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Endothelial cells modulate the vasoinhibitory effect of NKY-722, a Ca2+channel antagonist., in canine mesenteric arteries
Europeari Jowrzal of Phurmacologv, 196 ! 199!) 197- 199 0 1991 Elsevier Science Publishers B.V. OOM-2999/91/%03.50 ADONIS 0014299991OO321J
197
Short communication
Hisanobu Shimaji, Hiroaki Shirahase ‘, Mamoru Kanda I, Seimei Osumi ’ and Kazuyoshi Kurahasti Pharmacology
Di~~isis,or.Radioisofupe
Research Center, Kyo~o Universi
kd.,
Kyoro 604, Japan
Received 11 Ekcember 1990. accepted 26 February 1991
Modulation of the vasoinhibitory effect of NKY-722 by vascular endothelial cells was studied in canine mesenteric arteries. A high conctiIltration of NKY-722 accumulated in the endothelium-intact arteries and its accumulation in endothelium-removed arteries was significantly less. The vasoinhibitory effect of NKY-722 in endothelium-intact arteries was significantly weaker than that in endothelium-removed arteries. These results suggest that endothelial cells can attenuate the vasoinhibitory effect of NKY-722.
Endothelial cell (vascular); NKY-722; Ca *+ channel antagonist; (Accumulation)
1. Introduction
thelial cells can modulate the vasoinhibitory NKY-722 in canine mesenteric arteries.
effect of
In vascular smooth muscle, nitrendipine Sperelakis, Sperelakis,
1984). verapamil, 1983), and benidipine
(Pang and bepridil (Pang and (Karasawa and Kubo.
1988) are accumulated, while nifedipine and diltiazem are not accumulated (Pang and Sperelakis. 1984). Recently, we reported that 3-(4-allyl-1-piperazinyl)-2,2-dimethylpropyl-3,5-pyridine dicarboxylate dihydrochloride (NKY - ILL), a wa:er-soluble vasoselective Ca” antagonist, showed persistent accumuialion in canine mesenteric arteries (Shimaji et al., 1990). NKY-722 exerts a potent and long-iasting antihypertensive effect in spon!aneously hypertensive rats and has a vasoselective effect in isolated, blood-perfused canine heart preparations (Imagawa et al., 1989; Osumi et al., 1988; 1990). We have previously suggested that the accumulation of NKY-722 in the vascular wall may contribute to the long duration of its antihypertensive effect (Shimaji et al., 1990). It has not yet been reported whether accumulation of Ca2+ antagonists in the vascular wall can be influenced by endothelial cells. The present experiments were undertaken to investigate whether or not endo-
Correspondence to: K. Kurahashi, Pharmacology Division. Radioisotope Research Center, Kyoto University, Kyoto 606. Japan.
2. Materials and methods 2.1. Accumultirion
of NKY-722
in endothelium-intact
or
-removed canine mesenteric arteries
Mongrel dogs of either sex weighing lo-24 kg were bled to death from the common carotid arteries under sodium pentobarbital anesthesia (25 mg/kg i.v.). The mesenteric arterics were then removed and were cut into helical strips. Endothelium-in!act or -removed nesenteric arteries were incubared for 6ti min at 37OC in Krebs-Henseleit solution bubbled with a &xtu:e of 95% O,-5% CO, containing lo-’ M (0.1 pCi/ml), iO_’ M (0.01 pCi/ml), or 10m9 M (0.001 @i/ml) NKY-722. The solution consisted of (mM): NaCl 120; KC1 4.7; MgSO,, 1.2; KH2P04 1.2; CaCl, 2.5; NaHCO, 25; and glucose 10. The preparations were then incubated for 10 min in ice-cold Krebs-Henseleit solution, gently blotted with fiiter paper, weighed, and solubilized overnight at room temperature with 3 ml of Foluene@-350 (Packard Japan Co. Ltd.. Tokyo, Japan). The solubilized preparations were mixed in vials with 1 mi of acetic acid and 10 ml of the scintillation cocktail. The radioactivity of the solubilized preparations was determined using a liquid
nter gAloka LSC-900. Aloka Co. Ltd., Accumulation of NKY-722 was ex~~e~rn~iurn (T/M) ratioe of the changes in the vasoinhibitolr3: effect 722
in e~dot~~~~~-int~~t or -removed canine
ArteriJ strips were fixed vertically between hooks in an organ bath containing Krebs-Henseleit solution, which was maintained at 37OC and bubbled with a O,-58 CO,. The ends of the strips were attached to the lever of a Fo~e~spla~ment transducer (NIX San-Ei instrument Co. Ltd., Tokyo, Japan), which was connected to an ink-writing oscillograph (NIX SanEi Inst~ment Co. Ltd., Tokyo, Japan} on which the isometric tension changes were recorded. The applied tension was adjusted to 1.5 g. Mesenteric arteries were incubate with NKY-722 {lO_‘, 10-a. or 10s7 Ml for 60 mitt and then washed out repeatedly for 30. 90, 180, or 300 min. KC1 (30 mM) was applied to the arteries after each wash-out.
2.3. Drugs NKY-722 was a gift from Kyoto Pharmaceutical Industries, Ltd. (Kyoto, Japan). The [3HlllKY-722 (88 Ci/mmol) was tritiated by Amersham International Plc. ~Buc~n~ams~re, U.K.). 2.4. Statistics All data were expressed as means f S.E.M. and statistical significance was assessed using Student’s t-test for unpaired data.
0
I 01,
in endotheIj~m-intact
(6)
‘
10-Q
10-s
10”
Coon. of NKY-722 (M) Fig. 1. Accumulation (T/M ratio) of NKY-722 in endothe~um-intact or -removed canine mesenteric arteries. The number of experiments is shown in parentheses. Each value represents the mean f S.E.M. * * P -z0.01. Student’s t-test for unpaired data.
3.2. Time course of the changes in the uasoinhibitory effect of IVKY-722 in ettdothelium-intact or -removed canine mese~rer~~ arteries
After mesenteric arteries were incubated with NKY722 (lo+, lo-‘, or lo-’ M) for 60 min. its vasoinhibitory effect in the endothelium-intact arteries was compared with that in the endothelium-removed arteries. The va~i~bito~ effect of NKY-722 at lo- ’ M in endothelium-removed arteries was similar to that in endothelium-intact arteries. In contrast, its vasoinhibitory effect at lo-* M was significantly greater in endothelium-removed arteries than that in endothelium-intact arteries. At lo-’ M, the vasoinhibitory effect of NKY-722 in both endothelium-intact and -removed arteries was nearly 100% up to 180 min of the wash-out period, and there were no significant differences. However, the vasoinhibitory effect after 300 min of wash-out 1OOr
3.1. Accumtdation of NKY-722
removed
T
or
-removed canine mesenteric arteries
Pndothelium-intact or -removed canine mesenteric arteries were incubated with NKY-722 (10e9, 10-r, or iG_’ M) for 60 min. The T/M ratio in the endothelium-intact arteries was 17 f 1 (n = 6) at 10e9 M, 18 f 2 (n = 6) at 10m8 M, and 20 ir 2 (n = 6) at lo-’ M. The T/M ratio in the endothelium-removed arteries was 15 f 1 (n = 6) at 10m9 M, 11 f 1 (n = 6) at lo-’ M, and 10f 1 In =6) at lo-’ M. The a~umulation of NKY-722 in endothelium-removed arteries was similar to that in endothelium-intact arterie-J at 10F9 $M, but was si~i~cantly less in the e~dothelium-remove arteries at 10d8 and IO-’ M (fig. 1).
180 300 Time (mid Fig. 2. Time course of the changes in the vasoinbibitory effect 01 NKY-722 on KCI-induced contraction in endothelium-intact f + E; -) or endothe~ium.remov~ ( - E; - - - - - -) canine mesenterie arteries ch.uing repeated wash-out. (0) NKY-722 (10e9 M) (n = 8); (A) NKY-722 (10-s M) (JI = 9); (0) NKY-722 (lo-’ M) (n = 10). The contraction produced by 30 mM KC1 in the absence of NKY-722 was regarded as 100%. Each value represents the meanf !&EM. * P c 0.05, l * P < 0.01, Student’s t-test for unpaired data. 030
90
199
was significantly greater in the endothelium-removed arteries (fig. 2).
4. Discussion We have previously reported that NKY-722 is accumulated in canine mesenteric arteries and have suggested that its accumulation in the vascular wall may contribute to the long duration of its antihypertensive effect (Shimaji et al., 1990). It has been reported that endothelial cells are an important site for the uptake and inactivation of circulating vasoactive amines (Shepro and Dunham, 1986), and that a cytochrome P-450-dependent monooxygenase is present in the endothelial cells (Abraham et al., 1985). When the mesenteric arteries were incubated with NKY-722 at lo-’ M for 60 mitt, its accumulation in endothelium-intact arteries was not different from that in endothelium-removed arteries, suggesting that endothelial cells did not accumulate NKY-722. In contrast, when mesenteric arteries were incubated with NKY-722 at 10s8 and lo-’ M for 60 min, its accumulation was significantly less in endothelium-removed arteries than in endothelium-intact arteries. This clp~ly indicated that the endothelial cells of the mesenteric arteries could accumulate NKY-722. The time course changes of the vasoinhibitory effect on KCl-induced contraction were similar in both endothelium-intact and -removed arteries during wash-out after incubation with NKY-722 (10e9 M for 60 min). This was compatible with the fmding that the accumulation of NKY-722 was similar in bot’n endothelium-intact and -removed arteries at 10s9 M. However, the time course changes of the vrsoinhibitory effect of NKY-722 (10s8 and lo-’ M) on KCl,-induced contraction in endothelium-removed arteries jhowed that it was significantly more potent in these than in endotheliumintact arteries. This was so throughout the wash-out period after incubation at 10V8 M and from 180 to 300 min of the wash-out period after incubation at lo-’ M. It has been reported that nitrendipine, which can accu-
mulate in the vascular wall, promotes the formation of prostacyclin in arteries and inhibits the development of atherosclerosis (Grodxinska et al., 1987). The present study demonstrates that NKY-722 is accumulated in both vascular smooth muscle cells and endothelial cells and that its accumulation in endothelial cells attenuates its vasoinhibitoq effect.
References Abraham, N.G.. A. Pinto. KM. Mullane, RD. Levere and E. Spokas. 1985, Pmsence of cytochrome P45Odependent monooxygenasc in intimal cells of ho8 aorta, H-on 7.899. Grodzinska, L., M. Basista, E. Basista, M. Slawinski, J. Swies, J. Stachura and R. Ohlro~ge, 1987. Nitrendipine-stimulated release of prostacyclin-like substana in normal and atherosclerotic animals, Arzneim. Forsch. 37.412. Imagawa, J., K. Satoh and N. Taira, 1989. Coronary vasodilator and cardiac effects of NKY-722, a novel hydrophilic l&diiydropyridine derivative, in the blood-perfused do8 heart, Cardiovasc. DN~S Ther. 3, 81. Karasawa, A. and K. Kubo. 1988, Calcium antagonistic effects and the in vitro duration of actions of KW-3049. a new l&dihydropyridine derivative, in isolated canine coronary arteries, Jap. J. Pharmacol. 47. 35. Osumi. S., S. Morishita. K. Wada, H. Usui. M. Kanda, H. Matsui and N. Kakeya, 1988, Antihypertensive effect of NKY-722, a new water-soluble l&dihydropyridme derivative, on conscious sponmatiusly hypertensive rats, Tohoku J. Exp. Med. 155,205. Osumi. S.. H. Sbirahase, H. Shimaji, S. Morishita. K. Wada, M. Kanda, Y. Hashizume and N. Kakeya, 1990. Antihypertensive effects of NKY-722, a watt:-solitibie, dihydropyddine-i;ype Cz” &Ulta~OniSt, European J. Pbarmacol. 183.1323. Pang, D.C. and N. Sperelakis. 1983. Nifedipine, dihiarem. bepridil and verapamil uptakes into cardiac and smooth muscles. European J. Phannacol. 87, 199. Pang, D.C. and N. Sperelakis. 1984, Uptake of calcium antagonistic drugs into muscles as related to their lipid solubihties. B&hem. Pharmacol. 33, 821. Shepro. D. and B. Dunham. 1986. Endothelid cell metabohsm of biogenic amines, Ann. Rev. Physiol. 48, 335. Shimaji, H., H. Shirahase, M. Kanda, S. Osumi, K. Kumha~hi and M. Fujiwara, 1990, Accumulation of NKY-722. a vasosefective Ca*+anlagonist in canine mesenteric arteries, European J. pharmatx% 183.1252.
197
Short communication
Hisanobu Shimaji, Hiroaki Shirahase ‘, Mamoru Kanda I, Seimei Osumi ’ and Kazuyoshi Kurahasti Pharmacology
Di~~isis,or.Radioisofupe
Research Center, Kyo~o Universi
kd.,
Kyoro 604, Japan
Received 11 Ekcember 1990. accepted 26 February 1991
Modulation of the vasoinhibitory effect of NKY-722 by vascular endothelial cells was studied in canine mesenteric arteries. A high conctiIltration of NKY-722 accumulated in the endothelium-intact arteries and its accumulation in endothelium-removed arteries was significantly less. The vasoinhibitory effect of NKY-722 in endothelium-intact arteries was significantly weaker than that in endothelium-removed arteries. These results suggest that endothelial cells can attenuate the vasoinhibitory effect of NKY-722.
Endothelial cell (vascular); NKY-722; Ca *+ channel antagonist; (Accumulation)
1. Introduction
thelial cells can modulate the vasoinhibitory NKY-722 in canine mesenteric arteries.
effect of
In vascular smooth muscle, nitrendipine Sperelakis, Sperelakis,
1984). verapamil, 1983), and benidipine
(Pang and bepridil (Pang and (Karasawa and Kubo.
1988) are accumulated, while nifedipine and diltiazem are not accumulated (Pang and Sperelakis. 1984). Recently, we reported that 3-(4-allyl-1-piperazinyl)-2,2-dimethylpropyl-3,5-pyridine dicarboxylate dihydrochloride (NKY - ILL), a wa:er-soluble vasoselective Ca” antagonist, showed persistent accumuialion in canine mesenteric arteries (Shimaji et al., 1990). NKY-722 exerts a potent and long-iasting antihypertensive effect in spon!aneously hypertensive rats and has a vasoselective effect in isolated, blood-perfused canine heart preparations (Imagawa et al., 1989; Osumi et al., 1988; 1990). We have previously suggested that the accumulation of NKY-722 in the vascular wall may contribute to the long duration of its antihypertensive effect (Shimaji et al., 1990). It has not yet been reported whether accumulation of Ca2+ antagonists in the vascular wall can be influenced by endothelial cells. The present experiments were undertaken to investigate whether or not endo-
Correspondence to: K. Kurahashi, Pharmacology Division. Radioisotope Research Center, Kyoto University, Kyoto 606. Japan.
2. Materials and methods 2.1. Accumultirion
of NKY-722
in endothelium-intact
or
-removed canine mesenteric arteries
Mongrel dogs of either sex weighing lo-24 kg were bled to death from the common carotid arteries under sodium pentobarbital anesthesia (25 mg/kg i.v.). The mesenteric arterics were then removed and were cut into helical strips. Endothelium-in!act or -removed nesenteric arteries were incubared for 6ti min at 37OC in Krebs-Henseleit solution bubbled with a &xtu:e of 95% O,-5% CO, containing lo-’ M (0.1 pCi/ml), iO_’ M (0.01 pCi/ml), or 10m9 M (0.001 @i/ml) NKY-722. The solution consisted of (mM): NaCl 120; KC1 4.7; MgSO,, 1.2; KH2P04 1.2; CaCl, 2.5; NaHCO, 25; and glucose 10. The preparations were then incubated for 10 min in ice-cold Krebs-Henseleit solution, gently blotted with fiiter paper, weighed, and solubilized overnight at room temperature with 3 ml of Foluene@-350 (Packard Japan Co. Ltd.. Tokyo, Japan). The solubilized preparations were mixed in vials with 1 mi of acetic acid and 10 ml of the scintillation cocktail. The radioactivity of the solubilized preparations was determined using a liquid
nter gAloka LSC-900. Aloka Co. Ltd., Accumulation of NKY-722 was ex~~e~rn~iurn (T/M) ratioe of the changes in the vasoinhibitolr3: effect 722
in e~dot~~~~~-int~~t or -removed canine
ArteriJ strips were fixed vertically between hooks in an organ bath containing Krebs-Henseleit solution, which was maintained at 37OC and bubbled with a O,-58 CO,. The ends of the strips were attached to the lever of a Fo~e~spla~ment transducer (NIX San-Ei instrument Co. Ltd., Tokyo, Japan), which was connected to an ink-writing oscillograph (NIX SanEi Inst~ment Co. Ltd., Tokyo, Japan} on which the isometric tension changes were recorded. The applied tension was adjusted to 1.5 g. Mesenteric arteries were incubate with NKY-722 {lO_‘, 10-a. or 10s7 Ml for 60 mitt and then washed out repeatedly for 30. 90, 180, or 300 min. KC1 (30 mM) was applied to the arteries after each wash-out.
2.3. Drugs NKY-722 was a gift from Kyoto Pharmaceutical Industries, Ltd. (Kyoto, Japan). The [3HlllKY-722 (88 Ci/mmol) was tritiated by Amersham International Plc. ~Buc~n~ams~re, U.K.). 2.4. Statistics All data were expressed as means f S.E.M. and statistical significance was assessed using Student’s t-test for unpaired data.
0
I 01,
in endotheIj~m-intact
(6)
‘
10-Q
10-s
10”
Coon. of NKY-722 (M) Fig. 1. Accumulation (T/M ratio) of NKY-722 in endothe~um-intact or -removed canine mesenteric arteries. The number of experiments is shown in parentheses. Each value represents the mean f S.E.M. * * P -z0.01. Student’s t-test for unpaired data.
3.2. Time course of the changes in the uasoinhibitory effect of IVKY-722 in ettdothelium-intact or -removed canine mese~rer~~ arteries
After mesenteric arteries were incubated with NKY722 (lo+, lo-‘, or lo-’ M) for 60 min. its vasoinhibitory effect in the endothelium-intact arteries was compared with that in the endothelium-removed arteries. The va~i~bito~ effect of NKY-722 at lo- ’ M in endothelium-removed arteries was similar to that in endothelium-intact arteries. In contrast, its vasoinhibitory effect at lo-* M was significantly greater in endothelium-removed arteries than that in endothelium-intact arteries. At lo-’ M, the vasoinhibitory effect of NKY-722 in both endothelium-intact and -removed arteries was nearly 100% up to 180 min of the wash-out period, and there were no significant differences. However, the vasoinhibitory effect after 300 min of wash-out 1OOr
3.1. Accumtdation of NKY-722
removed
T
or
-removed canine mesenteric arteries
Pndothelium-intact or -removed canine mesenteric arteries were incubated with NKY-722 (10e9, 10-r, or iG_’ M) for 60 min. The T/M ratio in the endothelium-intact arteries was 17 f 1 (n = 6) at 10e9 M, 18 f 2 (n = 6) at 10m8 M, and 20 ir 2 (n = 6) at lo-’ M. The T/M ratio in the endothelium-removed arteries was 15 f 1 (n = 6) at 10m9 M, 11 f 1 (n = 6) at lo-’ M, and 10f 1 In =6) at lo-’ M. The a~umulation of NKY-722 in endothelium-removed arteries was similar to that in endothelium-intact arterie-J at 10F9 $M, but was si~i~cantly less in the e~dothelium-remove arteries at 10d8 and IO-’ M (fig. 1).
180 300 Time (mid Fig. 2. Time course of the changes in the vasoinbibitory effect 01 NKY-722 on KCI-induced contraction in endothelium-intact f + E; -) or endothe~ium.remov~ ( - E; - - - - - -) canine mesenterie arteries ch.uing repeated wash-out. (0) NKY-722 (10e9 M) (n = 8); (A) NKY-722 (10-s M) (JI = 9); (0) NKY-722 (lo-’ M) (n = 10). The contraction produced by 30 mM KC1 in the absence of NKY-722 was regarded as 100%. Each value represents the meanf !&EM. * P c 0.05, l * P < 0.01, Student’s t-test for unpaired data. 030
90
199
was significantly greater in the endothelium-removed arteries (fig. 2).
4. Discussion We have previously reported that NKY-722 is accumulated in canine mesenteric arteries and have suggested that its accumulation in the vascular wall may contribute to the long duration of its antihypertensive effect (Shimaji et al., 1990). It has been reported that endothelial cells are an important site for the uptake and inactivation of circulating vasoactive amines (Shepro and Dunham, 1986), and that a cytochrome P-450-dependent monooxygenase is present in the endothelial cells (Abraham et al., 1985). When the mesenteric arteries were incubated with NKY-722 at lo-’ M for 60 mitt, its accumulation in endothelium-intact arteries was not different from that in endothelium-removed arteries, suggesting that endothelial cells did not accumulate NKY-722. In contrast, when mesenteric arteries were incubated with NKY-722 at 10s8 and lo-’ M for 60 min, its accumulation was significantly less in endothelium-removed arteries than in endothelium-intact arteries. This clp~ly indicated that the endothelial cells of the mesenteric arteries could accumulate NKY-722. The time course changes of the vasoinhibitory effect on KCl-induced contraction were similar in both endothelium-intact and -removed arteries during wash-out after incubation with NKY-722 (10e9 M for 60 min). This was compatible with the fmding that the accumulation of NKY-722 was similar in bot’n endothelium-intact and -removed arteries at 10s9 M. However, the time course changes of the vrsoinhibitory effect of NKY-722 (10s8 and lo-’ M) on KCl,-induced contraction in endothelium-removed arteries jhowed that it was significantly more potent in these than in endotheliumintact arteries. This was so throughout the wash-out period after incubation at 10V8 M and from 180 to 300 min of the wash-out period after incubation at lo-’ M. It has been reported that nitrendipine, which can accu-
mulate in the vascular wall, promotes the formation of prostacyclin in arteries and inhibits the development of atherosclerosis (Grodxinska et al., 1987). The present study demonstrates that NKY-722 is accumulated in both vascular smooth muscle cells and endothelial cells and that its accumulation in endothelial cells attenuates its vasoinhibitoq effect.
References Abraham, N.G.. A. Pinto. KM. Mullane, RD. Levere and E. Spokas. 1985, Pmsence of cytochrome P45Odependent monooxygenasc in intimal cells of ho8 aorta, H-on 7.899. Grodzinska, L., M. Basista, E. Basista, M. Slawinski, J. Swies, J. Stachura and R. Ohlro~ge, 1987. Nitrendipine-stimulated release of prostacyclin-like substana in normal and atherosclerotic animals, Arzneim. Forsch. 37.412. Imagawa, J., K. Satoh and N. Taira, 1989. Coronary vasodilator and cardiac effects of NKY-722, a novel hydrophilic l&diiydropyridine derivative, in the blood-perfused do8 heart, Cardiovasc. DN~S Ther. 3, 81. Karasawa, A. and K. Kubo. 1988, Calcium antagonistic effects and the in vitro duration of actions of KW-3049. a new l&dihydropyridine derivative, in isolated canine coronary arteries, Jap. J. Pharmacol. 47. 35. Osumi. S., S. Morishita. K. Wada, H. Usui. M. Kanda, H. Matsui and N. Kakeya, 1988, Antihypertensive effect of NKY-722, a new water-soluble l&dihydropyridme derivative, on conscious sponmatiusly hypertensive rats, Tohoku J. Exp. Med. 155,205. Osumi. S.. H. Sbirahase, H. Shimaji, S. Morishita. K. Wada, M. Kanda, Y. Hashizume and N. Kakeya, 1990. Antihypertensive effects of NKY-722, a watt:-solitibie, dihydropyddine-i;ype Cz” &Ulta~OniSt, European J. Pbarmacol. 183.1323. Pang, D.C. and N. Sperelakis. 1983. Nifedipine, dihiarem. bepridil and verapamil uptakes into cardiac and smooth muscles. European J. Phannacol. 87, 199. Pang, D.C. and N. Sperelakis. 1984, Uptake of calcium antagonistic drugs into muscles as related to their lipid solubihties. B&hem. Pharmacol. 33, 821. Shepro. D. and B. Dunham. 1986. Endothelid cell metabohsm of biogenic amines, Ann. Rev. Physiol. 48, 335. Shimaji, H., H. Shirahase, M. Kanda, S. Osumi, K. Kumha~hi and M. Fujiwara, 1990, Accumulation of NKY-722. a vasosefective Ca*+anlagonist in canine mesenteric arteries, European J. pharmatx% 183.1252.