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Adrenotensin: An ADM gene product with the opposite effects of ADM
Life Scicncer, Vol. 57, No. 8, pp. PL 87-90,199S Copyright 0 199s E?kvier Seiencc Ltd Printed in the USA. All rights rrr~rved om4-3205/95 $950 t .oo
Pergamou
0024-3245(95)02012-8 PEt4RU4COLOGY L.ElTERs Accelerated Communication ADRENOTENSIN:
AN ADM GENE PRODUCT
WITH THE OPPOSITE
EFFECTS
OF ADM
‘Bulent Gumusel, 2Jaw-Kang Chang, ‘Albert Hyman, and lp3HowardLippton ‘Department of Surgery, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana, 70112, USA. 2Phoenix Laboratories, USA, 3Department of Pharmacology, Louisiana State University School of Medicine, New Orleans, Louisiana, 70112, USA. (SubmittedMarch 20, 1995; accepted April 28, 1995; received in final form May 30, 1995)
Abstract:The purpose of the present study was to investigate the effects of putative products of the ADM gene, other than ADM including, prodepin, proADUls.st and proADM1s3.ra on cat pulmonary arterial (PA) rings with or without precontraction with U46619. Addition of proADMrs3_rss(3x10~‘“-10%l) increased tension in a concentration-dependent manner in cat PA rings without precontraction. When vessels were precontracted with U46619, ADM(3~10~‘~-10%4) produced a concentration-dependent vasorelaxant response, whereas proADMr33_185 produced a weak concentration-dependent contractile response. Prodepin and proADM5.B up to 10dM had no activity on PA rings. Since proADM 153 _185, similar to ADM, would be expected to be released in free form following endopeptidase-induced cleavage, the present data suggest proADM undergoes proteolytic processing to release peptides with divergent vascular effects.Thus, the present data also suggest that proADMrss_r85may represent a novel product of the ADM gene and term this putative new substance “adrenotensin”.
Key Words: adrenomedullingene, adrenotensin,vasoconstriction
Introduction Adrenomedullin (ADM) is a newly-discovered peptide in normal human plasma (1). mRNA blot analysis indicates that ADM is expressed in normal human, rat and porcine tissues of the lung, heart, kidney, adrenal gland, duodenum, and spleen (2,3,4). ADM decreases systemic arterial pressure with little or no effect on cardiac performance (5). The discovery and initial isolation of ADM was based, in large measure, on proteolytic processing of proADM (3). Specifically, endopeptidase cleavage of proADM at sites of basic aminoacids including proADMB.93and proADM14s_r49 would be expected to form free ADM. Such endopeptidase cleavage sites also flank the following three other distinct portions of proADM: proADM=_,r , proADM5.92, and proADMra.ra. The presence of one of these truncated sequences of proADM, proADM2wr termed prodepin (6) , has recently been reported to be present in human plasma (7). However, the effects of these predicted free proADM fragments on isolated blood vessels has not been determined. Address Correspondence: Howard Lippton, M.D, Department of Surgery, Tulane University School ofMedicine, New Orleans, LA,701 12, USA, Fax:(S04) 584 1838.
PL88
Divergent Effects of ADM
Vol. 57, No. 8, 1995
The present study was undertaken to investigate the effects of human ADM, prodepin, proADI&_92 and proADMrs3.rason isolated cat pulmonary arterial (PA) rings with or without
precontraction with U46619, a thromboxane AZ mimic(S).
Methods
Cats of both sexes, weighing 2.5-3.2 kg were sedated with ketamine (lo-15 mg/kg i.m) and anesthetized with pentobarbital sodium (30-35 mg/kg i.p). The cat lungs were quickly removed and immersed in cold (4’C) Krebs-Henseleit (K-H) solution (composition mM:NaCl 118, KC1 4.7, Cat& 2.5, KI-I*PO.+ 1.2, NaHCOs 25, MgSO, 1.2, and dextrose 10). PA rings were isolated and excess fat and connective tissue were removed. Vessels were cut into rings of about 2.5-3 mm in length and were mounted in organ baths containing 5 ml K-H solution. The tissue bath solution was maintained at 37’C and gassed with a 95%02 - 5%CO2mixture. Two stainless steel hooks were inserted into the lumen of PA, one was fixed while the other was connected to a transducer. The pulmonary blood vessels were equilibrated for 60 min with three changes of K-H solution and an optimal tension of 1 gm. applied. Contractions were measured isometrically with a force displacement transducer (FT03, Grass, USA) and were recorded on a Grass model 7 polygraph. The contractile ability of each ring was then examined by exposure to 60 mM KCl, then washed and allowed to dilate to baseline tension. Only when two reproducible contractions could be elicited was the individual ring used in further studiesThe integrity of the endothelium was determined by obtaining a full vasorelaxant response to acetylcholine. The EC50 for U46619 on isolated cat PA rings is 15 nM. All peptides were added directly to the organ bath and cumulative concentration-response curves contracted. The concentrations of all drugs were reported as the final molar concentration (M) in organ chambers. Results are expressed as the mean+S.E.M. N equals the number of rings from different cats (i.e. one ring was taken from each cat). Results
Data showing the effects of ADM, prodepin, proADMs_92and proADM15J.la on isolated cat PA rings without precontraction are illustrated in figure 1. Addition of proADMrn.rgs (3~10~‘~-10”M) increased tension in isolated cat PA rings in a concentration-dependent manner. Under similar conditions addition of ADM, prodepin and proADMs.92 up to 1OdMhad no effect (see Fig. 1). In order to determine if pre-existing tension of isolated cat PA rings influenced the vascular response to the four peptides studied, additional experiments were performed using isolated cat PA rings precontracted with U46619 and data from these experiments are illustrated in figure 2. When cat PA rings were precontracted with 15 &I U46619, ADM (3~10~lo-lo6 M) produced a concentration-dependent vasorelaxant responseIn contrast, proADM r3j.ra5under similar conditions produced a concentration-dependent contractile response that was somewhat less than the contractile response observed with this peptide on cat PA rings without precontraction (see fig.1 and 2). Prodepin and proAIX&92 (up to 10” M) had no effect on cat PA rings precontracted with U46619.
PL-89
Divergent Effects of ADM
Vol. 57, No. 8, 1995
loo-
prodepin ---1--~~ proADM 45-92 --..+-ADM -!I-
F
.s E
60 -
E t s ._
i
t
s g 8
I-”
----•---
proADM 153-165
n=6
60-
”
,’+
___---+
$/;;
40-
8’ ,**’ a*’
6 is
o-
= -9.5
-
-
-e--.-s___--=
-9
-6.5
-6
=
=
-7.5
Concentration
-7
-
-6.5
-
-6
’
(log M)
Fig.1 Effects of prodepin, proADM.,S_92, AbM and P~oADM~~~_~~~ on cat pulmonary arterial rings with endothelium. n= number of animals. Data expressed as mean+ S.E.M. 20-
-20 -
.._. ““-0. .....*... “3 ......_..
-40 -
’w --. . . .. .. 0 _______.___ 0 -60 -
*
---..---60 -
prodepin proADM 45-92
-----@-- ADM ----•---
proADM
153-165
n=6 -100 -9.5
-9.0
-6.5
-6.0
-7.5
Concentration
-7.0
-6.5
H -6.0
(log M)
Fig.2 on cat pulmonary arterial rings with _185 Effects of prodepin, proAD&s_92, ADM and proADM153 endothelium precontractedwith 15 nM U46619. n= number of animals. Data expressed as mean+ S.E.M.
PL-90
DivergentEffects of ADM
Vol. 57, No. 8, 1995
Discussion
Results of the present study are the first reported comparison of the biological effects of truncated sequences of proADM predicted to exist in free form. ProADM i53_r85possesses marked contractile activity on cat PA and this property albeit quantitatively reduced is retained when PA vessels are precontracted. In contrast, ADM produced the opposite response by exclusively relaxing PA. The present data are consistent with earlier work in the intact cat demonstrating that ADM dilates the pulmonary vascular bed precontracted by U46619 (7). Taken together both studies suggest ADM possesses similar vasorelaxant activity on conductance and resistance segments of the pulmonary vascular bed of the same species. The divergent properties of proADMi~~.i~~and ADM on isolated cat PA rings are novel in that both peptides originate from the same proADM sequence. Furthermore, the contractile properties of proADMis3.rss are markedly enhanced in vessels without precontraction, however, the mechanism responsible for the “tone-dependent” response to proADMir3_i8sis uncertain. Since the pulmonary vascular bed functions at a low or vasodilated may play an important role in regulating the state, the present data suggest that proADM 153_18s pulmonary vascular bed under resting conditions. Although prodepin has been shown to dilate the rat systemic vascular bed (6) , this peptide along with proADM45_92appears to have little direct effect on isolated cat PA conductance vessels. Prodepin, unlike ADM, appears to act in a speciesspesific manner (6). Since proADMls3_iss is an ADM gene product possessing contractile properties on blood vessels, the authors propose the term “adrenotensin” to refer to this novel vasoconstrictor peptide.
References
K. KITAMURA, K. KANGAWA, M. KAWAMOTO, Y. ICHIKI, S. NAKAMURE, H. MATSUO, AND T.ETO. Biochem and Biophys Res. Comm 192553-560 (1993). 2. Y. ICHIKI, K. KITAMURA, K. KANGAWA, M. KAWAMOTO, H. MATSUO, AND T. ETO. FEBS 338 6-10 (1994). 3. J. SAKATA, T.SHIMOKUBO, K. KITAMURA, S. NAKAMURq K. KANGAWA, HMATSUO, AND T.ETO. Biochem and Biophys Res. Comm 195 921-927 (1993). 4. K. KITAMURA, K. KANGAWA, M. KOJIMA, Y. ICHIKI, H. MATSUO AND T. ETO. FEBS 338 306-310 (1994). 5. M. PERRET, H. BROUSSARD, T. LEGROS, A. BURNS, J. CHANG,W. SUMMER A.HYMAN AND H. LIPPTON. Life Sciences 53377-379 (1993). 6. H. LIPPTON, Y. GAO, B. LIN, J. HEATON, J. FERRARA, M. DEVITO, T. GRANGER, J.PIGOTT, J.CHANG AND A. HYMAN. Life Sciences 54409-4 12 (1994). 7. M. JOUGASAKI, C-M. WEI. AND J.C. BURNETT. Clinical Research 42 410A (1994). 8. H. LIPPTON, J. CHANG, Q. HAO, W. SUMMER AND A. HYMAN. J. Appl. Physiol. 76 2154-2156 (1994). 1.
Pergamou
0024-3245(95)02012-8 PEt4RU4COLOGY L.ElTERs Accelerated Communication ADRENOTENSIN:
AN ADM GENE PRODUCT
WITH THE OPPOSITE
EFFECTS
OF ADM
‘Bulent Gumusel, 2Jaw-Kang Chang, ‘Albert Hyman, and lp3HowardLippton ‘Department of Surgery, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana, 70112, USA. 2Phoenix Laboratories, USA, 3Department of Pharmacology, Louisiana State University School of Medicine, New Orleans, Louisiana, 70112, USA. (SubmittedMarch 20, 1995; accepted April 28, 1995; received in final form May 30, 1995)
Abstract:The purpose of the present study was to investigate the effects of putative products of the ADM gene, other than ADM including, prodepin, proADUls.st and proADM1s3.ra on cat pulmonary arterial (PA) rings with or without precontraction with U46619. Addition of proADMrs3_rss(3x10~‘“-10%l) increased tension in a concentration-dependent manner in cat PA rings without precontraction. When vessels were precontracted with U46619, ADM(3~10~‘~-10%4) produced a concentration-dependent vasorelaxant response, whereas proADMr33_185 produced a weak concentration-dependent contractile response. Prodepin and proADM5.B up to 10dM had no activity on PA rings. Since proADM 153 _185, similar to ADM, would be expected to be released in free form following endopeptidase-induced cleavage, the present data suggest proADM undergoes proteolytic processing to release peptides with divergent vascular effects.Thus, the present data also suggest that proADMrss_r85may represent a novel product of the ADM gene and term this putative new substance “adrenotensin”.
Key Words: adrenomedullingene, adrenotensin,vasoconstriction
Introduction Adrenomedullin (ADM) is a newly-discovered peptide in normal human plasma (1). mRNA blot analysis indicates that ADM is expressed in normal human, rat and porcine tissues of the lung, heart, kidney, adrenal gland, duodenum, and spleen (2,3,4). ADM decreases systemic arterial pressure with little or no effect on cardiac performance (5). The discovery and initial isolation of ADM was based, in large measure, on proteolytic processing of proADM (3). Specifically, endopeptidase cleavage of proADM at sites of basic aminoacids including proADMB.93and proADM14s_r49 would be expected to form free ADM. Such endopeptidase cleavage sites also flank the following three other distinct portions of proADM: proADM=_,r , proADM5.92, and proADMra.ra. The presence of one of these truncated sequences of proADM, proADM2wr termed prodepin (6) , has recently been reported to be present in human plasma (7). However, the effects of these predicted free proADM fragments on isolated blood vessels has not been determined. Address Correspondence: Howard Lippton, M.D, Department of Surgery, Tulane University School ofMedicine, New Orleans, LA,701 12, USA, Fax:(S04) 584 1838.
PL88
Divergent Effects of ADM
Vol. 57, No. 8, 1995
The present study was undertaken to investigate the effects of human ADM, prodepin, proADI&_92 and proADMrs3.rason isolated cat pulmonary arterial (PA) rings with or without
precontraction with U46619, a thromboxane AZ mimic(S).
Methods
Cats of both sexes, weighing 2.5-3.2 kg were sedated with ketamine (lo-15 mg/kg i.m) and anesthetized with pentobarbital sodium (30-35 mg/kg i.p). The cat lungs were quickly removed and immersed in cold (4’C) Krebs-Henseleit (K-H) solution (composition mM:NaCl 118, KC1 4.7, Cat& 2.5, KI-I*PO.+ 1.2, NaHCOs 25, MgSO, 1.2, and dextrose 10). PA rings were isolated and excess fat and connective tissue were removed. Vessels were cut into rings of about 2.5-3 mm in length and were mounted in organ baths containing 5 ml K-H solution. The tissue bath solution was maintained at 37’C and gassed with a 95%02 - 5%CO2mixture. Two stainless steel hooks were inserted into the lumen of PA, one was fixed while the other was connected to a transducer. The pulmonary blood vessels were equilibrated for 60 min with three changes of K-H solution and an optimal tension of 1 gm. applied. Contractions were measured isometrically with a force displacement transducer (FT03, Grass, USA) and were recorded on a Grass model 7 polygraph. The contractile ability of each ring was then examined by exposure to 60 mM KCl, then washed and allowed to dilate to baseline tension. Only when two reproducible contractions could be elicited was the individual ring used in further studiesThe integrity of the endothelium was determined by obtaining a full vasorelaxant response to acetylcholine. The EC50 for U46619 on isolated cat PA rings is 15 nM. All peptides were added directly to the organ bath and cumulative concentration-response curves contracted. The concentrations of all drugs were reported as the final molar concentration (M) in organ chambers. Results are expressed as the mean+S.E.M. N equals the number of rings from different cats (i.e. one ring was taken from each cat). Results
Data showing the effects of ADM, prodepin, proADMs_92and proADM15J.la on isolated cat PA rings without precontraction are illustrated in figure 1. Addition of proADMrn.rgs (3~10~‘~-10”M) increased tension in isolated cat PA rings in a concentration-dependent manner. Under similar conditions addition of ADM, prodepin and proADMs.92 up to 1OdMhad no effect (see Fig. 1). In order to determine if pre-existing tension of isolated cat PA rings influenced the vascular response to the four peptides studied, additional experiments were performed using isolated cat PA rings precontracted with U46619 and data from these experiments are illustrated in figure 2. When cat PA rings were precontracted with 15 &I U46619, ADM (3~10~lo-lo6 M) produced a concentration-dependent vasorelaxant responseIn contrast, proADM r3j.ra5under similar conditions produced a concentration-dependent contractile response that was somewhat less than the contractile response observed with this peptide on cat PA rings without precontraction (see fig.1 and 2). Prodepin and proAIX&92 (up to 10” M) had no effect on cat PA rings precontracted with U46619.
PL-89
Divergent Effects of ADM
Vol. 57, No. 8, 1995
loo-
prodepin ---1--~~ proADM 45-92 --..+-ADM -!I-
F
.s E
60 -
E t s ._
i
t
s g 8
I-”
----•---
proADM 153-165
n=6
60-
”
,’+
___---+
$/;;
40-
8’ ,**’ a*’
6 is
o-
= -9.5
-
-
-e--.-s___--=
-9
-6.5
-6
=
=
-7.5
Concentration
-7
-
-6.5
-
-6
’
(log M)
Fig.1 Effects of prodepin, proADM.,S_92, AbM and P~oADM~~~_~~~ on cat pulmonary arterial rings with endothelium. n= number of animals. Data expressed as mean+ S.E.M. 20-
-20 -
.._. ““-0. .....*... “3 ......_..
-40 -
’w --. . . .. .. 0 _______.___ 0 -60 -
*
---..---60 -
prodepin proADM 45-92
-----@-- ADM ----•---
proADM
153-165
n=6 -100 -9.5
-9.0
-6.5
-6.0
-7.5
Concentration
-7.0
-6.5
H -6.0
(log M)
Fig.2 on cat pulmonary arterial rings with _185 Effects of prodepin, proAD&s_92, ADM and proADM153 endothelium precontractedwith 15 nM U46619. n= number of animals. Data expressed as mean+ S.E.M.
PL-90
DivergentEffects of ADM
Vol. 57, No. 8, 1995
Discussion
Results of the present study are the first reported comparison of the biological effects of truncated sequences of proADM predicted to exist in free form. ProADM i53_r85possesses marked contractile activity on cat PA and this property albeit quantitatively reduced is retained when PA vessels are precontracted. In contrast, ADM produced the opposite response by exclusively relaxing PA. The present data are consistent with earlier work in the intact cat demonstrating that ADM dilates the pulmonary vascular bed precontracted by U46619 (7). Taken together both studies suggest ADM possesses similar vasorelaxant activity on conductance and resistance segments of the pulmonary vascular bed of the same species. The divergent properties of proADMi~~.i~~and ADM on isolated cat PA rings are novel in that both peptides originate from the same proADM sequence. Furthermore, the contractile properties of proADMis3.rss are markedly enhanced in vessels without precontraction, however, the mechanism responsible for the “tone-dependent” response to proADMir3_i8sis uncertain. Since the pulmonary vascular bed functions at a low or vasodilated may play an important role in regulating the state, the present data suggest that proADM 153_18s pulmonary vascular bed under resting conditions. Although prodepin has been shown to dilate the rat systemic vascular bed (6) , this peptide along with proADM45_92appears to have little direct effect on isolated cat PA conductance vessels. Prodepin, unlike ADM, appears to act in a speciesspesific manner (6). Since proADMls3_iss is an ADM gene product possessing contractile properties on blood vessels, the authors propose the term “adrenotensin” to refer to this novel vasoconstrictor peptide.
References
K. KITAMURA, K. KANGAWA, M. KAWAMOTO, Y. ICHIKI, S. NAKAMURE, H. MATSUO, AND T.ETO. Biochem and Biophys Res. Comm 192553-560 (1993). 2. Y. ICHIKI, K. KITAMURA, K. KANGAWA, M. KAWAMOTO, H. MATSUO, AND T. ETO. FEBS 338 6-10 (1994). 3. J. SAKATA, T.SHIMOKUBO, K. KITAMURA, S. NAKAMURq K. KANGAWA, HMATSUO, AND T.ETO. Biochem and Biophys Res. Comm 195 921-927 (1993). 4. K. KITAMURA, K. KANGAWA, M. KOJIMA, Y. ICHIKI, H. MATSUO AND T. ETO. FEBS 338 306-310 (1994). 5. M. PERRET, H. BROUSSARD, T. LEGROS, A. BURNS, J. CHANG,W. SUMMER A.HYMAN AND H. LIPPTON. Life Sciences 53377-379 (1993). 6. H. LIPPTON, Y. GAO, B. LIN, J. HEATON, J. FERRARA, M. DEVITO, T. GRANGER, J.PIGOTT, J.CHANG AND A. HYMAN. Life Sciences 54409-4 12 (1994). 7. M. JOUGASAKI, C-M. WEI. AND J.C. BURNETT. Clinical Research 42 410A (1994). 8. H. LIPPTON, J. CHANG, Q. HAO, W. SUMMER AND A. HYMAN. J. Appl. Physiol. 76 2154-2156 (1994). 1.