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Effect of Captopril on Converting Enzyme Activity in Chemically Sympathectomized, Spontaneously Hypertensive Rats
Effect
of Captopril
on Converting
Sympathectomized, Munavvar School
of
Abdul
Pharmaceutical
Enzyme
Spontaneously SATTAR, Sciences,
Aishah
Universiti
Accepted
June
Activity Hypertensive
LATIFF and Sains 24,
in Chemically
Malaysia,
Ee-Kiang Minden,
Rats GAN*
Penang,
Malaysia
1985
Abstract-Effect of subacute angiotensin converting enzyme (ACE) blockade on the converting enzyme activity (ACE activity) in plasma, aorta, lung, kidney and whole brain was evaluated in chemically-sympathectomized (with 6-hydroxy dopamine) normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) using captopril given peripherally via the intraperitoneal (i.p) route and centrally through intracerebroventricular (l.c.v.) administration. Daily i.p. injection of 25 mg/kg for 8 days reduced the blood pressure of both WKY rats and SHR, and the ACE activity in the aorta, lung and plasma of both WKY rats and SHR were correspondingly depressed. The brain ACE activity remained unaltered in both strain of rats. The ACE activity in the kidney of WKY was depressed, while that of SHR remained unchanged. These observations are independent of peripheral sympathectomy with 6-hydroxydopamine (6-OHDA). Daily central captopril administration at a dose of 2 mg/kg, i.c.v., for 8 days significantly reduced the blood pressure of SHR but not WKY rats, whereas the ACE activity of the whole brain of both WKY and SHR were depressed. Central sympathectomy with 6-OHDA did not alter these responses. It is concluded that captopril exerts its antihypertensive effect not only via reduction of the ACE activity in the plasma and lungs as reported earlier, but also that of other organs, principally the aorta, and that these effects are in dependent of the sympathetic nervous system. Angiotensin converting enzyme (ACE) was first isolated and purified from plasma in 1956 by Skeggs et al. (1). Subsequent studies confirmed the presence of this enzyme in a variety of tissues and organs in the body (2). Relatively high concentration of ACE is found in the lung (3). The presence of ACE has also been reported in the brain (4) and may constitute a component of a separate brain renin-angiotensin system. ACE, the key enzyme in the renin angiotensin system, is known to play an important role in the regulation of blood pressure and in the pathogenesis of hyper tension. This is because of its ability to convert the inactive decapeptide angiotensin I to the potent vasopressor octapeptide angiotensin II, principally in the pulmonary circulation (5) and its ability to inactivate bradykinin, the * To
whom
correspondence
should
be
addressed
.
hypotensive nonapeptide product of kallikrein (6). Specific inhibition of ACE by orally active captopril results in marked reduction in blood pressure (7) and captopril has proven to be an efficacious antihyper tensive agent. Blockade of conversion of angiotensin I to angiotensin II cannot account for its anti hypertensive efficacy (8); yet an anti hypertensive mechanism for captopril unrelated to ACE activity has not been directly demonstrated (9). The present study is aimed at examining the effect of captopril on ACE activity in the aorta, lung, plasma, kidney and whole brain of chemically sympa thectomized normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). This is deemed important in view of the close relationship that exists between the renin-angiotensin system and the sympa thetic nervous system in the regulation of normal blood pressure and in hypertensive
conditions (10, 11 ). Experiments were therefore conducted in central sympa thectomized and peripheral sympathec tomized WKY and SHR. Materials and Methods Male WKY and SHR weighing 200-300 g were used in the present investigation. Rats that were to receive peripheral administration of drugs were housed in groups of six in a cage and rats that were to receive central administration of drugs were housed individually. All rats were allowed free access to commercial food pellets and drank tap water ad libitum. Experimental sessions were conducted in the same room as the home cages were located, and the room was relatively quiet. All experiments were con ducted at room temperature, and no at tempts were made to control the humidity of the room. Central administration: In central adminis tration, injections were made via the i.c.v. route adapted from Hayden et al. (12). General anesthesia was achieved by intra peritoneal injection of sodium pentobarbitone (Nembutal, Abbot Laboratories), 40 mg/kg, and then aseptic surgery was performed. Implantation of cannula for i.c.v. injection was performed using a micro-manipulator in the stereotaxic instrument (Stoelting Co., U.S.A.). For this purpose, a 20 G needle cannula guide was fitted into the base of the plexiglass block that had a cavity filled with sealant. Dental cement was used to hold the needle in position. After surgery, the rats were given a recovery period of 7 days. All drugs used for central administration were dissolved in sterile saline. Central i.c.v. administration of captopril was made daily at a dose of 2 mg/kg for 8 days, whereas central sympathectomy was achieved by adminis tration of 6-OHDA, i.c.v.: 1200 ,,tg/kg on day 1, 2400 gig/kg on day 2 and 1200 /ag/kg on days 5 and 8, modified for central adminis tration as described by Simpson (13). 6 OHDA was prepared freshly each time in sterile saline containing 0.01 M ascorbic acid to prevent oxidation. Peripheral administration: In peripherally treated animals, injections of captopril were made via the intraperitoneal route at a dose of
25 mg/kg daily for a period of 8 days, whereas peripheral sympathectomy was achieved through administration of 6-OHDA via the tail vein at a dose of 50 mg/kg on day 1, 100 mg/kg on day 2 and 500 mg/kg each on days 5 and 8 (13). Blood pressure determination: Blood pressure was measured by tail plethysom graphy, connected to a physiograph in prewarmed (40°C for 15 min) rats, as reported by Krakoff et al. (14). Conscious systolic blood pressure was measured before and after treatment. Experimental protocol: Experiments in volved both central and peripheral treatment, and for these purposes, rats were divided into 4 groups: Group 1, serving as controls Group 2, captopril treated Group 3, 6-O H DA treated Group 4, both captopril and 6-OHDA treated All animals were sacrificed after day 8, 24 hr after the last treatment by drug. For centrally treated rats, the whole brain was removed and angiotensin converting enzyme activity determined. For peripherally treated rats, the thoracic region was quickly opened to expose the heart directly after sacrificing the animal. Blood was obtained from the heart by a direct heart puncture using a 1 ml syringe fitted to size 26 needle which had been previously moistened with heparinized saline. The blood was then centrifuged to obtain the plasma. The aorta, lung, kidney and whole brain were immediately removed for the determination of converting enzyme activity. Determination of converting enzyme activity: The converting enzyme activity was determined according to the method of Cushman and Cheung (15). One unit of ACE activity was defined as the amount that catalyzes the formation of 1 icmole of hip puric acid from hippuryl-L-histidyl-L-leucine (HHL) in 1 min at 37°C and atmospheric pressure. ACE activity was expressed as mU/ mg protein. Total protein measurement was made using a Biorad Kit, a-gent" (Abbott Laboratories). Statistics: Student's t-test was used to determine significant differences between the blood pressure before and after treat
ment. In comparing the effects of captopril on the ACE activity in the various organs/tissues and plasma of the SHR and the WKY rats, Student's t-test was also used to determine the significance between control and treated animals. All values are expressed as means ±S.E.M. An asterisk indicates values that are significantly different from the control group: *P<0 .05, **P<0.01, ***P<0.001.
aorta of the two strains of rat. However, in the plasma and in the kidney of SHR, the ACE
Results Captopril, 6-OHDA, and combination of captopril and 6-OHDA given via the i.p. route significantly reduced the blood pressure of SHR and WKY rats as shown in Table 1. When similar experiments were repeated with central administration via the i.c.v. route, only the blood pressure of SHR was sig nificantly reduced (Table 2), no significant changes in blood pressure were detected in WKY rats. Figure 1 shows the ACE activity in the various tissues of WKY and SHR. It can be seen that there was no difference in the ACE activity both in the whole brain and in the Table
1.
SHR
and
Table
2.
and
WKY
Effect WKY
Effect rats
of
captopril,
6-OHDA
and
both
Fig. 1. various
Histogram comparing ACE tissues of WKY 0 and SHR
are the
mean+S.E.M.
captopril
and
6-OHDA
for 6 animals.
on
systolic
blood
activity in . Values
***P<0.001.
pressure
of
rats
of captopril,
6-OHDA
and
both
captopril
and
6-OHDA
on
systolic
blood
pressure
of SHR
Table
3.
ing enzyme
Table verting
4.
Effect of captopril, activity
Effect enzyme
of
in various
captopril,
activity
6-OHDA
and both captopril
tissues
of SHR
6-OHDA
and
in various
tissues
activities were significantly lower, in the lung, the ACE activity of SHR in comparison with WKY rats.
both
of the
captopril WKY
and 6-OHDA
and
6-OHDA
treatment
treatment
on angiotensin
on
angiotensin
convert
con
rat
whereas is higher
Subacute intraperitoneal administration of captopril at 25 mg/kg daily for 8 days sig nificantly reduced the ACE activity in the plasma, the aorta and the lungs of both the SHR (Table 3) and WKY rats (Table 4). Similarly, subacute intraperitoneal adminis tration of captopril reduced the ACE activity in the kidney of WKY rats. However this effect is not seen in SHR where the ACE activity is unaffected by captopril. All these observations were independent of peripheral sympathectomy achieved through the admin istration of 6-OHDA via the tail vein. Results involving central captopril treat ment are shown in Fig. 2. Intracerebroven tricular administration of captopril, 2 mg/kg daily for 8 days, significantly depressed the ACE activity measured in the whole brain of both SHR and WKY rats. In neither of these strains did central sympathectomy by 6 OHDA exert any effect on the ACE activity.
Fig. 2. Histogram comparing ACE activity in the brain of the control 0, Captopril treated 6-OH DA treated , and both Captopril and 6 OHDA treated ;'Li WKY rats and SHR rats. Admin istration of drugs were made centrally via the i.c.v. route. Values are the mean±S.E.M. for 6 animals. *P<0 .05, **P<0.01, ***P<0.001.
Discussion The renin-angiotensin system in the is generally known to be in a depressed as can be seen from the lowered
SH R state ACE
activity in the plasma and the kidney of SHR. This is in spite of the higher ACE activity in the lung of SHR. These findings concur with
earlier observations (16). The present findings infer that there may be a functional renin angiotensin system in these tissues that plays an important role in the regulation of normal blood pressure and in the hypertensive con dition both in WKY and SHR. The lung appeared to be the predominant site where ACE activity is found in the body and is certainly the main organ that is respon sible for the conversion of inactive angiotensin I to active angiotensin II (5, 17). This is generally viewed to be the main mechanism through which blood pressure reduction is achieved following peripheral captopril administration in both the WKY and SHR. However the drastic reduction in the ACE activity both in the aorta of WKY and SHR following captopril treatment suggests that the blood pressure lowering effect of captopril could also be the consequence of this action. Evidence is available to suggest that the local renin-angiotensin system within the arterial walls may play a role in the main tenance of blood pressure (18, 19). Inhibition of angiotensin II production within the aortic wall and peripheral arteries will reduce the degree of vasoconstriction and thus peripheral resistance (20). Further evidence that the vascular tissue may be the target for angiotensin converting enzyme inhibitor is provided by Antonaccio et al. (21). They showed that the antihypertensive response after captopril treatment was as sociated with an increase in arterial wall renin which may indicate inhibition of angio tensin converting enzyme. Of particular interest is the present observation that this effect is independent of the sympathetic nervous system. It infers that the functional role of the renin-angiotensin system is operative even in the absence of the sympa thetic nervous system and that the sympa thetic nervous system is in no way affecting the converting enzyme inhibitory effect of captopril. This applies to both the lung and the aorta for both strain of rats.
had also been implicated (24, 25). The characteristically low ACE activity in the kidney of SHR as compared to that of WKY rats suggests a differentiating role of the renin-angiotensin system in the two strains of rat. The fact that the ACE activity is selectively depressed in the WKY rats further strengthened this claim and may suggest the existence of a mechanism for exerting control over the amount of ACE activity present in the SHR that differs from that of WKY particularly consequent to subacute captopril administration. How this difference arises is not known with certainty. It is of interest to note that no prerequisite of a functioning peripheral sympathetic nervous system is essential for the ACE activity in spite of the close relationship of angiotensin II with noradrenaline and the sympathetic nervous system, especially in relation to blood pressure regulation (10, 11 ). Central subacute captopril administration reduced the ACE activity in the whole brain of WKY and SHR. Peripheral subacute administration of captopril exerts no influence on the ACE activity of the whole brain of both WKY and SHR, indicating that the peri pherally administered captopril is not able to cross the blood-brain barrier. Alternatively, it may be that the amount of captopril reaching the brain following peripheral administration is negligible for affecting the brain ACE activity. The ability of peripheral captopril to cross the blood-brain barrier is a subject of much controversy. However, it is possible that the differences in the blood brain barrier among the different species and strains of rats coupled with the relative instability of tissue captopril may account for the conflicting results on the ability of peripherally administered captopril entering the brain (8, 26). Whatever it is, the central reduction of ACE activity in the brain brought about by i.c.v. captopril is independent of the intact central sympathetic nervous system. In summary, the present data suggest that captopril exerts its inhibition of ACE activity at least at two principle sites, the lung (also reflected through the plasma) and the aorta that may contribute towards its efficacious antihypertensive effect. Captopril is also able to inhibit the ACE activity in the brain.
However, the role of the brain renin angiotensin system in the regulation of blood pressure remains to be elucidated as central inhibition of ACE activity is not a requirement for acute blood pressure regulation by captopril. This is in agreement with obser vations made by Cohen and Kurz (27). It is concluded that the inhibitory effect of captopril on ACE activity is independent of the sympathetic nervous system. Acknowledgements: The authors are grateful to Professor K. Okamoto, Kinki University School of Medicine, Osaka, Japan for the generous gift of WKY and SHR. We would like to thank Squibb and Sons, Princeton, New Jersey, U.S.A.for the donation of captopril, and we thank Mrs. Alice Yap for typing the manuscript. References 1 Skeggs, L.T., Kahn, J.R. and Shumway, N.P.: Preparation and function of hypertensin con verting enzyme. J. Exp. Med. 105, 295-299 (1956) 2 Cushman, D.W. and Cheung, H.S.: Concen tration of angiotensin converting enzyme in tissue of rats. Biochim. Biophys. Acta 250, 261-265 (1971) 3 Cushman, D.W. and Cheung, H.S.: Studies on in vitro of angiotensin converting enzyme of lung and other tissues. In Hypertension, Edited by Genest, J., p. 532, Springer, Berlin, Heidelberg and New York (1972) 4 Ganten, D. and Speck, G.: The brain renin angiotensin system: a model for the synthesis of peptides in the brain. Br. J. Pharmacol. 27, 2379-2389 (1978) 5 Ng, K.K.F. and Vane, J.R.: The conversion of angiotensin I to angiotensin II. Nature 216, 762-766 (1967) 6 Rubin, B., Laffan, R.J., Kotler. D.G., O'Keefe, E.H., Demaio, D.A. and Goldberg, M.E.: S 14225 (D-3-mercapto-2-methyl-propanoyl-L Qproline), a- novel orally active inhibitor of angio tensin I-converting enzyme. J. Pharmacol. Exp. Ther. 204, 271-280 (1978) 7 Rubin, B. and Antonaccio, M.J.: Captopril. In Pharmacology of Antihypertensive Drugs, Edited by Scriabine, A., p. 21-42, Ravern Press, New York (1980) 8 Vollmer, R.R., Boccaguo, J.A.. Harris, D.N. and Murthy V.S.: Hypotension induced by inhibition of angiotensin-converting enzyme in pento barbital-anesthetized dogs. Eur. J. Pharmacol. 51, 39-45 (1978)
9 Antonaccio, M.J., Asaad, M., Rubin, B. and Horovitz, Z.P.: Captopril: Factors involved in its mechanism of action. In Angiotensin Converting Enzyme Inhibitors, Edited by Horovitz, Z.P., p. 161, Urban and Schwarzenberg, Baltimore and Munich (1981) 10 Khairallah, P.A., Davila, D., Papanicolaou, N., Glende, N.M. and Meyer, Ph.: Effects of angio tensin infusion on catecholamine uptake and reactivity in blood vessels. Circ. Res. 28/29, Suppl. 88, 96-106 (1971) 11 Panisset, J.C. and Bourdois, P.: Effect of angiotensin on the response to noradrenaline and sympathetic nerve stimulation and on 3H noradrenaline uptake in cat mesenteric blood vessels. Can. J. Physiol. Pharmacol. 46, 125-131 (1968) 12 Hayden, J.F., Johnson, L.R. and Maickel, R.O.: Construction and implantation of a permanent cannula for making injections into lateral ven tricle of the rat brain. Life Sci. 5, 1509-1514 (1966) 13 Simpson, L.L.: The effect of chemical sympa thectomy on vascular responses in spon taneously hypertensive rats. Neuropharmacology 13, 895-909 (1974) 14 Krakoff, L.R., Selvadural, R. and Sutter, E.: Effect of methyl-prednisolone upon arterial pressure and the renin-angiotensin system in the rat. Am. J. Physiol. 228, 613-617 (1975) 15 Cushman, D.W. and Cheung, H.S.: Spectro photometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637-1648 (1970) 16 Polaky-Cynkin, R., Reichlin, S. and Famburg, B.L.: Angiotensin I-converting enzyme activity in spontaneously hypertensive rat. Proc. Soc. Exp. Biol. Med. 164, 242-247 (1980) 17 Ng, K.K.F. and Vane, J.R.: Fate of angiotensin I to angiotensin II. Nature 216, 762-766 (1967) 18 Collis, M.G. and Keddie, J.R.: Captopril at tenuates adrenergic vasoconstriction in rat mesenteric arteries by angiotensin-dependent and -independent mechanisms. Clin. Sci. 61, 281-286 (1981) 19 Okuno, T., Kondo, K., Konishi, K., Saruta, T. and Kato, E.: SQ-14225 attenuates the vascular response to norepinephrine in the rat mesenteric arteries. Life Sci. 25, 1343-1350 (1979) 20 Hatton, R. and Clough, D.P.: Captopril interferes with neurogenic vasoconstriction in the pithed rat by angiotensin-dependent mechanism. J. Cardiovasc. Pharmacol. 4, 116-123 (1982) 21 Antonaccio, M.J., Asaad, M., Rubin, B. and Horovitz, Z.P.: Captopril: Factors involved in its
mechanism
of
Enzyme p.
In
K.,
Boucher, substrate in the
Munich
J.M., Genest,
angiotensin of rats.
Z.P.,
Schwarzenberg,
(1981)
and
and lymph
Horovitz,
and
Rojo-Ortega, R.
Converting
by
Urban and
Horky,
Angiotensin
Edited
161-180,
Baltimore 22
action.
Inhibitors,
Rodriguez, J.:
J.,
Renin,
renin
I•\converting
Am.
J.
enzyme
Physiol.
220,
307-311
(1977) 23
Hosie,
K.F.,
A.F.,
Brown,
MacAdam,
Robertson, renal Sci. 24
J.I.S.:
circulation 38.
Hall,
157-174
Lohmeier, Jr.:
release
the
A.M.,
Lever, J.
of
renin
anaesthetized
into dog.
and the Clin.
(1970) Guyton,
T.E.,
Intrarenal
angiotensin
Harper, MacGregor,
The of
J.E.,
J.J., R.F.,
A.C.,
McCaa, control
II.
Am.
R.E. of
J.
Trippodo, and
electrolyte
Physiol.
N.C.,
Cowley, excretion 232,
F538-F54
A.W., by
4
(1977) Ther. Lohmeier, A.and 220, 25Ganten, 63-69 T.E., D,: Cowley, (1981 Effects ) A.W., of anPhysiol. Jr., with 233, orally F388-F395 captopril or (1977) Trippodo, MK-421. Hall, Mann, J.active J.F.E., J.E. Pharmacol. Rascher, 26 and Exp. W., inhibitor, Dietz. N.C., Guyton, converting-enzy R., SQ-14225 A.C.: Schoning, Effects onofrespons pressor endog angio to an II
of Captopril
on Converting
Sympathectomized, Munavvar School
of
Abdul
Pharmaceutical
Enzyme
Spontaneously SATTAR, Sciences,
Aishah
Universiti
Accepted
June
Activity Hypertensive
LATIFF and Sains 24,
in Chemically
Malaysia,
Ee-Kiang Minden,
Rats GAN*
Penang,
Malaysia
1985
Abstract-Effect of subacute angiotensin converting enzyme (ACE) blockade on the converting enzyme activity (ACE activity) in plasma, aorta, lung, kidney and whole brain was evaluated in chemically-sympathectomized (with 6-hydroxy dopamine) normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) using captopril given peripherally via the intraperitoneal (i.p) route and centrally through intracerebroventricular (l.c.v.) administration. Daily i.p. injection of 25 mg/kg for 8 days reduced the blood pressure of both WKY rats and SHR, and the ACE activity in the aorta, lung and plasma of both WKY rats and SHR were correspondingly depressed. The brain ACE activity remained unaltered in both strain of rats. The ACE activity in the kidney of WKY was depressed, while that of SHR remained unchanged. These observations are independent of peripheral sympathectomy with 6-hydroxydopamine (6-OHDA). Daily central captopril administration at a dose of 2 mg/kg, i.c.v., for 8 days significantly reduced the blood pressure of SHR but not WKY rats, whereas the ACE activity of the whole brain of both WKY and SHR were depressed. Central sympathectomy with 6-OHDA did not alter these responses. It is concluded that captopril exerts its antihypertensive effect not only via reduction of the ACE activity in the plasma and lungs as reported earlier, but also that of other organs, principally the aorta, and that these effects are in dependent of the sympathetic nervous system. Angiotensin converting enzyme (ACE) was first isolated and purified from plasma in 1956 by Skeggs et al. (1). Subsequent studies confirmed the presence of this enzyme in a variety of tissues and organs in the body (2). Relatively high concentration of ACE is found in the lung (3). The presence of ACE has also been reported in the brain (4) and may constitute a component of a separate brain renin-angiotensin system. ACE, the key enzyme in the renin angiotensin system, is known to play an important role in the regulation of blood pressure and in the pathogenesis of hyper tension. This is because of its ability to convert the inactive decapeptide angiotensin I to the potent vasopressor octapeptide angiotensin II, principally in the pulmonary circulation (5) and its ability to inactivate bradykinin, the * To
whom
correspondence
should
be
addressed
.
hypotensive nonapeptide product of kallikrein (6). Specific inhibition of ACE by orally active captopril results in marked reduction in blood pressure (7) and captopril has proven to be an efficacious antihyper tensive agent. Blockade of conversion of angiotensin I to angiotensin II cannot account for its anti hypertensive efficacy (8); yet an anti hypertensive mechanism for captopril unrelated to ACE activity has not been directly demonstrated (9). The present study is aimed at examining the effect of captopril on ACE activity in the aorta, lung, plasma, kidney and whole brain of chemically sympa thectomized normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). This is deemed important in view of the close relationship that exists between the renin-angiotensin system and the sympa thetic nervous system in the regulation of normal blood pressure and in hypertensive
conditions (10, 11 ). Experiments were therefore conducted in central sympa thectomized and peripheral sympathec tomized WKY and SHR. Materials and Methods Male WKY and SHR weighing 200-300 g were used in the present investigation. Rats that were to receive peripheral administration of drugs were housed in groups of six in a cage and rats that were to receive central administration of drugs were housed individually. All rats were allowed free access to commercial food pellets and drank tap water ad libitum. Experimental sessions were conducted in the same room as the home cages were located, and the room was relatively quiet. All experiments were con ducted at room temperature, and no at tempts were made to control the humidity of the room. Central administration: In central adminis tration, injections were made via the i.c.v. route adapted from Hayden et al. (12). General anesthesia was achieved by intra peritoneal injection of sodium pentobarbitone (Nembutal, Abbot Laboratories), 40 mg/kg, and then aseptic surgery was performed. Implantation of cannula for i.c.v. injection was performed using a micro-manipulator in the stereotaxic instrument (Stoelting Co., U.S.A.). For this purpose, a 20 G needle cannula guide was fitted into the base of the plexiglass block that had a cavity filled with sealant. Dental cement was used to hold the needle in position. After surgery, the rats were given a recovery period of 7 days. All drugs used for central administration were dissolved in sterile saline. Central i.c.v. administration of captopril was made daily at a dose of 2 mg/kg for 8 days, whereas central sympathectomy was achieved by adminis tration of 6-OHDA, i.c.v.: 1200 ,,tg/kg on day 1, 2400 gig/kg on day 2 and 1200 /ag/kg on days 5 and 8, modified for central adminis tration as described by Simpson (13). 6 OHDA was prepared freshly each time in sterile saline containing 0.01 M ascorbic acid to prevent oxidation. Peripheral administration: In peripherally treated animals, injections of captopril were made via the intraperitoneal route at a dose of
25 mg/kg daily for a period of 8 days, whereas peripheral sympathectomy was achieved through administration of 6-OHDA via the tail vein at a dose of 50 mg/kg on day 1, 100 mg/kg on day 2 and 500 mg/kg each on days 5 and 8 (13). Blood pressure determination: Blood pressure was measured by tail plethysom graphy, connected to a physiograph in prewarmed (40°C for 15 min) rats, as reported by Krakoff et al. (14). Conscious systolic blood pressure was measured before and after treatment. Experimental protocol: Experiments in volved both central and peripheral treatment, and for these purposes, rats were divided into 4 groups: Group 1, serving as controls Group 2, captopril treated Group 3, 6-O H DA treated Group 4, both captopril and 6-OHDA treated All animals were sacrificed after day 8, 24 hr after the last treatment by drug. For centrally treated rats, the whole brain was removed and angiotensin converting enzyme activity determined. For peripherally treated rats, the thoracic region was quickly opened to expose the heart directly after sacrificing the animal. Blood was obtained from the heart by a direct heart puncture using a 1 ml syringe fitted to size 26 needle which had been previously moistened with heparinized saline. The blood was then centrifuged to obtain the plasma. The aorta, lung, kidney and whole brain were immediately removed for the determination of converting enzyme activity. Determination of converting enzyme activity: The converting enzyme activity was determined according to the method of Cushman and Cheung (15). One unit of ACE activity was defined as the amount that catalyzes the formation of 1 icmole of hip puric acid from hippuryl-L-histidyl-L-leucine (HHL) in 1 min at 37°C and atmospheric pressure. ACE activity was expressed as mU/ mg protein. Total protein measurement was made using a Biorad Kit, a-gent" (Abbott Laboratories). Statistics: Student's t-test was used to determine significant differences between the blood pressure before and after treat
ment. In comparing the effects of captopril on the ACE activity in the various organs/tissues and plasma of the SHR and the WKY rats, Student's t-test was also used to determine the significance between control and treated animals. All values are expressed as means ±S.E.M. An asterisk indicates values that are significantly different from the control group: *P<0 .05, **P<0.01, ***P<0.001.
aorta of the two strains of rat. However, in the plasma and in the kidney of SHR, the ACE
Results Captopril, 6-OHDA, and combination of captopril and 6-OHDA given via the i.p. route significantly reduced the blood pressure of SHR and WKY rats as shown in Table 1. When similar experiments were repeated with central administration via the i.c.v. route, only the blood pressure of SHR was sig nificantly reduced (Table 2), no significant changes in blood pressure were detected in WKY rats. Figure 1 shows the ACE activity in the various tissues of WKY and SHR. It can be seen that there was no difference in the ACE activity both in the whole brain and in the Table
1.
SHR
and
Table
2.
and
WKY
Effect WKY
Effect rats
of
captopril,
6-OHDA
and
both
Fig. 1. various
Histogram comparing ACE tissues of WKY 0 and SHR
are the
mean+S.E.M.
captopril
and
6-OHDA
for 6 animals.
on
systolic
blood
activity in . Values
***P<0.001.
pressure
of
rats
of captopril,
6-OHDA
and
both
captopril
and
6-OHDA
on
systolic
blood
pressure
of SHR
Table
3.
ing enzyme
Table verting
4.
Effect of captopril, activity
Effect enzyme
of
in various
captopril,
activity
6-OHDA
and both captopril
tissues
of SHR
6-OHDA
and
in various
tissues
activities were significantly lower, in the lung, the ACE activity of SHR in comparison with WKY rats.
both
of the
captopril WKY
and 6-OHDA
and
6-OHDA
treatment
treatment
on angiotensin
on
angiotensin
convert
con
rat
whereas is higher
Subacute intraperitoneal administration of captopril at 25 mg/kg daily for 8 days sig nificantly reduced the ACE activity in the plasma, the aorta and the lungs of both the SHR (Table 3) and WKY rats (Table 4). Similarly, subacute intraperitoneal adminis tration of captopril reduced the ACE activity in the kidney of WKY rats. However this effect is not seen in SHR where the ACE activity is unaffected by captopril. All these observations were independent of peripheral sympathectomy achieved through the admin istration of 6-OHDA via the tail vein. Results involving central captopril treat ment are shown in Fig. 2. Intracerebroven tricular administration of captopril, 2 mg/kg daily for 8 days, significantly depressed the ACE activity measured in the whole brain of both SHR and WKY rats. In neither of these strains did central sympathectomy by 6 OHDA exert any effect on the ACE activity.
Fig. 2. Histogram comparing ACE activity in the brain of the control 0, Captopril treated 6-OH DA treated , and both Captopril and 6 OHDA treated ;'Li WKY rats and SHR rats. Admin istration of drugs were made centrally via the i.c.v. route. Values are the mean±S.E.M. for 6 animals. *P<0 .05, **P<0.01, ***P<0.001.
Discussion The renin-angiotensin system in the is generally known to be in a depressed as can be seen from the lowered
SH R state ACE
activity in the plasma and the kidney of SHR. This is in spite of the higher ACE activity in the lung of SHR. These findings concur with
earlier observations (16). The present findings infer that there may be a functional renin angiotensin system in these tissues that plays an important role in the regulation of normal blood pressure and in the hypertensive con dition both in WKY and SHR. The lung appeared to be the predominant site where ACE activity is found in the body and is certainly the main organ that is respon sible for the conversion of inactive angiotensin I to active angiotensin II (5, 17). This is generally viewed to be the main mechanism through which blood pressure reduction is achieved following peripheral captopril administration in both the WKY and SHR. However the drastic reduction in the ACE activity both in the aorta of WKY and SHR following captopril treatment suggests that the blood pressure lowering effect of captopril could also be the consequence of this action. Evidence is available to suggest that the local renin-angiotensin system within the arterial walls may play a role in the main tenance of blood pressure (18, 19). Inhibition of angiotensin II production within the aortic wall and peripheral arteries will reduce the degree of vasoconstriction and thus peripheral resistance (20). Further evidence that the vascular tissue may be the target for angiotensin converting enzyme inhibitor is provided by Antonaccio et al. (21). They showed that the antihypertensive response after captopril treatment was as sociated with an increase in arterial wall renin which may indicate inhibition of angio tensin converting enzyme. Of particular interest is the present observation that this effect is independent of the sympathetic nervous system. It infers that the functional role of the renin-angiotensin system is operative even in the absence of the sympa thetic nervous system and that the sympa thetic nervous system is in no way affecting the converting enzyme inhibitory effect of captopril. This applies to both the lung and the aorta for both strain of rats.
had also been implicated (24, 25). The characteristically low ACE activity in the kidney of SHR as compared to that of WKY rats suggests a differentiating role of the renin-angiotensin system in the two strains of rat. The fact that the ACE activity is selectively depressed in the WKY rats further strengthened this claim and may suggest the existence of a mechanism for exerting control over the amount of ACE activity present in the SHR that differs from that of WKY particularly consequent to subacute captopril administration. How this difference arises is not known with certainty. It is of interest to note that no prerequisite of a functioning peripheral sympathetic nervous system is essential for the ACE activity in spite of the close relationship of angiotensin II with noradrenaline and the sympathetic nervous system, especially in relation to blood pressure regulation (10, 11 ). Central subacute captopril administration reduced the ACE activity in the whole brain of WKY and SHR. Peripheral subacute administration of captopril exerts no influence on the ACE activity of the whole brain of both WKY and SHR, indicating that the peri pherally administered captopril is not able to cross the blood-brain barrier. Alternatively, it may be that the amount of captopril reaching the brain following peripheral administration is negligible for affecting the brain ACE activity. The ability of peripheral captopril to cross the blood-brain barrier is a subject of much controversy. However, it is possible that the differences in the blood brain barrier among the different species and strains of rats coupled with the relative instability of tissue captopril may account for the conflicting results on the ability of peripherally administered captopril entering the brain (8, 26). Whatever it is, the central reduction of ACE activity in the brain brought about by i.c.v. captopril is independent of the intact central sympathetic nervous system. In summary, the present data suggest that captopril exerts its inhibition of ACE activity at least at two principle sites, the lung (also reflected through the plasma) and the aorta that may contribute towards its efficacious antihypertensive effect. Captopril is also able to inhibit the ACE activity in the brain.
However, the role of the brain renin angiotensin system in the regulation of blood pressure remains to be elucidated as central inhibition of ACE activity is not a requirement for acute blood pressure regulation by captopril. This is in agreement with obser vations made by Cohen and Kurz (27). It is concluded that the inhibitory effect of captopril on ACE activity is independent of the sympathetic nervous system. Acknowledgements: The authors are grateful to Professor K. Okamoto, Kinki University School of Medicine, Osaka, Japan for the generous gift of WKY and SHR. We would like to thank Squibb and Sons, Princeton, New Jersey, U.S.A.for the donation of captopril, and we thank Mrs. Alice Yap for typing the manuscript. References 1 Skeggs, L.T., Kahn, J.R. and Shumway, N.P.: Preparation and function of hypertensin con verting enzyme. J. Exp. Med. 105, 295-299 (1956) 2 Cushman, D.W. and Cheung, H.S.: Concen tration of angiotensin converting enzyme in tissue of rats. Biochim. Biophys. Acta 250, 261-265 (1971) 3 Cushman, D.W. and Cheung, H.S.: Studies on in vitro of angiotensin converting enzyme of lung and other tissues. In Hypertension, Edited by Genest, J., p. 532, Springer, Berlin, Heidelberg and New York (1972) 4 Ganten, D. and Speck, G.: The brain renin angiotensin system: a model for the synthesis of peptides in the brain. Br. J. Pharmacol. 27, 2379-2389 (1978) 5 Ng, K.K.F. and Vane, J.R.: The conversion of angiotensin I to angiotensin II. Nature 216, 762-766 (1967) 6 Rubin, B., Laffan, R.J., Kotler. D.G., O'Keefe, E.H., Demaio, D.A. and Goldberg, M.E.: S 14225 (D-3-mercapto-2-methyl-propanoyl-L Qproline), a- novel orally active inhibitor of angio tensin I-converting enzyme. J. Pharmacol. Exp. Ther. 204, 271-280 (1978) 7 Rubin, B. and Antonaccio, M.J.: Captopril. In Pharmacology of Antihypertensive Drugs, Edited by Scriabine, A., p. 21-42, Ravern Press, New York (1980) 8 Vollmer, R.R., Boccaguo, J.A.. Harris, D.N. and Murthy V.S.: Hypotension induced by inhibition of angiotensin-converting enzyme in pento barbital-anesthetized dogs. Eur. J. Pharmacol. 51, 39-45 (1978)
9 Antonaccio, M.J., Asaad, M., Rubin, B. and Horovitz, Z.P.: Captopril: Factors involved in its mechanism of action. In Angiotensin Converting Enzyme Inhibitors, Edited by Horovitz, Z.P., p. 161, Urban and Schwarzenberg, Baltimore and Munich (1981) 10 Khairallah, P.A., Davila, D., Papanicolaou, N., Glende, N.M. and Meyer, Ph.: Effects of angio tensin infusion on catecholamine uptake and reactivity in blood vessels. Circ. Res. 28/29, Suppl. 88, 96-106 (1971) 11 Panisset, J.C. and Bourdois, P.: Effect of angiotensin on the response to noradrenaline and sympathetic nerve stimulation and on 3H noradrenaline uptake in cat mesenteric blood vessels. Can. J. Physiol. Pharmacol. 46, 125-131 (1968) 12 Hayden, J.F., Johnson, L.R. and Maickel, R.O.: Construction and implantation of a permanent cannula for making injections into lateral ven tricle of the rat brain. Life Sci. 5, 1509-1514 (1966) 13 Simpson, L.L.: The effect of chemical sympa thectomy on vascular responses in spon taneously hypertensive rats. Neuropharmacology 13, 895-909 (1974) 14 Krakoff, L.R., Selvadural, R. and Sutter, E.: Effect of methyl-prednisolone upon arterial pressure and the renin-angiotensin system in the rat. Am. J. Physiol. 228, 613-617 (1975) 15 Cushman, D.W. and Cheung, H.S.: Spectro photometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol. 20, 1637-1648 (1970) 16 Polaky-Cynkin, R., Reichlin, S. and Famburg, B.L.: Angiotensin I-converting enzyme activity in spontaneously hypertensive rat. Proc. Soc. Exp. Biol. Med. 164, 242-247 (1980) 17 Ng, K.K.F. and Vane, J.R.: Fate of angiotensin I to angiotensin II. Nature 216, 762-766 (1967) 18 Collis, M.G. and Keddie, J.R.: Captopril at tenuates adrenergic vasoconstriction in rat mesenteric arteries by angiotensin-dependent and -independent mechanisms. Clin. Sci. 61, 281-286 (1981) 19 Okuno, T., Kondo, K., Konishi, K., Saruta, T. and Kato, E.: SQ-14225 attenuates the vascular response to norepinephrine in the rat mesenteric arteries. Life Sci. 25, 1343-1350 (1979) 20 Hatton, R. and Clough, D.P.: Captopril interferes with neurogenic vasoconstriction in the pithed rat by angiotensin-dependent mechanism. J. Cardiovasc. Pharmacol. 4, 116-123 (1982) 21 Antonaccio, M.J., Asaad, M., Rubin, B. and Horovitz, Z.P.: Captopril: Factors involved in its
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