity in the aqueous humor (Ikemoto and Yamamoto, 1978) and the angiotensinconverting enzyme (ACE) activity in retina (Ferraridries et al., 1988 ; Igic and Kojovic, 1980), choroid (Igic and Kojovic, 1980), ciliary body (Igic and Kojovic, 1980), aqueous humor (Weinreb et al., 1985; Vita et al., 1981) and tear (Vita et al., 1981) have been detected, functional significance of these enzymes has not been elucidated in the ocular tissues. The present study was thus undertaken to compare the actions of ANG II and its precursors on dog sphincter muscles in vitro and in vivo, in order to clarify the local generation and action of ANG II in the ocular tissues.
Preparation of Isolated Sphincter Pupillae
*For correspondence at: Department of Pharmacology, Shiga University of Medical Sciences, Seta, Ohtsu 520-21. Japan. 0014-4835/92/100563 + 06 $08.00/0
Mongrel dogs of both sexes, weighing 8-15 kg, were anesthetized with intravenous injections of sodium pentobarbital (30 mg kg -1) and killed by bleeding from the common carotid arteries. Eyes were rapidly removed. One strip of the iris sphincter was isolated from each eye. The strip was fixed vertically between hooks in the muscle bath of 20 ml capacity containing the modified Ringer-Locke solution, which was maintained at 37+_0-3°C and aerated with a mixture of 95% 02 and 5% CO2. The hook anchoring the upper end of the strip was connected to the lever of a forcedisplacement transducer (Nihonkohden Kogyo Co., Tokyo, ]apan). The resting tension was adjusted to 50 rag. Constituents of the solution were as follows (raM): NaC1, 120; KC1, 5.4; NaHCO 3, 25.0; CaC12, 2.2; MgCI~, 1.0; and dextrose, 5"6. The pH of the solution was 7.3-7"4. Before the start of experiments, the strips were allowed to equilibrate for 6 0 - 9 0 rain in control media, during which time the solutions were replaced every 10-15 rain.
Tension Recording Isometric contractions were recorded on an inkwriting oscillograph (Nihonkohden Kogyo Co.). Test drugs were added directly to the bathing media in cumulative concentrations (acetylcholine) or in single concentrations (tetradecapeptide, ANG I and ANG II) because of tachyphylaxis by cumulative applications. The contractile response to 30 mM K÷ was first obtained and the preparations were washed three times with control media and equilibrated for 4 0 -
© 1992 Academic Press Limited
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50 rain. Then, the concentration-contraction curve for acetylcholine was obtained. Contractions induced by 10 -4 M acetylcholine were taken as a standard for comparison on the contractile responses to the peptides. To determine the concentration-response relationship for ANG I or II, the response to 10-VM ANG II was obtained twice and then the response to one of the concentrations (5 x 10 -9 to 2 x 10 -6 M) of ANG II was obtained. Preparations were treated for 20 rain with pharmacological inhibitors, before the response to the agonist was obtained.
Measurement of Angiotensin-converting Enzgme (ACE) Activity Tissue ACE activity was determined by using the modified method of Cushman and Cheung (1970) as previously described (Okamura et al., 1986). Briefly, flesh tissues (dog iris sphincters, mesenteric arteries with and without the endothelium and lungs, and monkey lungs; wet weight 3 0 - 5 0 0 mg) were homogenized with ten volumes of homogenization buffer. This solution consisted of 20 mM Tris-HC1 buffer (pH 8-3), 5 mM magnesium acetate, 30 mM KC1, 0-25 M sucrose and 0'5% Nonidet P-40 (Okamura et al., 1986). The homogenized sample was stored overnight at 4°C and then centrifuged for 20 rain at 2 0 0 0 0 g and 4°C. The supernatant was then incubated in the presence and absence of captopril, an ACE inhibitor, at 37°C for 30 min with hippuryl-L-histidylr-leucine (5 raM) in lOmM potassium phosphate buffer, pH 8.3, containing 800 m ~ or 600 mM sodium chloride for dog or monkey tissues, respectively (Miyazaki et al., 1984). The enzyme reaction was stopped by the addition of 3 % metaphosphate. The reaction mixture then was centrifuged at 12 000 g for 10 min and the supernatant was applied to a reversed phase column and eluted at 38°C with lOmM KH~PQ:methanol (1:1, pH3"O) at a rate of 0.7 ml rain -1. Hippurate was detected by ultraviolet absorbance at 228 nm. The activity of ACE was expressed as specific activity (mUmg -1 protein) using tissue protein concentration values determined by the modified method of Bradford (1976).
Measurement of PGF2~ Two sphincter muscle strips obtained from eyes of the same dogs were used for the paired analysis of PGF~ release in the bathing media. After 20 rain preincubation at 37°C, one of the strips was treated with 10 7 M ANG II and the other with vehicle. The preparation was incubated for 15 rain, before adding indomethacin (10 -~ M) to stop the further production of PGF~. The bathing medium was then quickly collected by aspiration and stored frozen at below - 6 0 ° C until the assay. Samples were concentrated ten times by SEP-PAK C18 column (Waters, MA, U.S.A.) prior to the assay. PGF2~ was measured by
radioimmunoassay using a commercial kit (PGF~[3H] assay system, Amersham, U.K.).
Miotic Response In Vivo Mongrel dogs were anesthetized with intravenous administration of sodium pentobarbital as described above. Before topical applications of testing drugs, both eyes received a couple of drops of Mydrin P~ (0'5% tropicamide and 0"5% phenylephrine hydrochloride) in order to obtain sufficient dilatation of iris sphincter muscles. After stabilization, a small part of cornea was incised with a razor blade. All drugs (100 #1) were diluted with saline and injected into the anterior chamber through the incision site of either eye and saline was injected into the other eye, as a control. Whenever the drug was injected, 100 #1 of aqueous humor was taken just before the injection. Time-dependent changes of the pupil sizes were photographed. Miotic responses were quantitatively analysed by measuring the pupil size which was calculated as a ratio of' white-to-white' a n d ' black-toblack' diameters.
Statistics and Drugs Results shown in the text, table and figures are expressed as mean+s.E.M. Statistical analyses were made using the Student's paired and unpaired t-test and Tukey's method after one-way analysis of variance. Drugs used were PGF~ (Ono Pharmaceutical Co., Osaka, Japan), indomethacin and tetradecapeptide (Sigma, St Louis, MO, U.S.A.), angiotensin I, angiotensin I[, [Sar 1, AlaS]angiotensin II (saralasin) and hippuryl-L-histydyl-L-leucine (Peptide Institute Inc., Minoh, Japan), acetylsalicylic acid (Nacalai Tesque, Kyoto, Japan) acetylcholine chloride (Daiichi Pharmaceutical Co., Tokyo, Japan), captopril (Sankyo Pharmaceutical Co., Tokyo), KRI-1314 (Kissei Pharmaceutical Co., Nagano, Japan), Mydrin P® (0"5% tropicamide and 0.5 % phenylephrine, Santen Pharmaceutical Co., Osaka) and sodium pentobarbital (Abbott Lab., North Chicago, IL, U.S.A.).
3. Results Responses to ANG I and ANG II The addition of ANG I (2 x 10 -s to 2 x 10 -6 M) and ANG II (5 x 10 -9 to 10 -6 M) caused a concentrationrelated contraction in the dog isolated sphincter muscle (Fig. 1). Mean values of the median effective concentration (EC60) of ANG I and ANG II were ( 3 " 6 7 _ 1 - 0 0 ) × 1 0 - 7 M ( n = 7 ) and (3.96+1.08) x 10-8M (n = 7), respectively. Maximum contractions caused by these peptides were obtained at 10 -6 M and the magnitude of the contractions were almost the same. The contractile response to 5 x lO-TM ANG I was almost abolished by treatment with lO-VM
RENIN-ANGIOTENSIN
SYSTEM
IN IRIS
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Soralasin Captopril Indomefhocin KRI 1514
FIG. 3. Modification by 10 .7 M saralasin (n = 7), 10 -s M captopril (n = 6), 10-6M indomethacin (n = 7) and 5 x 10-5M KPd-1314 (n = 11) of the tetradecapeptide (5 x 10 -7 M)-induced contraction in iris sphincter strips. Contractions induced by tetradecapeptide in control preparations (n = 23) were taken as 100%; the m e a n absolute values was 30.0 _+4.9 mg. * Significantly different from controls at P < 0"01. Vertical hines indicate S.E.M.
8
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FIG. 1. Concentration-response curves for angiotensin [ (C), n = 7) and angiotensin II ( 0 , n = 7) in the dog iris sphincter muscle. Contractions induced by 10-4M acetylcholine were taken as 100 %; the m e a n absolute value was 141"2 +- 13"0 mg (n = 14). Vertical lines indicate S.E.M.
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o Control Soralasin Captopril Indomethacin Aspirin FIG. 2. Modification by 10 -7 M saralasin (n = 11), 10 5 M captopril (n = 10), 10 -6 M indomethacin (n = 8) and 5 x 10-SM aspirin (n = 10) of angiotensin I (5X10-TM) induced contraction in iris sphincter strips. Contractions induced by angiotensin I in control preparations (n = 10) were taken as 100%; the m e a n absolute value was 54.6 +_9.6 mg. * Significantly different from controls at P < 0-001. Vertical lines indicate S.E.M.
s a r a l a s i n , ]O-SM captopril, 1 0 - 6 M i n d o m e t h a c i n or 5 x 10 -5 M a s p i r i n (Fig. 2). The i n h i b i t i o n w a s reversed by r e p e a t e d r i n s i n g of t h e p r e p a r a t i o n w i t h fresh m e d i a , T r e a t m e n t w i t h e a c h a n t a g o n i s t did n o t s i g n i f i c a n t l y alter t h e c o n t r a c t i o n i n d u c e d b y 30 mM K + (n = 3 ) .
Fro. 4. Effects of KRI-1314 on the angiotensin lI (O, n = 5) and tetradecapeptide-induced contractions (©, n = 5) in iris sphincter strips. Contractions induced by angiotensin II and tetradecapeptide in the absence of KRI1314 were taken as 100%; the m e a n absolute values were 65'7+-_14"0 mg and 26.1+-,4.2 mg, respectively. *Significantly different from controls at P < 0'01. Vertical lines indicate S.E.M.
Responses to Tetradecapeptide T e t r a d e c a p e p t i d e (5 x 1 0 _7 M), a s y n t h e t i c r e n i n substrate, contracted the sphincter muscle preparat i o n s ; m e a n v a l u e s were ] 2 . 9 + 1 ' 5 % of t h e c o n t r a c t i o n i n d u c e d by 1 0 - 4 M a c e t y l c h o l i n e . The c o n tractile r e s p o n s e w a s a l m o s t a b o l i s h e d by t r e a t m e n t w i t h i n d o m e t h a c i n a n d m a r k e d l y suppressed b y s a r a l a s i n , captopril or 5 x 1 0 -5 M K R I - 1 3 1 4 (Fig. 3). K R I - 1 3 1 4 ( 1 0 - 7 - 1 0 -5 M) i n h i b i t e d t h e t e t r a d e c a p e p -
566
T. O K A M U R A ET AL. I00
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FIG. 5. Concentration-inhibition curves for captopril on the ACE activities in (A) monkey lung (C), n = 3), dog lung (O, n = 3) and dog mesenteric artery (&, n = 3) and in (B) dog iris sphincter muscle (&, n = 3). ACE activities in monkey lung, dog lung, dog mesenteric artery and iris sphincter without captopril were taken as 100%: the mean absolute values were 109"4+ 16'8, 27.6_+ 3"1, 4.9 + 1-2 and 0"5 _+0'1 mU mg-~ protein, respectively. tide-induced contraction in a concentration-related manner, but did not significantly affect the contractile response to ANG II at the concentrations used (Fig. 4).
Release of PGF2~ by Angiotensin II Paired comparisons were made of the amount of PGF~ released in the bathing media in which sphincter muscle preparations from the same dogs were treated
I00
Tissue ACE Activity The ACE activity in dog and monkey tissues measured is summarized in Table I. The enzyme activity in dog sphincter muscle preparations was low but significantly detectable; the value was similar to that in mesenteric arteries without the endothelium and approximately 1/70 of that in the lung. Captopril (10-9-10 -7 M) dose-dependently inhibited the ACE activity in dog sphincter muscles as well as the activities in the monkey lung, dog lung and mesenteric artery (Fig. 5). Mean values of the median inhibitory concentration (IC~0) of captopril were [8.1_+2.3], [9.7 1.6], [12.0+_2.6] and [14.4+_3"8]x10 -gM in dog sphincter muscles (n -= 3), monkey lung (n = 3), dog mesenteric arteries (n = 3) and dog lung (n = 3), respectively; the differences in these values are statistically not significant.
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TABLE I
Angiotensin-converting enzyme (ACE) activities in various tissues Animal Dog
Monkey
Tissue Iris sphincter Mesenteric EM (+) artery EM ( - ) Lung Lung
EM, Endothelium.
n
ACE activity (mU mg -1 protein)
8 6 3 14 3
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0
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Time (rain) FIG. 6. Typical time-dependent changes in pupil size induced by injection of saline (O), 5 x 10 -5 M angiotensin I (A) and 5 x 10 5M angiotensin II ( I ) into the anterior chamber of anesthetized dogs. Pupil size was expressed as a ratio of'white-to-white' diameter (A) and 'black-to-black' diameter (B) from the photographs. Eyes were topically treated with 0"5 % tropicamide and 0-5 % phenylephrine. One-hundred microliters of the aqueous humor was drawn before the drug injection (100 #1).
RENIN-ANGIOTENSIN SYSTEM IN IRIS
with either ANG II (10 -v M) or its vehicle. The amount of PGF.a~ released from control preparations averaged 1 5 9 + 3 5 pg mg -1 wet tissue weight (n = 5). Treatment with ANG II significantly increased the release by 18_+6% (n = 5, P < 0.025). Miosis Induced by ANG I and ANG II
Miotic responses were studied by the direct injection of a drug into the anterior chamber. The ratio of 'black-to-black' diameter and 'white-to-white' diameter was in a range between 0"59-0-77 ( 0 . 6 6 + 0"02, n = 10) under control conditions. ANG I or ANG II at 5 x 10 -~ M caused a rapid, marked miosis. The typical time-courses are illustrated in Fig. 6. Maximal responses to the peptides were obtained at 1 0 - 2 0 min and gradually recovered to the control level until 60 min after injection. Mean ratios of the maximal response to ANG II and ANG I were 0"20_+0.01 ( 2 6 ' 7 + 0 " 9 % of control, n = 3 ) and 0"27+0"02 (31.7 + 4" 1%, n = 3), respectively. The peak response to ANG II was obtained at 10 rain after injection, which tended to be earlier than that to ANG I. The injection of saline did not significantly alter the ratio for at least 60 rain. 4. Discussion The synthetic renin substrate and ANG I contracted the dog isolated sphincter muscle in a concentrationrelated manner. The contractions by both peptides appear to be mediated by activation of the ANG II receptor, as the responses to these ANG precursors were almost abolished by treatment with saralasin and significantly suppressed by captopril (for tetradecapeptide and ANG I) and KRI-1314 (for tetradecapeptide). Treatment with cyclooxygenase inhibitors such as indomethacin or aspirin also almost abolished the contractions by tetradecapeptide, ANG I (present study) and ANG II (Lu et al., 1988), but did not alter the PGF2~-induced contraction (Lu et al., 1988). These findings suggest that the contractions induced by ANG II and its precursors via the ANG II receptor are mediated by PGs, possibly synthesized by cyclooxygenase from arachidonic acid and demonstrated in the vasculature (McGiff et al., 1970; Toda and Miyazaki, 1981; Toda, Ayajiki and Okamura, 1990) and the cultured vascular smooth muscle cells (Alexander and Gimbrone, 1976). in the present study, ANG II was found to stimulate the release of PGF2~ from isolated sphincter muscle. PGF~ is one of the most potent constrictor PGs in the sphincter muscle (Lu et al., 1988), therefore, the PG may be responsible for the contraction caused by ANG I and tetradecapeptide. ANG I appears to be converted to ANG II mainly by ACE located in the sphincter muscle, since the contractile response to ANG I was almost abolished by captopril. The ACE activity was chemically detected in the homogenate of the sphincter muscle. The activity
567
was similar to that in the arteries without the endothelium and the affinity of captopril to sphincter muscle ACE was comparable with that to vascular or lung ACE. ACE activity has also been detected in the vascular smooth muscle layer (Velletri and Bean, 1982). Tetradecapeptide seems to be converted to ANG II via ANG I by catalyses of renin and ACE, since the contractions by tetradecapeptide were markedly attenuated by treatment with captopril or KR[-1314, a specific renin inhibitor (Miyazaki et al., 1989), in concentrations in which the ANG II-induced contraction was not attenuated. Tonin in rat tissues (Thibault and Genest, 1981), unlike renin, converts tetradecapeptide directly to A N G / [ ; however, this is not the case in dog sphincter muscle. ANG I caused miosis in vivo; the potency was comparable with that of ANG IL Since ACE activity is present not only in the sphincter muscle (present study) but also in the choroid, ciliary body (Igic and Kojovic, 1980) and aqueous humor (Weinreb et al., 1985; Vita et al., 1981), ANG I seems to be converted rapidly and effectively to ANG II in the anterior chamber and then constrict the sphincter muscle. Irnmunohistochemical evidence suggests the involvement of ACE located in the ciliary process internal epithelium in aqueous humor secretion, but not in reabsorption (Laliberte et al., 1988). Further study is underway to determine whether or not ANG II generated intracamerally is involved in the regulation of the volume of aqueous humor. The present study revealed that the contractile responses of iris sphincter to tetradecapeptide and ANG I were suppressed by inhibitors of renin and ACE and abolished by saralasin, it seems likely that the renin-angiotensin system exists in the dog iris sphincter muscle. We hypothesize that ANG II synthesized locally from its substrates plays a physiological role in the regulation of the muscle tone by generating the constrictor PGs, possibly PGF2~.
Acknowledgements We thank Drs Kenji Sasamoto and Wei Lu for their excellent technical assistance.
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and Anderson, D.R. (1988). Angiotensin-converting enzyme in bovine, feline and human ocular tissues. Invest. OphthalmoI. Vis. Sci. 29, 876-81. Igic, R. and Kojovic, V. (1980). Angiotensin I converting enzyme (kininase II) in ocular tissues. Exp. Eye Res. 30, 299-303. Ikemoto, F. and Yamamoto, K. (1978). Renin-angiotensin system in the aqueous humor of rabbits, dogs and monkeys. Exp. Eye Res. 27, 723-5. Laliberte, M. F., Laliberte, F., Alhenc-Gelas, F. and Chevillard, C. (1988). Immunohistochemistry of angiotensin Pconverting enzyme in rat eye structures involved in aqueous humor regulation. Lab. Invest. 59, 263-70. Lu, W., Okamura, T., Bian, K., Inatomi, A. and Toda, N. (1988). Prostaglandins involved in contractions by angiotensin II and bradykinin of isolated dog sphincter pupillae. Br. J. Pharmacol. 95, 544-50. Macri, F. ]. (1965). The action of angiotensin on intraocular pressure. Arch. Ophthalmol. 73, 528-39. McGiff, J. C., Crowshaw, K., Terragno, N.A. and Lonigro, A. J. (1970). Release of a prostaglandin-like substance into renal venous blood in response to angiotensin II. Circ. Res. 26 & 27 (Suppl. I) I121-30. Miyazaki, M., Etoh, Y., Iizuka, K. and Toda, N. (1989). An orally active renin inhibitor: cyclohexylnorstatinecontaining dipeptide (KRI-1314). J. Hypertens. 7 (Suppl. 2). S25-7. Miyazaki, M., Okunishi, H., Nishimura, K. and Toda, N. (1984). Vascular angiotensin-converting enzyme activity in man and other species. Clin. Sci. 66, 39-45. Nakamaru, M., Misono, K. S., Naruse, M., Workman, R. ]. and Inagami, T. (1985). A role for the adrenal renin-angiotensin system in the regulation of potassium-stimulated aldosterone production. Endocrinol. 117, 1772-8. Okamura, T., Miyazaki, M , Inagami, T. and Toda, N. (1986). Vascular renin-angiotensin system in two-
T. OKAMURA ET AL.
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