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Dissociation of calcium- and barium-induced contractions of the rat duodenum smooth muscle
Gen. Pharmac. Vol. 14, No. 6, pp. 643-647, 1983 Printed in Great Britain. All rights reserved
0306-3623/8353.00 + 0.00 Copyright © 1983 Pergamon Press Ltd
DISSOCIATION OF CALCIUM- A N D BARIUM-INDUCED CONTRACTIONS OF THE RAT D U O D E N U M SMOOTH MUSCLE Y. YASHUDA 1, A. M. CABRAL2, J. G. P. PIRES 2 a n d A. ANTONIO3. *Departamento de Ci6ncias Fisiolbgicas, Faculdade de Farmficia e Odontologia de Araraquara, Sao Paulo, Brazil 2Laborat6rio de Farmacologia do Departamento de Biologia, Universidade Federal do Espirito Santo, Vit6ria, Espirito Santo, Brazil, and 3Departamento de Farmacologia, Faculdade de Medicina de Ribeirgo Preto, Universidade de Silo Paulo, Brazil
(Tel: 634-6035) (Received 26 January 1983) Abstract--1. The contractile responses elicited by calcium (Ca) and barium (Ba) upon the isolated rat d u o d e n u m immersed in a Ca-free Tyrode solution are not nervously mediated and are differently affected by epinephrine (E), cocaine (C) and verapamil (V). 2. E antagonizes Ca but not Ba while C inhibits only Ba. The antagonism of V is greater towards Ba than Ca. 3. The different influence of the drugs disappear in the excess potassium-depolarized duodenum. Thus, C and V block equally Ca and Ba, while E is only slightly effective against the two cations.
INTRODUCTION
MATERIALS AND METHODS
T h e m u s c u l a r tone n o r m a l l y exhibited by the isolated d u o d e n u m of the rat is completely lost when the tissue is i m m e r s e d in a calcium-free medium. A contractile response ensues w h e n calcium is reint r o d u c e d ( A n t o n i o , 1968; Valette a n d Leclair, 1975, 19~16). Interestingly, the tension developed by the d u o d e n u m to the re-addition of calcium has been reported to be increased in the presence of local anaesthetics (Valette a n d Leclair, 1975), a result which is a p p a r e n t l y in disaccord with the well k n o w n effect o f these drugs in blocking the calcium influx a n d consequently m u s c u l a r c o n t r a c t i o n (Feinstein, 1966; A n t o n i o et al., 1970). It should be recalled t h a t the c o n t r a c t i o n of the rat d u o d e n u m to calcium seems to be a peculiarity o f the s m o o t h muscles which show a very high t o n u s in n o r m a l conditions. Accordingly, the muscles which in n o r m a l e n v i r o n m e n t have n o t o n u s do n o t c o n t r a c t to the re-addition o f calcium when immersed in a calcium-free medium, unless they are depolarized by excess p o t a s s i u m or in the presence o f c o n v e n t i o n a l agonists ( E d m a n a n d Schild, 1962; Feinstein, 1966; A n t o n i o et al., 1973). W e t h o u g h t it of interest to further analyse the c o n t r a c t i o n o f the isolated rat d u o d e n u m elicited by calcium as well as the influence of local anaesthetics a n d other drugs k n o w n to interfere with intestinal s m o o t h muscle c o n t r a c t i o n a n d calcium transport. Barium, which has been reported to p r o d u c e a contraction o f the isolated rat d u o d e n u m i n d e p e n d e n t o f the presence o f calcium in the m e d i u m (Back a n d Charlier, 1963; A n t o n i o , 1968) was used for comparison.
Adult albino rats of either sex were 'killed by a blow on the head and bled. The duodenum was set up in a 10-ml organ bath containing calcium-free Tyrode solution (composition in mM/1 of glass-distilled water: NaC1 137.0, KCI 3.0, MgCI 2 1.0, NaH2PO 4 0.4, NarlCO 3 12.0 and glucose 5.5) at 35°C and continuously bubbled with air. The responses of the duodenum, magnified six times, were recorded isotonically on a smoked paper. The total load on the tissue was approximately 1 g. The preparation was repeatedly washed during a 2-hr period of stabilization. After this time the duodenum was usually considered to be "free" of calcium since the characteristic tonus and spontaneous activity were not seen; moreover, no contraction was obtained by the addition of 2 × 10-TM acetylcholine. In the experiments to be described, the "agonists" which elicited contractile responses were Ca 2÷ and Ba2÷. They were added directly to the organ bath in volumes never exceeding 0.2 ml. The time cycle between successive additions was 3 min and included the contraction, washings and relaxation. Freshly made antagonists were also added directly to the muscle chamber 90 sec before either Ca 2+ or Ba2+. The potassium depolarizing solution was made up by replacing 70~o of the NaCI with equimolar amounts of KC1. In other experiments, variable amounts of the NaC1 of the Tyrode solution were replaced with twice the molar concentration of sucrose. The following drugs were used: l-epinephrine bitartrate (Mann Research Laboratories, U.S.A.), cocaine hydrochloride and atropine sulphate (Merck Darmstadt, Germany), verapamil hydrochloride (Knoll, Brazil), tetrod0toxin (Calbiochem, U.S.A.). All other reagents were of analytical grade. RESULTS
*Author to whom all correspondence should be addressed.
Following the 2-hr period o f stabilization, the 643
644
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Fig. 1. Dose-response curves for calcium and barium in the rat duodenum immersed in a calcium-free Tyrode solution. In ordinates the per cent of the maximal contractile response obtained wiht 12.8 x 10-3 M barium. The points represent the mean_ SE of 6 experiments. Panel A shows the dose-response curves in the 3 mM KCI solution. Panel B in the 70 mM KCI solution. tissue responds to successive additions of calcium with highly reproducible contractions. These responses which are readily reversible by washing increase with the dose up to a final concentration of calcium of the order of 0.8-1.6 mM. If the concentration of calcium is further increased, a decline in response is observed (Fig. 1A). It should be mentioned that a vigorous contraction follows the washing of the higher concentrations of calcium and that in this case a longer time is needed for the complete relaxation of the tissue. The same figure shows the dose-response curve for Ba 2÷ with the contractions increasing with the concentration up to the maximal response of the preparation. Tachyphylaxis develops with successive additions of barium. As shown in Fig. 2, the development of tachyphylaxis is inversely related to the concentration of Ba 2+. Thus, when successive additions of doses of barium of 1.6 mM are employed, practically no tachyphylaxis is observed. Moreover, if Ca 2+ and Ba 2÷ are added alternatively even smaller doses of barium are no longer tachyphylactic. For this reason the two ions were always added alternatively when constructing the dose-response curves and testing the influence of antagonists. The responses to Ca 2+ and Ba 2+ are not nervously
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mediated since they are not blocked by neither atropine (3 x 10-7 M) or tetrodotoxin (2 × 10-7 M). The contractions to Ca 2÷ and Ba 2÷ are differently affected by epinephrine and cocaine. While epinephrine specifically blocks the response to Ca 2+, the local anaesthetic inhibits the response to Ba 2÷ without decreasing, and in higher doses even facilitating the action of Ca 2+ (Table 1). The recovery from inhibition is immediate for both epinephrine and cocaine upon washing the tissue. The influence of verapamil was not so clear cut as that observed with epinephrine and cocaine. As illustrated in Fig. 3, the antagonism which is relatively slow in onset and resistant to recovery after washing is greater towards Ba 2÷ than Ca 2+. In the potassium depolarizing Tyrode solution the dose-response curves to Ca 2÷ and Ba2÷ are much alike without any decrement in the responses with increasing doses of Ca 2÷ (Fig. IB) and complete absence of tachyphylaxis to Ba2+; the two ions are now equally affected by the antagonists. As summarized in Table 1, in the depolarized preparation the contraction to the two ions are equally blocked by cocaine and practically unaffected by the small range of doses of epinephrine. Only very high doses of epinephrine produce a partial blockade of the responses. The replacement of NaCI by sucrose induces a rapid decline in the responses to Ca 2÷ with practically no influence in the contractions elicited by Ba 2÷ (Fig. 4).
"9.-.~aq
12
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Time (rnin) Fig. 2. Isotonic contractile responses of the rat duodenum immersed in calcium-free Tyrode solution to successive additions of the final concentrations of O. 1, 0.2, 0.4, 0.8 and
1.6mM barium chloride. The points represent the mean _+SE of 4 experiments.
It has been proposed that the smooth muscle tonus which is regulated by the free intracellular calcium (Rfiegg, 1971) is due to spontaneous spike discharges; these spikes are probably the result of an increased sodium permeability consequent to a poor adsorption of Ca 2+ at the cell membrane (Bueding and Biilbring, 1964). It is also possible that calcium itself can carry the current of the action potential in addition to its function in initiating the contraction (Bfilbring and Tomita, 1969b; Fleckenstein, 1977). Whatever the underlying mechanism might be, the tone of the isolated rat duodenum is completely lost following the removal of calcium of the Tyrode solution. Under these circumstances a bell-shaped dose-response
Calcium- and barium-induced contractions
645
Table I. The inhibitory effect of epinephrine, cocaine and verapamil on the contractile responses elicited by calcium and barium upon the isolated rat duodenum immersed in normal and excess-potassium calcium-free Tyrode solutions. The results are expressed as the mean per cent change of the control contraction _+SE of 4-9 experiments Normal potassium Antagonist x 10 6M Epinephrine Epinephrine Epinephrine Cocaine Cocaine Verapamil Verapamil
0.4 4.0 40.0 30.0 60,0 0,01 0.05
Excess potassium
Calcium
Barium
Calcium
Barium
92.9 ___1.1 99.1 ___1.0 -+2.4___0.8 + 3.6 ___0.6 9.0 -+ 2,3 39.8 _+ 2.1
+4,3 + 1,0 + 5.4 + 1,5
9.6 _+ 1.5 29.4 + 3.5 48.2 + 3.3 81.2+2.6 98.6 + 1.0 34.6 -+ 2.6 83.1 _+ 2.8
8.4 + 1.3 24.5 + 3.4 37.8 + 3.2 83.9_+2.3 99.2 + 1.1 36.8 _+ 3.1 85,2 _+ 3,7
67.7_+2,5 97.4 -+ 1,5 47.3 + 3,4 88,2 + 2,7
The sign + before some of the per cent change values denotes a facilitatory action.
curve is obtained by re-adding calcium to the organ bath. While the left side of the curve, up to 1.0-1.6 mM calcium, is indicative of the contractile function of calcium and therefore of the maintenance of the smooth muscle tonus, the right side of the curve (above 1.0 to 1.6mM) is indicative of the well-known stabilizing effect of calcium which probably has the same underlying mechanism of the inhibitory action of epinephrine (Bileding and Billbring, 1964; Billbring and Tomita, 1969a). In the present study we tried to analyse only the contractile action of calcium. Having in mind the aforementioned stabilizing effect, we did not persist in overcoming a drug-induced blockade of the contractions by increasing the calcium concentration. Under the same conditions barium was more potent than calcium in eliciting the contraction and did not show any decrement in the response with increasing concentrations. Contrary to the guinea-pig ileum immersed in a calcium-free Tyrode solution where its effect is strongly tachyphylactic (Antonio et al., 1973), the successive addition of 0.5-1.0mM Ba 2÷ induces only a moderate and slowly developing tachyphylaxis upon the rat duodenum. Even so we circumvent such a possible source of raisin-
1.0 rnM Co 2+
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terpretation by alternating the additions of barium and calcium. Under these conditions no tachyphylaxis was observed. This result is in agreement with reports showing the dependence of at least part of the effect of barium on a cellular store of calcium (Antonio et al., 1973; Rahwan et aL, 1977; Wikberg, 1981; Slegers et aL, 1981). In the case of the rat duodenum the dependence for calcium seems to be greater for the smaller doses of barium. The contractile response to calcium was quantitatively blocked by epinephrine in doses which did not affect the responses to barium. The inhibition of calcium contraction is obtained with the same concentration of the sympathomimetic reported to relax the rat duodenum immersed in normal-calcium Tyrode solution (Antonio, 1968; Miachon et al., 1979). The inhibitory action of epinephrine is probably due to a greater adsorption of calcium at the cell membrane (Billbring and Tomita, 1969a) a mechanism which seems to be linked to an enhanced Na+/K ÷ active transport (Scheid et al., 1979). On the other hand, cocaine which is known to prevent the passage of calcium from extracellular to intracellular sites in other smooth muscle preparations (Feinstein, 1966), abolishes the responses to barium without interfering and sometimes even facilitating the contractions due to calcium. The potentiation of the responses to calcium by cocaine have
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Fig. 3. Isolated rat duodenum immersed in a calcium-free Tyrode solution. Inhibitory action of verapamil (VP) on the contractions elicited by calcium and barium. The antagonist was added directly into the muscle chamber 90 sees before each addition of the ions during the time shown between the arrows. Observe the greater inhibition and slower recovery of barium contractions. The results are the mean _+SE of 4 experiments.
0
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Fig. 4. The influence of the replacement of NaCI of the calcium-free Tyrode solution by sucrose on the contractions elicited by calcium and barium on the isolated rat duodenum. The points represent the mean _ SE of 4 experiments.
646
Y. YASHUDAet al.
already been reported by Valette and Leclair (1975) and as they postulate could be due to a blockade of calcium uptake by intracellular organelles since it is also obtained with several metabolic inhibitors (Valette and Leclair, 1976). Another possibility suggested by Bianchi and Bolton (1967) could be an intracellular release of calcium by the local anesthetic. It is also interesting that the blockade by cocaine of the responses to barium is not obtained with tetrodotoxin thus discarding the participation of the fast sodium channels. The calcium antagonistic drug verapamil (Fleckenstein, 1977) in very small doses was able to antagonize the responses to both calcium and barium. The antagonism produced by verapamil towards barium was more pronounced and of slower recovery which is suggestive of a more efficient or an additional system to transport calcium from the extra-to the intracellular space in the rat duodenum. The differences in the responses to calcium and barium practically disappear when the preparation is depolarized by the replacement of NaCI by KCI: (1) their potencies are about the same, (2) no decrement in the responses is observed when the calcium concentration is increased, (3) unless very high doses are used, epinephrine has no action on either calcium or barium-induced contractions, (4) cocaine is equally effective against both cations. These results support the view of a common mechanism for the transport of calcium and barium in the depolarized duodenum. Such a transport is probably passive and the same one involved in the contraction elicited by barium in the polarized muscle. The concomitant disappearnace of the stabilizing action of calcium and of the inhibitory effect of epinephrine in the potassium-depolarizing solution is an argument in favour of a common mechanism for epinephrine and excess calcium-induced relaxations as already suggested by Bueding and Biilbring (1964). On the other hand, the mechanisms by which calcium within a very limited range of concentration (left side of the bell-shaped curve) in the extracellular space is transported to the interior of the cell and triggers the contraction remains to be determined. As pointed out, the spontaneous electrical activity of some intestinal smooth muscle and the resultant tonic activity does not seem to require nervous inputs (Daniel and Sarna, 1978). This seems to be the case of the rat duodenum tone which is not inhibited by either cocaine or tetrodotoxin. However, other types of chemical mediation by a locally produced substance cannot be exlcuded since the contractile responses to calcium decrease as increasing amounts of sodium are replaced by sucrose. SUMMARY
A contractile response is obtained by the readdition of 0.1 to 1.6 × 10 -3 M calcium (Ca) to a previously decalcified rat duodenum. If higher concentrations of Ca are added, the contractions decrease in amplitude. Under the same conditions, no decrement in the contractile response is observed with barium (Ba) over a wider range of concentration (0.1 to 12.8 × 10 -3 M). The contractions to either Ca or Ba are not affected by atropine (3.0 x 10 -7 M) or
tetrodotoxim (2.0 x 10-TM). The responses to Ca but not those to Ba are quantitatively blocked by epinephrine (0.4 to 4.0 × 10-7M). Cocaine (3.0 to 6.0 × 10 5 M), on the other hand, inhibits only the responses to Ba. Verapamil (5.0 x 10 -8 M) produces an antagonism which is more intense and of slower recovery towards Ba rather than Ca. These differences between Ba and Ca practically disappear in the depolarized rat duodenum. Thus, the replacement of the NaC1 of the Tyrode solution by KCI makes the responses to both Ca and Ba become almost identical with those obtained with Ba in the polarized preparation, i.e. a continuous increase in the response with the dose, blockade by cocaine and only a slight influence of epinephrine. The replacement of NaCI by sucrose abolishes the responses to Ca without affecting the contractions to Ba. These results are suggestive of a non-neurogenic nature of the contractions elicited by Ca and Ba in the normal polarized preparation. Moreover, although the two cations may be using the same channel, the transport of Ca seems to be chemically mediated while that of Ba depends only on its inwardly directed gradient.
REFERENCES
Antonio A. (1968) The relaxing effect of bradykinin on intestinal smooth muscle. Br. J. Pharmac. 32, 78-86. Antonio A., Rocha e Silva M. and Yashuda Y. (1970) Influence of pH on the inhibitory action of local anaesthetics on smooth muscle contraction. Br. J. Pharmac. 40, 501-507. Antonio A., Rocha e Silva M. and Yashuda Y. (1973) The tachyphylactic effect of barium on intestinal smooth muscle. Archs int. Pharmacodyn. Thbr. 2114, 260-267. Back Z. M. and Charlier R. (1963) Action des ions alcalinoterreux sur la musculature lisse. In Ions alcalino-terreux (Edited by Back Z. M.) pp. 435-481. Springer-Verlag, Berlin. Bianchi C. P. and Bolton T. C. (1967) Action of local anaesthetics on coupling systems in muscle. J. Pharmac. exp. Ther. 157, 388-405. Bueding E. and Bulbring E. (1964) The inhibitory action of adrenaline. Biochemical and biophysical observations. In Pharmacology o f Smooth Muscle (Edited by Biilbring E.) pp. 37. Pergamon Press, London. Biilbring E. and Tomita T. (1969a) Suppression of Spohtaneous spike generation by catecholamines in the smooth muscle of the guinea pig taenia coli. Proc. R. Soc. B 172, 103-119. Bfilbring E. and Tomita T. (1969b) Effect of calcium, barium and manganese on the action of adrenaline in the smooth muscle of the guinea-pig taenia coli. Proc. R. Soc. B 172, 121-136. Daniel E. E. and Sarna S. (1978) The generation and conduction of activity in smooth muscle. A. Rev. pharmac. toxic. 18, 145-166. Edman K. A. P. and Schild H. O. (1962) The need for calcium in the contractile responses induced by acetylcholine and potassium in the rat uterus. J. Physiol., Lond. 161, 424-441. Feinstein M. B. (1966) Inhibition of contraction and calcium exchangeability in rat uterus by local anesthetics. J. Pharmac. exp. Ther. 152, 516-524. Fleckenstein A. (1977) Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle. A. Rev. pharmac, toxic. 17, 149-166. Miachon S., Peyrin L. and Cier J. F. (1979) Action des pr6eurseurs et des m6tabolites des cat~cholamines sur la
Calcium- and barium-induced contractions motricit~ du duod6num isol~ de rat. Arch. int. Physiol. Biochim. 87, 899-914. Rahwan R. G., Faust M. M. and Witiak D. T. (1977) Pharmacological evaluation of new calcium antagonists: 2-substituted 3-dimethylamino-5,6-methylenedioxyindenes. J. Pharrnac. exp. Ther. 201, 126-137. Riiegg J. C. (1971) Smooth muscle tone. Physiol. Rev. 51, 201-248. Scheid C. R., Honeyman T. W. and Fay F. S. (1979) Mechanism of/3-adrenergic relaxation of smooth muscle. Nature Lond. 277, 32-36. Slegers J. F. G., F6rster T. G. and Moons W. M. (1981) Inhibition of calcium release by diazoxide studied in the
647
isolated rat kidney. Archs int. Pharmacodyn. ThOr. 250, 147-163. Valette G. and Leclair M,-F. (1975) Effects de la cocaine sur les 6changes de calcium observes sur le duod+num de rat. C.r. hebd. S~anc. Acad. Sci. 281, 949-950. Valette G. and Leclair M.-F. (1976) Actions des inhibiteurs m&aboliques sur les r~ponses m~caniques d6veloppbes par le duodenum isol~ de rat sous les effects des variations du taux de calcium du milieu. J. Pharmac., Paris 7, 549-562. Wikberg J. E. S. (1981) Reversal of at-receptor mediated relaxation in intestinal smooth muscle. Acta physiol. scand. 111, 385-395.
0306-3623/8353.00 + 0.00 Copyright © 1983 Pergamon Press Ltd
DISSOCIATION OF CALCIUM- A N D BARIUM-INDUCED CONTRACTIONS OF THE RAT D U O D E N U M SMOOTH MUSCLE Y. YASHUDA 1, A. M. CABRAL2, J. G. P. PIRES 2 a n d A. ANTONIO3. *Departamento de Ci6ncias Fisiolbgicas, Faculdade de Farmficia e Odontologia de Araraquara, Sao Paulo, Brazil 2Laborat6rio de Farmacologia do Departamento de Biologia, Universidade Federal do Espirito Santo, Vit6ria, Espirito Santo, Brazil, and 3Departamento de Farmacologia, Faculdade de Medicina de Ribeirgo Preto, Universidade de Silo Paulo, Brazil
(Tel: 634-6035) (Received 26 January 1983) Abstract--1. The contractile responses elicited by calcium (Ca) and barium (Ba) upon the isolated rat d u o d e n u m immersed in a Ca-free Tyrode solution are not nervously mediated and are differently affected by epinephrine (E), cocaine (C) and verapamil (V). 2. E antagonizes Ca but not Ba while C inhibits only Ba. The antagonism of V is greater towards Ba than Ca. 3. The different influence of the drugs disappear in the excess potassium-depolarized duodenum. Thus, C and V block equally Ca and Ba, while E is only slightly effective against the two cations.
INTRODUCTION
MATERIALS AND METHODS
T h e m u s c u l a r tone n o r m a l l y exhibited by the isolated d u o d e n u m of the rat is completely lost when the tissue is i m m e r s e d in a calcium-free medium. A contractile response ensues w h e n calcium is reint r o d u c e d ( A n t o n i o , 1968; Valette a n d Leclair, 1975, 19~16). Interestingly, the tension developed by the d u o d e n u m to the re-addition of calcium has been reported to be increased in the presence of local anaesthetics (Valette a n d Leclair, 1975), a result which is a p p a r e n t l y in disaccord with the well k n o w n effect o f these drugs in blocking the calcium influx a n d consequently m u s c u l a r c o n t r a c t i o n (Feinstein, 1966; A n t o n i o et al., 1970). It should be recalled t h a t the c o n t r a c t i o n of the rat d u o d e n u m to calcium seems to be a peculiarity o f the s m o o t h muscles which show a very high t o n u s in n o r m a l conditions. Accordingly, the muscles which in n o r m a l e n v i r o n m e n t have n o t o n u s do n o t c o n t r a c t to the re-addition o f calcium when immersed in a calcium-free medium, unless they are depolarized by excess p o t a s s i u m or in the presence o f c o n v e n t i o n a l agonists ( E d m a n a n d Schild, 1962; Feinstein, 1966; A n t o n i o et al., 1973). W e t h o u g h t it of interest to further analyse the c o n t r a c t i o n o f the isolated rat d u o d e n u m elicited by calcium as well as the influence of local anaesthetics a n d other drugs k n o w n to interfere with intestinal s m o o t h muscle c o n t r a c t i o n a n d calcium transport. Barium, which has been reported to p r o d u c e a contraction o f the isolated rat d u o d e n u m i n d e p e n d e n t o f the presence o f calcium in the m e d i u m (Back a n d Charlier, 1963; A n t o n i o , 1968) was used for comparison.
Adult albino rats of either sex were 'killed by a blow on the head and bled. The duodenum was set up in a 10-ml organ bath containing calcium-free Tyrode solution (composition in mM/1 of glass-distilled water: NaC1 137.0, KCI 3.0, MgCI 2 1.0, NaH2PO 4 0.4, NarlCO 3 12.0 and glucose 5.5) at 35°C and continuously bubbled with air. The responses of the duodenum, magnified six times, were recorded isotonically on a smoked paper. The total load on the tissue was approximately 1 g. The preparation was repeatedly washed during a 2-hr period of stabilization. After this time the duodenum was usually considered to be "free" of calcium since the characteristic tonus and spontaneous activity were not seen; moreover, no contraction was obtained by the addition of 2 × 10-TM acetylcholine. In the experiments to be described, the "agonists" which elicited contractile responses were Ca 2÷ and Ba2÷. They were added directly to the organ bath in volumes never exceeding 0.2 ml. The time cycle between successive additions was 3 min and included the contraction, washings and relaxation. Freshly made antagonists were also added directly to the muscle chamber 90 sec before either Ca 2+ or Ba2+. The potassium depolarizing solution was made up by replacing 70~o of the NaCI with equimolar amounts of KC1. In other experiments, variable amounts of the NaC1 of the Tyrode solution were replaced with twice the molar concentration of sucrose. The following drugs were used: l-epinephrine bitartrate (Mann Research Laboratories, U.S.A.), cocaine hydrochloride and atropine sulphate (Merck Darmstadt, Germany), verapamil hydrochloride (Knoll, Brazil), tetrod0toxin (Calbiochem, U.S.A.). All other reagents were of analytical grade. RESULTS
*Author to whom all correspondence should be addressed.
Following the 2-hr period o f stabilization, the 643
644
Y. YASHUDAet I00
- A
g.-
J,-~-=--=
al.
- =
d;.;.._l-.
BO
.!
60
-
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x`
40
~/(
~f'!'~
20 o
I 10-2M
I
10-4
10-3
10-4
• Barium
I A calou~ 10-3
10-2M
Borium or colciurn
Fig. 1. Dose-response curves for calcium and barium in the rat duodenum immersed in a calcium-free Tyrode solution. In ordinates the per cent of the maximal contractile response obtained wiht 12.8 x 10-3 M barium. The points represent the mean_ SE of 6 experiments. Panel A shows the dose-response curves in the 3 mM KCI solution. Panel B in the 70 mM KCI solution. tissue responds to successive additions of calcium with highly reproducible contractions. These responses which are readily reversible by washing increase with the dose up to a final concentration of calcium of the order of 0.8-1.6 mM. If the concentration of calcium is further increased, a decline in response is observed (Fig. 1A). It should be mentioned that a vigorous contraction follows the washing of the higher concentrations of calcium and that in this case a longer time is needed for the complete relaxation of the tissue. The same figure shows the dose-response curve for Ba 2÷ with the contractions increasing with the concentration up to the maximal response of the preparation. Tachyphylaxis develops with successive additions of barium. As shown in Fig. 2, the development of tachyphylaxis is inversely related to the concentration of Ba 2+. Thus, when successive additions of doses of barium of 1.6 mM are employed, practically no tachyphylaxis is observed. Moreover, if Ca 2+ and Ba 2÷ are added alternatively even smaller doses of barium are no longer tachyphylactic. For this reason the two ions were always added alternatively when constructing the dose-response curves and testing the influence of antagonists. The responses to Ca 2+ and Ba 2+ are not nervously
,oo
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'
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r.
= .oi k
DISCUSSION
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6
mediated since they are not blocked by neither atropine (3 x 10-7 M) or tetrodotoxin (2 × 10-7 M). The contractions to Ca 2÷ and Ba 2÷ are differently affected by epinephrine and cocaine. While epinephrine specifically blocks the response to Ca 2+, the local anaesthetic inhibits the response to Ba 2÷ without decreasing, and in higher doses even facilitating the action of Ca 2+ (Table 1). The recovery from inhibition is immediate for both epinephrine and cocaine upon washing the tissue. The influence of verapamil was not so clear cut as that observed with epinephrine and cocaine. As illustrated in Fig. 3, the antagonism which is relatively slow in onset and resistant to recovery after washing is greater towards Ba 2÷ than Ca 2+. In the potassium depolarizing Tyrode solution the dose-response curves to Ca 2÷ and Ba2÷ are much alike without any decrement in the responses with increasing doses of Ca 2÷ (Fig. IB) and complete absence of tachyphylaxis to Ba2+; the two ions are now equally affected by the antagonists. As summarized in Table 1, in the depolarized preparation the contraction to the two ions are equally blocked by cocaine and practically unaffected by the small range of doses of epinephrine. Only very high doses of epinephrine produce a partial blockade of the responses. The replacement of NaCI by sucrose induces a rapid decline in the responses to Ca 2÷ with practically no influence in the contractions elicited by Ba 2÷ (Fig. 4).
"9.-.~aq
12
18
t 24
J -27
J
33
Time (rnin) Fig. 2. Isotonic contractile responses of the rat duodenum immersed in calcium-free Tyrode solution to successive additions of the final concentrations of O. 1, 0.2, 0.4, 0.8 and
1.6mM barium chloride. The points represent the mean _+SE of 4 experiments.
It has been proposed that the smooth muscle tonus which is regulated by the free intracellular calcium (Rfiegg, 1971) is due to spontaneous spike discharges; these spikes are probably the result of an increased sodium permeability consequent to a poor adsorption of Ca 2+ at the cell membrane (Bueding and Biilbring, 1964). It is also possible that calcium itself can carry the current of the action potential in addition to its function in initiating the contraction (Bfilbring and Tomita, 1969b; Fleckenstein, 1977). Whatever the underlying mechanism might be, the tone of the isolated rat duodenum is completely lost following the removal of calcium of the Tyrode solution. Under these circumstances a bell-shaped dose-response
Calcium- and barium-induced contractions
645
Table I. The inhibitory effect of epinephrine, cocaine and verapamil on the contractile responses elicited by calcium and barium upon the isolated rat duodenum immersed in normal and excess-potassium calcium-free Tyrode solutions. The results are expressed as the mean per cent change of the control contraction _+SE of 4-9 experiments Normal potassium Antagonist x 10 6M Epinephrine Epinephrine Epinephrine Cocaine Cocaine Verapamil Verapamil
0.4 4.0 40.0 30.0 60,0 0,01 0.05
Excess potassium
Calcium
Barium
Calcium
Barium
92.9 ___1.1 99.1 ___1.0 -+2.4___0.8 + 3.6 ___0.6 9.0 -+ 2,3 39.8 _+ 2.1
+4,3 + 1,0 + 5.4 + 1,5
9.6 _+ 1.5 29.4 + 3.5 48.2 + 3.3 81.2+2.6 98.6 + 1.0 34.6 -+ 2.6 83.1 _+ 2.8
8.4 + 1.3 24.5 + 3.4 37.8 + 3.2 83.9_+2.3 99.2 + 1.1 36.8 _+ 3.1 85,2 _+ 3,7
67.7_+2,5 97.4 -+ 1,5 47.3 + 3,4 88,2 + 2,7
The sign + before some of the per cent change values denotes a facilitatory action.
curve is obtained by re-adding calcium to the organ bath. While the left side of the curve, up to 1.0-1.6 mM calcium, is indicative of the contractile function of calcium and therefore of the maintenance of the smooth muscle tonus, the right side of the curve (above 1.0 to 1.6mM) is indicative of the well-known stabilizing effect of calcium which probably has the same underlying mechanism of the inhibitory action of epinephrine (Bileding and Billbring, 1964; Billbring and Tomita, 1969a). In the present study we tried to analyse only the contractile action of calcium. Having in mind the aforementioned stabilizing effect, we did not persist in overcoming a drug-induced blockade of the contractions by increasing the calcium concentration. Under the same conditions barium was more potent than calcium in eliciting the contraction and did not show any decrement in the response with increasing concentrations. Contrary to the guinea-pig ileum immersed in a calcium-free Tyrode solution where its effect is strongly tachyphylactic (Antonio et al., 1973), the successive addition of 0.5-1.0mM Ba 2÷ induces only a moderate and slowly developing tachyphylaxis upon the rat duodenum. Even so we circumvent such a possible source of raisin-
1.0 rnM Co 2+
,oo
terpretation by alternating the additions of barium and calcium. Under these conditions no tachyphylaxis was observed. This result is in agreement with reports showing the dependence of at least part of the effect of barium on a cellular store of calcium (Antonio et al., 1973; Rahwan et aL, 1977; Wikberg, 1981; Slegers et aL, 1981). In the case of the rat duodenum the dependence for calcium seems to be greater for the smaller doses of barium. The contractile response to calcium was quantitatively blocked by epinephrine in doses which did not affect the responses to barium. The inhibition of calcium contraction is obtained with the same concentration of the sympathomimetic reported to relax the rat duodenum immersed in normal-calcium Tyrode solution (Antonio, 1968; Miachon et al., 1979). The inhibitory action of epinephrine is probably due to a greater adsorption of calcium at the cell membrane (Billbring and Tomita, 1969a) a mechanism which seems to be linked to an enhanced Na+/K ÷ active transport (Scheid et al., 1979). On the other hand, cocaine which is known to prevent the passage of calcium from extracellular to intracellular sites in other smooth muscle preparations (Feinstein, 1966), abolishes the responses to barium without interfering and sometimes even facilitating the contractions due to calcium. The potentiation of the responses to calcium by cocaine have
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z
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, "I/
o H , , , , , , , , , , , 0
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Fig. 3. Isolated rat duodenum immersed in a calcium-free Tyrode solution. Inhibitory action of verapamil (VP) on the contractions elicited by calcium and barium. The antagonist was added directly into the muscle chamber 90 sees before each addition of the ions during the time shown between the arrows. Observe the greater inhibition and slower recovery of barium contractions. The results are the mean _+SE of 4 experiments.
0
I 20 %
I 40
I 60
80
I O0
NaCI replaced by sucrose
Fig. 4. The influence of the replacement of NaCI of the calcium-free Tyrode solution by sucrose on the contractions elicited by calcium and barium on the isolated rat duodenum. The points represent the mean _ SE of 4 experiments.
646
Y. YASHUDAet al.
already been reported by Valette and Leclair (1975) and as they postulate could be due to a blockade of calcium uptake by intracellular organelles since it is also obtained with several metabolic inhibitors (Valette and Leclair, 1976). Another possibility suggested by Bianchi and Bolton (1967) could be an intracellular release of calcium by the local anesthetic. It is also interesting that the blockade by cocaine of the responses to barium is not obtained with tetrodotoxin thus discarding the participation of the fast sodium channels. The calcium antagonistic drug verapamil (Fleckenstein, 1977) in very small doses was able to antagonize the responses to both calcium and barium. The antagonism produced by verapamil towards barium was more pronounced and of slower recovery which is suggestive of a more efficient or an additional system to transport calcium from the extra-to the intracellular space in the rat duodenum. The differences in the responses to calcium and barium practically disappear when the preparation is depolarized by the replacement of NaCI by KCI: (1) their potencies are about the same, (2) no decrement in the responses is observed when the calcium concentration is increased, (3) unless very high doses are used, epinephrine has no action on either calcium or barium-induced contractions, (4) cocaine is equally effective against both cations. These results support the view of a common mechanism for the transport of calcium and barium in the depolarized duodenum. Such a transport is probably passive and the same one involved in the contraction elicited by barium in the polarized muscle. The concomitant disappearnace of the stabilizing action of calcium and of the inhibitory effect of epinephrine in the potassium-depolarizing solution is an argument in favour of a common mechanism for epinephrine and excess calcium-induced relaxations as already suggested by Bueding and Biilbring (1964). On the other hand, the mechanisms by which calcium within a very limited range of concentration (left side of the bell-shaped curve) in the extracellular space is transported to the interior of the cell and triggers the contraction remains to be determined. As pointed out, the spontaneous electrical activity of some intestinal smooth muscle and the resultant tonic activity does not seem to require nervous inputs (Daniel and Sarna, 1978). This seems to be the case of the rat duodenum tone which is not inhibited by either cocaine or tetrodotoxin. However, other types of chemical mediation by a locally produced substance cannot be exlcuded since the contractile responses to calcium decrease as increasing amounts of sodium are replaced by sucrose. SUMMARY
A contractile response is obtained by the readdition of 0.1 to 1.6 × 10 -3 M calcium (Ca) to a previously decalcified rat duodenum. If higher concentrations of Ca are added, the contractions decrease in amplitude. Under the same conditions, no decrement in the contractile response is observed with barium (Ba) over a wider range of concentration (0.1 to 12.8 × 10 -3 M). The contractions to either Ca or Ba are not affected by atropine (3.0 x 10 -7 M) or
tetrodotoxim (2.0 x 10-TM). The responses to Ca but not those to Ba are quantitatively blocked by epinephrine (0.4 to 4.0 × 10-7M). Cocaine (3.0 to 6.0 × 10 5 M), on the other hand, inhibits only the responses to Ba. Verapamil (5.0 x 10 -8 M) produces an antagonism which is more intense and of slower recovery towards Ba rather than Ca. These differences between Ba and Ca practically disappear in the depolarized rat duodenum. Thus, the replacement of the NaC1 of the Tyrode solution by KCI makes the responses to both Ca and Ba become almost identical with those obtained with Ba in the polarized preparation, i.e. a continuous increase in the response with the dose, blockade by cocaine and only a slight influence of epinephrine. The replacement of NaCI by sucrose abolishes the responses to Ca without affecting the contractions to Ba. These results are suggestive of a non-neurogenic nature of the contractions elicited by Ca and Ba in the normal polarized preparation. Moreover, although the two cations may be using the same channel, the transport of Ca seems to be chemically mediated while that of Ba depends only on its inwardly directed gradient.
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647
isolated rat kidney. Archs int. Pharmacodyn. ThOr. 250, 147-163. Valette G. and Leclair M,-F. (1975) Effects de la cocaine sur les 6changes de calcium observes sur le duod+num de rat. C.r. hebd. S~anc. Acad. Sci. 281, 949-950. Valette G. and Leclair M.-F. (1976) Actions des inhibiteurs m&aboliques sur les r~ponses m~caniques d6veloppbes par le duodenum isol~ de rat sous les effects des variations du taux de calcium du milieu. J. Pharmac., Paris 7, 549-562. Wikberg J. E. S. (1981) Reversal of at-receptor mediated relaxation in intestinal smooth muscle. Acta physiol. scand. 111, 385-395.