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VASODILATING EFFECTS OF TETRAZEPAM IN ISOLATED VASCULAR SMOOTH MUSCLES: COMPARISON WITH CROMAKALIM AND DILTIAZEM
Pharmacological Research, Vol. 36, No. 3, 1997
VASODILATING EFFECTS OF TETRAZEPAM IN ISOLATED VASCULAR SMOOTH MUSCLES: COMPARISON WITH CROMAKALIM AND DILTIAZEM U ´ ´ C. PEREZ-GUERRERO , J. SUAREZ, M.D. HERRERA and E. MARHUENDA
Laboratorio de Farmacologıa, ´ Facultad de Farmacia, Uni®ersidad de Se®illa, Se®illa, Espana ˜ Accepted 28 July 1997
The vasodilating effects of tetrazepam Ž1,4-benzodiazepine derivative. were studied and compared with those of the K-channel activator, cromakalim and the Ca-channel blocker, diltiazem, in rat aorta smooth muscle and on the spontaneous contractile activity of the rat portal vein. In the aorta, tetrazepam Ž3 = 10y7 ]10y4 M. and diltiazem Ž10y8 ]3 = 10y6 M. concentration-dependently relaxed aortic rings contracted by 30 mM as well as 80 mM KCl. Although cromakalim Ž10y8 ]3 = 10y6 M. concentration-dependently relaxed aortic rings contracted by 30 mM KCl, it did not relax those contrated by 80 mM KCl. In the presence of the ATP-sensitive K-channel blocker, glibenclamide Ž10y6 and 3 = 10y6 M., 30 mM KCl concentration]response curves for the relaxant effect of tetrazepam and diltiazem were unaffected but cromakalim caused a progressive shift of these curves upwards. In the portal vein, tetrazepam inhibited spontaneous contractions, decreased amplitude and increased frequency. Similar behaviour was shown with diltiazem Ž10y8 ]10y5 M. and in both cases, pre-treatment with glibenclamide Ž10y6 M. was ineffective. Although cromakalim Ž10y5 ]10y6 M. decreased both amplitude and frequency, this effect was blocked by glibenclamide. These results indicate that the vasodilator action of tetrazepam is not mediated to the opening of ATP-sensitive K-channels, unlike cromakalim. This may be mediated, like those of diltiazem, by the blockade of calcium movements across the cell Q 1997 The Italian Pharmacological Society membrane. KEY
WORDS:
tetrazepam, benzodiazepine, rat aorta, rat portal vein, vasodilators.
INTRODUCTION This study investigated the effect of tetrazepam on vascular smooth muscle. Tetrazepam is a 1,4-benzodiazepine derivative clinically used as a muscle relaxant. However, unlike most 1,4-benzodiazepines, tetrazepam binds to central and peripheral benzodiazepine binding sites ŽPBS. w1x. The physiological roles of the ŽPBS. remains unclear. It has been suggested that these sites may be linked to voltagedependent Ca2q channels and that ligands which interact at these sites serve as Ca2q channel regulators w2]5x. Previous studies in our laboratory have evaluated the effects of this compound on several types of smooth muscle; rat uterus w6x, rat and rabbit aorta w7x, rat and guinea-pig trachea w8x and rat duodenum and guinea-pig ileum w9x. Tetrazepam produced a relaxation of contractions in vascular and non-vascuU
Corresponding author. Laboratorio de Farmacologıa, ´ Facultad de Farmacia, Cr Prof Garcıa ´ Gonzalez srn 41012, Sevilla, Espana. ˜ 1043]6618r97r090237]06r$25.00r0rfr970221
lar smooth muscles. The results suggest that its relaxant action can be related to calcium movements and these events are not related to high-affinity perypheral benzodiazepine binding sites. The present study was therefore undertaken to again further information into the mechanisms implicated in its vasodilator effects in rat vascular smooth muscle, comparing its effects with those obtained with a calcium entry blocker, diltiazem, as there may be sufficient structural similarity between diltiazem Žbenzothiazepine. and tetrazepam Žbenzodiazepine.. On the other hand, the K channel openers, such as cromakalim, would thus act as indirect Ca2q entry blockers and would be expected to have a pharmacological profile similar to that of the Ca2q antagonist w10x. K channel openers increase the permeability of the cell membrane to Kq and induce membrane hyperpolarization in vascular smooth muscle w11x. Membrane hyperpolarization inhibits the opening of the voltage-dependent Ca channels. It is thus hypothesized that K channel openers indiQ1997 The Italian Pharmacological Society
Pharmacological Research, Vol. 36, No. 3, 1997
238
rectly act as Ca channel antagonists. K channel openers, such as cromakalim may, therefore, be considered to belong to a novel class of vasodilatoy agents and the vasodilating effects for K channel openers have been shown in a number of experimental models w12]16x. Competitive antagonism by glibenclamide, an ATP-sensitive K channel blocker, indicates that the vasodilating effects of cromakalim are mediated by the opening of glibenclamide-sensitive K channels w12, 15, 17x. A number of studies reported the rat portal vein to be useful in evaluating the K channel-opening activity w11, 12, 17]19x. K channel openers decrease the frequency of spontaneous rhythmic contractions in this vein by enhancing Kq ion efflux, leading to a hyperpolarization of the cell membrane w11, 18, 19x. The aim of the present study thus was to investigate the possible K channel-opening activity of tetrazepam by measuring its inhibitory action in rat isolated portal vein. The vasorelaxant profiles of the compound in the rat aorta were also investigated and compared with those of the Ca2q channel antagonist, diltiazem.
MATERIAL AND METHODS
Tissue preparation Wistar rats of either sex, weighing 250]300 g, were killed by a blow to the head. The descending thoracic aorta and portal veins were rapidly dissected and placed in Krebs bicarbonate solution ŽKBS. of the following composition Žin mM.: NaCl 116, KCl 4.75, CaCl 2 1.8, MgCl 2 1.2, KH 2 PO4 1.2, NaHCO3 25 and glucose 11. After an excess of fat and conective tissue had been removed, the aortas were cut into transverse rings, aproximately 2]3 mm wide. These rings were suspended between two stainless steel hooks under a basal tension of 1 g in organ baths containing 30 ml of KBS maintained at 378C and administered with 95% O 2 and 5% CO 2 . In the same way, the portal vein segments Ž15 mm in length. were mounted vertically under a basal tension of 1 g in 30 ml organ baths containing KBS. Aortic rings and portal vein segments were attached to a force-displacement transducer ŽHarvard UF-1. and recorded on an Omniscribe polygraph to measure isometric contractile force. Each preparation was allowed to equilibrate for at least 90 min before the initiation of experimental procedures and during this period the incubation media was changed every 15 min and the resting tension adjusted. After equilibration the following experiments were performed.
stable, tetrazepam was added in progressively increasing cumulative concentrations Ž3 = 10y7 ]10y4 M. at 15-min intervals since preliminary experiments demonstrated that this was the time needed to produce a steady-state relaxation. The experiment was repeated with diltiazem Ž10y8 ]3 = 10y6 M. and cromakalim Ž10y8 ]3 = 10y6 M.. In aditional experiments, aortic rings were incubated with glibenclamide Ž10y6 M or 3 = 10y6 M. 20 min prior to the addition of KCl Ž30 or 80 mM.. Only one agonist was used in each experiment. These results were expressed as a percentage of the maximal control of agonist-induced responses.
Spontaneous contractions in the rat portal ®ein After the spontaneous rhythmic contractions were equilibrated for 90 min, test drugs were added cumulatively to the preparation every 15 min. In separate experiments, glibenclamide 10y6 M was added and allowed to equilibrate for 20 min, before beginning the concentration-response curves for test drugs. This study was undertaken to examine the inhibition of amplitude as the frequency of spontaneous contractions produced by test drugs. The frequency of rhythmic contractions was counted for the last 5 min of every 15-min interval. The ordinate of the concentration]response curve is expressed as a percentage of amplitude or frequency of contractions during the pre-drug period. The doses of tetrazepam used were in the range of blood concentrations as the benzodiazepines have been reported to attain high levels Ž10y7 ]10y5 M. w20x.
Drugs All drugs except tetrazepam, were provided by Sigma Chemical Company ŽSt. Louis, MO, USA.. Potassium chloride was obtained from E. Merck; Tetrazepam was generously provided by Sanofi Winthrop. All drugs were dissolved in 0.9% NaCl except cromakalim and glibenclamide as they were dissolved in distilled water containing 20% dimethyl sulfoxide and 20% ethanol. The final concentration of dimethyl sulfoxide in the tissue bath was less than 0.01% which was shown to have no effect on the basal tonus of the preparation. Tetrazepam was initially dissolved in 1 ml of Tween 80 and diluted further in saline to prepare a 2 = 10y3 M stock solution. The final Tween 80 concentration in the bath did not affect the results, as we confirmed in parallel experiments.
Statisticals analysis Contractions induced in aortic rings In some experiments aortic rings were exposed to a single concentration of KCl Ž80 mM or 30 mM.. When the contractile response to each agonist was
Results are expressed as mean " SEM of six or more preparations obtained from different animals. The statistical significance of differences between the means was assessed using the Student’s t-test for
Pharmacological Research, Vol. 36, No. 3, 1997
unpaired data; P- 0.05 values were considered to represent a significant difference. The concentration needed to produce 50% inhibition ŽIC 50 . was obtained from the regression plot and a mean IC 50 " 95% confidence interval was calculated for each dose assessed.
239
The vasorelaxant effect of tetrazepam, diltiazem and cromakalim were evaluated in the developed
tension before and after treatment with these compounds in rat pre-concentrated with KCl. In the rat isolated aorta pre-concentrated with 80 mM and 30 mM KCl, tetrazepam Ž3 = 10y6 ]10y4 M. and diltiazem Ž10y8 ]3 = 10y6 M. produced a concentration-dependent relaxation wFig. 1 ŽA, B.; Fig. 2 ŽA, B.x. In the presence of glibenclamide Ž10y6 and 3 = 10y5 M. the concentration]response curves were not significantly different. However, cromakalim showed itself to be more potent against 30 mM KCl induced contractions than those evoked by 80 mM KCl and in the presence of glibenclamide, the concentration]response curves shifted upwards in a parallel fashion wFig. 3 ŽA, B.x. Table I shows the IC 50 values obtained in all
Fig. 1. Tetrazepam-induced relaxation in the rat aorta contracted by KCl Ž80 mM. A and Ž30 mM. B. In the absence Ž`. and presence of glibenclamide, 1 m M Žv. and 3 m M Ž'.. Contractions are expressed as percentages of the maximum control responses. Each point represents the mean " SEM of six experiments.
Fig. 2. Diltiazem-induced relaxation in the rat aorta contracted by KCl Ž80 mM. A and Ž30 mM. B. In the absence Ž`. and presence of glibenclamide 1 m M Žv. and 3 m M Ž'.. Contractions are expressed as percentages of maximum control responses. Each point represents the mean " SEM of six experiments.
RESULTS
Effects on KCl (80 and 30 m M ) induced contraction on isolated rat aorta
240
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progressive increase in the rate of spontaneous contractions. Pre-treatment with the specific inhibitor of ATP-dependent Kq channels, glibenclamide Ž10y6 M., had no significant effects on tetrazepam and diltiazem-induced inhibition of spontaneous contractions ŽIC 50 : 3.1= 10y6 M and 3.1= 10y7 M, respectively.. However, cromakalim Ž10y8 ]3 = 10y6 M. decreased in a concentration-dependent manner both the amplitude and frequency of these contractions ŽIC 50 : 3.3= 10y7 M. and at 10y6 M it suppressed spontaneous activity. Furthermore, these effects of cromakalim were blocked by glibenclamide Ž10y6 M..
CONCLUSIONS
Fig. 3. Cromakalim-induced relaxation in the rat aorta contracted by KCl Ž80 mM. A and Ž30 mM. B. In the absence Ž`. and presence of glibenclamide, 1 m M Žv. and 3 m M Ž'.. Contractions are expressed as percentages of the maximum control responses. Each point represents the mean " SEM of six experiments.
experiments, in the absence and presence of glibenclamide.
Effects on spontaneous contractions of the rat portal ®ein The frequency and amplitude of spontaneous rhythmic contractions was unchanged for over 2.5 h after the equilibration period Ždata not shown.. Therefore, the effects of tetrazepam, diltiazem and cromakalim were evaluated by measuring changes in the amplitude and frequency before and after treatment with these compounds. Tetrazepam Ž10y8 ]10y4 M. and diltiazem Ž10y8 ]10y5 M. inhibited the amplitude of these contractions in a concentration-dependent manner ŽIC 50 : 6.3= 10y6 M and 3.2= 10y7 M, respectively. and at 10y4 M and 10y5 M, respectively, it suppressed spontaneous activity. This effect was accompanied by a
The contractile responses induced by high concentrations of KCl Ž80 mM. are due to the influx of extracellular Ca2q through L-type voltage-sensitive channels ŽVOCs. and have been used to provide a simple means of studying drugs Žtetrazepam. with possible Ca2q-entry blocking properties. Tetrazepam inhibited these contractile responses in a concentration-dependent manner, which suggested that it inhibited Ca2q through VOCs. In the same way, a similar behaviour was found with the calcium antagonist, diltiazem. One of the major characteristics of the Kq channel openers is that they relax the contractions induced by the low concentration Ž30 mM. of KCl and their vasorelaxant effect is abolished in media containing high concentrations of Kq Ž) 50 mM. w11x. In the rat isolated aorta, cromakalim inhibited the contractions induced by low concentrations Ž30 mM. more effectively than those induced by high concentrations Ž80 mM. of KCl. However, tetrazepam and diltiazem showed a similar effect on both KCl Ž30 and 80 mM.-induced contractions. The relaxant effects of tetrazepam and diltiazem were resistant to potassium channel blockade by glibenclamide Ž10y6 M. even at a 30 mM concentration in the rat aorta. Although cromakalim on KCl Ž30 mM.-induced contractions were competitively antagonized, suggesting that the tetrazepam and diltiazem vasorelaxant effects were not associated to the opening of ATP-sensitive Kq channels. The portal vein develops spontaneous rhythmic transient membrane depolarizations coupled to Ca2q-dependent contractions. This myogenic activity can be inhibited by the removal of extemal Ca2q, Ca2q channel blockers and Kq channel openers, suggesting that myogenic activity depends on Ca2q influx probably through VOCs w21, 22x. K channel openers decrease the frequency of spontaneous rhythmic contractions in this vein by enhancing Kq ion efflux, leading to a hyperpolariza-
Pharmacological Research, Vol. 36, No. 3, 1997
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Table I IC 50 values of tetrazepam, diltiazem and cromakalim in the absence and presence of glibenclamide in rat aorta and rat portal vein. Mean values " SEM obtained from six preparations from different animals Rat aorta
Tetrazepam
Diltiazem
Cromakalim
U
Rat portal ®ein
Contractions induced by KCl 80 m M
Contractions induced by KCl 30 m M
Spontaneous contractions
In absence of Gly In presence of Gly 1 m M In presence of Gly 3 m M
1.7" 0.3= 10y5 1.2" 0.4= 10y5
2.5" 0.8= 10y5 1.9" 0.2= 10y5
6.3" 0.8= 10y6 3.1 "0.9= 10y6
3.0" 0.2= 10y5
1.2" 0.7= 10y5
}
In absence of Gly In presence of Gly 1 m M In presence of Gly 3 m M
1.1" 0.9= 10y7 2.5" 1.4= 10y7
1.0" 0.8= 10y7 1.2" 0.3= 10y7
3.2" 0.8= 10y7 3.1" 0.7= 10y7
3.3" 0.1= 10y7
1.1" 1.4= 10y7
}
In absence of Gly In presence of Gly 1 m M In presence of Gly 3 m M
1.9" 0.8= 10y6 }
1.5" 0.6= 10y7 U 3.9" 1.2= 10y7
3.3" 0.1= 10y7 U 1.9" 0.2= 10y7
}
7.9" 1.3= 10y7
U
}
P- 0.01 significantly different from controls.
Fig. 4. Effects of tetrazepam Ž10y8 ]10y4 M., added cummulatively on amplitude, in the absence Ž`. and presence of glibenclamide, 1 m M Žv. and on frequency, in the absence Ž^. and presence of glibenclamide 1 m M Ž'. of portal vein spontaneous contractions. Ordinate scale: percent of control values. Absissa scale: tetrazepam concentration ŽM.. Each point represents the mean " SEM of six experiments.
Fig. 5. Effects of diltiazem Ž10y8 ]3 = 10y6 M., added cummulatively on amplitude, in the absence Ž`. and presence of glibenclamide 1 m M Žv. and on frequency, in the absence Ž^. and presence of glibenclamide, 1 m M Ž'. of portal vein spontaneous contractions. Ordinate scale: percent of control values. Absissa scale: tetrazepam concentration ŽM.. Each point represents the mean " SEM of six experiments.
tion of the cell membrane w11, 18, 19x. In spontaneously-contracting veins, tetrazepam reduced the size of the contractions with a progressive increase in its frequency. Under the same experimental conditions, diltiazem also inhibited these contractions. Moreover, the inhibition of spontaneous activity persisted after pre-treatment with glibenclamide. In contrast, cromakalim decreased both amplitude and frequency and this effect was blocked by glibenclamide. Hamilton et al. w11x, have clearly shown in
microelectrode experiments that the decrease in the frequency of spontaneous rhythmic contractions is a consequence of the hyperpolarizing effect of K channel openers, which again suggested that its inhibitory effect is not mediated by activation of ATP-dependent Kq channels but may be explained by its ability to decrease Ca2q through VOCs, like those of diltiazem. These results may suggest that, in contrast to cromakalim, the relaxant effects of tetrazepam are
Pharmacological Research, Vol. 36, No. 3, 1997
242
7. 8.
9. 10. 11.
Fig. 6. Effects of cromakalim Ž10y8 ]3 = 10y6 M., added cummulatively on amplitude, in the absence Ž`. and presence of glibenclamide 1 m M Žv. and on frequency, in the absence Ž^. and presence of glibenclamide 1 m M Ž'. of portal vein spontaneous contractions. Ordinate scale: percent of control values. Absissa scale: tetrazepam concentration ŽM.. Each point represents the mean " SEM of six experiments.
12.
13.
14.
not mediated by activation of ATP-dependent Kq channels but may be explained through the blockade of Ca2q entry across the cell membrane like diltiazem.
REFERENCES 1. Groh B, Muller WE. A comparison of the relative in vitro and in vivo binding affinities of various benzodiazepines and related compounds for the benzodiazepine receptor and for peripheral benzodiazepines binding sites. Res Commum Chem Pathol Pharmacol 1985; 49: 463]6. 2. Taft WC, DeLorenzo RJ. Micromolar-affinity benzodiazepine receptors regulated voltage-sensitive calcium channels in nerve terminal preparations. Proc Natl Acad Sci USA 1984; 81: 3118. 3. Mestre M, Carriot C, Belin C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Doble A, Le Fur G. Electrophysiological and pharmacological evidences that peripheral type benzodiazepine receptors are couple to calcium channels in heart. Life Sci 1985; 36: 391]400. 4. Bender AS, Hertz L. Pharmacological evidence that the non-neuronal diazepam binding sites in primary cultures of glial cells is associated with a calcium channels. Eur J Pharmacol 1985; 110: 278]88. 5. Rampe D, Triggle D. Benzodiazepine interactions at neuronal and smooth muscle Ca2q channels. Eur J Pharmacol 1987; 134: 189]95. 6. Perez-Guerrero C, Herrera MD, Marhuenda E. Relaxant effect of tetrazepam on rat uterine smooth
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muscle: role of calcium movement. J Pharm Pharmacol 1996; 48: 1169]73. Perez-Guerrero C, Herrera MD, Marhuenda E. Effects of tetrazepam on vascular smooth muscle of rat and rabbit aorta. Pharma Sci 1996; 2: 1]3. Herrerra MD, Perez-Guerrero C, Marhuenda E. Smooth muscle relaxant effects of tetrazepam on isolated guinea-pig and rat trachealis. J Auton Pharmacol 1996; 16: 105]10. Perez-Guerrero C, Herrera MD, Marhuenda E. Spasmolytic effects of tetrazepam on rat duodenum and guinea-pig ileum. Pharmacol Res 1997; 31: 173]7. Quast U. Do the channel openers relax smooth muscle by opening Kq channels? TIPs 1993; 14: 332]7. Hamilton TC, Weir SW, Wesston AH. Comparison of the effects of BRL-34915 and verapamil on electrical and mechanical activity in rat portal vein. Br J Pharmacol 1986; 88: 103]11. Buckingham RE, Clapham JC. Hamilton TC, Longman SD, Norton J, Poyser RH. Comparison of effects on blood pressure other hemodynamic parameters with those of nifedipine in animal models. J Cardio®asc Pharmacol 1986; 8: 798]804. Buckingham RE, Hamilton TC, Howlett DR, Mootoo S, Wilson C. Inhibition by glibenclamide of the vasorelaxant action of cromakalim in the rat. Br J Pharmacol 1989; 97: 57]64. Cook NS, Weir SW, Danzeisen MC. Antivasoconstrictor effects of the Kq channel opener cromakalim on the rabbit aorta. Comparison with the calcium antagonist isradipine. Br J Pharmacol 1988; 95: 741]52. Cavero I, Mondot S, Mestre M. The vasorelaxant effects of cromakalim in rats are mediated by glibenclamide-sensitive potassioum channels. J Pharmacol Exp Ther 1989; 248: 1261]8. Richer C, Mulder P, Doussau MP, Giudicelli JF. Agonistes potassiques: profil vasodilateur regional shez le rat. Arch Mal Coeur 1989; 82: 1333]7. Winquist RJ, Heaney LA, Wallace AA, Baskin EP. Stein RB, Garcia ML, Kaczorowski GJ. Glyburide blocks the relaxation response to BRL 34915 Žcromakalim., minoxidil sulfate and diazoxide in vascular smooth muscle. J Pharmacol Exp Ther 1989; 248: 149]56. Weir SW, Weston AH. The effects of BRL 34915 and nicorandil on electrical and mechanical activity on 86 Rb efflux in rat blood vessels. Br J Pharmacol 1986; 88: 121]8. Weir SW, Weston AH. Effect of apamin on responses to BRL 34915, nicorandil and other relaxant in guinea-pig taenia caeci. Br J Pharmacol 1986; 88: 113]20. Kaplan FA. Pharmacokinetics of benzodiazepines. In: Priest RG, Vianna Filho U, Amrin R, Skreta M, eds. Benzodiazepines Today and Tomorrow. Baltimore: University Park Press, 1980: pp. 47]70. Cauvin C, Louutzenhiser R, Van Bremem C. Mechanism of calcium antagonist-induced vasodilatation. Ann Re® Pharmacol Toxicol 1983; 23: 373]8. Dacquet C, Mironneau C, Mironneau J. Effects of calcium-dependent contractions of rat portal vein. Br J Pharmacol 1987; 92: 203]9.
VASODILATING EFFECTS OF TETRAZEPAM IN ISOLATED VASCULAR SMOOTH MUSCLES: COMPARISON WITH CROMAKALIM AND DILTIAZEM U ´ ´ C. PEREZ-GUERRERO , J. SUAREZ, M.D. HERRERA and E. MARHUENDA
Laboratorio de Farmacologıa, ´ Facultad de Farmacia, Uni®ersidad de Se®illa, Se®illa, Espana ˜ Accepted 28 July 1997
The vasodilating effects of tetrazepam Ž1,4-benzodiazepine derivative. were studied and compared with those of the K-channel activator, cromakalim and the Ca-channel blocker, diltiazem, in rat aorta smooth muscle and on the spontaneous contractile activity of the rat portal vein. In the aorta, tetrazepam Ž3 = 10y7 ]10y4 M. and diltiazem Ž10y8 ]3 = 10y6 M. concentration-dependently relaxed aortic rings contracted by 30 mM as well as 80 mM KCl. Although cromakalim Ž10y8 ]3 = 10y6 M. concentration-dependently relaxed aortic rings contracted by 30 mM KCl, it did not relax those contrated by 80 mM KCl. In the presence of the ATP-sensitive K-channel blocker, glibenclamide Ž10y6 and 3 = 10y6 M., 30 mM KCl concentration]response curves for the relaxant effect of tetrazepam and diltiazem were unaffected but cromakalim caused a progressive shift of these curves upwards. In the portal vein, tetrazepam inhibited spontaneous contractions, decreased amplitude and increased frequency. Similar behaviour was shown with diltiazem Ž10y8 ]10y5 M. and in both cases, pre-treatment with glibenclamide Ž10y6 M. was ineffective. Although cromakalim Ž10y5 ]10y6 M. decreased both amplitude and frequency, this effect was blocked by glibenclamide. These results indicate that the vasodilator action of tetrazepam is not mediated to the opening of ATP-sensitive K-channels, unlike cromakalim. This may be mediated, like those of diltiazem, by the blockade of calcium movements across the cell Q 1997 The Italian Pharmacological Society membrane. KEY
WORDS:
tetrazepam, benzodiazepine, rat aorta, rat portal vein, vasodilators.
INTRODUCTION This study investigated the effect of tetrazepam on vascular smooth muscle. Tetrazepam is a 1,4-benzodiazepine derivative clinically used as a muscle relaxant. However, unlike most 1,4-benzodiazepines, tetrazepam binds to central and peripheral benzodiazepine binding sites ŽPBS. w1x. The physiological roles of the ŽPBS. remains unclear. It has been suggested that these sites may be linked to voltagedependent Ca2q channels and that ligands which interact at these sites serve as Ca2q channel regulators w2]5x. Previous studies in our laboratory have evaluated the effects of this compound on several types of smooth muscle; rat uterus w6x, rat and rabbit aorta w7x, rat and guinea-pig trachea w8x and rat duodenum and guinea-pig ileum w9x. Tetrazepam produced a relaxation of contractions in vascular and non-vascuU
Corresponding author. Laboratorio de Farmacologıa, ´ Facultad de Farmacia, Cr Prof Garcıa ´ Gonzalez srn 41012, Sevilla, Espana. ˜ 1043]6618r97r090237]06r$25.00r0rfr970221
lar smooth muscles. The results suggest that its relaxant action can be related to calcium movements and these events are not related to high-affinity perypheral benzodiazepine binding sites. The present study was therefore undertaken to again further information into the mechanisms implicated in its vasodilator effects in rat vascular smooth muscle, comparing its effects with those obtained with a calcium entry blocker, diltiazem, as there may be sufficient structural similarity between diltiazem Žbenzothiazepine. and tetrazepam Žbenzodiazepine.. On the other hand, the K channel openers, such as cromakalim, would thus act as indirect Ca2q entry blockers and would be expected to have a pharmacological profile similar to that of the Ca2q antagonist w10x. K channel openers increase the permeability of the cell membrane to Kq and induce membrane hyperpolarization in vascular smooth muscle w11x. Membrane hyperpolarization inhibits the opening of the voltage-dependent Ca channels. It is thus hypothesized that K channel openers indiQ1997 The Italian Pharmacological Society
Pharmacological Research, Vol. 36, No. 3, 1997
238
rectly act as Ca channel antagonists. K channel openers, such as cromakalim may, therefore, be considered to belong to a novel class of vasodilatoy agents and the vasodilating effects for K channel openers have been shown in a number of experimental models w12]16x. Competitive antagonism by glibenclamide, an ATP-sensitive K channel blocker, indicates that the vasodilating effects of cromakalim are mediated by the opening of glibenclamide-sensitive K channels w12, 15, 17x. A number of studies reported the rat portal vein to be useful in evaluating the K channel-opening activity w11, 12, 17]19x. K channel openers decrease the frequency of spontaneous rhythmic contractions in this vein by enhancing Kq ion efflux, leading to a hyperpolarization of the cell membrane w11, 18, 19x. The aim of the present study thus was to investigate the possible K channel-opening activity of tetrazepam by measuring its inhibitory action in rat isolated portal vein. The vasorelaxant profiles of the compound in the rat aorta were also investigated and compared with those of the Ca2q channel antagonist, diltiazem.
MATERIAL AND METHODS
Tissue preparation Wistar rats of either sex, weighing 250]300 g, were killed by a blow to the head. The descending thoracic aorta and portal veins were rapidly dissected and placed in Krebs bicarbonate solution ŽKBS. of the following composition Žin mM.: NaCl 116, KCl 4.75, CaCl 2 1.8, MgCl 2 1.2, KH 2 PO4 1.2, NaHCO3 25 and glucose 11. After an excess of fat and conective tissue had been removed, the aortas were cut into transverse rings, aproximately 2]3 mm wide. These rings were suspended between two stainless steel hooks under a basal tension of 1 g in organ baths containing 30 ml of KBS maintained at 378C and administered with 95% O 2 and 5% CO 2 . In the same way, the portal vein segments Ž15 mm in length. were mounted vertically under a basal tension of 1 g in 30 ml organ baths containing KBS. Aortic rings and portal vein segments were attached to a force-displacement transducer ŽHarvard UF-1. and recorded on an Omniscribe polygraph to measure isometric contractile force. Each preparation was allowed to equilibrate for at least 90 min before the initiation of experimental procedures and during this period the incubation media was changed every 15 min and the resting tension adjusted. After equilibration the following experiments were performed.
stable, tetrazepam was added in progressively increasing cumulative concentrations Ž3 = 10y7 ]10y4 M. at 15-min intervals since preliminary experiments demonstrated that this was the time needed to produce a steady-state relaxation. The experiment was repeated with diltiazem Ž10y8 ]3 = 10y6 M. and cromakalim Ž10y8 ]3 = 10y6 M.. In aditional experiments, aortic rings were incubated with glibenclamide Ž10y6 M or 3 = 10y6 M. 20 min prior to the addition of KCl Ž30 or 80 mM.. Only one agonist was used in each experiment. These results were expressed as a percentage of the maximal control of agonist-induced responses.
Spontaneous contractions in the rat portal ®ein After the spontaneous rhythmic contractions were equilibrated for 90 min, test drugs were added cumulatively to the preparation every 15 min. In separate experiments, glibenclamide 10y6 M was added and allowed to equilibrate for 20 min, before beginning the concentration-response curves for test drugs. This study was undertaken to examine the inhibition of amplitude as the frequency of spontaneous contractions produced by test drugs. The frequency of rhythmic contractions was counted for the last 5 min of every 15-min interval. The ordinate of the concentration]response curve is expressed as a percentage of amplitude or frequency of contractions during the pre-drug period. The doses of tetrazepam used were in the range of blood concentrations as the benzodiazepines have been reported to attain high levels Ž10y7 ]10y5 M. w20x.
Drugs All drugs except tetrazepam, were provided by Sigma Chemical Company ŽSt. Louis, MO, USA.. Potassium chloride was obtained from E. Merck; Tetrazepam was generously provided by Sanofi Winthrop. All drugs were dissolved in 0.9% NaCl except cromakalim and glibenclamide as they were dissolved in distilled water containing 20% dimethyl sulfoxide and 20% ethanol. The final concentration of dimethyl sulfoxide in the tissue bath was less than 0.01% which was shown to have no effect on the basal tonus of the preparation. Tetrazepam was initially dissolved in 1 ml of Tween 80 and diluted further in saline to prepare a 2 = 10y3 M stock solution. The final Tween 80 concentration in the bath did not affect the results, as we confirmed in parallel experiments.
Statisticals analysis Contractions induced in aortic rings In some experiments aortic rings were exposed to a single concentration of KCl Ž80 mM or 30 mM.. When the contractile response to each agonist was
Results are expressed as mean " SEM of six or more preparations obtained from different animals. The statistical significance of differences between the means was assessed using the Student’s t-test for
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unpaired data; P- 0.05 values were considered to represent a significant difference. The concentration needed to produce 50% inhibition ŽIC 50 . was obtained from the regression plot and a mean IC 50 " 95% confidence interval was calculated for each dose assessed.
239
The vasorelaxant effect of tetrazepam, diltiazem and cromakalim were evaluated in the developed
tension before and after treatment with these compounds in rat pre-concentrated with KCl. In the rat isolated aorta pre-concentrated with 80 mM and 30 mM KCl, tetrazepam Ž3 = 10y6 ]10y4 M. and diltiazem Ž10y8 ]3 = 10y6 M. produced a concentration-dependent relaxation wFig. 1 ŽA, B.; Fig. 2 ŽA, B.x. In the presence of glibenclamide Ž10y6 and 3 = 10y5 M. the concentration]response curves were not significantly different. However, cromakalim showed itself to be more potent against 30 mM KCl induced contractions than those evoked by 80 mM KCl and in the presence of glibenclamide, the concentration]response curves shifted upwards in a parallel fashion wFig. 3 ŽA, B.x. Table I shows the IC 50 values obtained in all
Fig. 1. Tetrazepam-induced relaxation in the rat aorta contracted by KCl Ž80 mM. A and Ž30 mM. B. In the absence Ž`. and presence of glibenclamide, 1 m M Žv. and 3 m M Ž'.. Contractions are expressed as percentages of the maximum control responses. Each point represents the mean " SEM of six experiments.
Fig. 2. Diltiazem-induced relaxation in the rat aorta contracted by KCl Ž80 mM. A and Ž30 mM. B. In the absence Ž`. and presence of glibenclamide 1 m M Žv. and 3 m M Ž'.. Contractions are expressed as percentages of maximum control responses. Each point represents the mean " SEM of six experiments.
RESULTS
Effects on KCl (80 and 30 m M ) induced contraction on isolated rat aorta
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progressive increase in the rate of spontaneous contractions. Pre-treatment with the specific inhibitor of ATP-dependent Kq channels, glibenclamide Ž10y6 M., had no significant effects on tetrazepam and diltiazem-induced inhibition of spontaneous contractions ŽIC 50 : 3.1= 10y6 M and 3.1= 10y7 M, respectively.. However, cromakalim Ž10y8 ]3 = 10y6 M. decreased in a concentration-dependent manner both the amplitude and frequency of these contractions ŽIC 50 : 3.3= 10y7 M. and at 10y6 M it suppressed spontaneous activity. Furthermore, these effects of cromakalim were blocked by glibenclamide Ž10y6 M..
CONCLUSIONS
Fig. 3. Cromakalim-induced relaxation in the rat aorta contracted by KCl Ž80 mM. A and Ž30 mM. B. In the absence Ž`. and presence of glibenclamide, 1 m M Žv. and 3 m M Ž'.. Contractions are expressed as percentages of the maximum control responses. Each point represents the mean " SEM of six experiments.
experiments, in the absence and presence of glibenclamide.
Effects on spontaneous contractions of the rat portal ®ein The frequency and amplitude of spontaneous rhythmic contractions was unchanged for over 2.5 h after the equilibration period Ždata not shown.. Therefore, the effects of tetrazepam, diltiazem and cromakalim were evaluated by measuring changes in the amplitude and frequency before and after treatment with these compounds. Tetrazepam Ž10y8 ]10y4 M. and diltiazem Ž10y8 ]10y5 M. inhibited the amplitude of these contractions in a concentration-dependent manner ŽIC 50 : 6.3= 10y6 M and 3.2= 10y7 M, respectively. and at 10y4 M and 10y5 M, respectively, it suppressed spontaneous activity. This effect was accompanied by a
The contractile responses induced by high concentrations of KCl Ž80 mM. are due to the influx of extracellular Ca2q through L-type voltage-sensitive channels ŽVOCs. and have been used to provide a simple means of studying drugs Žtetrazepam. with possible Ca2q-entry blocking properties. Tetrazepam inhibited these contractile responses in a concentration-dependent manner, which suggested that it inhibited Ca2q through VOCs. In the same way, a similar behaviour was found with the calcium antagonist, diltiazem. One of the major characteristics of the Kq channel openers is that they relax the contractions induced by the low concentration Ž30 mM. of KCl and their vasorelaxant effect is abolished in media containing high concentrations of Kq Ž) 50 mM. w11x. In the rat isolated aorta, cromakalim inhibited the contractions induced by low concentrations Ž30 mM. more effectively than those induced by high concentrations Ž80 mM. of KCl. However, tetrazepam and diltiazem showed a similar effect on both KCl Ž30 and 80 mM.-induced contractions. The relaxant effects of tetrazepam and diltiazem were resistant to potassium channel blockade by glibenclamide Ž10y6 M. even at a 30 mM concentration in the rat aorta. Although cromakalim on KCl Ž30 mM.-induced contractions were competitively antagonized, suggesting that the tetrazepam and diltiazem vasorelaxant effects were not associated to the opening of ATP-sensitive Kq channels. The portal vein develops spontaneous rhythmic transient membrane depolarizations coupled to Ca2q-dependent contractions. This myogenic activity can be inhibited by the removal of extemal Ca2q, Ca2q channel blockers and Kq channel openers, suggesting that myogenic activity depends on Ca2q influx probably through VOCs w21, 22x. K channel openers decrease the frequency of spontaneous rhythmic contractions in this vein by enhancing Kq ion efflux, leading to a hyperpolariza-
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241
Table I IC 50 values of tetrazepam, diltiazem and cromakalim in the absence and presence of glibenclamide in rat aorta and rat portal vein. Mean values " SEM obtained from six preparations from different animals Rat aorta
Tetrazepam
Diltiazem
Cromakalim
U
Rat portal ®ein
Contractions induced by KCl 80 m M
Contractions induced by KCl 30 m M
Spontaneous contractions
In absence of Gly In presence of Gly 1 m M In presence of Gly 3 m M
1.7" 0.3= 10y5 1.2" 0.4= 10y5
2.5" 0.8= 10y5 1.9" 0.2= 10y5
6.3" 0.8= 10y6 3.1 "0.9= 10y6
3.0" 0.2= 10y5
1.2" 0.7= 10y5
}
In absence of Gly In presence of Gly 1 m M In presence of Gly 3 m M
1.1" 0.9= 10y7 2.5" 1.4= 10y7
1.0" 0.8= 10y7 1.2" 0.3= 10y7
3.2" 0.8= 10y7 3.1" 0.7= 10y7
3.3" 0.1= 10y7
1.1" 1.4= 10y7
}
In absence of Gly In presence of Gly 1 m M In presence of Gly 3 m M
1.9" 0.8= 10y6 }
1.5" 0.6= 10y7 U 3.9" 1.2= 10y7
3.3" 0.1= 10y7 U 1.9" 0.2= 10y7
}
7.9" 1.3= 10y7
U
}
P- 0.01 significantly different from controls.
Fig. 4. Effects of tetrazepam Ž10y8 ]10y4 M., added cummulatively on amplitude, in the absence Ž`. and presence of glibenclamide, 1 m M Žv. and on frequency, in the absence Ž^. and presence of glibenclamide 1 m M Ž'. of portal vein spontaneous contractions. Ordinate scale: percent of control values. Absissa scale: tetrazepam concentration ŽM.. Each point represents the mean " SEM of six experiments.
Fig. 5. Effects of diltiazem Ž10y8 ]3 = 10y6 M., added cummulatively on amplitude, in the absence Ž`. and presence of glibenclamide 1 m M Žv. and on frequency, in the absence Ž^. and presence of glibenclamide, 1 m M Ž'. of portal vein spontaneous contractions. Ordinate scale: percent of control values. Absissa scale: tetrazepam concentration ŽM.. Each point represents the mean " SEM of six experiments.
tion of the cell membrane w11, 18, 19x. In spontaneously-contracting veins, tetrazepam reduced the size of the contractions with a progressive increase in its frequency. Under the same experimental conditions, diltiazem also inhibited these contractions. Moreover, the inhibition of spontaneous activity persisted after pre-treatment with glibenclamide. In contrast, cromakalim decreased both amplitude and frequency and this effect was blocked by glibenclamide. Hamilton et al. w11x, have clearly shown in
microelectrode experiments that the decrease in the frequency of spontaneous rhythmic contractions is a consequence of the hyperpolarizing effect of K channel openers, which again suggested that its inhibitory effect is not mediated by activation of ATP-dependent Kq channels but may be explained by its ability to decrease Ca2q through VOCs, like those of diltiazem. These results may suggest that, in contrast to cromakalim, the relaxant effects of tetrazepam are
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242
7. 8.
9. 10. 11.
Fig. 6. Effects of cromakalim Ž10y8 ]3 = 10y6 M., added cummulatively on amplitude, in the absence Ž`. and presence of glibenclamide 1 m M Žv. and on frequency, in the absence Ž^. and presence of glibenclamide 1 m M Ž'. of portal vein spontaneous contractions. Ordinate scale: percent of control values. Absissa scale: tetrazepam concentration ŽM.. Each point represents the mean " SEM of six experiments.
12.
13.
14.
not mediated by activation of ATP-dependent Kq channels but may be explained through the blockade of Ca2q entry across the cell membrane like diltiazem.
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