GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
The Vasorelaxant
78,42X’
(19%)
Effect of Atrial Natriuretic
Peptide in the Frog
K. W. CHIU, Y. C. LEE, AND P. K. T. PANG* Department of Biology, The Chinese University of Hong Kong, Hong Kong, and *Department of Physiology, University of Alberta, Edmonton, Alberta, Canada Accepted June 12, 1989 The vasorelaxant effect of atrial natriuretic peptide (ANP) in two species of frogs, Rana catesbeiana and R. tigrina, were studied in vitro. Isolated arterial strips were prepared from the aortic loop, dorsal aorta, iliac, and femoral arteries in the frog. These were stimulated to contract with KCl, norepinephrine, or arginine vasotocin. When maximal contraction was attained, ANP was added to assess if it could relax the strips. Data show that ANP is vasorelaxant in these preparations. The sensitivity of these tissues depends on the contractile agents, e.g., KCl-stimulated preparations from R. catesbeiana did not respond to ANP. Data also suggest that ANP action elicited in the frog vascular tissue is due to an inhibitory effect on the mobilization of the intracellular calcium store and/or calcium influx from extracellular space to initiate contraction. 8 1990Academic press, Inc.
In mammals, atria1 natriuretic factor (ANF) has a pronounced effect on the cardiovascular activity (de Bold, 1985; Atlas et al., 1986; Bussian et al., 1986; Cogan, 1986; Winquist, 1986). It relaxes preconstricted smooth muscle and decreases heart rate and tension. Immunocytochemical and radioimmunological studies indicate presence of ANF in almost all major classes of vertebrates (de Bold and Salerno, 1983; Chapeau et al., 1985; Netchitailo ef al., 1986, 1988; Reinecke et al., 1987), and in the fish and turtle, homologous heart extracts produce renal and pressure responses (Duff and Olson, 1986; Lee and Malvin, 1987; Cho et al., 1988). Recent reports on isolated arteries and perfused organs in trouts (Olson and Meisheri, 1989) and isolated atria from amphibians and snakes (K. W. Chiu et al., unpublished data) indicate that ANF also has an effect on the cardiovascular system in the lower vertebrates. We have been studying the vasorelaxant property of the calcitonin gene-related peptide in the vertebrates (Kline and Pang, 1988; Kline et al., 1988) and have since extended the study to ANP. In this report, the vas-
cular effects of ANP in two species of frogs are presented. MATERIALS
Bullfrogs (Rana catesbeiana) were obtained from a commercial supplier in the United States and tiger frogs (R. t&ha) were purchased from local markets in Hong Kong. These frogs were pithed and the viscera cut open. The aortic loop, dorsal aorta, iliac, and femoral arteries were removed en bloc and were placed in frog Ringer’s solution (IRS) (80 n& NaCl, 2.5 m&f KCl, 1.8 m&f CaCl,, 24 mM NaHCO,, 0.12 m&f NaH,PO,, and 1.1 n&f glucose) which was oxygenated with 95% 0,:5% CO2 maintained at room temperature. The various portions of the blood vessels were separated, cleared of connective tissues, cut helically, and the strips were secured in an organ bath containing 10 ml FRS. A resting tension of 0.7 g was set. Subsequent changes in the force developed by the helical strips were measured by a force displacement transducer and recorded on a polygraph. The procedure to examine the vasodilatory effect of ANP on the helical strips followed that described by Kline et al. (1988). Isolated strips were equilibrated for 45 min prior to being tested. Each strip was exposed to 60 m&f KC1 and the amount of tension was monitored. This was followed by three changes of bathing FRS and a lO-min rest before repeating the test. Each strip was tested three times. A tension of 0.5 g or more was required. The strip was then equilibrated for 30 min. 42
001~6480/90 $1.50 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.
AND METHODS
VASORBLAXANT
EFFECT OF ANP IN FROG
The substances used to stimulate contraction were 60 mM KCI, 3 x 10m6 M norepinephrine (NE), and 30 rig/ml arginine vasotocin (AVT). The stimulating drug to be used was added to the bath. Once maximum tension was obtained, one of the four doses (10, 30, 100, and 300 t&ml in R. catesbeinana, and 1, 3, 10, and 30 rig/ml in R. ligrina) of ANP [atrial natriuretic peptide, rat (28 amino acids) PATR 20, Bachem] was added to the bath. Both the maximum tension and the amount of relaxation, if any, were recorded. The organ bath was rinsed three times with FRS and the tissue was equilibrated for 30 min if either KC1 or NE was used, or 60 mm if AVT was used. This process was repeated. At the end of the experiment, 60 m&f KC1 was added to the chamber and a final tension to ensure that no tachyphylaxis occurred. The role of intracellular Ca2+ and the vasorelaxant effect of ANP was also tested in the bullfrog. After being tested for use, the strips were placed in FRS containing no Ca” and 2 rnkf EGTA (Sigma). After 2 min, NE was added to the bath and the tension was recorded. The bath was rinsed three times with normal FRS. The strips were then equilibrated in normal FRS for 45 min. The procedure was repeated. To test the effects of ANP, it was added to the bath containing no Ca2+ and 2 mM EGTA prior to the addition of NE. The procedure was repeated for each dose of ANP after 45 min of equilibrium in FRS. To verify the setup and protocol, the vasorelaxant effect of ANP on the dorsal aorta strips from rats was
similarly assessed. The dorsal aorta (2 to 3 cm) was removed from anesthetized rats. They were transferred to Krebs-Henseleit solution (KHS) (115 mkf NaCl, 5 mil4 KCI, 2.1 m&f CaCl,, 1.2 m&I MgSO,, 1.2 mM NaH,PO,, 25 mM NaHCO,, and 11 mM glucose), oxygenated with 95% 0,:5% C02, cleared of connective tissue, cut helically, and mounted in an organ bath which was set at 37”. All data were presented in mean 2 SEM. Both sexes of bullfrogs were used, but only females of tiger frogs were chosen as they are much larger than males and yield larger blood vessels.
RESULTS
R. catesbeiana. Results of the vascular studies using the bullfrog aortic loop, aorta, iliac, and femoral arteries indicated a varying degree of sensitivity to the vasorelaxant effect of ANP as well as the contractile agents used. ANP had no significant relaxing effect on KCl-stimulated contraction in all preparations. When AVT was used to stimulate contraction, it had no significant relaxing effect on the femoral artery strips but had a more pronounced effect on the aortic loop and iliac artery (Fig. 1). A de-
Cont.
(rig/ml)
0
20
1 n 0-0
-m
aortic
43
loop
iliac
FIG. 1. The relaxant effect of ANP on AVT-stimulated represent SE.
contraction in R. catesbeiuna. Vertical bars
44
CHIU,
LEE,
crease of about 60% at 30 rig/ml in the latter preparation was seen. Large individual variations were noted. When NE was used to stimulate the contraction prior to ANP (Fig. 2), the vasorelaxant effect was apparent at the initial dose of ANP 10 rig/ml, and except in the femoral preparation, all other preparations showed a relaxation of about 40%. The femoral strip showed only a relaxation of about 15%. The maximum of 60% was achieved with all preparations, except in the femoral artery. R. tigrina. The pattern of vasorelaxant responses to ANP showed variations and differences as compared with the bullfrog. With KC1 preconstriction, all preparations of dorsal aorta, iliac, and femoral arteries showed dose-dependent relaxation (Fig. 3), noting of course that the dose of ANP used had been reduced. The femoral preparation was the most sensitive of all preparations, with the aortic loop not sensitive to KC1 and the other two contractile agents NE and AVT, and therefore the effect of ANP could not be tested and accordingly is not
cont.
4
dp
I
10 I
AND
PANG
shown in this and subsequent figures. With AVT, all preparations responded in a similar manner among different preparations and with a similar range of relaxation (Fig. 4). When the preparations were preconstricted with NE, they all showed, as expected, dose-related relaxation (Fig. 5). They were all far more sensitive to ANP than they were preconstricted with AVT or KCl. Further, these dose-dependent relaxation curves are present as parallel sets of lines, indicating therefore that the iliac was the most sensitive of all three preparations. In view of the discrepancy in the vasorelaxant response of the isolated strips to ANP under KC1 preconstriction between the two species of frogs, a repeat of this set of the experiment was performed using the tiger frog with lower ANP doses (0.1,0.3,1, and 3 r&ml). The strips yielded similar relaxation responses to ANP. The effects of ANP on intracellular calcium-dependent contraction in bullfrogs are shown in Fig. 6. The percentage inhibition of the developed tension versus the doses
(ngfml)
30
100 I
300
60
m-m
aortic
~00~
l -
n--o
dorsal
aorta
A-A
FIG. 2. The relaxant effect of ANP on NE-stimulated
l
iliac femoral
contraction in R. catesbeiana.
VASORELAXANT
Cont.
1
EFFECT
OF ANP
IN
FROG Cont.
(rig/ml)
3
45
10
30
1
(rig/ml) 3
10
30
0
10
20 E .2 + B 2 8 *
30 30 40
50
0
Femoral
*
dorsal
aorta I
60 FIG. 3. The relaxant effect of ANP on KClstimulated contraction in R. figrina.
of ANP indicated that the sensitivity of the iliac, dorsal aorta, femoral, and aortic loop to the peptide was in this order. A dosedependent response to ANP was readily apCont.
(rig/ml)
70
80
90
*
dorsal
aorta
100
Ol
FIG. 5. The relaxant effect of ANP stimulated contraction in R. tigrina.
10
on NE-
parent with the dorsal aorta and aortic loop. A maximal inhibition of about 60% was observed in the iliac, femoral, and dorsal aorta preparations.
20
DISCUSSION q 50
60
1
l
iliac
*
dorsal
I aorta
FIG. 4. The relaxant effect of ANP on AVTstimulated contraction in R. tigrina.
Results from the present study demonstrated a vasorelaxant effect of ANP in two species of frogs, R. catesbeiana and R. tigrina. This finding is in accord with the relaxant effect of ANP in isolated arteries and perfused organs in the trout, Salmo gairdneri, under NE stimulation (Olson and
46
CHIU,
LEE,
AND
Cont.
PANG
(rig/ml)
Oh
60
80
n
aortic loop
0
iliac
0
dorsal
A
femoral
FIG. 6. Intracellular calcium-dependent containing no calcium and 2 mM EGTA.
aorta
contraction. NE was used to stimulate helical
Meisheri, 1989). In the chondrichthyes, Squalus acanthias, Scyliorhinus canicula, Raja clavata, and Chimaera monstrosa, it
has been reported that purified extracts prepared from the hearts produced dosedependent relaxation of preconstricted mammalian (rabbit) aorta (Reinecke et al., 1987). The occurrence of ANP, and possibly its vasorelaxant property, must have evolved at an early stage of the vertebrates. The present data also show differences in the vasorelaxant effect between the two species of frogs. For example, KClstimulated dorsal aorta, iliac, and femoral artery strips of R. tigrina responded to ANP with relaxation, but no response was observed with similar preparations from R. catesbeiana. Regional and species differences in the vasorelaxant response of the vascular tissue to ANP in mammals have, however, been documented (Winquist, 1985, 1986). Garcia et al. (1984) reported that rat atrial extract relaxes rabbit aortic
stripsin FRS
and renal but not mesenteric helical strips stimulated by NE and ANG II. Using isolated rabbit vessels, Faison et al. (1985) noted that large central ones respond best to ANF, more distal ones such as iliac and femoral arteries only partially respond, and finally the peripheral ones are unresponsive at all. On the mechanism of this vasorelaxant effect, data from the present intracellular calcium experiment in the bulIfrog strongly indicate that vasorelaxation is due to an interference on the mobilization of intracellular calcium from storage sites, as AVT and NE stimulate muscle contraction by making available intracellular Ca*’ from these sites (P. K. T. Pang, unpublished data; Droogmans et al., 1977; Maigaard et al., 1986). This mode of action of ANF in vasorelaxation has recently been reported for the trout (Olson and Meisheri, 1989) and reportedly is similar to that of CGRP for the bullfrog (Kline et al., 1388). It is likely that
VASORELAXANT
EFFECT OF ANP IN FROG
this vascular effect of ANP and its mechanism of action are similar in the fish, amphibia, and mammal.
REFERENCES Atlas, S. A., Volpe, M., Sosa, R. E., Laragh, J. H., Camergo, M. J. F., and Maack, T. (1986). Effects of atrial natriuretic factor on blood pressure and the renin-angiotensin-aldosterone system. Fed. Proc. 45, 2115-2121. Bussien, J. P., Biollaz, J., Waeber, B., Nussberger, J., Turini, G. A., Brunner, H. R., BrunnerFerber, F., Gomez, H. J., and Otterbein, E. S. (1986). Dose-dependent effect of atrial natriuretic peptide on blood pressure, heart rate, and skin blood flow of normal volunteers. J. Cardiovas. Pharmacol. 8, 216-220. Chapeau, C., Gutkowska, J., Schiller, P. W., Mime, R. W., Thibault, G., Garcia, R., Genest, J., and Cantin, M. (1985). Localization of immunoreactive synthetic atrial natriuretic factor (ANF) in the heart of various animal species. J. Histochem. Cyrochem. 33, 541-550. Cho, K. W., Kim, S. H., Koh, G. Y., and Seul, K. H. (1988). Renal and hormonal responses to atrial natriuretic peptide and turtle atrial extract in the freshwater turtle, Amyda japonica. J. Exp. Zool. 247, 139-145. Cogan, M. G. (1986). Atria1 natriuretic factor. West. J. Med. 144, 591-595. de Bold, A. J. (1985). Atrial natriuretic factor: A hormone produced by the heart. Science 230, 767770. de Bold, A. J., and Salerno, T. A. (1983). Natriuretic activity of extracts obtained from hearts of different species and from various rat tissues. Canad. J. Physiol. Pharmacol. 61, 127-130. Droogmans, G., Raeymaekers, L., and Casteels, R. (1977). Electra- and pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery. J. Gen. Physiol. 70, 129-148. DuB, W. D., and Olson, K. R. (1986). Trout vascular and renal responses to atrial natriuretic factor and heart extracts. Amer. J. Physiol. 251, R639-R642. Faison, E. P., Siegl, P. K. S., Morgan, G., and Winquist, R. J. (1985). Regional vasorelaxant selec-
47
tivity of atrial natriuretic factor in isolated rabbit vessels. Life Sci. 37, 1073-1079. Garcia, R., Thibauh, G., Nutt, R. F., Cantin, M., and Genest, J. (1984). Comparative vasoactive effects of native and synthetic atrial natriuretic factor (ANF). Biochem. Biophys. Res. Commun. 119, 658-688. Kline, L., and Pang, P. (1988). Calcitonin gene-related peptide relaxed rat tail artery helical strips in vitro in an intracellular calcium-dependent manner. Eur. 1. Pharmacol. 150, 233-238. Kline, L. W-, Kaneko, T., Chiu, K. W., Harvey, S., and Pang, P. K. T. (1988). Calcitonin gene-related peptide in the bullfrog, Rana catesbeiana: Localization and vascular actions. Gen. Comp. Endocrinol. 72, 123-129. Lee, J., and Malvin, R. L. (1987). Natriuretic response to homologous heart extract in aglomerular toad&h. Amer. J. Physiol. 252, R1055-Rl058. Maigaard, S., Forman, S., Brogaard-Hansen, K. P., and Andersson, K. E. (1986). Inhibitory effects of nitrendipine of myometrial and vascular smooth muscle in human pregnant uterus and placenta. Acta Pharmacol. Toxicol. 59, l-10. Netchitailo, P., Feuilloley, M., Pelletier, G., Cantin, M., De Lean, A., Leboulenger, F., and Vaudry, H. (1986). Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium. Peptides 7, 573-579. Netchitailo, P., Feuilloley, L. M., Pelletier, G., De Lean, A., Ong, H., Cantin, M., Gutkowska, J., Leboulenger, F., and Vat&y, H. (1988). Localization and identification of immunoreactive atrial natriuretic factor (ANF) in the frog ventricle. Peptides 9, 1-6. Olson, K. R., and Meisheri, K. D. (1989). Effects of atrial natriuretic factor on isolated arteries and perfused organs of trout. Amer. J. Physiol. 256, RlO-R18. Reinecke, M., Betzler, D., Forssmann, W. G., Thomdyke, M., Askensten, U., and Falkmer, S. (1987). Electron microscopic, immunohistochemical, immunocytochemical and biological evidence for the occurrence of cardiac hormones (ANP/CDD) in chondrichthyes. Histochemistry 87, 531-538. Winquist, R. J. (1985). The relaxant effects of atrial natriuretic factor on smooth muscle. Life Sci. 37, 1081-1087. Winquist, R. J. (1986). Possible mechanisms underlying the vasorelaxant response to atrial natriuretic factor. Fed. Proc. 45, 2371-2375.
AND
COMPARATIVE
ENDOCRINOLOGY
The Vasorelaxant
78,42X’
(19%)
Effect of Atrial Natriuretic
Peptide in the Frog
K. W. CHIU, Y. C. LEE, AND P. K. T. PANG* Department of Biology, The Chinese University of Hong Kong, Hong Kong, and *Department of Physiology, University of Alberta, Edmonton, Alberta, Canada Accepted June 12, 1989 The vasorelaxant effect of atrial natriuretic peptide (ANP) in two species of frogs, Rana catesbeiana and R. tigrina, were studied in vitro. Isolated arterial strips were prepared from the aortic loop, dorsal aorta, iliac, and femoral arteries in the frog. These were stimulated to contract with KCl, norepinephrine, or arginine vasotocin. When maximal contraction was attained, ANP was added to assess if it could relax the strips. Data show that ANP is vasorelaxant in these preparations. The sensitivity of these tissues depends on the contractile agents, e.g., KCl-stimulated preparations from R. catesbeiana did not respond to ANP. Data also suggest that ANP action elicited in the frog vascular tissue is due to an inhibitory effect on the mobilization of the intracellular calcium store and/or calcium influx from extracellular space to initiate contraction. 8 1990Academic press, Inc.
In mammals, atria1 natriuretic factor (ANF) has a pronounced effect on the cardiovascular activity (de Bold, 1985; Atlas et al., 1986; Bussian et al., 1986; Cogan, 1986; Winquist, 1986). It relaxes preconstricted smooth muscle and decreases heart rate and tension. Immunocytochemical and radioimmunological studies indicate presence of ANF in almost all major classes of vertebrates (de Bold and Salerno, 1983; Chapeau et al., 1985; Netchitailo ef al., 1986, 1988; Reinecke et al., 1987), and in the fish and turtle, homologous heart extracts produce renal and pressure responses (Duff and Olson, 1986; Lee and Malvin, 1987; Cho et al., 1988). Recent reports on isolated arteries and perfused organs in trouts (Olson and Meisheri, 1989) and isolated atria from amphibians and snakes (K. W. Chiu et al., unpublished data) indicate that ANF also has an effect on the cardiovascular system in the lower vertebrates. We have been studying the vasorelaxant property of the calcitonin gene-related peptide in the vertebrates (Kline and Pang, 1988; Kline et al., 1988) and have since extended the study to ANP. In this report, the vas-
cular effects of ANP in two species of frogs are presented. MATERIALS
Bullfrogs (Rana catesbeiana) were obtained from a commercial supplier in the United States and tiger frogs (R. t&ha) were purchased from local markets in Hong Kong. These frogs were pithed and the viscera cut open. The aortic loop, dorsal aorta, iliac, and femoral arteries were removed en bloc and were placed in frog Ringer’s solution (IRS) (80 n& NaCl, 2.5 m&f KCl, 1.8 m&f CaCl,, 24 mM NaHCO,, 0.12 m&f NaH,PO,, and 1.1 n&f glucose) which was oxygenated with 95% 0,:5% CO2 maintained at room temperature. The various portions of the blood vessels were separated, cleared of connective tissues, cut helically, and the strips were secured in an organ bath containing 10 ml FRS. A resting tension of 0.7 g was set. Subsequent changes in the force developed by the helical strips were measured by a force displacement transducer and recorded on a polygraph. The procedure to examine the vasodilatory effect of ANP on the helical strips followed that described by Kline et al. (1988). Isolated strips were equilibrated for 45 min prior to being tested. Each strip was exposed to 60 m&f KC1 and the amount of tension was monitored. This was followed by three changes of bathing FRS and a lO-min rest before repeating the test. Each strip was tested three times. A tension of 0.5 g or more was required. The strip was then equilibrated for 30 min. 42
001~6480/90 $1.50 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.
AND METHODS
VASORBLAXANT
EFFECT OF ANP IN FROG
The substances used to stimulate contraction were 60 mM KCI, 3 x 10m6 M norepinephrine (NE), and 30 rig/ml arginine vasotocin (AVT). The stimulating drug to be used was added to the bath. Once maximum tension was obtained, one of the four doses (10, 30, 100, and 300 t&ml in R. catesbeinana, and 1, 3, 10, and 30 rig/ml in R. ligrina) of ANP [atrial natriuretic peptide, rat (28 amino acids) PATR 20, Bachem] was added to the bath. Both the maximum tension and the amount of relaxation, if any, were recorded. The organ bath was rinsed three times with FRS and the tissue was equilibrated for 30 min if either KC1 or NE was used, or 60 mm if AVT was used. This process was repeated. At the end of the experiment, 60 m&f KC1 was added to the chamber and a final tension to ensure that no tachyphylaxis occurred. The role of intracellular Ca2+ and the vasorelaxant effect of ANP was also tested in the bullfrog. After being tested for use, the strips were placed in FRS containing no Ca” and 2 rnkf EGTA (Sigma). After 2 min, NE was added to the bath and the tension was recorded. The bath was rinsed three times with normal FRS. The strips were then equilibrated in normal FRS for 45 min. The procedure was repeated. To test the effects of ANP, it was added to the bath containing no Ca2+ and 2 mM EGTA prior to the addition of NE. The procedure was repeated for each dose of ANP after 45 min of equilibrium in FRS. To verify the setup and protocol, the vasorelaxant effect of ANP on the dorsal aorta strips from rats was
similarly assessed. The dorsal aorta (2 to 3 cm) was removed from anesthetized rats. They were transferred to Krebs-Henseleit solution (KHS) (115 mkf NaCl, 5 mil4 KCI, 2.1 m&f CaCl,, 1.2 m&I MgSO,, 1.2 mM NaH,PO,, 25 mM NaHCO,, and 11 mM glucose), oxygenated with 95% 0,:5% C02, cleared of connective tissue, cut helically, and mounted in an organ bath which was set at 37”. All data were presented in mean 2 SEM. Both sexes of bullfrogs were used, but only females of tiger frogs were chosen as they are much larger than males and yield larger blood vessels.
RESULTS
R. catesbeiana. Results of the vascular studies using the bullfrog aortic loop, aorta, iliac, and femoral arteries indicated a varying degree of sensitivity to the vasorelaxant effect of ANP as well as the contractile agents used. ANP had no significant relaxing effect on KCl-stimulated contraction in all preparations. When AVT was used to stimulate contraction, it had no significant relaxing effect on the femoral artery strips but had a more pronounced effect on the aortic loop and iliac artery (Fig. 1). A de-
Cont.
(rig/ml)
0
20
1 n 0-0
-m
aortic
43
loop
iliac
FIG. 1. The relaxant effect of ANP on AVT-stimulated represent SE.
contraction in R. catesbeiuna. Vertical bars
44
CHIU,
LEE,
crease of about 60% at 30 rig/ml in the latter preparation was seen. Large individual variations were noted. When NE was used to stimulate the contraction prior to ANP (Fig. 2), the vasorelaxant effect was apparent at the initial dose of ANP 10 rig/ml, and except in the femoral preparation, all other preparations showed a relaxation of about 40%. The femoral strip showed only a relaxation of about 15%. The maximum of 60% was achieved with all preparations, except in the femoral artery. R. tigrina. The pattern of vasorelaxant responses to ANP showed variations and differences as compared with the bullfrog. With KC1 preconstriction, all preparations of dorsal aorta, iliac, and femoral arteries showed dose-dependent relaxation (Fig. 3), noting of course that the dose of ANP used had been reduced. The femoral preparation was the most sensitive of all preparations, with the aortic loop not sensitive to KC1 and the other two contractile agents NE and AVT, and therefore the effect of ANP could not be tested and accordingly is not
cont.
4
dp
I
10 I
AND
PANG
shown in this and subsequent figures. With AVT, all preparations responded in a similar manner among different preparations and with a similar range of relaxation (Fig. 4). When the preparations were preconstricted with NE, they all showed, as expected, dose-related relaxation (Fig. 5). They were all far more sensitive to ANP than they were preconstricted with AVT or KCl. Further, these dose-dependent relaxation curves are present as parallel sets of lines, indicating therefore that the iliac was the most sensitive of all three preparations. In view of the discrepancy in the vasorelaxant response of the isolated strips to ANP under KC1 preconstriction between the two species of frogs, a repeat of this set of the experiment was performed using the tiger frog with lower ANP doses (0.1,0.3,1, and 3 r&ml). The strips yielded similar relaxation responses to ANP. The effects of ANP on intracellular calcium-dependent contraction in bullfrogs are shown in Fig. 6. The percentage inhibition of the developed tension versus the doses
(ngfml)
30
100 I
300
60
m-m
aortic
~00~
l -
n--o
dorsal
aorta
A-A
FIG. 2. The relaxant effect of ANP on NE-stimulated
l
iliac femoral
contraction in R. catesbeiana.
VASORELAXANT
Cont.
1
EFFECT
OF ANP
IN
FROG Cont.
(rig/ml)
3
45
10
30
1
(rig/ml) 3
10
30
0
10
20 E .2 + B 2 8 *
30 30 40
50
0
Femoral
*
dorsal
aorta I
60 FIG. 3. The relaxant effect of ANP on KClstimulated contraction in R. figrina.
of ANP indicated that the sensitivity of the iliac, dorsal aorta, femoral, and aortic loop to the peptide was in this order. A dosedependent response to ANP was readily apCont.
(rig/ml)
70
80
90
*
dorsal
aorta
100
Ol
FIG. 5. The relaxant effect of ANP stimulated contraction in R. tigrina.
10
on NE-
parent with the dorsal aorta and aortic loop. A maximal inhibition of about 60% was observed in the iliac, femoral, and dorsal aorta preparations.
20
DISCUSSION q 50
60
1
l
iliac
*
dorsal
I aorta
FIG. 4. The relaxant effect of ANP on AVTstimulated contraction in R. tigrina.
Results from the present study demonstrated a vasorelaxant effect of ANP in two species of frogs, R. catesbeiana and R. tigrina. This finding is in accord with the relaxant effect of ANP in isolated arteries and perfused organs in the trout, Salmo gairdneri, under NE stimulation (Olson and
46
CHIU,
LEE,
AND
Cont.
PANG
(rig/ml)
Oh
60
80
n
aortic loop
0
iliac
0
dorsal
A
femoral
FIG. 6. Intracellular calcium-dependent containing no calcium and 2 mM EGTA.
aorta
contraction. NE was used to stimulate helical
Meisheri, 1989). In the chondrichthyes, Squalus acanthias, Scyliorhinus canicula, Raja clavata, and Chimaera monstrosa, it
has been reported that purified extracts prepared from the hearts produced dosedependent relaxation of preconstricted mammalian (rabbit) aorta (Reinecke et al., 1987). The occurrence of ANP, and possibly its vasorelaxant property, must have evolved at an early stage of the vertebrates. The present data also show differences in the vasorelaxant effect between the two species of frogs. For example, KClstimulated dorsal aorta, iliac, and femoral artery strips of R. tigrina responded to ANP with relaxation, but no response was observed with similar preparations from R. catesbeiana. Regional and species differences in the vasorelaxant response of the vascular tissue to ANP in mammals have, however, been documented (Winquist, 1985, 1986). Garcia et al. (1984) reported that rat atrial extract relaxes rabbit aortic
stripsin FRS
and renal but not mesenteric helical strips stimulated by NE and ANG II. Using isolated rabbit vessels, Faison et al. (1985) noted that large central ones respond best to ANF, more distal ones such as iliac and femoral arteries only partially respond, and finally the peripheral ones are unresponsive at all. On the mechanism of this vasorelaxant effect, data from the present intracellular calcium experiment in the bulIfrog strongly indicate that vasorelaxation is due to an interference on the mobilization of intracellular calcium from storage sites, as AVT and NE stimulate muscle contraction by making available intracellular Ca*’ from these sites (P. K. T. Pang, unpublished data; Droogmans et al., 1977; Maigaard et al., 1986). This mode of action of ANF in vasorelaxation has recently been reported for the trout (Olson and Meisheri, 1989) and reportedly is similar to that of CGRP for the bullfrog (Kline et al., 1388). It is likely that
VASORELAXANT
EFFECT OF ANP IN FROG
this vascular effect of ANP and its mechanism of action are similar in the fish, amphibia, and mammal.
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