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Effects of nitro compounds, isosorbide dinitrate, 5-isosorbide mononitrate and glyceryl trinitrate on Ca-uptake into Ca-stores and Ca-release from Ca-stores in rabbit isolated femoral veins and femoral arteries
Gen. Pharmac. Vol. 18, No. 1, pp. 95-97, 1987 Printed in Great Britain
0306-3623/87 $3.00+0.00 Pergamon Journals Ltd
EFFECTS OF NITRO COMPOUNDS, ISOSORBIDE DINITRATE, 5-ISOSORBIDE MONONITRATE A N D GLYCERYL TRINITRATE ON Ca-UPTAKE INTO Ca-STORES A N D Ca-RELEASE FROM Ca-STORES IN RABBIT ISOLATED FEMORAL VEINS A N D FEMORAL ARTERIES JIRO TOYODA, TETSUHIRO HISAYAMA and ISSEI TAKAYANAGI* Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan [Tel: (0474) 72-1141]
(Received 3 April 1986) Abstract--1. In organ bath studies, effects of isosorbide dinitrate (ISDN), 5-isosorbide mononitrate (ISMN), a major metabolite of ISDN, and glyceryl trinitrate (GTN) on Ca-uptake into Ca-stores and Ca-release from Ca-stores were tested in the rabbit isolated femoral veins and femoral arteries. 2. ISDN 00 -4 M) and GTN 0 0 -4 M) inhibited Ca-uptake in the femoral veins but not in the femoral arteries. The selectivity to the femoral veins was not observed in ISMN (10 -~ M) and GTN (3 x 10 -~ M). 3. All the nitro compounds inhibited Ca-release from Ca-stores more effectively in the femoral veins than in the femoral arteries. 4. The present results may explain the selectivity of the nitro coml=.ounds to the femoral veins.
an angle of 35° with respect to the axis of the blood vessels, and suspended in a 20 ml organ bath. The composition of the organ bath solution was as follows (in raM): NaCI 154, KC1 5.6, CaC12 2.5, MgCI2 2.1, glucose 12 and HEPES/NaOH 12.5. The bath solution was gassed with oxygen giving a pH of 7.4 and kept at 37°C. The vascular preparations were attached to isometric force transducers under an initial tension of 100 rag. Responses to test drugs were recorded isometrically. To inhibit an uptake of norepinephrine, desipramine (10 -7 M) and normethanephrine (10-6M) were added to the bath solution. Propranolol (10-6M) was also added to the bath solution to block fl-adrenoceptors.
INTRODUCTION
Nitro compounds reduce the left ventricular enddiastolic pressure and volume. This action is considered to be one o f the mechanisms responsible for the antianginal effect of these compounds. The reduction in the left ventricular end-diastolic pressure and volume is thought to be due to a decrease in venous return, which is accompanied by relaxation of veins and venules (Armstrong et al., 1975; I m h o f et al., 1980; Taira et al., 1980). On the other hand, orthostatic hypotension was known as a side effect for these compounds, which is due to dilation of arteries (Ncedleman et al., 1985). It is generally recognized that myoplasmic free Ca ions play an important role in controlling the contraction-relaxation processes of smooth muscles. However, relatively little is known of effects of nitro compounds on Ca-movements in femoral veins and femoral arteries. In order to study their effects, we compared effects of nitro compounds, such as isosorbide dinitrate (ISDN), 5-isosorbide mononitrate (ISMN), a major metabolite of I S D N , and glyceryl trinitrate ( G T N ) on Ca-uptake into Ca-stores in cells and Ca-release from the stores in rabbit isolated femoral veins and femoral arteries.
I. Effects of nitro compounds on Ca-uptake into Ca-stores In order to confirm responses of vascular smooth muscle preparations to norepinephrine a control response to norepinephrine 0 0 -4 M) was obtained. After l0 rain incubation of the vascular preparations with Ca-free solution, which was made by omitting CaCl 2 from the normal bath solution and adding I mM EGTA, no response to a second application of norepinephrine (10 -4 M) was observed (Fig. 1). The preparations again responded to norepinephrine (10 -4 M) in Ca-free solution, after the preparations were incubated with CaCI2 (2.5 mM) for 1 min and immersed in Ca-free solution for 10 min. These observations suggest that the response to norepinephrine (10 -4 M) in Ca-free solution was mainly due to intracellular Ca ions. After a constant response to norepinephrine (10 -4 M) was obtained in Ca-free solution with these procedures, one of the nitro compounds was applied I min before the 1 rain incubation of the preparations with CaCI2 (2.5 mM) (Fig. 1). After the 2 min (sum of incubation times) incubation with one of the nitro compounds, the preparations were washed with Ca-free solution, equilibrated in Ca-free solution for 10 rain and norepinephrine (10 -4 M) was applied. Effects of a test drug was expressed as a ratio (%) of responses in the absence (a) and presence (b) of the test drug: b/a x I00 (Fig. 1).
MATERIALS AND M E T H O D S
Male rabbits weighing 2-3 kg were killed by a blow on the head and their femoral vein and femoral artery were excised. Helical strips, l-I.5 cm long and 1.5 mm broad, were cut at *To whom correspondence and reprint requests should be addressed. 95
JIRO TOYODA et al.
96 w T
a
b
_NC W Ca I
NE
I
I 1
I
I
I
NE
NE
NE
I
I
10
I
15
40
I 1
I 1
I 10
1 15
Fig. 1. Experimental procedures to determine effects of nitro compounds on Ca-uptake into Ca-stores. Experiments were carried out in Ca-free solution. NC: nitro compound, Ca: CaCI 2 (2.5 mM), NE: norepinephrine (10 -4 M), W: washed with Ca-free solution 1, 10 and 15: 1, I0 and 15 min, a and b: contractile responses to norepinephrine (10-4 M) in the absence (a) and presence (b) of nitro compounds. w o
xlO0
NC
W T
Ca
Ca
W
W
I NE
I
I 1
10
I
I
NE
15
I
NE
4O
I
1
I
10
, I
NE
15
I
Fig. 2. Experimental procedures to determine effects of nitro compounds on Ca-release from intracellular Ca-stores. Experiments were carried out in Ca-free solution. Ca: CaCI2 (2.5 mM), NE: norepinephrine (10-4M), W: washed with Ca-free solution, 1, I0 and 15: 15min, a and b: contractile responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of one of the nitro compounds.
2. Effects of nitro compounds on Ca-release from Ca-stores After Ca ions were stored in intracellular Ca-stores according to the procedures mentioned above, the contractile responses to norepinephrine (10 4 M) were obtained in the presence of one of the nitro compounds. Effects of test drugs were also expressed as a ratio (%) of the responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of the drugs: b/a x 100 (Fig. 2). Drugs used: Isosorbide dinitrate (ISDN; Toa Eiyo), 5-isosorbide mononitrate (ISMN; Toa Eiyo), glyceryl trinitrate (GTN; Nippon Kayaku), norepinephrine bitartrate (Wako), normethanephrine hydrochloride (Sigma), propranolol hydrochloride (Sigma), desipramine hydrochloride (Fujisawa), ethyleneglycol-bis-(fl-aminoethylether)-N,N'tetraacetic acid (EGTA; Wako) and N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; Wako). All drugs except ISDN were used as solutions in double-distilled water. ISDN was dissolved in 7% ethanol solution (the highest concentration of ethanol added to the organ bath was 0.35%, which did not affect biological responses). Other chemicals used were of analytical grade. RESULTS D o s e - i n h i b i t o r y response curves o f G T N in b o t h the vascular p r e p a r a t i o n s showed biphasic shapes
consisting o f the lower a n d higher c o m p o n e n t s while the curves o f I S D N a n d I S M N were r a t h e r m o n o phasic ( T o y o d a et al., 1986). Therefore, as the conc e n t r a t i o n s necessary for the m a x i m a l responses estim a t e d from the d o s e - r e s p o n s e curves o f I S D N a n d I S M N , a n d the lower a n d higher c o m p o n e n t s o f G T N , l0 4 M was used for I S D N , l0 -3 M for I S M N , 3 × 1 0 - 6 M for G T N in the lower c o m p o n e n t a n d l0 -4 M for G T N in the higher c o m p o n e n t ( T o y o d a et aL, 1986).
I. Effects of nitro compounds on Ca-uptake into Ca -stores I S D N (10 -4 M) a n d G T N 0 0 -4 M) inhibited Cau p t a k e in the femoral veins but n o t in the femoral arteries (Table l). Such a selectivity to the femoral veins was n o t observed in I S M N (10 -3 M ) a n d G T N (3 x l0 -6 M). (Table 1)
2. Effects o f nitro compounds on Ca-release from intracellular Ca-stores I S D N (10 4M), I S M N (10 a M ) a n d G T N (3 x l0 -6 a n d l0 4 M ) inhibited Ca-release from intracellular Ca-stores. All the nitro c o m p o u n d s used inhibited Ca-release from Ca-stores more effectively
Effects o f nitro c o m p o u n d s Table I. Effects of nitro compounds on Ca-uptake into Ca-stores Nitro compound (M)
Tissue
Ca-uptake (b/a x 100)I"
GTN 3 × 10-6
Vein Artery Vein Artery Vein Artery Vein Artery
53.1 +7.4 76.2 __.23.9 32.9 ___5.3 73.3 + 7.0 43.6 + 3.3 129.6 + 22.1 77.5 + 7.0 63.5 +_ 13.0
GTN 10-4 ISDN 10 4 ISMN 10-3
N 4 4 4 4 4 4 4 4
?A ratio (%) of the responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of the test drug. Each value is expressed as a mean _ SE. N: number of experiments. Table 2. Effects of nitro compounds on Ca-release from intraeellular Ca-stores
97
femoral arteries (Table 2). The minimum requirement of cyclic GMP for the regulation of Ca stored in the cell under physiological condition is not clarified. If cyclic GMP plays an important role in the relaxant actions of the nitro compounds, cyclic GMPproduction ability of the drugs used might be larger in the femoral veins than in the femoral arteries. ISDN (10 -4 M) and GTN (10-4M) inhibited Cauptake into Ca-stores in the femoral veins but not in the femoral arteries (Table 1). The nitro compounds, ISDN, ISMN and GTN were reported to have the selectivity to the femoral veins (Toyoda et al., 1986). The present results are considered to be available to explain the selectivity of the nitro compounds to the femoral veins.
Nitro compound (M)
Tissue
Ca-release (b/a x 100)t
N
REFERENCES
GTN 3 × 10-6
Vein Artery Vein Artery Vein Artery Vein Artery
54.1 + 3.8* 77.9 + 7.2 27.9 + 7.4* 68.4 __. 1.5 60,9 __.3.9* 89.2 _ 4.3 61,5 ___2.8* 86.7 + 8.3
4 4 4 4 4 4 4 4
Armstrong P. W., Walker D. C., Burton J. R. and Perker J. O. (1975) Vasodilator therapy in acute myocardial infarction. A comparison of sodium nitroprusside and nitroglycerin. Circulation 52, 1118-1122. Axelsson K. L., Andersson R. C. G. and Wikberg J. E. S. (1981) Correlation between vascular s m o o t h muscle relaxation and increase in cyclic G M P induced by some organic nitro esters. Acta Pharmac. Toxic 49, 270-276. Endoh M. and Taira N. (1983) Relationship between relaxation and cyclic G M P formation caused by nicorandil in canine mesenteric artery. Naunyn-Schmiedebergs Arch. Pharmac. 332, 319-321. I m h o f P. R., Ott B., Trankhauser P., C h u L. C. and Holder J. (1980) Difference in nitroglycerin dose-response in the venous and arterial beds. Eur. J. clin. Pharmac. 18, 455--460. Itoh T., K a n m u r a Y., Kuriyama H. and Sasaguri T, (1985) Nitroglycerin- and isoprenaline-induced vasodilation: assessment from the actions of cyclic nucleotides. Br. J. Pharmac. 84, 393--406. Kukovetz W. R., H a l z m a n n S., W u r m A. and Poch G. (1979) Evidence for cyclic GMP-mediated relaxant effects of nitro-compounds in coronary smooth muscle. NaunynSchmiedebergs Arch. Pharmac. 310, 129-139. Ncedleman P., Corr P. B. and John E. M. Jr (1985) Drugs used for treatment of angina; organic nitrates, calcium channel blockers and beta-adrenergic antagonists. In The Pharmacological Basis of Therapeutics (Edited by Goodm a n L. S. and Gilman A.), 7th edn. pp. 800-826. Macmillan, New York. Schultz K. D., Schultz K. and Schultz J. (1977) Sodium nitroprusside and other s m o o t h muscle relaxants increase cyclic G M P levels in rat ductus deferens. Nature 265, 750-751. Taira N., Imai Y. and Hiwatari M. (1980) Differential effects of nitroglycerin, Trimetadizine, verapamil and S K & F 2460 on venous return as revealed by the openloop method in dog. Jap. J. Pharmac. 30, 449~,61. Toyoda J., Hisayama T. and Takayanagi I. (1986) Nitro c o m p o u n d s (isosorbide dinitrate, 5-isosorbide m o n o nitrate and glyceryl trinitrate) on the femoral vein and femoral artery. Gen. Pharmac. 17, 89-91.
GTN 10-4 ISDN 10-4 ISMN 10-3
tA ratio (%) of the responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of the test drug. Each value is expressed as a mean __.SE. N: number of experiments. *Significant difference from the value for the artery at P < 0.05.
in the femoral veins than in the femoral arteries (Table 2). DISCUSSION
The mechanism of action of GTN on the smooth muscle cell of the coronary arteries of the pig is postulated to be due to a nonselective suppression of Ca-mobilization from Ca-stores with no noticeable change in membrane properties (Itoh et al., 1980). It was reported that nitro compounds including GTN and ISDN increased the amount of cyclic GMP in various vascular smooth muscles (Schultz et al., 1977; Kukovetz et al., 1979; Axelsson et al., 1981; Endoh and Taira, 1983). The main effect of cyclic GMP is activation of Ca-extrusion, thus reducing the amount of Ca stored in the cell. Itoh et al. (1985) mentioned that, if the main action of GTN is the production of cyclic GMP, GTN would reduce the free Ca and Ca stored in the cell, thus relaxing the tissue. In the present study all the nitro compounds used inhibited Ca-release from intracellular Ca-store sites. This action is thought to be due to accumulation of cyclic GMP in the cell. Furthermore, all the nitro compounds, ISDN, ISMN and GTN inhibited Ca-release more effectively in the femoral veins than in the
G,P, l S / l ~
0306-3623/87 $3.00+0.00 Pergamon Journals Ltd
EFFECTS OF NITRO COMPOUNDS, ISOSORBIDE DINITRATE, 5-ISOSORBIDE MONONITRATE A N D GLYCERYL TRINITRATE ON Ca-UPTAKE INTO Ca-STORES A N D Ca-RELEASE FROM Ca-STORES IN RABBIT ISOLATED FEMORAL VEINS A N D FEMORAL ARTERIES JIRO TOYODA, TETSUHIRO HISAYAMA and ISSEI TAKAYANAGI* Department of Chemical Pharmacology, Toho University School of Pharmaceutical Sciences, Funabashi, Chiba 274, Japan [Tel: (0474) 72-1141]
(Received 3 April 1986) Abstract--1. In organ bath studies, effects of isosorbide dinitrate (ISDN), 5-isosorbide mononitrate (ISMN), a major metabolite of ISDN, and glyceryl trinitrate (GTN) on Ca-uptake into Ca-stores and Ca-release from Ca-stores were tested in the rabbit isolated femoral veins and femoral arteries. 2. ISDN 00 -4 M) and GTN 0 0 -4 M) inhibited Ca-uptake in the femoral veins but not in the femoral arteries. The selectivity to the femoral veins was not observed in ISMN (10 -~ M) and GTN (3 x 10 -~ M). 3. All the nitro compounds inhibited Ca-release from Ca-stores more effectively in the femoral veins than in the femoral arteries. 4. The present results may explain the selectivity of the nitro coml=.ounds to the femoral veins.
an angle of 35° with respect to the axis of the blood vessels, and suspended in a 20 ml organ bath. The composition of the organ bath solution was as follows (in raM): NaCI 154, KC1 5.6, CaC12 2.5, MgCI2 2.1, glucose 12 and HEPES/NaOH 12.5. The bath solution was gassed with oxygen giving a pH of 7.4 and kept at 37°C. The vascular preparations were attached to isometric force transducers under an initial tension of 100 rag. Responses to test drugs were recorded isometrically. To inhibit an uptake of norepinephrine, desipramine (10 -7 M) and normethanephrine (10-6M) were added to the bath solution. Propranolol (10-6M) was also added to the bath solution to block fl-adrenoceptors.
INTRODUCTION
Nitro compounds reduce the left ventricular enddiastolic pressure and volume. This action is considered to be one o f the mechanisms responsible for the antianginal effect of these compounds. The reduction in the left ventricular end-diastolic pressure and volume is thought to be due to a decrease in venous return, which is accompanied by relaxation of veins and venules (Armstrong et al., 1975; I m h o f et al., 1980; Taira et al., 1980). On the other hand, orthostatic hypotension was known as a side effect for these compounds, which is due to dilation of arteries (Ncedleman et al., 1985). It is generally recognized that myoplasmic free Ca ions play an important role in controlling the contraction-relaxation processes of smooth muscles. However, relatively little is known of effects of nitro compounds on Ca-movements in femoral veins and femoral arteries. In order to study their effects, we compared effects of nitro compounds, such as isosorbide dinitrate (ISDN), 5-isosorbide mononitrate (ISMN), a major metabolite of I S D N , and glyceryl trinitrate ( G T N ) on Ca-uptake into Ca-stores in cells and Ca-release from the stores in rabbit isolated femoral veins and femoral arteries.
I. Effects of nitro compounds on Ca-uptake into Ca-stores In order to confirm responses of vascular smooth muscle preparations to norepinephrine a control response to norepinephrine 0 0 -4 M) was obtained. After l0 rain incubation of the vascular preparations with Ca-free solution, which was made by omitting CaCl 2 from the normal bath solution and adding I mM EGTA, no response to a second application of norepinephrine (10 -4 M) was observed (Fig. 1). The preparations again responded to norepinephrine (10 -4 M) in Ca-free solution, after the preparations were incubated with CaCI2 (2.5 mM) for 1 min and immersed in Ca-free solution for 10 min. These observations suggest that the response to norepinephrine (10 -4 M) in Ca-free solution was mainly due to intracellular Ca ions. After a constant response to norepinephrine (10 -4 M) was obtained in Ca-free solution with these procedures, one of the nitro compounds was applied I min before the 1 rain incubation of the preparations with CaCI2 (2.5 mM) (Fig. 1). After the 2 min (sum of incubation times) incubation with one of the nitro compounds, the preparations were washed with Ca-free solution, equilibrated in Ca-free solution for 10 rain and norepinephrine (10 -4 M) was applied. Effects of a test drug was expressed as a ratio (%) of responses in the absence (a) and presence (b) of the test drug: b/a x I00 (Fig. 1).
MATERIALS AND M E T H O D S
Male rabbits weighing 2-3 kg were killed by a blow on the head and their femoral vein and femoral artery were excised. Helical strips, l-I.5 cm long and 1.5 mm broad, were cut at *To whom correspondence and reprint requests should be addressed. 95
JIRO TOYODA et al.
96 w T
a
b
_NC W Ca I
NE
I
I 1
I
I
I
NE
NE
NE
I
I
10
I
15
40
I 1
I 1
I 10
1 15
Fig. 1. Experimental procedures to determine effects of nitro compounds on Ca-uptake into Ca-stores. Experiments were carried out in Ca-free solution. NC: nitro compound, Ca: CaCI 2 (2.5 mM), NE: norepinephrine (10 -4 M), W: washed with Ca-free solution 1, 10 and 15: 1, I0 and 15 min, a and b: contractile responses to norepinephrine (10-4 M) in the absence (a) and presence (b) of nitro compounds. w o
xlO0
NC
W T
Ca
Ca
W
W
I NE
I
I 1
10
I
I
NE
15
I
NE
4O
I
1
I
10
, I
NE
15
I
Fig. 2. Experimental procedures to determine effects of nitro compounds on Ca-release from intracellular Ca-stores. Experiments were carried out in Ca-free solution. Ca: CaCI2 (2.5 mM), NE: norepinephrine (10-4M), W: washed with Ca-free solution, 1, I0 and 15: 15min, a and b: contractile responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of one of the nitro compounds.
2. Effects of nitro compounds on Ca-release from Ca-stores After Ca ions were stored in intracellular Ca-stores according to the procedures mentioned above, the contractile responses to norepinephrine (10 4 M) were obtained in the presence of one of the nitro compounds. Effects of test drugs were also expressed as a ratio (%) of the responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of the drugs: b/a x 100 (Fig. 2). Drugs used: Isosorbide dinitrate (ISDN; Toa Eiyo), 5-isosorbide mononitrate (ISMN; Toa Eiyo), glyceryl trinitrate (GTN; Nippon Kayaku), norepinephrine bitartrate (Wako), normethanephrine hydrochloride (Sigma), propranolol hydrochloride (Sigma), desipramine hydrochloride (Fujisawa), ethyleneglycol-bis-(fl-aminoethylether)-N,N'tetraacetic acid (EGTA; Wako) and N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; Wako). All drugs except ISDN were used as solutions in double-distilled water. ISDN was dissolved in 7% ethanol solution (the highest concentration of ethanol added to the organ bath was 0.35%, which did not affect biological responses). Other chemicals used were of analytical grade. RESULTS D o s e - i n h i b i t o r y response curves o f G T N in b o t h the vascular p r e p a r a t i o n s showed biphasic shapes
consisting o f the lower a n d higher c o m p o n e n t s while the curves o f I S D N a n d I S M N were r a t h e r m o n o phasic ( T o y o d a et al., 1986). Therefore, as the conc e n t r a t i o n s necessary for the m a x i m a l responses estim a t e d from the d o s e - r e s p o n s e curves o f I S D N a n d I S M N , a n d the lower a n d higher c o m p o n e n t s o f G T N , l0 4 M was used for I S D N , l0 -3 M for I S M N , 3 × 1 0 - 6 M for G T N in the lower c o m p o n e n t a n d l0 -4 M for G T N in the higher c o m p o n e n t ( T o y o d a et aL, 1986).
I. Effects of nitro compounds on Ca-uptake into Ca -stores I S D N (10 -4 M) a n d G T N 0 0 -4 M) inhibited Cau p t a k e in the femoral veins but n o t in the femoral arteries (Table l). Such a selectivity to the femoral veins was n o t observed in I S M N (10 -3 M ) a n d G T N (3 x l0 -6 M). (Table 1)
2. Effects o f nitro compounds on Ca-release from intracellular Ca-stores I S D N (10 4M), I S M N (10 a M ) a n d G T N (3 x l0 -6 a n d l0 4 M ) inhibited Ca-release from intracellular Ca-stores. All the nitro c o m p o u n d s used inhibited Ca-release from Ca-stores more effectively
Effects o f nitro c o m p o u n d s Table I. Effects of nitro compounds on Ca-uptake into Ca-stores Nitro compound (M)
Tissue
Ca-uptake (b/a x 100)I"
GTN 3 × 10-6
Vein Artery Vein Artery Vein Artery Vein Artery
53.1 +7.4 76.2 __.23.9 32.9 ___5.3 73.3 + 7.0 43.6 + 3.3 129.6 + 22.1 77.5 + 7.0 63.5 +_ 13.0
GTN 10-4 ISDN 10 4 ISMN 10-3
N 4 4 4 4 4 4 4 4
?A ratio (%) of the responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of the test drug. Each value is expressed as a mean _ SE. N: number of experiments. Table 2. Effects of nitro compounds on Ca-release from intraeellular Ca-stores
97
femoral arteries (Table 2). The minimum requirement of cyclic GMP for the regulation of Ca stored in the cell under physiological condition is not clarified. If cyclic GMP plays an important role in the relaxant actions of the nitro compounds, cyclic GMPproduction ability of the drugs used might be larger in the femoral veins than in the femoral arteries. ISDN (10 -4 M) and GTN (10-4M) inhibited Cauptake into Ca-stores in the femoral veins but not in the femoral arteries (Table 1). The nitro compounds, ISDN, ISMN and GTN were reported to have the selectivity to the femoral veins (Toyoda et al., 1986). The present results are considered to be available to explain the selectivity of the nitro compounds to the femoral veins.
Nitro compound (M)
Tissue
Ca-release (b/a x 100)t
N
REFERENCES
GTN 3 × 10-6
Vein Artery Vein Artery Vein Artery Vein Artery
54.1 + 3.8* 77.9 + 7.2 27.9 + 7.4* 68.4 __. 1.5 60,9 __.3.9* 89.2 _ 4.3 61,5 ___2.8* 86.7 + 8.3
4 4 4 4 4 4 4 4
Armstrong P. W., Walker D. C., Burton J. R. and Perker J. O. (1975) Vasodilator therapy in acute myocardial infarction. A comparison of sodium nitroprusside and nitroglycerin. Circulation 52, 1118-1122. Axelsson K. L., Andersson R. C. G. and Wikberg J. E. S. (1981) Correlation between vascular s m o o t h muscle relaxation and increase in cyclic G M P induced by some organic nitro esters. Acta Pharmac. Toxic 49, 270-276. Endoh M. and Taira N. (1983) Relationship between relaxation and cyclic G M P formation caused by nicorandil in canine mesenteric artery. Naunyn-Schmiedebergs Arch. Pharmac. 332, 319-321. I m h o f P. R., Ott B., Trankhauser P., C h u L. C. and Holder J. (1980) Difference in nitroglycerin dose-response in the venous and arterial beds. Eur. J. clin. Pharmac. 18, 455--460. Itoh T., K a n m u r a Y., Kuriyama H. and Sasaguri T, (1985) Nitroglycerin- and isoprenaline-induced vasodilation: assessment from the actions of cyclic nucleotides. Br. J. Pharmac. 84, 393--406. Kukovetz W. R., H a l z m a n n S., W u r m A. and Poch G. (1979) Evidence for cyclic GMP-mediated relaxant effects of nitro-compounds in coronary smooth muscle. NaunynSchmiedebergs Arch. Pharmac. 310, 129-139. Ncedleman P., Corr P. B. and John E. M. Jr (1985) Drugs used for treatment of angina; organic nitrates, calcium channel blockers and beta-adrenergic antagonists. In The Pharmacological Basis of Therapeutics (Edited by Goodm a n L. S. and Gilman A.), 7th edn. pp. 800-826. Macmillan, New York. Schultz K. D., Schultz K. and Schultz J. (1977) Sodium nitroprusside and other s m o o t h muscle relaxants increase cyclic G M P levels in rat ductus deferens. Nature 265, 750-751. Taira N., Imai Y. and Hiwatari M. (1980) Differential effects of nitroglycerin, Trimetadizine, verapamil and S K & F 2460 on venous return as revealed by the openloop method in dog. Jap. J. Pharmac. 30, 449~,61. Toyoda J., Hisayama T. and Takayanagi I. (1986) Nitro c o m p o u n d s (isosorbide dinitrate, 5-isosorbide m o n o nitrate and glyceryl trinitrate) on the femoral vein and femoral artery. Gen. Pharmac. 17, 89-91.
GTN 10-4 ISDN 10-4 ISMN 10-3
tA ratio (%) of the responses to norepinephrine (10 -4 M) in the absence (a) and presence (b) of the test drug. Each value is expressed as a mean __.SE. N: number of experiments. *Significant difference from the value for the artery at P < 0.05.
in the femoral veins than in the femoral arteries (Table 2). DISCUSSION
The mechanism of action of GTN on the smooth muscle cell of the coronary arteries of the pig is postulated to be due to a nonselective suppression of Ca-mobilization from Ca-stores with no noticeable change in membrane properties (Itoh et al., 1980). It was reported that nitro compounds including GTN and ISDN increased the amount of cyclic GMP in various vascular smooth muscles (Schultz et al., 1977; Kukovetz et al., 1979; Axelsson et al., 1981; Endoh and Taira, 1983). The main effect of cyclic GMP is activation of Ca-extrusion, thus reducing the amount of Ca stored in the cell. Itoh et al. (1985) mentioned that, if the main action of GTN is the production of cyclic GMP, GTN would reduce the free Ca and Ca stored in the cell, thus relaxing the tissue. In the present study all the nitro compounds used inhibited Ca-release from intracellular Ca-store sites. This action is thought to be due to accumulation of cyclic GMP in the cell. Furthermore, all the nitro compounds, ISDN, ISMN and GTN inhibited Ca-release more effectively in the femoral veins than in the
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