Life Sciences, Vol. Printed in the USA
51, pp. 1317-1323
Pergamon Press
MANIPULATION OF DOPAMINE RECEPTORS ALTERS HYPOXIC PULMONARY VASOCONSTRICTION IN ISOLATED PERFUSED RAT LUNGS Mark J. Polak *, Lizabeth A. Kennedy **, and Wiila H. Drummond ** * Department of Pediatrics, West Virginia University School of Medicine, Health Science Center, Morgantown, WV, 26506. ** Departments of Pediatrics and Physiology, University of Florida College of Medicine, Box J-296 JHMHC, Gainesville, FL 32610. (Received in final form August 18, 1992)
Summarv
Using an isolated, perfused rat lung model, we examined the hypoxic pulmonary vasoconstriction (HPV). We studied the alterations in HPV induced by the selective DA 1 receptor agonist, fenoldopam, the selective DA1 antagonist, SCH 23390, as well as a combination of these agents. Fenoldopam significantly attenuated HPV. SCH 23390 had no effect on HPV, but was ableto block the effect of fenoldopam. These data confirm the presence of vasodilatory DA~ receptors in the pulmonary vascular bed. The data further suggest that ongoing DA 1 activity may be important in counterbalancing some pathologic pulmonary hypertensive states.
We have previously described vasodilating dopamine receptors in the pulmonary vasculature of rats. DA 1 receptor activation with fenoldopam reverses vasoconstriction induced by PGF2~ (1). The physiologic significance of vasodilating DA 1 receptors in the pulmonary vascular bed is somewhat unclear, however it is possible that these receptors may be involved in the maintenance of normal pulmonary vascular tone, counterbalancing the effects of norepinephrine, vasoactive prostanoids, and other compounds that tend to cause pulmonary vasoconstriction. We were interested in the effects of dopamine receptor manipulation on the development of hypoxic pulmonary vasoconstriction, which involves complex vascular Correspondence: Mark J. Polak, M.D., Department of Pediatrics, Health Science Center, North, West Virginia University School of Medicine, Morgantown, West Virginia 26506. USA.
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1318
Manipulation
of DA I R e c e p t o r s A l t e r H P V
Vol.
51, No.
17,
1992
interactions within the pulmonary vascular bed (2). Using an isolated per/used rat lung model, we investigated the the effects of DA 1 receptor stimulation with fenoldopam, and DA1 receptor blockade with SCH 23390, on the development of hypoxic pulmonary vasoconstriction. Material and Methods
Adult, male Sprague-Dawley rats weighing 200-380 grams were instrumented for isolated per/used lung studies. A tracheostomy was performed and mechanical ventilation begun using a Harvard rodent ventilator. The heart and lungs were exposed via a median sternotomy. A ligature was placed around the pulmonic and aortic trunks, and 200u heparin was injected into the right ventricle. A perfusion cannula was inserted into the main pulmonary artery through a small incision in the right ventricular outflow tract. The ligature was tightened around both the pulmonary artery and aorta, and an infusion of Earl's Balanced Salt Solution, with 21 mM NaHCO 3, 4% Ficoll, and 30 t~M meclofenamate was begun. The perfusate pH was adjusted to 7.40. The left ventricle was incised and a second cannula was introduced into the left atrium through the mitral valve and fixed into position with a ligature around both ventricles. The perfusate was pumped from a syringe reservoir, at constant flow rate greater than 50 mL/kg,min -~, through a heat exchanger (temp =37 ° C) and into the pulmonary artery, where it circulated through the lungs and returned to the reservoir via the left atrial catheter. Mean pulmonary artery pressure, and mean left atrial pressure, were continuously measured by pressure transducers connected to a physiologic recorder. Ventilatory gases (normoxic and hypoxic) were blended in a SiMet gas blender, humidified, and connected to the ventilator. Study Drugs Fenoldopam mesylate (SKF-82526, Smith, Kline, and Beecham Laboratories, Philadelphia Pa.) is a benzapine compound developed as an antihypertensive agent. It is a selective DA 1 agonist (3, 4). For DA 1 stimulation experiments, fenoldopam was added to the circulating perfusate to to give a concentration of 10 I~M. SCH 23390 (Schering Pharmaceuticals, Bloomfield, NJ) is a highly selective DA 1 receptor antagonist (5, 6). We have shown in the isolated perfused lung that a I~M concentration of SCH 23390 attenuates fenoldopam induced vasodilation by greater than 60% (1). ExPerimental Protocols The protocols, detailed in the table, were designed to investigate the effects of DA 1 receptor manipulation on the development of hypoxic pulmonary vasoconstriction. After the lungs were instrumented for study, the perfusate flow rate was set to between 50-56 mL/kg,min -1 and LAP was adjusted to 0 mm Hg. The lungs were ventilated with a room
Vol. 51, No. 17, 1992
Manipulation of DA I Receptors Alter HPV
1319
air mixture ( 0 2 = 21%, N 2 = 74%, CO 2 = 5%). SCH 23390 (1 p.M) or physiologic saline was added to the perfusate. After 3 minutes, 0, or 10.0 I~M fenolodopam was added to the perfusate and allowed to circulate. After another 3 minutes, PAP was recorded, and a sample of the perfusate was analyzed for pH and PO 2. A period of hypoxia was begun ( 02= 0%, N2 = 95%, 0 0 2 = 5% ). After six minutes, the change in PAP was recorded and perfusate was again sampled for pH and PO2. Ventilation with room air gas mixture was resumed. After a final 3 minute period of normoxia, a third perfusate sample was analyzed for pH and PO 2, the PAP was recorded, and the perfusion was terminated. At the end of the experiment the lungs were removed, weighed, dried in a drying oven and reweighed. Wet-to-dry lung weight ratios were calculated as a determination of lung edema. From our previous experience the wet/dry weight ratio ranges from 4-8 under conditions where edema development is minimal (1,7). Table 1 Outline of experimental protocols, stability data, and perfusate DH and PO~ for exoeriments Protocols Control fenoldopam SCH 23390 fen + SCH 10 I~M 1 ~M n 5 5 5 5 age 53.4_+0.09 63.5+1.4 57.0+1.9 55.6+3.7 (days) weight 2 4 8 . 4 + _ 2 1 . 0294.8-+9.1 236.0+11.1 292.0-+29.0 (grams) flow 52.3+0.6 53.1+0.8 52.9+0.3 51.8-+0.6 (ml_/kg/min) baseline PAP 8.6 + 0.6 7.9 + 0.6 8.8 + 0.7 9.6 +- 0.8 (mm Hg) W/D (lung 5 . 7 + - 0 . 0 9 5.2+0.2 5.3+0.2 5.2+-0.1 weight ratio) pH (pre) 7.34+0.02 7.33-+0.01 7.31+0.04 7.35+0.03 pH (hyp.) 7 . 2 8 _ + 0 . 0 4 7.25+0.01 7.24+0.06 7.25+0.03 p H (rec.) 7 . 3 7 + _ 0 . 0 5 7.30+0.01 7.27-+0.04 7.33-+0.04 P O 2 (pre) 1 4 8 . 6 + - 4 . 5 150.7+0.3 151.6+7.0 159.1+10.9
P 02 (hyp.) 70.9+7.9 * 70.3-+3.7 * 52.4-+4.3 * 74.5+9.0 * P 02 (rec.) 135.4+6.2 134.5+4.8 148.4+20 166.0+13.8 (all P02 measured in torr.) * hypoxic PO2, significantly (p < 0.05) different from baseline (pre) and recovery_ PO 2 as analvzed bv multifactor ANOVA for reoeated measures. Hypoxic pulmonary vasoconstriction under control conditons ( no fenoldopam, no SCH 23390 ) was considered the maximum, or reference value and was assigned a value of 1.0. Vasoconstriction attained with fenoldopam and/or SCH 23390 added was
1320
Manipulation
of DA I R e c e p t o r s
Alter HPV
Vol.
51, No.
17,
1992
considered as a percentage of the reference. Data was analyzed by single factor ANOVA, with treatment (control, fenoldopam, SCH 23390, fenoldopam + SCH 23390 ) as the independent variable and percent change in PAP as the dependent variable. The alpha level of significance was 0.05. All data presented as mean + S.E.M.
Results A total of 20 experiments were performed. The lung preparations remained stable throughout the experiments regardless of treatment group. Data detailing ages, weights, flow rates, wet/dry lung weight ratios and perfusate pH and PO 2 are detailed in the table. In control experiments, hypoxia caused an increase in PAP of 6.2 + 0.5 mm Hg. The percent change in PAP for the dopaminergic treatment groups compared to control are detailed in the figure. Pretreatment with fenoldopam significantly attenuated the development of HPV. Pretreatment with SCH 23390 does not affect HPV, and also blocks the attenuating effect of fenoldopam (SCH 23390+ fenoldopam). 160 {3_
51, pp. 1317-1323
Pergamon Press
MANIPULATION OF DOPAMINE RECEPTORS ALTERS HYPOXIC PULMONARY VASOCONSTRICTION IN ISOLATED PERFUSED RAT LUNGS Mark J. Polak *, Lizabeth A. Kennedy **, and Wiila H. Drummond ** * Department of Pediatrics, West Virginia University School of Medicine, Health Science Center, Morgantown, WV, 26506. ** Departments of Pediatrics and Physiology, University of Florida College of Medicine, Box J-296 JHMHC, Gainesville, FL 32610. (Received in final form August 18, 1992)
Summarv
Using an isolated, perfused rat lung model, we examined the hypoxic pulmonary vasoconstriction (HPV). We studied the alterations in HPV induced by the selective DA 1 receptor agonist, fenoldopam, the selective DA1 antagonist, SCH 23390, as well as a combination of these agents. Fenoldopam significantly attenuated HPV. SCH 23390 had no effect on HPV, but was ableto block the effect of fenoldopam. These data confirm the presence of vasodilatory DA~ receptors in the pulmonary vascular bed. The data further suggest that ongoing DA 1 activity may be important in counterbalancing some pathologic pulmonary hypertensive states.
We have previously described vasodilating dopamine receptors in the pulmonary vasculature of rats. DA 1 receptor activation with fenoldopam reverses vasoconstriction induced by PGF2~ (1). The physiologic significance of vasodilating DA 1 receptors in the pulmonary vascular bed is somewhat unclear, however it is possible that these receptors may be involved in the maintenance of normal pulmonary vascular tone, counterbalancing the effects of norepinephrine, vasoactive prostanoids, and other compounds that tend to cause pulmonary vasoconstriction. We were interested in the effects of dopamine receptor manipulation on the development of hypoxic pulmonary vasoconstriction, which involves complex vascular Correspondence: Mark J. Polak, M.D., Department of Pediatrics, Health Science Center, North, West Virginia University School of Medicine, Morgantown, West Virginia 26506. USA.
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1318
Manipulation
of DA I R e c e p t o r s A l t e r H P V
Vol.
51, No.
17,
1992
interactions within the pulmonary vascular bed (2). Using an isolated per/used rat lung model, we investigated the the effects of DA 1 receptor stimulation with fenoldopam, and DA1 receptor blockade with SCH 23390, on the development of hypoxic pulmonary vasoconstriction. Material and Methods
Adult, male Sprague-Dawley rats weighing 200-380 grams were instrumented for isolated per/used lung studies. A tracheostomy was performed and mechanical ventilation begun using a Harvard rodent ventilator. The heart and lungs were exposed via a median sternotomy. A ligature was placed around the pulmonic and aortic trunks, and 200u heparin was injected into the right ventricle. A perfusion cannula was inserted into the main pulmonary artery through a small incision in the right ventricular outflow tract. The ligature was tightened around both the pulmonary artery and aorta, and an infusion of Earl's Balanced Salt Solution, with 21 mM NaHCO 3, 4% Ficoll, and 30 t~M meclofenamate was begun. The perfusate pH was adjusted to 7.40. The left ventricle was incised and a second cannula was introduced into the left atrium through the mitral valve and fixed into position with a ligature around both ventricles. The perfusate was pumped from a syringe reservoir, at constant flow rate greater than 50 mL/kg,min -~, through a heat exchanger (temp =37 ° C) and into the pulmonary artery, where it circulated through the lungs and returned to the reservoir via the left atrial catheter. Mean pulmonary artery pressure, and mean left atrial pressure, were continuously measured by pressure transducers connected to a physiologic recorder. Ventilatory gases (normoxic and hypoxic) were blended in a SiMet gas blender, humidified, and connected to the ventilator. Study Drugs Fenoldopam mesylate (SKF-82526, Smith, Kline, and Beecham Laboratories, Philadelphia Pa.) is a benzapine compound developed as an antihypertensive agent. It is a selective DA 1 agonist (3, 4). For DA 1 stimulation experiments, fenoldopam was added to the circulating perfusate to to give a concentration of 10 I~M. SCH 23390 (Schering Pharmaceuticals, Bloomfield, NJ) is a highly selective DA 1 receptor antagonist (5, 6). We have shown in the isolated perfused lung that a I~M concentration of SCH 23390 attenuates fenoldopam induced vasodilation by greater than 60% (1). ExPerimental Protocols The protocols, detailed in the table, were designed to investigate the effects of DA 1 receptor manipulation on the development of hypoxic pulmonary vasoconstriction. After the lungs were instrumented for study, the perfusate flow rate was set to between 50-56 mL/kg,min -1 and LAP was adjusted to 0 mm Hg. The lungs were ventilated with a room
Vol. 51, No. 17, 1992
Manipulation of DA I Receptors Alter HPV
1319
air mixture ( 0 2 = 21%, N 2 = 74%, CO 2 = 5%). SCH 23390 (1 p.M) or physiologic saline was added to the perfusate. After 3 minutes, 0, or 10.0 I~M fenolodopam was added to the perfusate and allowed to circulate. After another 3 minutes, PAP was recorded, and a sample of the perfusate was analyzed for pH and PO 2. A period of hypoxia was begun ( 02= 0%, N2 = 95%, 0 0 2 = 5% ). After six minutes, the change in PAP was recorded and perfusate was again sampled for pH and PO2. Ventilation with room air gas mixture was resumed. After a final 3 minute period of normoxia, a third perfusate sample was analyzed for pH and PO 2, the PAP was recorded, and the perfusion was terminated. At the end of the experiment the lungs were removed, weighed, dried in a drying oven and reweighed. Wet-to-dry lung weight ratios were calculated as a determination of lung edema. From our previous experience the wet/dry weight ratio ranges from 4-8 under conditions where edema development is minimal (1,7). Table 1 Outline of experimental protocols, stability data, and perfusate DH and PO~ for exoeriments Protocols Control fenoldopam SCH 23390 fen + SCH 10 I~M 1 ~M n 5 5 5 5 age 53.4_+0.09 63.5+1.4 57.0+1.9 55.6+3.7 (days) weight 2 4 8 . 4 + _ 2 1 . 0294.8-+9.1 236.0+11.1 292.0-+29.0 (grams) flow 52.3+0.6 53.1+0.8 52.9+0.3 51.8-+0.6 (ml_/kg/min) baseline PAP 8.6 + 0.6 7.9 + 0.6 8.8 + 0.7 9.6 +- 0.8 (mm Hg) W/D (lung 5 . 7 + - 0 . 0 9 5.2+0.2 5.3+0.2 5.2+-0.1 weight ratio) pH (pre) 7.34+0.02 7.33-+0.01 7.31+0.04 7.35+0.03 pH (hyp.) 7 . 2 8 _ + 0 . 0 4 7.25+0.01 7.24+0.06 7.25+0.03 p H (rec.) 7 . 3 7 + _ 0 . 0 5 7.30+0.01 7.27-+0.04 7.33-+0.04 P O 2 (pre) 1 4 8 . 6 + - 4 . 5 150.7+0.3 151.6+7.0 159.1+10.9
P 02 (hyp.) 70.9+7.9 * 70.3-+3.7 * 52.4-+4.3 * 74.5+9.0 * P 02 (rec.) 135.4+6.2 134.5+4.8 148.4+20 166.0+13.8 (all P02 measured in torr.) * hypoxic PO2, significantly (p < 0.05) different from baseline (pre) and recovery_ PO 2 as analvzed bv multifactor ANOVA for reoeated measures. Hypoxic pulmonary vasoconstriction under control conditons ( no fenoldopam, no SCH 23390 ) was considered the maximum, or reference value and was assigned a value of 1.0. Vasoconstriction attained with fenoldopam and/or SCH 23390 added was
1320
Manipulation
of DA I R e c e p t o r s
Alter HPV
Vol.
51, No.
17,
1992
considered as a percentage of the reference. Data was analyzed by single factor ANOVA, with treatment (control, fenoldopam, SCH 23390, fenoldopam + SCH 23390 ) as the independent variable and percent change in PAP as the dependent variable. The alpha level of significance was 0.05. All data presented as mean + S.E.M.
Results A total of 20 experiments were performed. The lung preparations remained stable throughout the experiments regardless of treatment group. Data detailing ages, weights, flow rates, wet/dry lung weight ratios and perfusate pH and PO 2 are detailed in the table. In control experiments, hypoxia caused an increase in PAP of 6.2 + 0.5 mm Hg. The percent change in PAP for the dopaminergic treatment groups compared to control are detailed in the figure. Pretreatment with fenoldopam significantly attenuated the development of HPV. Pretreatment with SCH 23390 does not affect HPV, and also blocks the attenuating effect of fenoldopam (SCH 23390+ fenoldopam). 160 {3_
