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OMEPRAZOLE-INDUCED RELAXATION IN RAT AORTA IS PARTLY DEPENDENT ON ENDOTHELIUM
Pharmacological Research, Vol. 46, No. 4, 2002 doi:10.1016/S1043-6618(02)00124-X, available online at http://www.idealibrary.com on
OMEPRAZOLE-INDUCED RELAXATION IN RAT AORTA IS PARTLY DEPENDENT ON ENDOTHELIUM a , CAN PEKINER a,∗ , YUSUF SARIOGLU b and SERDAR UMA a ˘ ˙ KELICEN ˙ ˙ PELIN a Department of Pharmacology, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey, b Department of Pharmacology, Faculty of Medicine, Gazi University, 06330 Ankara, Turkey
Accepted 25 June 2002
We investigated the effect of omeprazole (1 × 10−5 –3 × 10−4 M), an inhibitor of H+ ,K+ -ATPase, on rat aortic rings pre-contracted with phenylephrine (10−6 M). Omeprazole relaxed the tissue in a concentration-dependent manner. Either removal of the endothelium or incubation with nitric oxide (NO) synthase inhibitor NG -nitro-l-arginine methyl ester (l-NAME, 3 × 10−5 M) significantly attenuated the relaxations. Pre-treatment with l-arginine (10−3 M), but not with d-arginine, reversed the inhibitory action of l-NAME. Indomethacin (10−6 M) and tetraethylammonium (TEA, 10−2 M) did not affect the relaxant responses to omeprazole indicating the lack of involvement of cyclooxygenase products and K+ channels, respectively. These results suggest a role of NO in the mechanism of action of omeprazole. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: omeprazole, endothelium, rat aorta, H+ ,K+ -ATPase.
INTRODUCTION
MATERIALS AND METHODS
The H+ ,K+ -ATPase was first described in the gastric parietal cell where it mediates gastric acid secretion into the stomach lumen [1]. It has been suggested that non-epithelial cells like vascular smooth muscle may also possess H+ ,K+ -ATPase activity [2]. Although the exact mechanism(s) of the effect of H+ ,K+ -ATPase inhibitors on vascular tone is unknown, the presence of a proton pump in the plasma membrane of smooth muscle cells may have implications for the regulation of contractile/relaxant function since changes in the intracellular pH are known to modulate smooth muscle tone [3]. It has been reported that vasorelaxant effect of leminoprazole, an H+ ,K+ -ATPase inhibitor, is in part due to inhibition of cGMP-phosphodiesterase in rat aortic rings [4]. H+ ,K+ -ATPase inhibitors also cause relaxation of guinea pig and human airway smooth muscle in vitro via epithelium-independent mechanism(s) [5]. Therefore, in this study we aimed to investigate the relaxant effect of omeprazole, a precursor of the inhibitors of the protonic pump H+ ,K+ -ATPase, in rat aorta and to test the influence of endothelium on this action.
Animals
∗ Corresponding
author. Department of Pharmacology, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey. E-mail: [email protected]
1043-6618/02/$ – see front matter
All rats used in the present study were cared for in accordance with the directory of Hacettepe University Animal Care Unit, which applies the guidelines of NIH on laboratory animal welfare.
Isolated rat aorta Male rats, 200–250 g were killed by stunning and bleeding. The thoracic aortas were removed and dissected free of fat and connective tissue. Aortic rings were mounted in 10 ml organ baths containing Krebs solution of the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2 PO4 1.2, NaHCO3 25, glucose 11. Solution was maintained at 37 ◦ C and aerated with 95% O2 –5% CO2 (pH 7.4). The preparations were equilibrated for 90 min under a resting tension of 2 g and were attached to a force-displacement transducer (Grass, FTO3). Changes in tension were recorded on a computer-based data acquisition system (MAY, Turkey).
Experimental procedure Concentration–response curve for omeprazole were constructed on strips pre-contracted with 10−6 M phenylephrine. In cumulative concentration–response curves on rat aortic rings, 10−6 M phenylephrine produced almost 70–80% of the maximum attainable contraction. The © 2002 Elsevier Science Ltd. All rights reserved.
322
endothelium integrity was verified by testing the relaxant responses to 10−6 M acetylcholine. When the contraction induced by 10−6 M phenylephrine reached a plateau, omeprazole was added to the organ bath in a cumulative manner (1 × 10−5 –3 × 10−4 M). The same procedure was repeated in endothelium denuded aortic rings. Denudation was performed by gently rubbing the internal surface of the vessels with a small wooden stick moistened with physiological salt solution. In another set of experiments, after obtaining the control responses to omeprazole the aortic rings were incubated with 3 × 10−5 M l-NAME for 15 min and omeprazole concentration–response curve was reconstructed. Both l-arginine (3×10−3 M) and d-arginine (3×10−3 M) were also added prior to incubation with l-NAME. In some experiments indomethacin (10−6 M) and TEA (10−2 M) were tested in order to elucidate the mechanism of action of omeprazole. At the end of the experiment maximal relaxation was induced in each aortic rings by the addition of papaverine (10−4 M). In some experiments the action of the vehicle (polyethylene glycol), that omeprazole was dissolved in, was also examined in rat aortic rings pre-contracted with phenylephrine.
Data analysis and statistics Omeprazole-induced relaxation was expressed as a percentage of reversal of the contraction produced by phenylephrine. Maximum response elicited by omeprazole and the concentration required to achieve half maximum contraction (EC50 ) were obtained from individual concentration–response curves. EC50 values are given as pD2 values which are defined as the negative logarithm of EC50 (pD2 : −log EC50 ) Results are expressed as mean ± sem of n experiments. Data were statistically evaluated by analysis of variance and paired samples were compared by Student’s t-test. P value of less than 0.05 was considered to be significant.
Pharmacological Research, Vol. 46, No. 4, 2002
Fig. 1. Effect of omeprazole on endothelium intact (䊏) and denuded (䊐) rat aortic rings. Relaxation was expressed as a percentage of reversal of pre-contraction induced by 10−6 M phenylephrine. Each point represents mean ± sem of tissues from n = 6 animals (∗ indicates P < 0.06).
change in pD2 values between endothelium-intact and -denuded rings was found (endothelium-intact pD2 : 3.92 ± 0.09 vs endothelium-denuded pD2 : 3.78 ± 0.11). Incubation with l-NAME (3 × 10−5 M) caused significant reduction in omeprazole-evoked relaxations of the endothelium-intact rat aortic rings (P < 0.05, Fig. 2). Maximal response to omeprazole at 3 × 10−4 M (59.5 ± 2.5%) was reduced to 27.1 ± 1.6% in the presence of l-NAME. The pD2 values between control (3.97±0.06) and l-NAME-treated groups (4.01 ± 0.08) were not
Chemicals Following drugs were used: omeprazole was obtained from Astra. Phenylephrine, acetylcholine, l-NAME, l-arginine, d-arginine, papaverine, TEA and indomethacin were all purchased from Sigma.
RESULTS Omeprazole (1×10−5 –3×10−4 M) caused concentrationdependent relaxation of the rat aortic rings pre-contracted with 10−6 M phenylephrine. Maximal relaxation obtained for omeprazole at 3×10−4 M was 60±2%. Similarly vehicle, which omeprazole was dissolved in, did not have any considerable effect on aortas pre-contracted with phenylephrine. Addition of 10−4 M papaverine produced a full relaxation. Removal of the endothelium markedly attenuated the relaxant effect of omeprazole (Fig. 1, P < 0.05). Maximal response to omeprazole at 3 × 10−4 M was decreased to 20.4±3.3% in these aortic rings. No significant
Fig. 2. Inhibition of omeprazole-induced relaxation (䊏) by 3×10−5 M l-NAME (䉲) and reversal of this effect by 3 mM l-arginine pre-treatment (䊊) but not by d-arginine (䊐) in rat aortic rings. Relaxation was expressed as a percentage of reversal of pre-contraction induced by 10−6 M phenylephrine. Each point represents mean ± sem of tissues from n = 6 animals (∗ indicates P < 0.05 l-NAME compared to omeprazole alone, ∗∗ indicates P < 0.05 l-arginine+l-NAME compared to l-NAME alone).
Pharmacological Research, Vol. 46, No. 4, 2002
significantly different. Pre-treatment with l-arginine (3 × 10−3 M) almost reversed the inhibitory effect of l-NAME, whereas incubation with d-arginine (3 × 10−3 M) was found ineffective (Fig. 2). In preparations incubated with indomethacin (10−6 M), omeprazole-induced relaxations were found unchanged (control pD2 : 3.90 ± 0.10 vs indomethacin-treated pD2 : 3.85 ± 0.11). Similarly TEA (10−2 M) had no significant effect on relaxant responses to omeprazole (control pD2 : 3.99±0.07 vs TEA-treated pD2 : 3.92±0.04). Both agents did not change the amplitude of the contractions evoked by phenylephrine.
DISCUSSION Although the H+ ,K+ -ATPase has been identified in vascular smooth muscle [2] the precise mechanism of the effect of proton pump inhibitors on vascular tone has not been clearly established. However, like omeprazole, another benzimidazole derivative NC-1300 B has been reported to prevent spontaneous and angiotensin II-induced hypertension in rats [6]. The results of the present study indicate that omeprazole, an H+ ,K+ -ATPase inhibitor, induces relaxation of rat aortic rings at concentrations similar to those used in guinea-pig and human airway smooth muscle [5]. Cyclooxygenase products do not seem to play a role in this relaxation since in the presence of indomethacin responses to omeprazole were unchanged. It has been reported that TEA acts as a non-specific inhibitor on K+ channels when used at concentrations higher than 5 mM [7]. Therefore, we can also exclude the possibility that K+ channels are involved, since TEA at 10 mM concentration did not alter the relaxation evoked by omeprazole in this study. The decrease in the relaxant effect of omeprazole in endothelium-denuded rat aortic rings and in vessels pre-incubated with l-NAME leads us to suggest that nitric oxide (NO) is involved. Reversal of the inhibitory effect of l-NAME by l-arginine, but not by d-arginine further supports our proposal. A similar vasodilatory action through endothelium has also been reported for leminoprazole in rat aorta [4]. This effect has been attributed partly to the inhibition of cGMP-phosphodiesterase depending on the observation that leminoprazole failed to relax the aortic rings pre-incubated with zaprinast, a cGMP-phosphodiesterase inhibitor. However, we were unable to obtain similar results, since phenylephrine could not maintain a sustained contraction when the vessels were incubated with zaprinast (data not shown). Okabe
323
et al. [4] have suggested that inhibition of voltage operated calcium channels might also be responsible from leminoprazole-induced relaxation of rat aorta. This may also be the case in our study because as can be seen in Figures 1 and 2 there is a residual relaxation occurring after either denudation of endothelium or pre-incubation with l-NAME indicating the involvement of additional mechanism(s) in omeprazole-induced relaxation of rat aorta. We do not know at present, whether H+ ,K+ -ATPase is involved in this endothelium-dependent relaxation caused by omeprazole. It seems unlikely to be related to the inhibition of proton pump, since the concentration of omeprazole required to cause maximal inhibition of H+ ,K+ -pump (<10−5 M) [8] is much less than the concentration required for maximal inhibition of the contractile responses in the present study (>3 × 10−4 M) suggesting a non-specific action of omeprazole. On the other hand it should also be kept in mind that high concentrations of proton pump inhibitors have the potential of affecting other ion-sensitive ATPases like Na+ ,K+ -ATPase and the vacuolar H+ -ATPase [9]. In conclusion, the results of the present study introduces the first in vitro evidence that omeprazole-induced relaxation in rat aortic rings is partly dependent on endothelium. Further experiments are needed to clarify the full mechanisms(s) underlying the endothelium independent part of the relaxant action of omeprazole. REFERENCES 1. Sachs G, Chang HH, Rabon E, Schackman R, Lewin M, Saccomani G. A nonelectrogenic H+ pump in plasma membranes of hog stomach. J Biol Chem 1976; 251: 7690–8. 2. McCabe RD, Young DB. Evidence of a H+ ,K+ -ATPase in vascular smooth muscle cells. Am J Physiol 1992; 262: H1955–8. 3. Wray S. Smooth muscle intracellular pH: measurement, regulation and function. Am J Physiol 1988; 2254: C213–25. 4. Okabe S, Amagase K, Fujita H, Iwata K, Satake N, Shibata S. Vasoinhibitory effect of leminoprazole, a H+ ,K+ -ATPase inhibitor, on rat aortic rings. Gen Pharmacol 1996; 27: 117–21. 5. Rhoden KJ, Tallini G, Douglas JS. H+ ,K+ -ATPase inhibitors cause relaxation of guinea-pig and human airway smooth muscle in vitro. J Pharmacol Exp Ther 1996; 276: 897–903. 6. McCabe RD, Kassner D, Young DB. A H+ ,K+ -ATPase inhibitor prevents hypertension (Abstract). FASEB J 1994; 9: A75. 7. Vanhoutte PM. Endothelium-derived free radicals: for worse and better. J Clin Invest 2001; 107: 23–5. 8. Okabe S, Akimoto Y, Yamasaki S, Kuwahara K. Effects of a new benzimidazole derivative, NC-1300-B, on gastric secretion and gastroduodenal lesions in rats. Jpn J Pharmacol 1991; 55: 477–91. 9. Keeling DJ, Fallowfield C, Milliner KJ, Tingley SK, Ife RJ, Underwood AH. Studies on the mechanism of action of omeprazole. Biochem Pharmacol 1985; 16: 2967–73.
OMEPRAZOLE-INDUCED RELAXATION IN RAT AORTA IS PARTLY DEPENDENT ON ENDOTHELIUM a , CAN PEKINER a,∗ , YUSUF SARIOGLU b and SERDAR UMA a ˘ ˙ KELICEN ˙ ˙ PELIN a Department of Pharmacology, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey, b Department of Pharmacology, Faculty of Medicine, Gazi University, 06330 Ankara, Turkey
Accepted 25 June 2002
We investigated the effect of omeprazole (1 × 10−5 –3 × 10−4 M), an inhibitor of H+ ,K+ -ATPase, on rat aortic rings pre-contracted with phenylephrine (10−6 M). Omeprazole relaxed the tissue in a concentration-dependent manner. Either removal of the endothelium or incubation with nitric oxide (NO) synthase inhibitor NG -nitro-l-arginine methyl ester (l-NAME, 3 × 10−5 M) significantly attenuated the relaxations. Pre-treatment with l-arginine (10−3 M), but not with d-arginine, reversed the inhibitory action of l-NAME. Indomethacin (10−6 M) and tetraethylammonium (TEA, 10−2 M) did not affect the relaxant responses to omeprazole indicating the lack of involvement of cyclooxygenase products and K+ channels, respectively. These results suggest a role of NO in the mechanism of action of omeprazole. © 2002 Elsevier Science Ltd. All rights reserved. Key wo rds: omeprazole, endothelium, rat aorta, H+ ,K+ -ATPase.
INTRODUCTION
MATERIALS AND METHODS
The H+ ,K+ -ATPase was first described in the gastric parietal cell where it mediates gastric acid secretion into the stomach lumen [1]. It has been suggested that non-epithelial cells like vascular smooth muscle may also possess H+ ,K+ -ATPase activity [2]. Although the exact mechanism(s) of the effect of H+ ,K+ -ATPase inhibitors on vascular tone is unknown, the presence of a proton pump in the plasma membrane of smooth muscle cells may have implications for the regulation of contractile/relaxant function since changes in the intracellular pH are known to modulate smooth muscle tone [3]. It has been reported that vasorelaxant effect of leminoprazole, an H+ ,K+ -ATPase inhibitor, is in part due to inhibition of cGMP-phosphodiesterase in rat aortic rings [4]. H+ ,K+ -ATPase inhibitors also cause relaxation of guinea pig and human airway smooth muscle in vitro via epithelium-independent mechanism(s) [5]. Therefore, in this study we aimed to investigate the relaxant effect of omeprazole, a precursor of the inhibitors of the protonic pump H+ ,K+ -ATPase, in rat aorta and to test the influence of endothelium on this action.
Animals
∗ Corresponding
author. Department of Pharmacology, Faculty of Pharmacy, Hacettepe University, 06100 Sıhhiye, Ankara, Turkey. E-mail: [email protected]
1043-6618/02/$ – see front matter
All rats used in the present study were cared for in accordance with the directory of Hacettepe University Animal Care Unit, which applies the guidelines of NIH on laboratory animal welfare.
Isolated rat aorta Male rats, 200–250 g were killed by stunning and bleeding. The thoracic aortas were removed and dissected free of fat and connective tissue. Aortic rings were mounted in 10 ml organ baths containing Krebs solution of the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2 PO4 1.2, NaHCO3 25, glucose 11. Solution was maintained at 37 ◦ C and aerated with 95% O2 –5% CO2 (pH 7.4). The preparations were equilibrated for 90 min under a resting tension of 2 g and were attached to a force-displacement transducer (Grass, FTO3). Changes in tension were recorded on a computer-based data acquisition system (MAY, Turkey).
Experimental procedure Concentration–response curve for omeprazole were constructed on strips pre-contracted with 10−6 M phenylephrine. In cumulative concentration–response curves on rat aortic rings, 10−6 M phenylephrine produced almost 70–80% of the maximum attainable contraction. The © 2002 Elsevier Science Ltd. All rights reserved.
322
endothelium integrity was verified by testing the relaxant responses to 10−6 M acetylcholine. When the contraction induced by 10−6 M phenylephrine reached a plateau, omeprazole was added to the organ bath in a cumulative manner (1 × 10−5 –3 × 10−4 M). The same procedure was repeated in endothelium denuded aortic rings. Denudation was performed by gently rubbing the internal surface of the vessels with a small wooden stick moistened with physiological salt solution. In another set of experiments, after obtaining the control responses to omeprazole the aortic rings were incubated with 3 × 10−5 M l-NAME for 15 min and omeprazole concentration–response curve was reconstructed. Both l-arginine (3×10−3 M) and d-arginine (3×10−3 M) were also added prior to incubation with l-NAME. In some experiments indomethacin (10−6 M) and TEA (10−2 M) were tested in order to elucidate the mechanism of action of omeprazole. At the end of the experiment maximal relaxation was induced in each aortic rings by the addition of papaverine (10−4 M). In some experiments the action of the vehicle (polyethylene glycol), that omeprazole was dissolved in, was also examined in rat aortic rings pre-contracted with phenylephrine.
Data analysis and statistics Omeprazole-induced relaxation was expressed as a percentage of reversal of the contraction produced by phenylephrine. Maximum response elicited by omeprazole and the concentration required to achieve half maximum contraction (EC50 ) were obtained from individual concentration–response curves. EC50 values are given as pD2 values which are defined as the negative logarithm of EC50 (pD2 : −log EC50 ) Results are expressed as mean ± sem of n experiments. Data were statistically evaluated by analysis of variance and paired samples were compared by Student’s t-test. P value of less than 0.05 was considered to be significant.
Pharmacological Research, Vol. 46, No. 4, 2002
Fig. 1. Effect of omeprazole on endothelium intact (䊏) and denuded (䊐) rat aortic rings. Relaxation was expressed as a percentage of reversal of pre-contraction induced by 10−6 M phenylephrine. Each point represents mean ± sem of tissues from n = 6 animals (∗ indicates P < 0.06).
change in pD2 values between endothelium-intact and -denuded rings was found (endothelium-intact pD2 : 3.92 ± 0.09 vs endothelium-denuded pD2 : 3.78 ± 0.11). Incubation with l-NAME (3 × 10−5 M) caused significant reduction in omeprazole-evoked relaxations of the endothelium-intact rat aortic rings (P < 0.05, Fig. 2). Maximal response to omeprazole at 3 × 10−4 M (59.5 ± 2.5%) was reduced to 27.1 ± 1.6% in the presence of l-NAME. The pD2 values between control (3.97±0.06) and l-NAME-treated groups (4.01 ± 0.08) were not
Chemicals Following drugs were used: omeprazole was obtained from Astra. Phenylephrine, acetylcholine, l-NAME, l-arginine, d-arginine, papaverine, TEA and indomethacin were all purchased from Sigma.
RESULTS Omeprazole (1×10−5 –3×10−4 M) caused concentrationdependent relaxation of the rat aortic rings pre-contracted with 10−6 M phenylephrine. Maximal relaxation obtained for omeprazole at 3×10−4 M was 60±2%. Similarly vehicle, which omeprazole was dissolved in, did not have any considerable effect on aortas pre-contracted with phenylephrine. Addition of 10−4 M papaverine produced a full relaxation. Removal of the endothelium markedly attenuated the relaxant effect of omeprazole (Fig. 1, P < 0.05). Maximal response to omeprazole at 3 × 10−4 M was decreased to 20.4±3.3% in these aortic rings. No significant
Fig. 2. Inhibition of omeprazole-induced relaxation (䊏) by 3×10−5 M l-NAME (䉲) and reversal of this effect by 3 mM l-arginine pre-treatment (䊊) but not by d-arginine (䊐) in rat aortic rings. Relaxation was expressed as a percentage of reversal of pre-contraction induced by 10−6 M phenylephrine. Each point represents mean ± sem of tissues from n = 6 animals (∗ indicates P < 0.05 l-NAME compared to omeprazole alone, ∗∗ indicates P < 0.05 l-arginine+l-NAME compared to l-NAME alone).
Pharmacological Research, Vol. 46, No. 4, 2002
significantly different. Pre-treatment with l-arginine (3 × 10−3 M) almost reversed the inhibitory effect of l-NAME, whereas incubation with d-arginine (3 × 10−3 M) was found ineffective (Fig. 2). In preparations incubated with indomethacin (10−6 M), omeprazole-induced relaxations were found unchanged (control pD2 : 3.90 ± 0.10 vs indomethacin-treated pD2 : 3.85 ± 0.11). Similarly TEA (10−2 M) had no significant effect on relaxant responses to omeprazole (control pD2 : 3.99±0.07 vs TEA-treated pD2 : 3.92±0.04). Both agents did not change the amplitude of the contractions evoked by phenylephrine.
DISCUSSION Although the H+ ,K+ -ATPase has been identified in vascular smooth muscle [2] the precise mechanism of the effect of proton pump inhibitors on vascular tone has not been clearly established. However, like omeprazole, another benzimidazole derivative NC-1300 B has been reported to prevent spontaneous and angiotensin II-induced hypertension in rats [6]. The results of the present study indicate that omeprazole, an H+ ,K+ -ATPase inhibitor, induces relaxation of rat aortic rings at concentrations similar to those used in guinea-pig and human airway smooth muscle [5]. Cyclooxygenase products do not seem to play a role in this relaxation since in the presence of indomethacin responses to omeprazole were unchanged. It has been reported that TEA acts as a non-specific inhibitor on K+ channels when used at concentrations higher than 5 mM [7]. Therefore, we can also exclude the possibility that K+ channels are involved, since TEA at 10 mM concentration did not alter the relaxation evoked by omeprazole in this study. The decrease in the relaxant effect of omeprazole in endothelium-denuded rat aortic rings and in vessels pre-incubated with l-NAME leads us to suggest that nitric oxide (NO) is involved. Reversal of the inhibitory effect of l-NAME by l-arginine, but not by d-arginine further supports our proposal. A similar vasodilatory action through endothelium has also been reported for leminoprazole in rat aorta [4]. This effect has been attributed partly to the inhibition of cGMP-phosphodiesterase depending on the observation that leminoprazole failed to relax the aortic rings pre-incubated with zaprinast, a cGMP-phosphodiesterase inhibitor. However, we were unable to obtain similar results, since phenylephrine could not maintain a sustained contraction when the vessels were incubated with zaprinast (data not shown). Okabe
323
et al. [4] have suggested that inhibition of voltage operated calcium channels might also be responsible from leminoprazole-induced relaxation of rat aorta. This may also be the case in our study because as can be seen in Figures 1 and 2 there is a residual relaxation occurring after either denudation of endothelium or pre-incubation with l-NAME indicating the involvement of additional mechanism(s) in omeprazole-induced relaxation of rat aorta. We do not know at present, whether H+ ,K+ -ATPase is involved in this endothelium-dependent relaxation caused by omeprazole. It seems unlikely to be related to the inhibition of proton pump, since the concentration of omeprazole required to cause maximal inhibition of H+ ,K+ -pump (<10−5 M) [8] is much less than the concentration required for maximal inhibition of the contractile responses in the present study (>3 × 10−4 M) suggesting a non-specific action of omeprazole. On the other hand it should also be kept in mind that high concentrations of proton pump inhibitors have the potential of affecting other ion-sensitive ATPases like Na+ ,K+ -ATPase and the vacuolar H+ -ATPase [9]. In conclusion, the results of the present study introduces the first in vitro evidence that omeprazole-induced relaxation in rat aortic rings is partly dependent on endothelium. Further experiments are needed to clarify the full mechanisms(s) underlying the endothelium independent part of the relaxant action of omeprazole. REFERENCES 1. Sachs G, Chang HH, Rabon E, Schackman R, Lewin M, Saccomani G. A nonelectrogenic H+ pump in plasma membranes of hog stomach. J Biol Chem 1976; 251: 7690–8. 2. McCabe RD, Young DB. Evidence of a H+ ,K+ -ATPase in vascular smooth muscle cells. Am J Physiol 1992; 262: H1955–8. 3. Wray S. Smooth muscle intracellular pH: measurement, regulation and function. Am J Physiol 1988; 2254: C213–25. 4. Okabe S, Amagase K, Fujita H, Iwata K, Satake N, Shibata S. Vasoinhibitory effect of leminoprazole, a H+ ,K+ -ATPase inhibitor, on rat aortic rings. Gen Pharmacol 1996; 27: 117–21. 5. Rhoden KJ, Tallini G, Douglas JS. H+ ,K+ -ATPase inhibitors cause relaxation of guinea-pig and human airway smooth muscle in vitro. J Pharmacol Exp Ther 1996; 276: 897–903. 6. McCabe RD, Kassner D, Young DB. A H+ ,K+ -ATPase inhibitor prevents hypertension (Abstract). FASEB J 1994; 9: A75. 7. Vanhoutte PM. Endothelium-derived free radicals: for worse and better. J Clin Invest 2001; 107: 23–5. 8. Okabe S, Akimoto Y, Yamasaki S, Kuwahara K. Effects of a new benzimidazole derivative, NC-1300-B, on gastric secretion and gastroduodenal lesions in rats. Jpn J Pharmacol 1991; 55: 477–91. 9. Keeling DJ, Fallowfield C, Milliner KJ, Tingley SK, Ife RJ, Underwood AH. Studies on the mechanism of action of omeprazole. Biochem Pharmacol 1985; 16: 2967–73.