Gen Pharmac Vol 23, No 6, pp 1171-1175, 1992 Pnnted m Great Bntmn All rights reserved
0306-3623/92 $5 00 + 0 00 Copyright © 1992Pergamon Press Ltd
VASOCONSTRICTIVE EFFECTS OF ANGIOTENSIN I A N D II IN CAT FEMORAL ARTERIES. ROLE OF ENDOTHELIUM M F~.RER, ~A ENCABO,t J MARIN2 and G BALFAC-~Nt* l Departamento de F1slologia and 2Departamento de Farmacologia y Terapeutaea, Facultad de Medlcma, Umversldad Aut6noma de Madrid, C/Arzoblspo Morcatlo, 4, 28029-Madrid, Spmn (Recewed 9 March 1992) Al~traet--1 AI and AII reduced concentration-dependent eontractaons ]n cat femoral artery segments, the potency of All being greater than that of AI 2 The antagomst of All receptors, saralasm (1 and I0 nM), mlublted the AI and AII responses 3 Indomethacm (I0 #M) and endothehum removal mereased the responses to AII, whereas those induced by AI were barely affected 4 The response reduced by AI was reduced by captopnl (200/aM) 5 These data suggest (1) the contraetaons reduced by AII are medmted by AII receptors and endothehal products derived from ¢yclooxygenase, (2) the response to AI is, m part, produced by its conversion into AII
INTRODUCTION Anglotensm II (AII) induces potent contractde responses m &fferent vascular preparataons by &reet stunulataon of specafic AII receptors present m the smooth muscle (Ferrano, 1990), and m&rectly by actavataon of presynaptac AII receptors of sympathcue nerve terrmnals facahtatang the noradrenahne release (Chne, 1984, Zlogas et al, 1985, Yasuda et al, 1987, Moldenngs et al, 1988) Exadence has been shown that the vessels possess the ang~otensm convertang enzyme (ACE), which transforms AI into AII (Whalley et al, 1983, Whalley and Wahl, 1988, Ferrano, 1990) Tlus enzyme is located m the vascular wall (Andre et al, 1990, Engl~me et al, 1990, Mfiller-Schwelmtzer and Olea-Baza, 1990), but mainly m the ¢ndothehum (Ryan et ai, 1976, Paulson et al, 1988) On the other hand, the endothehum produces a negatave modulataon of the vasocontractale responses ehcated by AI and AII m some vessels (Bullock et al, 1986, Gruetter et al, 1988; Engl~me et al, 1990, Yen et al, 1990) Tlus is due to anglotensms mducmg the release of relaxant substances, such as endothehum-denved relaxmg factor (EDRF, ldentafied as mtnc oxade, NO) (Gruetter et al , 1988; Engl~me et al , 1990, Yen et al , 1990) or prostacychn (PGI2) (Gruetter et al, 1988, Mmaml and Toda, 1988, Mfiller-Schwelmtzer and Olea-Baza, 1990, Yen et al, 1990) In other vessels, the endothehum does not affect the responses ehclted by these agents (Saye et al, 1984; Gruetter et al, 1988) or produces a posmve modulataon, since its removal reduces the contractaons caused by ang~otensms, which ~s m part due to the release of endothehal eontraetang factors, such as thromboxane A2 (Manabe et al, 1989, Yen et al, 1990)
*To whom all correspondence should be addressed
In wew of these data, the present study was undertaken m cat femoral arteries to analyze the ablhty of AI and AII to reduce contractde responses, the receptors revolved, the vascular presence of ACE, and the endothehal partaclpataon on the responses caused by both angtotensms MATERIALSAND METHODS Vascular reactwtty Cats of either sex (1 5-3 kg) were anaesthetazed vath sodmm pentobarbltal (35 mg/kg, 1p ) and killed by bleedmg Femoral arteries were isolated and placed m a Petn &sh contalmng Krebs-Henselelt solutaon (KHS) at 4°C and ¢hwded into nngs of 4 mm m length Each nng was set up for Isometric tension recording m an organ bath according to the method of Nlelscn and Owman (1971) The organ bath contmned 5 ml of KHS at 37°C contanuously bubbled wath a 95% 02-5% CO 2 rmxture, which gave a pH of 7 4 Two stainless steel pros were passed through the lumen of the nng One pm was fixed to the organ bath wall while the other was connected to a strain gauge for ~sometnc tension reeorchng The latter pm was m parallel posmon with the former and It was movable, pcrtmttang the application of resting tension m a perpendtcular plane to the long axas of the vascular cyhnder The ~sometnc contractaon was recorded through a force-displacement transducer (Grass FTO3C), connected to a Grass model 7D polygraph A restang tension of I g (optamal resting tone, i e the tone wluch produces maxnnal response to 75raM K +) was apphed to cyhndneal segments Tlus tension was readjusted every 15mm dunng 60-90rmn eqmhbrataon period (for base hne stabdxzataon), before addmon of drugs The segments were mmaUy exposed to 75 mM K + to check thelr functaonal mtegmy Afterwards, the bath medmm was changed several tames untal the restang tone recovery Then, a concentrataon-response curve to AI or AII was earned out Endothehum influence on the responses to angwtensms was analyzed by its removal (by gently rubbing) from arterial nngs The effectave removal was tested by the inability of acetylchohne (0 1 ttM) to produce vasoddataon m arteries precontracted vath noradrenahne
1171
1172
M FERRERet al
(1 #M) To examine the effect of saralasm (1 and 10nM), mdomethacm (10#M) and captopnl (200#M) on the actmns of angmtensms, they were added to the bath 10, 20 and 30 mm m advance Solutions, drugs and stattsttcal methods The composmon of KHS was as follows (mM) NaCI 115, CaC12 2 5, KC1 46, KH2PO 4 1 2, MgSO4 7H2 O 1 2, NaHCO 3 25, glucose 11 1, Na2 EDTA 0 03 (to prevent the ox~datmn of unstable substances) Stock solutions of AI, AII and saralasm (all at 1 raM) and captopnl (0 1 M) were made m bid]stilled water, while that of mdomethacm was achieved m a solutmn with 1 5 mM of sodmm carbonate and kept at -20°C Ahquots of these solutmns were diluted just before use with KHS to obtain the desired concentrations Drugs used were ang]otensm I, angmtensm II, saralas]n and mdomethacm (Sigma, U S A ) and captopnl (Sqmbb, Spmn) Contractmns were expressed as a percentage of responses reduced by 75 mM K + Results are given as mean _+SEM Statistical analysis was done by means of the Student's t-test for unpmred experiments, a P value of less than 0 05 was considered s]gmficant
E+ a o~ 4 rain Er-
• 9
lo
10 9
All, -log [M] ~
0306-3623/92 $5 00 + 0 00 Copyright © 1992Pergamon Press Ltd
VASOCONSTRICTIVE EFFECTS OF ANGIOTENSIN I A N D II IN CAT FEMORAL ARTERIES. ROLE OF ENDOTHELIUM M F~.RER, ~A ENCABO,t J MARIN2 and G BALFAC-~Nt* l Departamento de F1slologia and 2Departamento de Farmacologia y Terapeutaea, Facultad de Medlcma, Umversldad Aut6noma de Madrid, C/Arzoblspo Morcatlo, 4, 28029-Madrid, Spmn (Recewed 9 March 1992) Al~traet--1 AI and AII reduced concentration-dependent eontractaons ]n cat femoral artery segments, the potency of All being greater than that of AI 2 The antagomst of All receptors, saralasm (1 and I0 nM), mlublted the AI and AII responses 3 Indomethacm (I0 #M) and endothehum removal mereased the responses to AII, whereas those induced by AI were barely affected 4 The response reduced by AI was reduced by captopnl (200/aM) 5 These data suggest (1) the contraetaons reduced by AII are medmted by AII receptors and endothehal products derived from ¢yclooxygenase, (2) the response to AI is, m part, produced by its conversion into AII
INTRODUCTION Anglotensm II (AII) induces potent contractde responses m &fferent vascular preparataons by &reet stunulataon of specafic AII receptors present m the smooth muscle (Ferrano, 1990), and m&rectly by actavataon of presynaptac AII receptors of sympathcue nerve terrmnals facahtatang the noradrenahne release (Chne, 1984, Zlogas et al, 1985, Yasuda et al, 1987, Moldenngs et al, 1988) Exadence has been shown that the vessels possess the ang~otensm convertang enzyme (ACE), which transforms AI into AII (Whalley et al, 1983, Whalley and Wahl, 1988, Ferrano, 1990) Tlus enzyme is located m the vascular wall (Andre et al, 1990, Engl~me et al, 1990, Mfiller-Schwelmtzer and Olea-Baza, 1990), but mainly m the ¢ndothehum (Ryan et ai, 1976, Paulson et al, 1988) On the other hand, the endothehum produces a negatave modulataon of the vasocontractale responses ehcated by AI and AII m some vessels (Bullock et al, 1986, Gruetter et al, 1988; Engl~me et al, 1990, Yen et al, 1990) Tlus is due to anglotensms mducmg the release of relaxant substances, such as endothehum-denved relaxmg factor (EDRF, ldentafied as mtnc oxade, NO) (Gruetter et al , 1988; Engl~me et al , 1990, Yen et al , 1990) or prostacychn (PGI2) (Gruetter et al, 1988, Mmaml and Toda, 1988, Mfiller-Schwelmtzer and Olea-Baza, 1990, Yen et al, 1990) In other vessels, the endothehum does not affect the responses ehclted by these agents (Saye et al, 1984; Gruetter et al, 1988) or produces a posmve modulataon, since its removal reduces the contractaons caused by ang~otensms, which ~s m part due to the release of endothehal eontraetang factors, such as thromboxane A2 (Manabe et al, 1989, Yen et al, 1990)
*To whom all correspondence should be addressed
In wew of these data, the present study was undertaken m cat femoral arteries to analyze the ablhty of AI and AII to reduce contractde responses, the receptors revolved, the vascular presence of ACE, and the endothehal partaclpataon on the responses caused by both angtotensms MATERIALSAND METHODS Vascular reactwtty Cats of either sex (1 5-3 kg) were anaesthetazed vath sodmm pentobarbltal (35 mg/kg, 1p ) and killed by bleedmg Femoral arteries were isolated and placed m a Petn &sh contalmng Krebs-Henselelt solutaon (KHS) at 4°C and ¢hwded into nngs of 4 mm m length Each nng was set up for Isometric tension recording m an organ bath according to the method of Nlelscn and Owman (1971) The organ bath contmned 5 ml of KHS at 37°C contanuously bubbled wath a 95% 02-5% CO 2 rmxture, which gave a pH of 7 4 Two stainless steel pros were passed through the lumen of the nng One pm was fixed to the organ bath wall while the other was connected to a strain gauge for ~sometnc tension reeorchng The latter pm was m parallel posmon with the former and It was movable, pcrtmttang the application of resting tension m a perpendtcular plane to the long axas of the vascular cyhnder The ~sometnc contractaon was recorded through a force-displacement transducer (Grass FTO3C), connected to a Grass model 7D polygraph A restang tension of I g (optamal resting tone, i e the tone wluch produces maxnnal response to 75raM K +) was apphed to cyhndneal segments Tlus tension was readjusted every 15mm dunng 60-90rmn eqmhbrataon period (for base hne stabdxzataon), before addmon of drugs The segments were mmaUy exposed to 75 mM K + to check thelr functaonal mtegmy Afterwards, the bath medmm was changed several tames untal the restang tone recovery Then, a concentrataon-response curve to AI or AII was earned out Endothehum influence on the responses to angwtensms was analyzed by its removal (by gently rubbing) from arterial nngs The effectave removal was tested by the inability of acetylchohne (0 1 ttM) to produce vasoddataon m arteries precontracted vath noradrenahne
1171
1172
M FERRERet al
(1 #M) To examine the effect of saralasm (1 and 10nM), mdomethacm (10#M) and captopnl (200#M) on the actmns of angmtensms, they were added to the bath 10, 20 and 30 mm m advance Solutions, drugs and stattsttcal methods The composmon of KHS was as follows (mM) NaCI 115, CaC12 2 5, KC1 46, KH2PO 4 1 2, MgSO4 7H2 O 1 2, NaHCO 3 25, glucose 11 1, Na2 EDTA 0 03 (to prevent the ox~datmn of unstable substances) Stock solutions of AI, AII and saralasm (all at 1 raM) and captopnl (0 1 M) were made m bid]stilled water, while that of mdomethacm was achieved m a solutmn with 1 5 mM of sodmm carbonate and kept at -20°C Ahquots of these solutmns were diluted just before use with KHS to obtain the desired concentrations Drugs used were ang]otensm I, angmtensm II, saralas]n and mdomethacm (Sigma, U S A ) and captopnl (Sqmbb, Spmn) Contractmns were expressed as a percentage of responses reduced by 75 mM K + Results are given as mean _+SEM Statistical analysis was done by means of the Student's t-test for unpmred experiments, a P value of less than 0 05 was considered s]gmficant
E+ a o~ 4 rain Er-
• 9
lo
10 9
All, -log [M] ~
