11-98
Endothelin Vascular Receptors and Responses in Deoxycorticosterone Acetate-Salt Hypertensive Rats Paul V. Nguyen, Angele Parent, Li Yuan Deng, Jean-Pierre Fliickiger, Ga6tan Thibault, and Ernesto L. Schiflrin The vasoconstrictor effect, the binding, and the response of inositol phosphates to endothelin-1 (ET-1) were investigated in blood vessels of deoxycorticosterone acetate (DOCA) -salt hypertensive rats within 2 weeks of development of hypertension and in uninephrectomized control rats. In DOCA-salt and uninephrectomized rats, plasma levels of endothelin were similar (1.2±0.1 fmol/ml). Thoracic aorta and mesenteric artery rings devoid of endothelium presented significantly decreased responses to increasing concentrations of ET-1. Binding of ET-1 to mesenteric artery membranes was significantly lower in DOCA-salt rats (106±22 fmol/mg protein) than in uninephrectomized rats (172 ±19 fmol/mg protein, p < 0.05), whereas affinity was similar. Phosphoinositide metabolism was examined in aorta and mesenteric arteries after incubation with [3H]myoinositol. Inositol phosphates were separated by high-performance liquid chromatography. In response to 100 nmol/1 ET-1, accumulation of inositol 1,4,5trisphosphate after 20 seconds and of inositol monophosphate, inositol bisphosphate, and inositol 1,3,4 -trisphosphate after 30 minutes (in the presence of 25 mmol/1 LiCI) were significantly lower in DOCA-salt hypertensive than in uninephrectomized control rats, in both aorta and mesenteric arteries. In conclusion, decreased density of ET-1 receptors in DOCA-salt hypertensive rats results in decreased activation of phospholipase C and, consequently, reduced vasoconstriction induced by ET-1. Because the decrease in vasoconstrictor effects of ET-1 is found in the absence of endothelium, it is likely that receptor downregulation rather than prior receptor occupancy underlies these findings. (Hypertension 1992;19[suppl II]:II-98—11-104)
A
possible factor contributing to the elevated vascular resistance seen in hypertension is . altered reactivity of blood vessels to vasoactive substances. Increased responsiveness to vasoconstrictor agents has been demonstrated in different models of experimental hypertension, such as the renovascular hypertensive rat1-2 and the deoxycorticosterone acetate (DOCA)-salt hypertensive rat.3 The endothelium synthesizes and releases paracrine hormones that regulate the contraction and physiology of vascular smooth muscle cells.4 In addition to the well-described vasorelaxant factors (i.e., endo-
From the Experimental Hypertension Laboratory, Clinical Research Institute of Montreal, Montreal, Canada. Supported by grant MT-10667 from the Medical Research Council of Canada. P.V.N. was supported by a Research Fellowship from the Canadian Heart Foundation and a Research Scholarship from the Canadian Hypertension Society. A.P. was supported by a Research Studentship from the Fonds pour Formation des Chercheurs et Aide a la Recherche. J.-P.F. was supported by the Swiss National Fund. Address for reprints: Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7.
thelium-derived relaxing factor and prostacyclin),4 it has been shown that the endothelium also releases vasoconstrictor compounds, and one of these is the peptide endothelin.5 Endothelin-1 (ET-1), originally isolated from the supernatant of cultured porcine aortic endothelial cells,5 is a 21-amino acid peptide and one of the most potent vasoconstrictor agents thus far described. It probably acts more as a local rather than a circulating hormone, and it might be involved in the control of blood flow and pressure because of its potent microvascular constrictor effect demonstrated in vivo. Greater reactivity to ET-1 has been observed in the isolated arteries of spontaneously hypertensive rats.6 Thus, enhanced reactivity to this agent might be expected in other models of experimental hypertension, such as the DOCA-salt hypertensive rat.7 Initial studies suggested that endothelins act on vascular smooth muscle cells by increasing calcium influx from the extracellular fluid through voltagedependent calcium channels.8 It later became evident that activation of phospholipase C and breakdown of membrane phosphoinositides, resulting in
Downloaded from http://hyper.ahajournals.org/ at Mercer University on March 25, 2015
Nguyen et al
release of calcium from the endoplasmic reticulum to produce a rise in cytosolic calcium and activation of protein kinase C, were as or more important in the intracellular signal transduction of endothelin receptors.9"12 Secondary activation of a nonselective cationic channel permeable to calcium may result in membrane potential changes that activate L-type Ca2+ channels.13 The role of phosphoinositide turnover and activation of protein kinase C appears to be critical in rabbit aorta, as shown by the inhibition of protein kinase C.12 In this study, we therefore have investigated the reactivity to ET-1 of blood vessels (aorta and mesenteric arteries) in DOCA-salt hypertensive rats, concurrently with the binding of ET-1 to vascular membranes and postreceptor mechanisms such as the production of inositol phosphates. Methods
DOCA-Salt Hypertension and Endothelin
11-99
1.0 g for mesenteric arteries. During preparation, the luminal surface was gently rubbed to eliminate the endothelial layer. The bathing medium was Krebs' bicarbonate with the following composition (mmol/1): NaCl 118, MgSO4 1.18, KH2PO4 1.18, dextrose 5.5, NaHCO3 25.0, CaCl2 2.5, and KC1 4.7. The solution was bubbled with a mixture of 95% Oj-5% CO2. Tissues were equilibrated for 90 minutes. They were challenged with 1 /imol/1 norepinephrine, and relaxation was attempted with 0.1 mmol/1 acetylcholine to verify absence of endothelium. A cumulative concentration-response curve (at 15-minute intervals between each dose) to ET-1 (10 pmol/1 to 100 nmol/1) was obtained. Isometric contractions were recorded as changes in grams of force on a Grass Model 7 polygraph. All drugs were prepared immediately before use in concentrations such that volumes no greater than 100 yX were added to tissue baths.
Materials ET-1 (endothelin 1-21) (human, porcine) was purchased from Peninsula Laboratories, Inc., Belmont, Calif. D-Myo-2-[3H]inositol, D-myo-l-[3H(N)]inositol 1,3,4-trisphosphate, and D-myo-2-[3H]inositol 1,3,4,5-tetrakisphosphate were obtained from Amersham, Oakville, Canada, and Dupont New England Nuclear, Boston, Mass. All reactives used were of the highest reagent grade available.
Endothelin-1 Binding Assay Mesenteric arteries were used to prepare vascular membranes for binding experiments as described previously for other binding studies.15"17 In brief, mesenteric arteries were homogenized with a polytron (setting 8 for 10 seconds, twice) in a 0.25 mol/1 sucrose solution. The homogenate was centrifuged at 1 ,OOQg for 10 minutes, the supernatant then centrifuged at 104,000g for 30 minutes, and the Animal Experiments pellet resuspended in 0.05 mol/1 Tris-HCl (pH 7.2) The study was approved by the Animal Care Comcontaining 5 mmol/1 MgCl2, 1 ^unol/l aprotinin, mittee of the Clinical Research Institute. DOCA-salt 0.1% bacitracin, and 0.5 mmol/1 phenylmethylsulfohypertension was induced by the method of Ormsbee nyl fluoride. After proteins were measured by the and Ryan.14 In brief, male Sprague-Dawley rats Coomassie blue method, bovine serum albumin was (Charles River Laboratories, St. Constant, Canada) added to a concentration of 0.5%. Fifty micrograms weighing 200 g were uninephrectomized under ether of membrane protein was used per tube, with 40 anesthesia. Silicone rubber impregnated with DOCA pmol/1 125I-ET-1 and increasing concentrations of (130 mg/rat) was implanted subcutaneousry, and rats unlabeled ET-1 (1 pmol/1 to 1 /nmol/1) in a final were offered 1% saline to drink. Rats were studied 1-2 volume of 0.25 ml. In preliminary experiments, it weeks after becoming hypertensive. Another group of was shown that binding was linear between 20 and uninephrectomized rats receiving silicone rubber with100 fjig protein per tube. 125I-ET-1 was prepared by out DOCA impregnation served as control. This group the lactoperoxidase method as previously dereceived tap water to drink. Blood pressure measure18 and had a specific activity of approxiscribed ments were by the tail-cuff method. With rats under mately 1,000 Ci/mmol. All assays were performed in light anesthesia, blood pressure was measured the day duplicate at 22°C for 120 minutes. At this time before experiments and recorded on a Model 7 polyinterval, binding was at steady state and there was graph (Grass Instrument Co., Quincy, Mass.) fitted no degradation of labeled ET-1 as judged by highwith a 7P8 preamplifier and a Grass Model 1010 crystal performance liquid chromatography. Separation of microphone as a pulse detector. The average of three bound and free radioactivities was achieved by pressure readings was obtained. rapid filtration through GF/C filters (Whatman Inc., Constrictor Response of Blood Vessels to Endothelin-1 Clifton, N.J.). The filters were washed twice with 3 ml Tris-HCl (pH 7.4), allowed to dry, and counted Segments of rat thoracic aorta and mesenteric in a Rackgamma LKB counter (Turku, Finland) arteries from both experimental groups were rewith 80% efficiency. In preliminary experiments, we moved and dissected free of connective tissue. The found that binding was specific for ET-1, with an first 0.5 cm of the proximal end of the aortic strips inhibition constant (/Cj) of 0.4 nmol/1, whereas was discarded, and rings (4 mm in length) were endothelin-3 had a K, of 2.5 nmol/1. Neither angioprepared from the next 2.5 cm for incubation in a tensin II, vasopressin, atrial natriuretic peptide, nor glass-jacketed, 15-ml tissue bath at 37°C. Rings were other unrelated peptides displaced ET-1 binding. subjected to a passive force of 2.0 g for aorta and
Downloaded from http://hyper.ahajournals.org/ at Mercer University on March 25, 2015
11-100
Supplement II Hypertension
Vol 19, No 2 February 1992
TABLE 1. Blood Pressure, Plasma Renin Activity, and EndotheUn Binding Sites in Mesenteric Blood Vessels of Deoxycorticosterone Acetate-Salt Hypertensive Rats
Group Uninephrectomized DOCA-salt hypertensive
Systolic blood pressure (mmHg) 105±3 166+4*
PRA (ng angiotensin Iml-'-hr 1 )
Plasma ET-1 (fmol/ml)
1.17±0.15 ND
1.16±0.06 1.24±0.10
B^ (fmol/mg protein) 172±19 106±22f
(pmol/1) 277±46 176±28
Values are mean±SEM. Number of binding experiments, 6. Total number of rats studied per group, 18. Total amount of protein from mesenteric arteries of each group of three rats studied in each binding experiment, 3.6±0.2 mg in controls and 4.1 ±0.4 mg in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. PRA, plasma renin activity; ET-1, endothelin-1; !}„„, density of binding sites; K4, dissociation constant; ND, not detectable. *p
Endothelin Vascular Receptors and Responses in Deoxycorticosterone Acetate-Salt Hypertensive Rats Paul V. Nguyen, Angele Parent, Li Yuan Deng, Jean-Pierre Fliickiger, Ga6tan Thibault, and Ernesto L. Schiflrin The vasoconstrictor effect, the binding, and the response of inositol phosphates to endothelin-1 (ET-1) were investigated in blood vessels of deoxycorticosterone acetate (DOCA) -salt hypertensive rats within 2 weeks of development of hypertension and in uninephrectomized control rats. In DOCA-salt and uninephrectomized rats, plasma levels of endothelin were similar (1.2±0.1 fmol/ml). Thoracic aorta and mesenteric artery rings devoid of endothelium presented significantly decreased responses to increasing concentrations of ET-1. Binding of ET-1 to mesenteric artery membranes was significantly lower in DOCA-salt rats (106±22 fmol/mg protein) than in uninephrectomized rats (172 ±19 fmol/mg protein, p < 0.05), whereas affinity was similar. Phosphoinositide metabolism was examined in aorta and mesenteric arteries after incubation with [3H]myoinositol. Inositol phosphates were separated by high-performance liquid chromatography. In response to 100 nmol/1 ET-1, accumulation of inositol 1,4,5trisphosphate after 20 seconds and of inositol monophosphate, inositol bisphosphate, and inositol 1,3,4 -trisphosphate after 30 minutes (in the presence of 25 mmol/1 LiCI) were significantly lower in DOCA-salt hypertensive than in uninephrectomized control rats, in both aorta and mesenteric arteries. In conclusion, decreased density of ET-1 receptors in DOCA-salt hypertensive rats results in decreased activation of phospholipase C and, consequently, reduced vasoconstriction induced by ET-1. Because the decrease in vasoconstrictor effects of ET-1 is found in the absence of endothelium, it is likely that receptor downregulation rather than prior receptor occupancy underlies these findings. (Hypertension 1992;19[suppl II]:II-98—11-104)
A
possible factor contributing to the elevated vascular resistance seen in hypertension is . altered reactivity of blood vessels to vasoactive substances. Increased responsiveness to vasoconstrictor agents has been demonstrated in different models of experimental hypertension, such as the renovascular hypertensive rat1-2 and the deoxycorticosterone acetate (DOCA)-salt hypertensive rat.3 The endothelium synthesizes and releases paracrine hormones that regulate the contraction and physiology of vascular smooth muscle cells.4 In addition to the well-described vasorelaxant factors (i.e., endo-
From the Experimental Hypertension Laboratory, Clinical Research Institute of Montreal, Montreal, Canada. Supported by grant MT-10667 from the Medical Research Council of Canada. P.V.N. was supported by a Research Fellowship from the Canadian Heart Foundation and a Research Scholarship from the Canadian Hypertension Society. A.P. was supported by a Research Studentship from the Fonds pour Formation des Chercheurs et Aide a la Recherche. J.-P.F. was supported by the Swiss National Fund. Address for reprints: Ernesto L. Schiffrin, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7.
thelium-derived relaxing factor and prostacyclin),4 it has been shown that the endothelium also releases vasoconstrictor compounds, and one of these is the peptide endothelin.5 Endothelin-1 (ET-1), originally isolated from the supernatant of cultured porcine aortic endothelial cells,5 is a 21-amino acid peptide and one of the most potent vasoconstrictor agents thus far described. It probably acts more as a local rather than a circulating hormone, and it might be involved in the control of blood flow and pressure because of its potent microvascular constrictor effect demonstrated in vivo. Greater reactivity to ET-1 has been observed in the isolated arteries of spontaneously hypertensive rats.6 Thus, enhanced reactivity to this agent might be expected in other models of experimental hypertension, such as the DOCA-salt hypertensive rat.7 Initial studies suggested that endothelins act on vascular smooth muscle cells by increasing calcium influx from the extracellular fluid through voltagedependent calcium channels.8 It later became evident that activation of phospholipase C and breakdown of membrane phosphoinositides, resulting in
Downloaded from http://hyper.ahajournals.org/ at Mercer University on March 25, 2015
Nguyen et al
release of calcium from the endoplasmic reticulum to produce a rise in cytosolic calcium and activation of protein kinase C, were as or more important in the intracellular signal transduction of endothelin receptors.9"12 Secondary activation of a nonselective cationic channel permeable to calcium may result in membrane potential changes that activate L-type Ca2+ channels.13 The role of phosphoinositide turnover and activation of protein kinase C appears to be critical in rabbit aorta, as shown by the inhibition of protein kinase C.12 In this study, we therefore have investigated the reactivity to ET-1 of blood vessels (aorta and mesenteric arteries) in DOCA-salt hypertensive rats, concurrently with the binding of ET-1 to vascular membranes and postreceptor mechanisms such as the production of inositol phosphates. Methods
DOCA-Salt Hypertension and Endothelin
11-99
1.0 g for mesenteric arteries. During preparation, the luminal surface was gently rubbed to eliminate the endothelial layer. The bathing medium was Krebs' bicarbonate with the following composition (mmol/1): NaCl 118, MgSO4 1.18, KH2PO4 1.18, dextrose 5.5, NaHCO3 25.0, CaCl2 2.5, and KC1 4.7. The solution was bubbled with a mixture of 95% Oj-5% CO2. Tissues were equilibrated for 90 minutes. They were challenged with 1 /imol/1 norepinephrine, and relaxation was attempted with 0.1 mmol/1 acetylcholine to verify absence of endothelium. A cumulative concentration-response curve (at 15-minute intervals between each dose) to ET-1 (10 pmol/1 to 100 nmol/1) was obtained. Isometric contractions were recorded as changes in grams of force on a Grass Model 7 polygraph. All drugs were prepared immediately before use in concentrations such that volumes no greater than 100 yX were added to tissue baths.
Materials ET-1 (endothelin 1-21) (human, porcine) was purchased from Peninsula Laboratories, Inc., Belmont, Calif. D-Myo-2-[3H]inositol, D-myo-l-[3H(N)]inositol 1,3,4-trisphosphate, and D-myo-2-[3H]inositol 1,3,4,5-tetrakisphosphate were obtained from Amersham, Oakville, Canada, and Dupont New England Nuclear, Boston, Mass. All reactives used were of the highest reagent grade available.
Endothelin-1 Binding Assay Mesenteric arteries were used to prepare vascular membranes for binding experiments as described previously for other binding studies.15"17 In brief, mesenteric arteries were homogenized with a polytron (setting 8 for 10 seconds, twice) in a 0.25 mol/1 sucrose solution. The homogenate was centrifuged at 1 ,OOQg for 10 minutes, the supernatant then centrifuged at 104,000g for 30 minutes, and the Animal Experiments pellet resuspended in 0.05 mol/1 Tris-HCl (pH 7.2) The study was approved by the Animal Care Comcontaining 5 mmol/1 MgCl2, 1 ^unol/l aprotinin, mittee of the Clinical Research Institute. DOCA-salt 0.1% bacitracin, and 0.5 mmol/1 phenylmethylsulfohypertension was induced by the method of Ormsbee nyl fluoride. After proteins were measured by the and Ryan.14 In brief, male Sprague-Dawley rats Coomassie blue method, bovine serum albumin was (Charles River Laboratories, St. Constant, Canada) added to a concentration of 0.5%. Fifty micrograms weighing 200 g were uninephrectomized under ether of membrane protein was used per tube, with 40 anesthesia. Silicone rubber impregnated with DOCA pmol/1 125I-ET-1 and increasing concentrations of (130 mg/rat) was implanted subcutaneousry, and rats unlabeled ET-1 (1 pmol/1 to 1 /nmol/1) in a final were offered 1% saline to drink. Rats were studied 1-2 volume of 0.25 ml. In preliminary experiments, it weeks after becoming hypertensive. Another group of was shown that binding was linear between 20 and uninephrectomized rats receiving silicone rubber with100 fjig protein per tube. 125I-ET-1 was prepared by out DOCA impregnation served as control. This group the lactoperoxidase method as previously dereceived tap water to drink. Blood pressure measure18 and had a specific activity of approxiscribed ments were by the tail-cuff method. With rats under mately 1,000 Ci/mmol. All assays were performed in light anesthesia, blood pressure was measured the day duplicate at 22°C for 120 minutes. At this time before experiments and recorded on a Model 7 polyinterval, binding was at steady state and there was graph (Grass Instrument Co., Quincy, Mass.) fitted no degradation of labeled ET-1 as judged by highwith a 7P8 preamplifier and a Grass Model 1010 crystal performance liquid chromatography. Separation of microphone as a pulse detector. The average of three bound and free radioactivities was achieved by pressure readings was obtained. rapid filtration through GF/C filters (Whatman Inc., Constrictor Response of Blood Vessels to Endothelin-1 Clifton, N.J.). The filters were washed twice with 3 ml Tris-HCl (pH 7.4), allowed to dry, and counted Segments of rat thoracic aorta and mesenteric in a Rackgamma LKB counter (Turku, Finland) arteries from both experimental groups were rewith 80% efficiency. In preliminary experiments, we moved and dissected free of connective tissue. The found that binding was specific for ET-1, with an first 0.5 cm of the proximal end of the aortic strips inhibition constant (/Cj) of 0.4 nmol/1, whereas was discarded, and rings (4 mm in length) were endothelin-3 had a K, of 2.5 nmol/1. Neither angioprepared from the next 2.5 cm for incubation in a tensin II, vasopressin, atrial natriuretic peptide, nor glass-jacketed, 15-ml tissue bath at 37°C. Rings were other unrelated peptides displaced ET-1 binding. subjected to a passive force of 2.0 g for aorta and
Downloaded from http://hyper.ahajournals.org/ at Mercer University on March 25, 2015
11-100
Supplement II Hypertension
Vol 19, No 2 February 1992
TABLE 1. Blood Pressure, Plasma Renin Activity, and EndotheUn Binding Sites in Mesenteric Blood Vessels of Deoxycorticosterone Acetate-Salt Hypertensive Rats
Group Uninephrectomized DOCA-salt hypertensive
Systolic blood pressure (mmHg) 105±3 166+4*
PRA (ng angiotensin Iml-'-hr 1 )
Plasma ET-1 (fmol/ml)
1.17±0.15 ND
1.16±0.06 1.24±0.10
B^ (fmol/mg protein) 172±19 106±22f
(pmol/1) 277±46 176±28
Values are mean±SEM. Number of binding experiments, 6. Total number of rats studied per group, 18. Total amount of protein from mesenteric arteries of each group of three rats studied in each binding experiment, 3.6±0.2 mg in controls and 4.1 ±0.4 mg in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. PRA, plasma renin activity; ET-1, endothelin-1; !}„„, density of binding sites; K4, dissociation constant; ND, not detectable. *p
