BLOOD PRESSURE
1992; 1: 50-56
Raised Plasma Concentrations of Endothelin-1 and -3 in Marmosets with Acute Aortic Stenosis: No Relation to the Renin-angiotensin System DAVID GUSTAFSSON,' MARGARETA ELG,' THOMAS HEDNER,* EVA JOHNSSON,3 MORGAN SOHTELL,' LENNART SVENSSON' and FREJ FYHRQUIST4
Blood Press Downloaded from informahealthcare.com by Chulalongkorn University on 01/10/15 For personal use only.
From I Astra-Hassle Preclinical Research Laboratories. Molndal. =Department of Clinical Pharmacology, Sahlgrenska University Hospital, Gothenburg, 'Department of Physiology. University of Goteborg, Gothenburg, Sweden and 4Minert.a Foundation Institute for Medical Research, Helsinki, Finland Gustnfsson D, Elg M, Hedner T, Johnsson E, Sohtell M, Svensson L, Fyhrquist F. Raisedpiasma concentrations of endothelin-1 and -3 in marmosets with acute aortic stenosis: No relation to the renin-angiotensin system. Blood Pressure 1992; I: 50-56.
Plasma levels of endothelin (ET), plasma renin activity (PRA) and angiotensin I1 (Ang 11) were measured in anaesthetized marmosets exposed to acute aortic stenosis proximal to the renal arteries. In vehicle experiments, ET rose from 5 k 2 to 38 & 4 pgml- I , PRA from 5 + 2 to 99k21 ng ml-' h-' and Ang I1 from 21 k4 to 213 k76 pgml-'. Administration ofrenin inhibitor and angiotensinconvertingenzyme inhibitor reduced PRA and Ang I1 to control levels, while the plasma levels of ET increased further (51 10 and 71 & 16 pg ml-l, respectively). During aortic stenosis the two isoforms ET-I and ET-3 appeared in the circulation, while in conscious control animals only ET-I was found. It is concluded that the increased plasma levels of ET in our primate model could not be ascribed to the increased circulating levels of PRA and Ang 11. Key words: angiotensin 11, blood pressure, endothelin. plasma renin activity
INTRODUCTION Endothelin-1 (ET-I) is a 21 amino acid peptide with 2 disulphide bonds isolated from the supernatant of serum-free cultured endothelial cells [I]. Two further isoforms have been characterized, endothelin-2 (ET-2) and endothelin-3 (ET-3), which are encoded by separate genes [2]. ET- 1 and ET-3 are probably present in all species, while to our knowledge, circulating levels of ET-2 and ET-3 have not.as yet been reported. ET-1 seems to be mainly produced by vascular endothelial cells [3], while ET-3 is derived from other cells in, for example, the central nervous system [4]. The dominating biological effect of ET-1 is a long-lasting vasoconstriction leading to a sustained increase in blood pressure [l]. The regulation of endothelial biosynthesis and release of ET-1 in uiuo is still largely unknown. Like the generation of other bioactive peptides, that of ET-1 includessynthesis of preproendothelin structures which are cleaved into the mature 21 amino acid sequence. Preproendothelin mRNA expression in cultured endothelial cells in vitro is enhanced by agents such as thrombin, angiotensin I1 (Ang IT), vasopressin, transforminggrowth factor /?,endotoxin, Ca+ ionophores, and phorbol esters, as well as fluid-mechanical shear stress [ l , 5, 61. Most likely, ET-1 is not stored in granulae but produced by de novo protein synthesis [1, 71. High levels of circulating ET-1 have been reported in conditions such as myocardial infarction [8], subarachnoidal haemorrhage [9] and uraemia [lo]. Although some mechanisms involved in the endothe+
lial regulation of ET-1 expression and release in uitro are known, the factors controlling the circulating levels of ET- 1 under physiological and patho-physiological conditions are mostly unknown. As the in uitro data suggested that increased circulating concentrations of Ang I1 might be related to increased plasma levels of ET-I, we wanted to test this concept in a marmoset model of acute aortic stenosis leading to activation of the renin-angiotensin system. This hypothesis could, however, not be substantiated by our model; on the contrary, we found that the circulating ET-I concentrations were related to the reduced pressure distal to the aortic stenosis. METHODS The primary aim of the experimental series was to evaluate the haemodynamic effects of a new renin inhibitor, H 218/54, in a primate model with increased levels of PRA and Ang 11. Since the experiments also allowed blood sampling for determinations of ET immunoreactivity, investigation of the possible influence of Ang I1 on ET biosynthesis became a secondary aim. Preparation Marmosets (Callithrix Jacchus) of both sexes (338 7 g, n=37) were obtained from Fisons Ltd, Loughborough, UK (n = 28) and Charles River Ltd, Margate, UK (n = 9). On the day of the experiment, the animals
Blood Press Downloaded from informahealthcare.com by Chulalongkorn University on 01/10/15 For personal use only.
Endothelin and angiotensin II in vivo
were anaesthetized with pentobarbital (NordVacc, Sweden) 35 mg kg-' as an intraperitoneal bolus dose followed by an intravenous infusion of 4 mg kg-' h-I. After tracheotomy, artificial respiration was started with about 1.5 ml of room air (10% oxygen added) given at 30 cycles min-I. Blood gases and pH were kept within the physiological range by small adjustments of the tidal volume, and only in 3 cases was it necessary to give bicarbonate. Body temperature was maintained at 38°C throughout the experiment. The baroreceptors in the aorta and carotid arteries were denervated bilaterally by sectioning the superior laryngeal nerves and by etching the carotid sinus with phenol in ethanol (0.2 g ml-'). The result of the barodenervation was validated by a test dose of phenylephrine (1 pg intravenously). A mechanical occluder was placed on the abdominal aorta, proximal to the renal arteries and distal to the coeliac and superior mesenteric arteries, and the position was verified after each experiment. The aorta was stenosed by fine adjustments of the pressure in a miniature plastic bag of an occluder that compressed the artery from above. Arterial blood pressures were measured continuously (Statham transducers, Gould Inc, Oxnard, CA, USA) from the left carotid and femoral arteries, and an electrocardiogram was obtained from skin and oesophageal electrodes. Signals were recorded on a paper chart (Grass Polygraph, Quincy, MA., USA) and by computer (Deskpro 386s, Compaq Computer Corporation, Houston, TX, USA). Analyses The radioimmunoassay of ET was performed as previously described by Fyhrquist et al. [l 11. In short, 1020 pg of synthetic ET-1 (Peptide Institute, London, UK) was used for monthly injections into popliteal lymph nodes of rabbits. The antiserum chosen showed
1992; 1: 50-56
Raised Plasma Concentrations of Endothelin-1 and -3 in Marmosets with Acute Aortic Stenosis: No Relation to the Renin-angiotensin System DAVID GUSTAFSSON,' MARGARETA ELG,' THOMAS HEDNER,* EVA JOHNSSON,3 MORGAN SOHTELL,' LENNART SVENSSON' and FREJ FYHRQUIST4
Blood Press Downloaded from informahealthcare.com by Chulalongkorn University on 01/10/15 For personal use only.
From I Astra-Hassle Preclinical Research Laboratories. Molndal. =Department of Clinical Pharmacology, Sahlgrenska University Hospital, Gothenburg, 'Department of Physiology. University of Goteborg, Gothenburg, Sweden and 4Minert.a Foundation Institute for Medical Research, Helsinki, Finland Gustnfsson D, Elg M, Hedner T, Johnsson E, Sohtell M, Svensson L, Fyhrquist F. Raisedpiasma concentrations of endothelin-1 and -3 in marmosets with acute aortic stenosis: No relation to the renin-angiotensin system. Blood Pressure 1992; I: 50-56.
Plasma levels of endothelin (ET), plasma renin activity (PRA) and angiotensin I1 (Ang 11) were measured in anaesthetized marmosets exposed to acute aortic stenosis proximal to the renal arteries. In vehicle experiments, ET rose from 5 k 2 to 38 & 4 pgml- I , PRA from 5 + 2 to 99k21 ng ml-' h-' and Ang I1 from 21 k4 to 213 k76 pgml-'. Administration ofrenin inhibitor and angiotensinconvertingenzyme inhibitor reduced PRA and Ang I1 to control levels, while the plasma levels of ET increased further (51 10 and 71 & 16 pg ml-l, respectively). During aortic stenosis the two isoforms ET-I and ET-3 appeared in the circulation, while in conscious control animals only ET-I was found. It is concluded that the increased plasma levels of ET in our primate model could not be ascribed to the increased circulating levels of PRA and Ang 11. Key words: angiotensin 11, blood pressure, endothelin. plasma renin activity
INTRODUCTION Endothelin-1 (ET-I) is a 21 amino acid peptide with 2 disulphide bonds isolated from the supernatant of serum-free cultured endothelial cells [I]. Two further isoforms have been characterized, endothelin-2 (ET-2) and endothelin-3 (ET-3), which are encoded by separate genes [2]. ET- 1 and ET-3 are probably present in all species, while to our knowledge, circulating levels of ET-2 and ET-3 have not.as yet been reported. ET-1 seems to be mainly produced by vascular endothelial cells [3], while ET-3 is derived from other cells in, for example, the central nervous system [4]. The dominating biological effect of ET-1 is a long-lasting vasoconstriction leading to a sustained increase in blood pressure [l]. The regulation of endothelial biosynthesis and release of ET-1 in uiuo is still largely unknown. Like the generation of other bioactive peptides, that of ET-1 includessynthesis of preproendothelin structures which are cleaved into the mature 21 amino acid sequence. Preproendothelin mRNA expression in cultured endothelial cells in vitro is enhanced by agents such as thrombin, angiotensin I1 (Ang IT), vasopressin, transforminggrowth factor /?,endotoxin, Ca+ ionophores, and phorbol esters, as well as fluid-mechanical shear stress [ l , 5, 61. Most likely, ET-1 is not stored in granulae but produced by de novo protein synthesis [1, 71. High levels of circulating ET-1 have been reported in conditions such as myocardial infarction [8], subarachnoidal haemorrhage [9] and uraemia [lo]. Although some mechanisms involved in the endothe+
lial regulation of ET-1 expression and release in uitro are known, the factors controlling the circulating levels of ET- 1 under physiological and patho-physiological conditions are mostly unknown. As the in uitro data suggested that increased circulating concentrations of Ang I1 might be related to increased plasma levels of ET-I, we wanted to test this concept in a marmoset model of acute aortic stenosis leading to activation of the renin-angiotensin system. This hypothesis could, however, not be substantiated by our model; on the contrary, we found that the circulating ET-I concentrations were related to the reduced pressure distal to the aortic stenosis. METHODS The primary aim of the experimental series was to evaluate the haemodynamic effects of a new renin inhibitor, H 218/54, in a primate model with increased levels of PRA and Ang 11. Since the experiments also allowed blood sampling for determinations of ET immunoreactivity, investigation of the possible influence of Ang I1 on ET biosynthesis became a secondary aim. Preparation Marmosets (Callithrix Jacchus) of both sexes (338 7 g, n=37) were obtained from Fisons Ltd, Loughborough, UK (n = 28) and Charles River Ltd, Margate, UK (n = 9). On the day of the experiment, the animals
Blood Press Downloaded from informahealthcare.com by Chulalongkorn University on 01/10/15 For personal use only.
Endothelin and angiotensin II in vivo
were anaesthetized with pentobarbital (NordVacc, Sweden) 35 mg kg-' as an intraperitoneal bolus dose followed by an intravenous infusion of 4 mg kg-' h-I. After tracheotomy, artificial respiration was started with about 1.5 ml of room air (10% oxygen added) given at 30 cycles min-I. Blood gases and pH were kept within the physiological range by small adjustments of the tidal volume, and only in 3 cases was it necessary to give bicarbonate. Body temperature was maintained at 38°C throughout the experiment. The baroreceptors in the aorta and carotid arteries were denervated bilaterally by sectioning the superior laryngeal nerves and by etching the carotid sinus with phenol in ethanol (0.2 g ml-'). The result of the barodenervation was validated by a test dose of phenylephrine (1 pg intravenously). A mechanical occluder was placed on the abdominal aorta, proximal to the renal arteries and distal to the coeliac and superior mesenteric arteries, and the position was verified after each experiment. The aorta was stenosed by fine adjustments of the pressure in a miniature plastic bag of an occluder that compressed the artery from above. Arterial blood pressures were measured continuously (Statham transducers, Gould Inc, Oxnard, CA, USA) from the left carotid and femoral arteries, and an electrocardiogram was obtained from skin and oesophageal electrodes. Signals were recorded on a paper chart (Grass Polygraph, Quincy, MA., USA) and by computer (Deskpro 386s, Compaq Computer Corporation, Houston, TX, USA). Analyses The radioimmunoassay of ET was performed as previously described by Fyhrquist et al. [l 11. In short, 1020 pg of synthetic ET-1 (Peptide Institute, London, UK) was used for monthly injections into popliteal lymph nodes of rabbits. The antiserum chosen showed
