Vol. 172, No. 2, 1990 October 30, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
INHIBITION
OF BIOLOGICAL
390-395
ACTIONS OF BIG ENDOTHELIN-1
BY PHOSPHORAMIDON Takahiro Fukuroda, Kazuhito Noguchi, Sonoko Tsuchida, Masaru Nishikibe, Fumihiko Ikemoto, Kenji Okada* and Mitsuo Yano*# Pharmacology and *Biochemistry , Central Research Laboratories, Banyu Pharmaceutical Co., Ltd. 2-9-3 Shimomeguro, Meguro-ku, Tokyo 153, Japan Received August 31, 1990
SUMMARY: Endothelin (ETFI and big ET-l both caused contraction of isolated porcine coronary arteries, but the potency of big ET-l was l/100- l/200 that of ET-l. These responses were independent of the vascular endothelium. Phosphoramidon blocked the vasoconstriction caused by 30 nM big ET-l, but was ineffective on the action of 0.3 nM ET-l. Also in viva, phosphoramidon had no effect on the ET-l-induced pressor actions, but blocked the pressor and airway-contractile responses to big ET-l in rats and/or guinea pigs. Thus, it is likely that the vascular responses to exogenous big ET-1 are at least in part due to its conversion to ET-l by a phosphoramidon-sensitive ET converting enzyme(s) in the vascular smooth muscle in vitro and in vivo. 0 1990 Academic Press, Inc.
A potent vasoconstrictor peptide, endothelin (ET), was first isolated from culture supernatant of porcine vascular endothelial cells (EC) (l), and later designated ET-l on the finding of two other ET family peptides, ET-2 and ET-3 (2). ET-l is the only ET to be made by EC by a sequential proteolysis of preproET-1
the formation of the intermediate big
via
ET-l by a combination of dibasic-pair endopeptidase and ET converting enzyme (1). Taken together, one can postulate that ET converting enzyme must exist in vascular EC, and actually, cathepsin D - like aspartic peptidase (3) and a neutral metallopeptidase (4) in cultured EC have been reported to form ET-l from big ET-l. membrane-bound
We have also found that a
100 kD metalloendopeptidase derived from cultured bovine vascular EC
caused a quantitative conversion of human big ET-l
into ET-l
at neutral pH. It
was
inhibited by phosphoramidon, and the conversion rate of big ET-l by this enzyme was 9 fold that of big ET- 3 (5). Due to a lack of crucial
in vivo
evidence, it is still unclear whether all or
any one of the enzymes proposed so far are really involved in the pathophysiological conversion of big ET-l. vitro
As reported (6), big ET-l has a weaker vasocontractile effect
but a similar pressor effect
different potencies
in vitro
in vivo,
and in
vivo
compared to ET-l. A possible explanation for the is likely that big ET-1 itself has a direct but weak
# To whom correspondence should be addressed. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Inc. reserved.
in
390
Vol.
172,
No.
BIOCHEMICAL
2, 1990
vasoconstrictor activity
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
accounting for the in vitro effect, but a conversion of big ET-l is
much easier in vivo than in vitro generating ET-l with full activity. In order to evaluate the physiological significance of the phosphoramidon-sensitive
ET
converting enzyme originally found in cultured EC (5), we investigated the effects of phosphoramidon on the contraction of isolated porcine coronary arteries in vitro and the pressor action and airway contraction in vivo in response to exogenously added big ET-l. MATERIALS
AND METHODS
Chemicals: ET-l, human big ET-l and phosphoramidon were purchased from Peptide Institute Inc., Osaka. All other chemicals used were of analytical or reagent grade. In vitro experiments: Left anterior descending coronary arteries were isolated from fresh porcine hearts. Connective tissues and adherent fats were removed, and the cleaned arteries cut into spiral strips about lo-mm long x l-mm wide. For removal of the vascular endothelium, the intimal surface of spiral strips was rubbed gently with wet filter paper. Each strip with or without the endothelium was placed in a 5-ml organ bath containing Krebs-Henseleit solution bubbled with 95% 02-5% CO2 at 37°C , and allowed to equilibrate for at least 1 hr under a 0.6 g resting-load before starting the experiments. Tension was recorded on a polygraph (Nihon Kohden RMP-6018, Tokyo) via isometric transducers (Nihon Kohden TB-651T). The presence or absence of functional endothelium was confirmed by the ability or disability, respectively, of O.lpM A-23187 to relax isolated coronary arteries after pretreatment with 2 PM PGFz,. The contractile activity was expressed as percent contraction calculated over the 100% obtained with 50 mM KCl. Concentrationresponse curves were obtained by cumulative addition of the peptides in 2-fold increments to the organ bath. In vivo experiments: Male Wistar Kyoto rats 15-16 weeks of age were anesthetized with sodium pentobarbital (60 mg/kg subcutaneously (s.c.)). Catheters were inserted into the lower abdominal aorta through the left femoral artery for measurement of blood pressure and the left femoral vein for drug administration. Each animal was placed in an individual cage for the continuous measurement of blood pressure and heart rate with a polygraph (Nihon Kohden RM-6000) 1 day after the surgical operation. The parameters were allowed to stabilize for >1 hr. Male Hartley guinea pigs of 350- 450 g weight were anesthetized with urethane (750 mg/kg intraperitoneally (ip.)) and a-chloralose (37.5 mg/kg i.p.). Animals were treated with succinylcholine (5 mg/kg SC.) to suppress spontaneous breathing, then connected with a ventilation pump (Ugo Basile, Italy) through a tracheal cannula (10ml air/kg body weight at 60 strokes/min). Catheters were inserted into the right carotid artery for measurement of blood pressure and the left jugular vein for drug administration. Changes in bronchopulmonary inflation pressure (BIP) were measured with a bronchospasm transducer (Ugo Basile Model 7020) according to the method of Konzett and Rossler (7), and expressed as percent changes calculated over the 100% obtained by clamping the trachea at the end of the experiments.
RESULTS In isolated porcine coronary arteries with and without the endothelium, both ET-l and big ET-1
caused concentration-dependent
contractions
(Fig.
1). Judged from
the
concentration-response curves using intact arteries, ET-1 (EC0 = 0.25 nM) was 100-200 times as potent as big ET-l, but their maximal contractions seem to be comparable. There was no significant shift of these curves in the presence or absence of the endothelium, thus suggesting
that the endothelium
is not involved
and big ET-1 in this system. 391
significantly
in the vascular
effects of ET-1
Vol. 172, No. 2,
-log(ET-l
1990
BIOCHEMICAL
or
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
bigET-1]
Fig. 1. Comparison of vasoconstrictor effects of ET-l and big ET-l. ET-1( 0 , l ) or big ET-I( q , n ) was added to isolated porcine coronary arteries with (open symbols) or without (closed symbols) the endothelium. Values representmeans ? S.E.M.(n= 5-6). Fig. 2. Effects of phosphoramidon
on contraction
induced
by ET-l
or big ET-l.
0.3 nM
ET-I( 0 , l ) or 30 nM big ET-l( 0 , n ) was added to denudedporcine coronary arteries in the presence(open) or absence(closed)of 1 mM phosphoramidon.Values representmeans + S.E.M.(n= 4). Fig. 2 shows time-response curves of vasoconstriction induced by 0.3 nM ET-l and 30 nM big ET-l. Compared to 0.3 nM ET-l, 30 nM big ET-l caused a slow-onset contraction that reached, however, almost the same level of the steady state. On these responses, 1 mM phosphoramidon had different effects; no stimulation and no inhibition of the response to ET-l, but substantial inhibition of the response to big ET-l. These results strongly suggest that
big
ET-l
causes
vasoconstriction
after
its
conversion
to
ET-1
by
a
phosphoramidon-sensitive enzyme in the isolated arteries. An intravenous (iv.) injection of ET-l at a dose of 1 nmol/kg in awake rats produced a sustained
pressor effect preceded by a rapid, transient depressor effect (Fig. 3), while the
n.
40
gsi
20
SE a5 C’
-20
%I 0
0 -40 -60
0 Time
20 after
ET-1
40 admlnlstrstion
60 (min)
Fig. 3. Effects of phosphoramidon on ET-l-induced changes in mean arterial blood pressure (MBP) and heart rate (HR) in rats. ET-l, 1 nmol/kg i.v., was given to conscious, unrestrained Wistar Kyoto (WKY) rats Smin after the administration of phosphoramidon, 5 mg/kg i.v.( 0) or none( 0 ). Values represent means + S.E.M.(n= 3- 8).
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-20-/ looO-100-
-200 4 0 Time
20 after
big
ET-1
40
60
administration
(min)
Time
after
big
ET-1
administration
(min)
Fig. 4.,Effects of phosphoramidon on big ET-l-induced changes in MBP and HR in rats. Big ET-l, 1 nmol/kg Lv., was given to conscious, unrestrained WKY rats 5min after the administration of phosphoramidon, 1 mg/kg i.v.( n ), 5 mg/kg i.v.( 0 ) or none( 0 ). Values represent means + S.E.M.(n= 3-4). Fig. 5. Effects of phosphoramidon on big ET-l-induced changes in MBP and bronchopulmonary inflation pressure (BIP) in guinea pigs. Big ET-l, 4 nmol/kg i.v., was given to anesthetized guinea pigs 3min after the administration of phosphoramidon, 10 mgikg i.v. (0 ) or none( 0). Values represent means + S.E.M.(n= 3).
same dose of big ET-l 4). Pretreatment mg/kg)
inhibited
one. Thus, unlike
the big ET-l-induced and quantitative
In anesthetized
conversion
with
conversion
of circulating
of big ET-1
increase
was also attenuated
and airway
phosphoramidon,
in blood
(1 mg/kg)
might
pressure
effect
(Fig.
or extensively
(5
the ET-l-induced
have occurred
in viva,
was accompanied
by a
by phosphoramidon.
4 nmol/kg
resistance
10 mg/kg
big ET-l
partially
depressor
pressor action, but did not inhibit
guinea pigs, big ET-l,
of blood pressure
pretreatment
pressor effect, but no initial
The big ET-l-induced
decrease in heart rate, which
elevations
a similar
with two i.v. doses of phosphoramidon
a rapid
in vitro.
exerted
i.v., caused remakable
(expressed
and sustained
as BIP), that were blocked
i.v. (Fig. 5). These findings
by a phosphoramidon-sensitive
only in blood vessels but also in the bronchopulmonary
by
suggest that the
enzymes
will
occur not
system .
DISCUSSION ET-l precursor
is the most potent peptide
with a potency
big ET-l
vasoconstrictor
also caused a sustained
of l/55 that of ET-1
but,
rats was almost the same as that of ET-l, not seen (6). As to the discrepancy used phosphoramidon
with
and thereby
between
contraction
in contrast, although
a sustained
pressor
of isolated
the initial,
393
clearly
(1).
The
rat thoracic
aorta
the pressor effect of big ET-l transient
depressor
the in vitro and in vivo potencies
demonstrated
effect
that big ET-l
effect was
of big ET-l,
itself
i.v. in
we
had no direct
Vol.
172, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
action on the isolated vessels (Fig. 2), but only a small portion of big ET-l was likely to be converted to ET-l, because the contractile potency of big ET-l was l/100-
l/200 that of
ET-l (Fig. 1). When given intravenously, big ET-1 was apparently equipotent to ET-l in raising the arterial blood pressure in rats, but the pressor action of big ET-l, not ET-l, was blocked by phosphoramidon (Fig. 3 and Fig. 4). Thus, big ET-1 can be converted in vivo in a rapid and quantitative manner by a phosphoramidon-sensitive enzyme. Generation of ET-1 from big ET-l in vitro seems to be limited within about 1% (Fig. 1 and Fig. 2).
One possible explanation was a nonspecific and extensive degradation of big
ET-1 and/or ET-l in the organ bath, but it was excluded; because the organ-bath fluid after 2-hr incubation with big ET-l, where the contraction reached a steady state, reproduced the original time-response curve when it was transferred to another organ bath with a fresh isolated artery. This means that a considerable amount of big ET-l still remained after 2-hr incubation (data not shown). As demonstrated in vitro, big ET-l is converted to ET-l, then causes vasoconstriction (Fig. 2), but the endothelium is unlikely to be involved in this response (Fig. 1). Therefore, it is strongly suggested that non-EC, most probably vascular smooth muscle cell (VSMC) may be responsible for the vascular conversion of big ET-l. Very recently, the presence of ET converting enzyme in VSMC has been suggested in a report for the induction of ET mRNA expression and synthesis of functional ET peptide in cultured human VSMC (8). Plasma levels of big ET-l several times higher than those of ET-1 in healthy humans and plasma levels of big ET-l twice as high in patients with acute myocardial infarction as in normal (9) may be of pathophysiological importance. Thus, it becomes highly possible that circulating big ET-l will be activated when in contact with VSMC and/or EC in vivo. If ET-l elaborated by EC acts as a paracrine on adjacent VSMC, ET-l synthesized by VSMC may function in an autocrine manner. As we have noted previously, phosphoramidon inhibits ET converting enzyme derived from EC (5). Here we propose a contribution phosphoramidon-sensitive
for the vascular ET conversion of a
enzyme to be present in VSMC. Therefore, phosphoramidon or
other similar agents, if any, capable of inhibiting both enzymes at once would be a powerful blocker of ET-l, and furthermore, quite useful for understanding the pathophysiologicsl roles of ET-1 in vascular as well as nonvascular tissues. Acknowledgment:
We express our thanks to Dr. J. S. Walker, Merck & Co., for his
critical reading of this manuscript.
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9.
Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature 332,411-415. moue, A., Yanagisawa,.M., Kimura, S.,Kasuya, Y., Miyauchi, T., Goto, K.and Masaki, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867. Matsumura, Y., Ikegawa, R., Takaoka, M. and Morimoto, S. (1990) Biochem. Biophys. Res. Commun. 167,203-210. Ohnaka, K., Takayanagi, R., Yamauchi, T., Okazaki, H., Ohashi, M., Umeda, F. and Nawata, H. (1990) Biochem. Biophys. Res. Commun. 168,1128-1136. Okada, K., Miyazaki, Y., Takada, J., Matsuyama, K., Yamaki, T. and Yano, M. (in press)Biochem. Biophys. Res. Commun. Kashiwabara, T., Inagaki, Y., Ohta, H., Iwamatsu, A., Nomizu, M., Morita, A and Nishikori, K., (1989) FEBS Lett. 247, 73-76. Konzett, H. and Rossler, R., (1940) Arch. Exp. Path. Pharmacol., 135, 71-74. Resink, T. J., Hahn, A. W. A., Scott- Burden, T., Powell, J., Weber, E. and Buhler, F.R. (1990) Biochem. Biophys. Res. Commun. 168, 1303-1310 Miyauchi, T., Yanagisawa, M., Tomizuka, T., Sugishita, Y., Suzuki, N., Fujino, M., Ajisaka, R., Goto, K. and Masaki, T. (1989) Lancet 2, 53-54.
395
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
INHIBITION
OF BIOLOGICAL
390-395
ACTIONS OF BIG ENDOTHELIN-1
BY PHOSPHORAMIDON Takahiro Fukuroda, Kazuhito Noguchi, Sonoko Tsuchida, Masaru Nishikibe, Fumihiko Ikemoto, Kenji Okada* and Mitsuo Yano*# Pharmacology and *Biochemistry , Central Research Laboratories, Banyu Pharmaceutical Co., Ltd. 2-9-3 Shimomeguro, Meguro-ku, Tokyo 153, Japan Received August 31, 1990
SUMMARY: Endothelin (ETFI and big ET-l both caused contraction of isolated porcine coronary arteries, but the potency of big ET-l was l/100- l/200 that of ET-l. These responses were independent of the vascular endothelium. Phosphoramidon blocked the vasoconstriction caused by 30 nM big ET-l, but was ineffective on the action of 0.3 nM ET-l. Also in viva, phosphoramidon had no effect on the ET-l-induced pressor actions, but blocked the pressor and airway-contractile responses to big ET-l in rats and/or guinea pigs. Thus, it is likely that the vascular responses to exogenous big ET-1 are at least in part due to its conversion to ET-l by a phosphoramidon-sensitive ET converting enzyme(s) in the vascular smooth muscle in vitro and in vivo. 0 1990 Academic Press, Inc.
A potent vasoconstrictor peptide, endothelin (ET), was first isolated from culture supernatant of porcine vascular endothelial cells (EC) (l), and later designated ET-l on the finding of two other ET family peptides, ET-2 and ET-3 (2). ET-l is the only ET to be made by EC by a sequential proteolysis of preproET-1
the formation of the intermediate big
via
ET-l by a combination of dibasic-pair endopeptidase and ET converting enzyme (1). Taken together, one can postulate that ET converting enzyme must exist in vascular EC, and actually, cathepsin D - like aspartic peptidase (3) and a neutral metallopeptidase (4) in cultured EC have been reported to form ET-l from big ET-l. membrane-bound
We have also found that a
100 kD metalloendopeptidase derived from cultured bovine vascular EC
caused a quantitative conversion of human big ET-l
into ET-l
at neutral pH. It
was
inhibited by phosphoramidon, and the conversion rate of big ET-l by this enzyme was 9 fold that of big ET- 3 (5). Due to a lack of crucial
in vivo
evidence, it is still unclear whether all or
any one of the enzymes proposed so far are really involved in the pathophysiological conversion of big ET-l. vitro
As reported (6), big ET-l has a weaker vasocontractile effect
but a similar pressor effect
different potencies
in vitro
in vivo,
and in
vivo
compared to ET-l. A possible explanation for the is likely that big ET-1 itself has a direct but weak
# To whom correspondence should be addressed. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
Inc. reserved.
in
390
Vol.
172,
No.
BIOCHEMICAL
2, 1990
vasoconstrictor activity
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
accounting for the in vitro effect, but a conversion of big ET-l is
much easier in vivo than in vitro generating ET-l with full activity. In order to evaluate the physiological significance of the phosphoramidon-sensitive
ET
converting enzyme originally found in cultured EC (5), we investigated the effects of phosphoramidon on the contraction of isolated porcine coronary arteries in vitro and the pressor action and airway contraction in vivo in response to exogenously added big ET-l. MATERIALS
AND METHODS
Chemicals: ET-l, human big ET-l and phosphoramidon were purchased from Peptide Institute Inc., Osaka. All other chemicals used were of analytical or reagent grade. In vitro experiments: Left anterior descending coronary arteries were isolated from fresh porcine hearts. Connective tissues and adherent fats were removed, and the cleaned arteries cut into spiral strips about lo-mm long x l-mm wide. For removal of the vascular endothelium, the intimal surface of spiral strips was rubbed gently with wet filter paper. Each strip with or without the endothelium was placed in a 5-ml organ bath containing Krebs-Henseleit solution bubbled with 95% 02-5% CO2 at 37°C , and allowed to equilibrate for at least 1 hr under a 0.6 g resting-load before starting the experiments. Tension was recorded on a polygraph (Nihon Kohden RMP-6018, Tokyo) via isometric transducers (Nihon Kohden TB-651T). The presence or absence of functional endothelium was confirmed by the ability or disability, respectively, of O.lpM A-23187 to relax isolated coronary arteries after pretreatment with 2 PM PGFz,. The contractile activity was expressed as percent contraction calculated over the 100% obtained with 50 mM KCl. Concentrationresponse curves were obtained by cumulative addition of the peptides in 2-fold increments to the organ bath. In vivo experiments: Male Wistar Kyoto rats 15-16 weeks of age were anesthetized with sodium pentobarbital (60 mg/kg subcutaneously (s.c.)). Catheters were inserted into the lower abdominal aorta through the left femoral artery for measurement of blood pressure and the left femoral vein for drug administration. Each animal was placed in an individual cage for the continuous measurement of blood pressure and heart rate with a polygraph (Nihon Kohden RM-6000) 1 day after the surgical operation. The parameters were allowed to stabilize for >1 hr. Male Hartley guinea pigs of 350- 450 g weight were anesthetized with urethane (750 mg/kg intraperitoneally (ip.)) and a-chloralose (37.5 mg/kg i.p.). Animals were treated with succinylcholine (5 mg/kg SC.) to suppress spontaneous breathing, then connected with a ventilation pump (Ugo Basile, Italy) through a tracheal cannula (10ml air/kg body weight at 60 strokes/min). Catheters were inserted into the right carotid artery for measurement of blood pressure and the left jugular vein for drug administration. Changes in bronchopulmonary inflation pressure (BIP) were measured with a bronchospasm transducer (Ugo Basile Model 7020) according to the method of Konzett and Rossler (7), and expressed as percent changes calculated over the 100% obtained by clamping the trachea at the end of the experiments.
RESULTS In isolated porcine coronary arteries with and without the endothelium, both ET-l and big ET-1
caused concentration-dependent
contractions
(Fig.
1). Judged from
the
concentration-response curves using intact arteries, ET-1 (EC0 = 0.25 nM) was 100-200 times as potent as big ET-l, but their maximal contractions seem to be comparable. There was no significant shift of these curves in the presence or absence of the endothelium, thus suggesting
that the endothelium
is not involved
and big ET-1 in this system. 391
significantly
in the vascular
effects of ET-1
Vol. 172, No. 2,
-log(ET-l
1990
BIOCHEMICAL
or
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
bigET-1]
Fig. 1. Comparison of vasoconstrictor effects of ET-l and big ET-l. ET-1( 0 , l ) or big ET-I( q , n ) was added to isolated porcine coronary arteries with (open symbols) or without (closed symbols) the endothelium. Values representmeans ? S.E.M.(n= 5-6). Fig. 2. Effects of phosphoramidon
on contraction
induced
by ET-l
or big ET-l.
0.3 nM
ET-I( 0 , l ) or 30 nM big ET-l( 0 , n ) was added to denudedporcine coronary arteries in the presence(open) or absence(closed)of 1 mM phosphoramidon.Values representmeans + S.E.M.(n= 4). Fig. 2 shows time-response curves of vasoconstriction induced by 0.3 nM ET-l and 30 nM big ET-l. Compared to 0.3 nM ET-l, 30 nM big ET-l caused a slow-onset contraction that reached, however, almost the same level of the steady state. On these responses, 1 mM phosphoramidon had different effects; no stimulation and no inhibition of the response to ET-l, but substantial inhibition of the response to big ET-l. These results strongly suggest that
big
ET-l
causes
vasoconstriction
after
its
conversion
to
ET-1
by
a
phosphoramidon-sensitive enzyme in the isolated arteries. An intravenous (iv.) injection of ET-l at a dose of 1 nmol/kg in awake rats produced a sustained
pressor effect preceded by a rapid, transient depressor effect (Fig. 3), while the
n.
40
gsi
20
SE a5 C’
-20
%I 0
0 -40 -60
0 Time
20 after
ET-1
40 admlnlstrstion
60 (min)
Fig. 3. Effects of phosphoramidon on ET-l-induced changes in mean arterial blood pressure (MBP) and heart rate (HR) in rats. ET-l, 1 nmol/kg i.v., was given to conscious, unrestrained Wistar Kyoto (WKY) rats Smin after the administration of phosphoramidon, 5 mg/kg i.v.( 0) or none( 0 ). Values represent means + S.E.M.(n= 3- 8).
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-20-/ looO-100-
-200 4 0 Time
20 after
big
ET-1
40
60
administration
(min)
Time
after
big
ET-1
administration
(min)
Fig. 4.,Effects of phosphoramidon on big ET-l-induced changes in MBP and HR in rats. Big ET-l, 1 nmol/kg Lv., was given to conscious, unrestrained WKY rats 5min after the administration of phosphoramidon, 1 mg/kg i.v.( n ), 5 mg/kg i.v.( 0 ) or none( 0 ). Values represent means + S.E.M.(n= 3-4). Fig. 5. Effects of phosphoramidon on big ET-l-induced changes in MBP and bronchopulmonary inflation pressure (BIP) in guinea pigs. Big ET-l, 4 nmol/kg i.v., was given to anesthetized guinea pigs 3min after the administration of phosphoramidon, 10 mgikg i.v. (0 ) or none( 0). Values represent means + S.E.M.(n= 3).
same dose of big ET-l 4). Pretreatment mg/kg)
inhibited
one. Thus, unlike
the big ET-l-induced and quantitative
In anesthetized
conversion
with
conversion
of circulating
of big ET-1
increase
was also attenuated
and airway
phosphoramidon,
in blood
(1 mg/kg)
might
pressure
effect
(Fig.
or extensively
(5
the ET-l-induced
have occurred
in viva,
was accompanied
by a
by phosphoramidon.
4 nmol/kg
resistance
10 mg/kg
big ET-l
partially
depressor
pressor action, but did not inhibit
guinea pigs, big ET-l,
of blood pressure
pretreatment
pressor effect, but no initial
The big ET-l-induced
decrease in heart rate, which
elevations
a similar
with two i.v. doses of phosphoramidon
a rapid
in vitro.
exerted
i.v., caused remakable
(expressed
and sustained
as BIP), that were blocked
i.v. (Fig. 5). These findings
by a phosphoramidon-sensitive
only in blood vessels but also in the bronchopulmonary
by
suggest that the
enzymes
will
occur not
system .
DISCUSSION ET-l precursor
is the most potent peptide
with a potency
big ET-l
vasoconstrictor
also caused a sustained
of l/55 that of ET-1
but,
rats was almost the same as that of ET-l, not seen (6). As to the discrepancy used phosphoramidon
with
and thereby
between
contraction
in contrast, although
a sustained
pressor
of isolated
the initial,
393
clearly
(1).
The
rat thoracic
aorta
the pressor effect of big ET-l transient
depressor
the in vitro and in vivo potencies
demonstrated
effect
that big ET-l
effect was
of big ET-l,
itself
i.v. in
we
had no direct
Vol.
172, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
action on the isolated vessels (Fig. 2), but only a small portion of big ET-l was likely to be converted to ET-l, because the contractile potency of big ET-l was l/100-
l/200 that of
ET-l (Fig. 1). When given intravenously, big ET-1 was apparently equipotent to ET-l in raising the arterial blood pressure in rats, but the pressor action of big ET-l, not ET-l, was blocked by phosphoramidon (Fig. 3 and Fig. 4). Thus, big ET-1 can be converted in vivo in a rapid and quantitative manner by a phosphoramidon-sensitive enzyme. Generation of ET-1 from big ET-l in vitro seems to be limited within about 1% (Fig. 1 and Fig. 2).
One possible explanation was a nonspecific and extensive degradation of big
ET-1 and/or ET-l in the organ bath, but it was excluded; because the organ-bath fluid after 2-hr incubation with big ET-l, where the contraction reached a steady state, reproduced the original time-response curve when it was transferred to another organ bath with a fresh isolated artery. This means that a considerable amount of big ET-l still remained after 2-hr incubation (data not shown). As demonstrated in vitro, big ET-l is converted to ET-l, then causes vasoconstriction (Fig. 2), but the endothelium is unlikely to be involved in this response (Fig. 1). Therefore, it is strongly suggested that non-EC, most probably vascular smooth muscle cell (VSMC) may be responsible for the vascular conversion of big ET-l. Very recently, the presence of ET converting enzyme in VSMC has been suggested in a report for the induction of ET mRNA expression and synthesis of functional ET peptide in cultured human VSMC (8). Plasma levels of big ET-l several times higher than those of ET-1 in healthy humans and plasma levels of big ET-l twice as high in patients with acute myocardial infarction as in normal (9) may be of pathophysiological importance. Thus, it becomes highly possible that circulating big ET-l will be activated when in contact with VSMC and/or EC in vivo. If ET-l elaborated by EC acts as a paracrine on adjacent VSMC, ET-l synthesized by VSMC may function in an autocrine manner. As we have noted previously, phosphoramidon inhibits ET converting enzyme derived from EC (5). Here we propose a contribution phosphoramidon-sensitive
for the vascular ET conversion of a
enzyme to be present in VSMC. Therefore, phosphoramidon or
other similar agents, if any, capable of inhibiting both enzymes at once would be a powerful blocker of ET-l, and furthermore, quite useful for understanding the pathophysiologicsl roles of ET-1 in vascular as well as nonvascular tissues. Acknowledgment:
We express our thanks to Dr. J. S. Walker, Merck & Co., for his
critical reading of this manuscript.
Vol.
172,
No.
2,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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