- Email: [email protected]
Study of the mechanism behind the relaxing effect of furosemide on vascular smooth muscle
1273
thickness of the spleen, liver and sartorius muscle, and the rate of rise in arterial pressure pulses in the caudal portion of abdominal aorta (Adp/dt). GTN given sublingnally (s.i.), did not effectively dilate the venous system which revealed by calibrating the diameter of the vena cava and the thickness of the liver, spleen and sartorius muscle. This was also supported indirectly by the observation that GTN never reduced CO and BFV. GTN s.l. essentially produced no effect on coronary resistance vessels, e.g. CBF was not increased, but efficiently dilated the large coronary artery. This action was apparent not only in coronary artery but also in all artery tested. It is therefore, proposed that this action of GTN can be called "selective conductance a~erial dilation". GTN s.l. did not essentially reduce the preload, as far as changes in heart rate (HR). and CO concerned. Reduction of preload, decrease of LVID with reduction of LVEDP, was noted only in cases exhibiting a moderate fall in systolic pressure with concomitant increase in HR and CO with a moderate large dose. Thus, the reduction of preload by GTN is a secondary feature resulting from shift of the blood in heart, lung and vein to the arterial side by the baroreceptor reflexes mediated cardio-accerelation in response to hypotension in normal subjects. Intravenous GTN caused increases in HR, CO and CBF and reductions in BP, LVEDP and LVlD transiently, suggesting participation of dilation of resistance vessels exposed to high concentration of GTN, and increases in arterial diameters lasted several minutes with decreasing Adp/dt and systolic pressure indicating increase in the volume of Windkessel. The present results indicate that the prime vascular action of GTN s.l. in a certain limited dose range is solely conductance arterial dilation. Other actions such as reduction of LVID and LVEDP and increase of HR and CO are mainly derived from the vasoreceptor reflexes and suggest that the therapeutically important effects of GTN in angina pectoris involve selective dilation of conductance vessels, including both coronary and general systemic arteries. The effect of the former would act directly on stenotic lesions in the epicardiai large coronary artery, improving blood flow to the ischemic part of the myocardium, whereas the latter might be beneficial for reducing cardiac work load and lowering systolic pressure by increasing the arterial compliance. Therefore, the heart regains its ventricular performance and propels stagnated blood in the heart, lungs and veins to the arterial side, resulting in a reduction of the elevated preload in anginal patients. This work was supported by a research grant from the Japanese Ministry of Education.
IpwoHsl Study of the mechanism behind the relaxing effect of furosemide on vascular smooth muscle Tian, R., Aalkjeer, C. and Andreasen, F. Institute of Pharmacology, University of Aarhus, DK-8000, Aarhus C, Denmark
A relaxing effect of furosemide on vascular smooth muscle in vitro has been observed although the mechanism responsible is only partly understood. It has been suggested by Deth et al. (1987) and Kreye et al. (1981) that furosemide via the inhibition of the Na ÷, K ÷, Cl--cotransport causes membrane hyperpolarization and consequent suppression of contraction. The present study was intended to evaluate the importance of the endothelium, the membrane potential and the transmembrane ion movement for the in vitro effect of furosemide. Se~,nients of the rabbit central ear artery (CEA) were mounted in a tissue bath with physiological saline solution (PSS) gassed with carbogen at 37°C and p H - 7.4. The effect of furosemide on isometric contraction was studied. Results are expressed as mean :l: s.e.m.(number of segments). Furosemide (0.06 mM-1.0 raM) caused a dose-dependent inhibition of contraction (8.7 :t: 1.5~ (10) to 33.3 :t: 7.7~ (8)) to electrical field stimulation. Removal of the endothelium did not affect the force development nor the relaxing effect of furosemide (p > 0. 05). A constant depression of contraction by furosemide was seen over the entire range of the K ÷ depolarization dose-response curve (p < 0.05). In fully depolarized segments ([K ÷] - 1 2 0 mM) furosemide (0.12 mM or 1.0 mM) did not alter the sensitivity of the vessel segments to external [Ca +*] a|though the maximal
1274 contraction was still suppressed by furosemide as expected. Nimodipine (5 ltM) however, caused a significant decrease in sensitivity (p < 0.05). S6Rb uptake by the vessel segments was measured by loading in S6Rb-PSS for 10 minutes. Both furosemide and ouabain (1.0 raM) reduced SeRb uptake significantly (p < 0.01). No statistically significant interaction between the two drugs was found (ANOVA, P > 0.5). Furthermore, no difference was found between the effects of 0.12 mM and 1.0 mM furosemide either when furosemide was used alone or in combination with ouabain (p > 0.5). In summary, we have shown a direct relaxing effect of furosemide on the isolated rabbit CEA which is not endothelium-dependent. Although the results of S6Rb uptake in this study were consistent with previous reports, the constant relaxing effect during K + depol~.-izafion observed here does not support the assumption that membrane hyperpolarization due to the inhibition of Na+, K+, CI-~cotransport is the sole m~ha_nJsm responsible for the effect of fumsemide. In addition, the evidence that relaxation increased over the dose range where no further inhibition of S6Rb uptake was achieved further suggests that a mechanism unrelated to the inhibition of the Na+, K +, CI--cotransport is involved in the vascular effect of furose~ide although a direct blockade of Ca++ entry channels seems to be unlikely.
Regerences Deth, R.C., R.A. Payn¢ and P.M. Peecher, 1987, Influence of furosemide on Rubidium-86 uptake and alpha-adrenergic responsivehess of arterial smooth muscle, Blood Vessels 24, 321. Kreye V.A.W., P.K. Bauer and I. Villhauer, 1981, Evidence for furosemide-sensifive active chloride transport in vascular smooth muscle, European J. Pharmacol. 73, 91.
5-Hydroxyt~ptamine receptor dissociation constant, K A, increases but receptor reserve decreases with age in isolated rat coronary arteries Nyborg, N.C.B. a n d Mikkelsen, E.O. Department of Pharmacology. Aarhus University, DK-8000 Aarhus C, Denmark
5-Hydroxytryptamine (5-HT) is an important coronary vasoconstrictor in normal as well as in atherosclerotic arteries. We have previously shown that rat coronary sensitivity and contraction to 5-hydroxytryptamine increases with age (Nyborg and Mikkelsen, 1988). The present experiments were designed to elucidate whether or not the change in vascular sensitivity was associated with changes in 5-HT receptor dissociation constant, KA, and/or receptor reselNe.
Hearts were taken from 3 months and 2 years old male Wistar rats. Ring segments from the proximal, epicardial, and distal, intramural, part of the left coronary artery were dissected free and mounted in a physiological saline solution, oxygenated with 5 CO2 in 02, at 37°C, pH 7.4, on an isometric myograph, which allowed direct measurement of vessel wall forces while the internal circumference was controlled. The maximal artery contractile capacity, Em~x, was determined for each vessel as the difference in vessel wall force when fully contracted and relaxed in Ca 2+-free PSS. Integrety of the endothelium was assessed by measuring the relaxing effect of 10-5 M acetylcholine (Ach) on 10 -s M PGF2~, precontracted vessels. 5-HT receptor dissociation constant, KA, was determined by partial irreversible ~ b i 6 o n of 5-HT receptors with 3-10 -s M phenoxybenzamine (PBZ) for 15 rain. A decrease in maximal vessel response to 5-HT to 70-80~ of control after PBZ was sought. K A was determined from computerized linear regression analysis of reciprocals of eq,.deffective concentrations of 5-HT before and after PBZ treatment, where KA = (slope - 1)/intercept. 5-HT uptake in neurons and smooth muscle was blocked with 3- 10 -6 M cocaine. The relative 5-HT receptor occupancy, RpfRT, was calculated for each vessel from the equation, RA/RT = A / ( A + K^), where A is concentration of 5-HT required to give half maximal response, ECs0(M), and K^ is dissociation constant, calculated as stated above. 5-HT does not induce release of endothelium derived relaxing factor in rat coronary arteries. The data were analyzed using a two-way analysis of variance with significance level set at p < 0.05. The maximal response induced by 5-HT increased (p < 0.05) with age and was greater in proximal than in the distal
thickness of the spleen, liver and sartorius muscle, and the rate of rise in arterial pressure pulses in the caudal portion of abdominal aorta (Adp/dt). GTN given sublingnally (s.i.), did not effectively dilate the venous system which revealed by calibrating the diameter of the vena cava and the thickness of the liver, spleen and sartorius muscle. This was also supported indirectly by the observation that GTN never reduced CO and BFV. GTN s.l. essentially produced no effect on coronary resistance vessels, e.g. CBF was not increased, but efficiently dilated the large coronary artery. This action was apparent not only in coronary artery but also in all artery tested. It is therefore, proposed that this action of GTN can be called "selective conductance a~erial dilation". GTN s.l. did not essentially reduce the preload, as far as changes in heart rate (HR). and CO concerned. Reduction of preload, decrease of LVID with reduction of LVEDP, was noted only in cases exhibiting a moderate fall in systolic pressure with concomitant increase in HR and CO with a moderate large dose. Thus, the reduction of preload by GTN is a secondary feature resulting from shift of the blood in heart, lung and vein to the arterial side by the baroreceptor reflexes mediated cardio-accerelation in response to hypotension in normal subjects. Intravenous GTN caused increases in HR, CO and CBF and reductions in BP, LVEDP and LVlD transiently, suggesting participation of dilation of resistance vessels exposed to high concentration of GTN, and increases in arterial diameters lasted several minutes with decreasing Adp/dt and systolic pressure indicating increase in the volume of Windkessel. The present results indicate that the prime vascular action of GTN s.l. in a certain limited dose range is solely conductance arterial dilation. Other actions such as reduction of LVID and LVEDP and increase of HR and CO are mainly derived from the vasoreceptor reflexes and suggest that the therapeutically important effects of GTN in angina pectoris involve selective dilation of conductance vessels, including both coronary and general systemic arteries. The effect of the former would act directly on stenotic lesions in the epicardiai large coronary artery, improving blood flow to the ischemic part of the myocardium, whereas the latter might be beneficial for reducing cardiac work load and lowering systolic pressure by increasing the arterial compliance. Therefore, the heart regains its ventricular performance and propels stagnated blood in the heart, lungs and veins to the arterial side, resulting in a reduction of the elevated preload in anginal patients. This work was supported by a research grant from the Japanese Ministry of Education.
IpwoHsl Study of the mechanism behind the relaxing effect of furosemide on vascular smooth muscle Tian, R., Aalkjeer, C. and Andreasen, F. Institute of Pharmacology, University of Aarhus, DK-8000, Aarhus C, Denmark
A relaxing effect of furosemide on vascular smooth muscle in vitro has been observed although the mechanism responsible is only partly understood. It has been suggested by Deth et al. (1987) and Kreye et al. (1981) that furosemide via the inhibition of the Na ÷, K ÷, Cl--cotransport causes membrane hyperpolarization and consequent suppression of contraction. The present study was intended to evaluate the importance of the endothelium, the membrane potential and the transmembrane ion movement for the in vitro effect of furosemide. Se~,nients of the rabbit central ear artery (CEA) were mounted in a tissue bath with physiological saline solution (PSS) gassed with carbogen at 37°C and p H - 7.4. The effect of furosemide on isometric contraction was studied. Results are expressed as mean :l: s.e.m.(number of segments). Furosemide (0.06 mM-1.0 raM) caused a dose-dependent inhibition of contraction (8.7 :t: 1.5~ (10) to 33.3 :t: 7.7~ (8)) to electrical field stimulation. Removal of the endothelium did not affect the force development nor the relaxing effect of furosemide (p > 0. 05). A constant depression of contraction by furosemide was seen over the entire range of the K ÷ depolarization dose-response curve (p < 0.05). In fully depolarized segments ([K ÷] - 1 2 0 mM) furosemide (0.12 mM or 1.0 mM) did not alter the sensitivity of the vessel segments to external [Ca +*] a|though the maximal
1274 contraction was still suppressed by furosemide as expected. Nimodipine (5 ltM) however, caused a significant decrease in sensitivity (p < 0.05). S6Rb uptake by the vessel segments was measured by loading in S6Rb-PSS for 10 minutes. Both furosemide and ouabain (1.0 raM) reduced SeRb uptake significantly (p < 0.01). No statistically significant interaction between the two drugs was found (ANOVA, P > 0.5). Furthermore, no difference was found between the effects of 0.12 mM and 1.0 mM furosemide either when furosemide was used alone or in combination with ouabain (p > 0.5). In summary, we have shown a direct relaxing effect of furosemide on the isolated rabbit CEA which is not endothelium-dependent. Although the results of S6Rb uptake in this study were consistent with previous reports, the constant relaxing effect during K + depol~.-izafion observed here does not support the assumption that membrane hyperpolarization due to the inhibition of Na+, K+, CI-~cotransport is the sole m~ha_nJsm responsible for the effect of fumsemide. In addition, the evidence that relaxation increased over the dose range where no further inhibition of S6Rb uptake was achieved further suggests that a mechanism unrelated to the inhibition of the Na+, K +, CI--cotransport is involved in the vascular effect of furose~ide although a direct blockade of Ca++ entry channels seems to be unlikely.
Regerences Deth, R.C., R.A. Payn¢ and P.M. Peecher, 1987, Influence of furosemide on Rubidium-86 uptake and alpha-adrenergic responsivehess of arterial smooth muscle, Blood Vessels 24, 321. Kreye V.A.W., P.K. Bauer and I. Villhauer, 1981, Evidence for furosemide-sensifive active chloride transport in vascular smooth muscle, European J. Pharmacol. 73, 91.
5-Hydroxyt~ptamine receptor dissociation constant, K A, increases but receptor reserve decreases with age in isolated rat coronary arteries Nyborg, N.C.B. a n d Mikkelsen, E.O. Department of Pharmacology. Aarhus University, DK-8000 Aarhus C, Denmark
5-Hydroxytryptamine (5-HT) is an important coronary vasoconstrictor in normal as well as in atherosclerotic arteries. We have previously shown that rat coronary sensitivity and contraction to 5-hydroxytryptamine increases with age (Nyborg and Mikkelsen, 1988). The present experiments were designed to elucidate whether or not the change in vascular sensitivity was associated with changes in 5-HT receptor dissociation constant, KA, and/or receptor reselNe.
Hearts were taken from 3 months and 2 years old male Wistar rats. Ring segments from the proximal, epicardial, and distal, intramural, part of the left coronary artery were dissected free and mounted in a physiological saline solution, oxygenated with 5 CO2 in 02, at 37°C, pH 7.4, on an isometric myograph, which allowed direct measurement of vessel wall forces while the internal circumference was controlled. The maximal artery contractile capacity, Em~x, was determined for each vessel as the difference in vessel wall force when fully contracted and relaxed in Ca 2+-free PSS. Integrety of the endothelium was assessed by measuring the relaxing effect of 10-5 M acetylcholine (Ach) on 10 -s M PGF2~, precontracted vessels. 5-HT receptor dissociation constant, KA, was determined by partial irreversible ~ b i 6 o n of 5-HT receptors with 3-10 -s M phenoxybenzamine (PBZ) for 15 rain. A decrease in maximal vessel response to 5-HT to 70-80~ of control after PBZ was sought. K A was determined from computerized linear regression analysis of reciprocals of eq,.deffective concentrations of 5-HT before and after PBZ treatment, where KA = (slope - 1)/intercept. 5-HT uptake in neurons and smooth muscle was blocked with 3- 10 -6 M cocaine. The relative 5-HT receptor occupancy, RpfRT, was calculated for each vessel from the equation, RA/RT = A / ( A + K^), where A is concentration of 5-HT required to give half maximal response, ECs0(M), and K^ is dissociation constant, calculated as stated above. 5-HT does not induce release of endothelium derived relaxing factor in rat coronary arteries. The data were analyzed using a two-way analysis of variance with significance level set at p < 0.05. The maximal response induced by 5-HT increased (p < 0.05) with age and was greater in proximal than in the distal