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Effect of changes in ionic environment on the action of bradykinin on the guinea-pig taenia coli
EUROPEAN JOURNAL OF PHARMACOLOGY 12 (1970) 348-358. NORTH-HOLLAND PUBLISHING COMPANY
EFFECT
OF CHANGES
IN IONIC ENVIRONMENT
ON THE ACTION OF
BRADYKININ ON T H E G U I N E A - P I G T A E N I A C O L I P. N. A A R S E N and M. VAN CASPEL-DE BRUYN Pharmacological Laboratory of the University of A msterdam, PoMerweg 104, Amsterdam, The Netherlands
Received 25 March 1970
Accepted 25 June 1970
P.N.AARSEN and M.VAN CASPEL-DE BRUYN, Effect of changes in ionic environment on the action of bradykinin on the guinea-pig taenia coli, European J. Pharmacol. 12 (1970) 348-358. The effect of changes in the concentration of sodium and calcium in the medium and the effect of adrenaline on the mechanical and electrical responses of the guinea-pig taenia coli to bradykinin were investigated using an organ bath and a sucrose-gap method. In normal Krebs solution the response to bradykinin was mostly biphasic: an acute initial phase charaterized by spike inhibition and decreased isometric tension, followed by a second phase with an increased frequency and amplitude of the spikes and membrane depolarization, accompanied by an increase in isometric tension. Omission of sodium at normal or twice the normal calcium concentration strengthened and lengthened the initial phase and suppressed the second phase. Lowering the calcium concentration abolished the initial phase of bradykinin, while the second phase, especially the increased electrical activity, was enhanced. After lowering both the calcium and the sodium concentration the spontaneous spike activity ceased almost immediately. Under these conditions bradykinin only caused slight hyperpolarization. The effect of substituting strontium for calcium was similar to that of lowering the calcium concentration. However, after a long period of exposure to strontium the initial phase of the response to bradykinin reappeared and was enhanced by doubling the strontium concentration. After a preceding dose of adrenaline, 5 #g, the initial phase of the bradykinin effect was lengthened, while the second phase was not significantly affected. It is suggested that bradykinin acts on more than one site at the cell membrane. The spike inhibition in the initial phase may be explained if it is assumed that bradykinin prevents the release of calcium superficially bound to the cell membrane and involved in the spike generation. Spike stimulation accompanied by depolarization in the second phase is thought to be due to an action of bradykinin at another site in the membrane. Bradykinin Adrenaline
Guinea-pig taenia coli
1. I N T R O D U C T I O N Bradykinin is known to act directly on smooth muscle cells (Khairallah and Page, 1963; Ohashi, Nonomura and Ohga, 1967; Schnieden and Weston, 1969). According to the smooth muscle preparation used, bradykinin can induce contraction, relaxation or a biphasic effect, i.e. an initial relaxation followed by a contraction (Elliott, Horton and Lewis, 1960). Since, in most pharmacological organ preparations bradykinin causes contraction (StiJrmer and Berde,
Sodium Calcium
1963), the question arises as to whether the bradykinin induced relaxation e.g. in the rat duodenum, is due to a different mode o f action or to a difference in the condition o f the cell membrane. Ohashi, Nonomura and Ohga (1967) showed that changes in the biphasic response of the guinea-pig taenia coli to bradykinin appeared to be dependent in some cases on the electrical membrane activity at the moment o f addition of bradykinin, whereas in several other cases no such relation was found. They suggested that bradykinin might have a dual action on the smooth
P.N,4arsen, M.van Caspel-deBruyn, The action o[ bradykinin muscle membrane. Antonio (1968) showed that lowering the external c~cium concentration changed the bradykinin induced relaxation of the rat duodenum into a biph~sic response. In |he im~ent imm~ig~tion the various responses of smooth muscle cells to bradykinin were investigated after changing their ionic environment and hence their electrical and mechanical activities. The guineapig taenia coli was chosen since: l)this preparation usually shows a biphasic reaction to bradykinin in Krebs solution with a normal calcium concentration; 2) both electrical and mechanical activities can be recorded in this preparation by using the sucrose-gap technique; 3)the taenia coli has been investigated very extensively.
2. METHODS Female guinea-pigs of about 500 g body weight were killed by a blow on the head. Strips of 15 to 20 × 0.5 mm and about 0.5 mm thick, were torn out of the superficial layers of the taenia coli. The strips were suspended either in a small organ bath or in a sucrose-gap. One side of the strip was connected by means of a thin nylon thread to an RCA5734 transducer. In all experiments, the tension of the strips was adjusted to 0.5 g. The organ bath and the active side of the sucrose-gap were continuously perfused with salt solution at 37°C with a rate of about 2 ml per min; the perfusion fluid could be changed by means of a multiway stopcock. During the control periods the salt solution used was a modified Krebs-Ringer solution (further indicated as normal Krebs), containing (mM): NaCI 118.4; KC1 4.7; CaC12 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCOs 15.5; glucose 11.1. This solution was prepared from stock solutions according to the method described by Umbreit, Burris and Staufer (1951). The following test solutions were used: Krebs solutions with 0.25 or 5 mM CaCI2 instead of 2.5 mM; a Krebs solution in which CaC12 was replaced by 2.5 mM SrCI2 and 0.1 mM EGTA (ethylenglycol-2-(2-aminoethyl)-tetracetic acid); a sodium-free and a sodium-deficient Krebs solution. In the case of the Krebs solution with double the Ca2+-ion concentration, 10 ml tris-HCl buffer (pH 7.3) was added per 1.3 1. The sodium-free Krebs solution, in which sod-
349
ium was substituted by tris-HCl buffer, was prepared according to Biilbring and Kuriyama (1963a). In the sodium-deficient Krebs solution containing 15.5 mM Na÷ instead of 133.9 mM, NaCI was replaced by an equivalent amount of tris-HCl buffer (pH 7.3). Unless stated otherwise 0.25 ml of a solution containing 1 jag of synthetic bradykinin (Sandoz BRS/640) per ml was added during 30 sec to the supply of perfusion fluid by a continuous infusion apparatus. Bradykinin reached the taenia coli strip within one second after the beginning of the injection period. 2.1. Organ bath method The strip of taenia coli was suspended in the cylindrical hole, 20 X 2 mm, of a perspex organ bath by means of nylon threads. The thread at the upper end of the strip was connected to the anode pin of the transducer RCA 5734. The changes of the isometric tension were recorded by a Kipp pen-recorder. In all experiments a constant dose of bradykinin (0.25/ag) was added to the organ preparation at 4 min intervals. The perfusion fluid under test was always changed immediately after the effect of the preceding dose of bradykinin had been recorded. 2.2. Sucrose-gap method Changes of the membrane potential and the electrical activity as well as the isometric tension were recorded using the sucrose-gap of St~impfli (1954), as modified by Biilbring and Burnstock (1960). In all experiments the inactive side of the gap was perfused with normal Krebs at 5 to IO°C. Changes in the membrane potential and in the electrical activity were measured differentially by using two unity gain preamplifiers and a differential oscilloscope. The changes in the isometric tension were recorded on the same oscilloscope.
3. RESULTS 3.1. The effect of varying the calcium concentration, in the presence and absence o f sodium, on the mechanical responses to bradykinin In three series of organ bath experiments the effects of the following changes in the perfusion fluid were studied: 1) absence of sodium (8 exp.); 2) double the normal calcium concentration (5 mM) in the
350
P.N.Aarsen, M.van Caspel~e Bruyn, The action o f bradykinin
presence and absence of sodium (8 exp.); 3)one tenth of the normal calcium concentration in the presence and absence of sodium (6 exp.). Each experiment was started with three successive additions of 0.25/ag of bradykinin to the taenia coli perfused with normal Krebs. Thereafter, the perfusion was changed either to a sodium-free solution (first series) or to a Krebs solution having an altered calcium concentration first in the presence, and second in the absence of sodium (2nd and 3rd series). The results of these three series of experiments are plotted in figs. la, lb and 2. The points on the drawn lines represent the mean isometric tension ( t o n e ) a t the moment bradykinin was added. The vertical broken and drawn lines represent respectively the mean initial relaxation and the following contraction induced by bradykinin. These figures show that usually bradykinin produces a biphasic effect in normal Krebs (2.5 mM Ca). In sodium-free solution, muscle tone and the relaxing activity of bradykinin increased considerably in the presence of either normal or twice normal calcium concentration (see figs. l a and b). However, the increased relaxation induced by bradykinin was not related to the increased tone; figs. la and lb show that at the end of the experiments relaxation decreased progressively despite increased tone. Fig. 1b further shows that twice normal calcium concentration in the presence of a normal concentration of sodium did not affect the mechanical response to bradykinin. At a low calcium concentration (0.25 mM) both the tone and the reaction to bradykinin were decreased considerably (see fig. 2). The relaxing activity of bradykinin almost completely disappeared within eight min after changing the perfusion fluid and bradykinin only produced contraction; the height of the contraction was decreased. In the absence of sodium the effect of lowering the calcium concentration on the response to bradykinin appeared earlier; the initial relaxation was almost completely abolished within four min. The inhibition of the second contractile phase also occurred more rapidly and was more pronounced. These findings demonstrate that the reaction to bradykinin can be changed by changing the external medium. Omission of sodium enhanced the initial, relaxing, phase, whereas the second, contractile, phase was suppressed. The predominant effect of reducing calcium to one tenth of the normal concen.
Isometric tension(g) 5 Ca (raM) 2.5 i 2.5.Na-free 4
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24
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Fig. 1. The effect of the calcium concentration, in the presence and absence of sodium, on the mean isometric tension (tone, o) of taenia coli and changes induced by a constant dose of bradykinin (0.25 #g) added at 4 min intervals. The vertical broken and drawn lines indicate respectively the mean initial decrease (relaxation) and increase (contraction) in tension caused by bradykinin added at the moments indicated on the abscissae. a) Tone and response to bradykinin of the taenia coli (8 exp.) perfused first with normal Krebs (2.5 mM Ca), and second with a sodium-free Krebs solution (2.5 mM Ca, Na-free). Omission of sodium causes an increase in tone, while the relaxation response to bradykinin increases. Eight min after changing perfusion the bradykinin-induced relaxation decreases progressively in spite of the fact that the tone remains
high. b) Tone and response to bradykinin of the taenia coli (8 exp.) perfused successivelywith normal Krebs, Krebs with twice the normal calcium concentration (5 mM), normal Krebs and sodium-free Krebs containing 5 mM calcium. Double the calcium concentration in the presence of sodium does not considerably affect both tone and response to bradykinin, whereas omission of sodium has about the same influence as at a normal calcium concentration.
P.N.Aarsen, M.van CaspelwleBruyn, The action o f bradykinin Isometric tension(g) 5-C,a (mM)2.5 0.25
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Fig. 2. Effect of calcium concentration, in the presence and absence of sodium, on the mean isometric tension (tone, s) of taenia coil and changes induced by a constant dose of bradykinin (0.25 ~g) added at 4 rain intervals. In 6 experiments the taenia coli was superfused with normal Krebs (2.5 mM Ca), Krebs with one tenth the normal calcium concentration (0.25 raM), normal Krebs and sodinm-free Krebs containing 0.25 mM calcium. Lowering the calcium concentration causes both a decrease in tone and in response to bradykinin. The relaxing activity of bradykinin almost completely disappeared within eight rain o f changing the perfusion fluid. In the absence of sodium the effect of lowering the calcium concentration on the response to bradykinin appeared earlier: the mean initial relaxation was almost completely abolished within four min. For further description see fig. 1.
tration was suppression of the relaxing phase. In a low calcium and sodium-free medium both phases of the response to bradykinin were suppressed. 3.2. The effect o f various calcium concentrations, in the presence and absence o f sodium, on the electrical response to bradykinin and its relation to the mechanical response The organ bath experiments were repeated in a similar way using the sucrose-gap method; each experiment was repeated at least five times. The response to bradykinin in normal Krebs was, in most cases, biphasic: an initial phase characterized by acute spike suppression and decreased isometric tension was followed by a second phase which often started with irregular rlring of spikes and an increase in isometric tension. Later, firing became more regular; frequency and amplitude of the spikes were increased. Depolari-
351
zation ensued and isometric tension increased further. In our experiments, the membrane potential did not always increase during the initial phase as stated by Ohashi et al. (1967). Apart from a slight increase observed in a few cases, no change or even a decrease in membrane potential was usually found. However, the effect of bradykinin depended on the conditions prevailing before treatment. In preparations with a fluctuating, unstable membrane potential (see control in fig. 4), the predominant response to bradykinin was contraction (second phase). In preparations with a stable membrane potential, coupled with regular spike activity, the initial relaxing phase of the response to bradykinin was dominant. Such a pattern is shown by the first control in fig. 3. Furthermore this figure shows that on repeated administration of bradykinin the initial phase decreased; in the second control the initial phase was interrupted by a few spikes, resulting in fluctuations of the isometric tension. 3.2.1. Sodium-free solution Fig. 3 shows an experiment in which the influence of sodium-free Krebs on the response to bradykinin was investigated. The most characteristic effect of omission of sodium was an increase and prolongation of the initial phase within four min after changing the sodium content. The duration of the spike inhibition, which was 53 sec during the first control experiment, increased to more than 135 sec within four min after the omission of sodium. At 12, 16 and 20 min after starting the perfusion with sodium-free Krebs, the spike inhibition lasted 70, I01 and 105 sec respectively and was accompanied by a concurrent increase in duration of the relaxation. It must be stressed that during this phase no hyperpolarization occurred. The second phase of the response to bradykinin was abolished in sodium-free medium: the spike frequency did not increase, and in most cases it remained lower than before the addition of bradykinin. The tone usually returned to its resting value. Furthermore, usually depolarization of the membrane potential was not observed. An exception is shown in fig. 3 where a slight depolarization occurred 12 min after the perfusion with sodium-free solution was started, accompanied by a slight increase in tone over the resting value.
P.N.Aarsen, M.van Caspel-de Bruyn, The action o f bradykinin
352
|
Control
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Fig. 3. The effect of bradykinin on spontaenous membrane activity recorded by means of the sucrose-gap method (upper traces), and on the isometric tension (lower traces) of the guinea-pig taenia coli. Horizontal bars indicate periods when 0.25 pg bradykinin (B) was added to the perfusion fluid at the active side of the gap, being either normal Krebs during the control period (control), or sodium-free Krebs (Na-free). Records were taken at 4, 12, 16 and 20 min respectively after changing from normal Krebs to sodium-free Krebs.
P.N.Aarsen, M. van Caspel-de Bruyn, The action o f bradykinin
3.2.2. The effect of varying the calcium concentration in the presence and absence of sodium Doubling the calcium concentration to 5 mM without changing the sodium Concentration had only a small effect on the electrical response to bradykinin. Fig. 4 shows that within four min after exposure to this concentration a short-lasting initial phase (inhibition of spikes) was observed, whereas in the preceding control experiment this initial phase was absent. After twelve min, however, the response was not different from that in the control period. In the second control period the response to bradykinin had become biphasic. Exposure to twice the normal calcium concentration, in the absence of sodium in this case, had a stabilizing effect on the spike firing mechanism. The response to bradykinin changed in a characteristic
353
way: a pronounced initial phase occurred, whereas' the second phase was suppressed. Exposure to a tenth of the normal calcium concentration (0.25 mM) had the following effect. The initial phase was suppressed and the second phase, especially its electrical phenomena, was activated (fig. 5). In spite of the increase in frequency and amplitude of the spikes during the second phase, the increase in isometric tension was smaller than in the control situation. After a second control period, in which the active side of the sucrose-gap was again perfused with normal Krebs, the muscle was exposed to a solution containing 0.25 mM calcium and 15.5 mM sodium. In spite of the fact that approximately 10% of the sodium remained, spontaneous activity ceased within four min and the tone was considerably decreased.
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Fig. 4. The effect of 0.25 tag bradykinin (B) on the spontaneous membrane activity recorded with the sucrose-gap method (upper traces), and on the isometric tension (lower traces) of the guinea-pig taenia coll. The effects were investigated using the same preparation superfused in succession with normal Krebs (control), Krebs with twice the normal calcium concentration (5 mM Ca) (at 4 rain and 12 rain after starting the perfusion), normal Krebs (control), and sodium-free Krebs with twice the normal calcium concentration (5 mM Ca, Na-free) (at 4 rain and 12 rain after starting the perfusion).
P.N.Aarsen, M.van Caspei-deBruyn, The action of bradykinin
354
Con t rol
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5 mV B
4'
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Control
12'
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Q25mM Ca,15.5 mM Na
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Fig. 5. The effects of 0.25 pg hradykinin (B) on the spontaneous membrane activity recorded with the sucrose-gap method and on the isometric tension of the guinea-pig taenia eoli superfused successively with normal Krebs (control), Krebs with one tenth the normal calcium concentration (0.25 mM Ca), normal Krebs (control), and sodium-deficient Krebs with one tenth the normal calcium concentration (0.25 mM Ca, 15.5 mM Na). For further description see figs. 3 and 4. Under these conditions bradykinin only produced hyperpolarization, which was increased after a longer exposure to the low calcium, low sodium solution (see fig. 5). In this case no change in the tension occurred; in some o f the other experiments a small increase in tension occurred after hyperpolarization.
3.4. The effect o f substituting strontium for calcium Replacement o f calcium by an equivalent amount o f strontium in the presence o f 0.1 mM EGTA produced a characteristic pattern o f changes in the firing o f spikes, i.e. the spike amplitude was increased and the spike frequency was increased initially but declined after some time (see fig. 6). Moreover, the spikes became more regular. At low frequencies (about 0.1/sec), a pronounced positive after-potential
could be demonstrated. Replacement of calcium by strontium changed response to bradykinin as follows: in the beginning, when the spike frequency was still high, bradykinin caused only stimulation, an increase in spike frequency and amplitude accompanied by a small depolarization. Later on this stimulation was preceded by a short4asting initial phase, consisting mainly o f a decrease in spike frequency. After exposure to twice the strontium concentration (5 mM) a pronounced initial phase appeared within four min (see fig. 6). During the second phase of the response to bradykinin a contraction still occurred, even after two hours exposure to the calcium-free medium. After lowering the sodium concentration (to 15.5 mM) o f the strontium containing medium, the electrical activity stopped almost immediately.
355
P.N.Aarsen, M.van Caspel-deBruyn, The action ol'bradykinin
Control
2.5mM Sr +0.1mM EGTA
5 mV I. lg t B
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Fig. 6. The effect of 0.25 #g bradykinin (B) on the spontaneous membrane activity recorded with the sucrose-gap method (upper traces), and on the isometric tension (lower traces) of the guinea-pig taenia eo5 superfused successively with normal Krebs (control), calcium-free, strontium containing Krebs (2.5 mM Sr + 0.1 mM EGTA), and calcium-free Krebs with twice the concentration of strontium (5 mM Sr + 0.1 mM EGTA). For further description see figs. 3 and 4. 3.5. The effect o f halving the calcium concentration and the effect o f exposure to adrenaline In these experiments the effect of 30 min exposure to half the normal calcium concentration on the electrical responses of the taenia coli to bradykinin was investigated and compared with that of a preceding dose of adrenaline in normal Krebs. Adrenaline was used as a pharmacological tool, because its action resembles that of excess calcium (Biilbring, 1957; BiJlbring and Kuriyama, 1963b). In these experiments a constant volume o f 0.1 ml of bradykinin solution (10/ag/ml) was injected into the drop counter placed in the supply tube at the active side of the sucrosegap. The adrenaline (5/ag) was added by means of a continuous infusion apparatus as follows: 0.2 ml per min o f an adrenaline solution (10/ag/ml) during 2.5 min. Four min after the end o f this addition, the
injection of bradykinin was started. In each experiment the electrical response to bradykinin was investigated successively in Krebs solutions with 2.5, 1.25 and 2.5 mM calcium. Thereafter the effect of a preceding exposure to adrenaline was determined. The mean (-+ S.E.M.) o f the electrical responses, i.e. the duration o f the initial spike inhibition, the secondary increase in spike frequency and the depolarization, are plotted in fig. 7. This figure shows that halving the calcium concentration reduced the mean duration of the initial spike inhibition from 30 -+ 6.2 (S.E.M.) sec to 2 -+ 1.6 (S.E.M.) sec; in nine out of ten determinations, this initial response disappeared with half the normal calcium concentration. After exposure to adrenaline the initial spike inhibiting effect o f bradykinin increased from 31 -+ 6.2 (S.E.M.) see to 78 + 21.5 (S.E.M.) sec. However, the
356
P.N.Aarsen, M.van Caspel-de Bruyn, The action of bradykinin
number of
sec 100
spikes/1OOsec
mV 100
Plh [ ] period of inhibition(sec) / r ] i n c r e a s e m number
oh / of spikes/lO0sec 80 2 /~] depolarization(mV)
80
60
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(10)
(6)
(6)
40
40
20 ~
20
0
2.5
1.25
2.5
0 2.5 mM Ca
after exposure to adrenaline
Fig. 7. The effects of halving the calcium concentration and of preceding exposure to adrenaline on the first (ph 1) and the second phase (ph 2) of electrical responses of the guinea-pig taenia coli induced by 1 pg of bradykinin. In each experiment the active side of the sucrose-gap was perfused in succession with Krebs containing 2.5, 1.25 and 2.5 mM calcium. In the last solution the effect of a preceding dose of 5 pg adrenaline on the response to bradykinin was studied. The heights of the columns indicate the means of the in brackets indicated number of determinations; the vertical bars indicate the S.E.M.
electrical phenomena of the second phase, increase in spike frequency and depolarization, were not affected by both halving the calcium concentration and preceding exposure to adrenaline.
4. DISCUSSION In normal Krebs solution, the response of the taenia coli to bradykinin was usually biphasic. An initial phase characterized by spike inhibition and a decrease in isometric tension was followed by a second phase with an increased frequency and amplitude of the spikes and membrane depolarization, accompanied by an increase in isometric tension. Spike inhibition and a decrease in isometric tension occurred simultaneously during the initial phase as did stimulation of spike activity and increase in isometric tension during the second phase. Less correlation was found between the mechanical response
and the change in membrane potential. In some cases the initial relaxation seemed to be accompanied by a small hyperpolarization, probably due to suppression of the superimposed spikes of numerous firing cells, ~ausing a slight decrease in the base-potential. In other cases either a depolarization or no change of the membrane potential was found during the relaxation. The maximum increase in isometric tension, observed in the second phase, occurred before membrane depolarization. Thus, bradykinin-induced changes in isometric tension must be a direct consequence o f changes in the spontaneous spike activity, which according to Bueding and Bi]lbring (1964) maintains tone. However, in some cases either predominantly inhibitory or predominantly stimulatory responses to bradykinin were f o u n d in normal Krebs, probably depending on the membrane activity before the addition of bradykinin. If the spontaneous spikes were regular and the resting membrane potential constant, clear-cut inhibition of the spikes commenced within 15 sec of adding bradykinin. This reaction was frequently followed by a period of increased spike activity. When the spontaneous spikes were irregular. and the resting membrane potential fluctuated, the response to bradykinin was predominantly a stimulation. Since Biilbring and Kuriyama (1963a) demonstrated that a solution containing excess calcium stabilizes the membrane activity, regardless of whether sodium is present or not, it was assumed that the differences in response of the taenia coli to bradykinin might depend on differences in the amount of calcium bound to the cell membrane. The more calcium is bound to the cell membrane, the stronger the initial response to bradykinin. This hypothesis is supported by the following findings. Lowering of the calcium concentration abolished the initial phase of spike inhibition, whereas the second phase of the electrical response started sooner and was more pronounced. A predominance of the initial inhibitory phase was also found after omission of sodium. This may be explained by an increase in the amount of calcium bound to the superficial anionic sites caused by a reduction in the concentration o f competing sodium, as postulated by Goodford (1967). The fact that doubling the calcium concentration at a normal sodium concentration had only a slight effect, is not at variance with this explanation, since the concentra-
P.N.Aarsen, M.van Caspel-cleBruyn, The action of bradykinin
tion of competing sodium ions is many times higher than that of calcium ions. After pretreatment with adrenaline the initial phase of the bradykinin effect was also lengthened, but the second phase was not suppressed. According to Bueding and Biilbring (1964), Biilbring, Goodford and Setekleiv (1966) and BiJlbring and Tomita (1969) adrenaline may cause an increase in the amount of calcium bound to the cell membrane. Therefore, the observed effect of adrenaline may also support the view that the occurrence of the initial phase of the response to bradykinin is somehow related to the amount of calcium bound to the cell membrane. However, adrenaline did not affect the second phase whereas sodium deficiency caused a reduction of the second phase in addition to its effect on the initial phase. This shows that a reduction of the second phase is not necessarily coupled with a predominance of the initial phase. The suppression of the second phase during sodium deficiency might therefore be caused by an additional effect not related to an increase in the amount of calcium bound at the anionic sites. The calcium ion is very important for spike activity in taenia smooth muscle, and under normal conditions probably carries most of the current during the action potential (Biilbring and Kuriyama, 1963a; Nonomura, Hotta and Ohashi, 1966; Brading and Tomita, 1968; Brading, BLilbring and Tomita, 1969a). According to Biilbring (personal communication) the results described by Brading, Biilbring and Tomita (1969a,b) favour superficially bound calcium as the source from which ions can be released to carry the inward current of the spike. Strontium and barium are supposed to replace calcium in its function of generating the spike (Hotta and Tsukui, 1968). The initial phase of the bradykinin action resembled the effect of adrenaline in so far as both compounds suppressed the spikes in normal Krebs solution and both effects were enhanced in excess calcium. However, the initial phase of bradykinin differs from the effect of adrenaline in the following respects: 1) in contrast to adrenaline bradykinin does not usually cause hyperpolarization; 2) in sodium-free solution, the effect of bradykinin is enhanced but the effect of adrenaline is decreased (Biilbring and Kuriyama, 1963b); 3)according to Biilbring and Tomita (1969) substitution of barium for calcium suppresses
357
the effect of adrenaline, whereas substitution of strontium for calcium never suppresses the initial phase of bradykinin action completely. The most likely explanation for the initial effect of bradykinin is that bradykinin prevents the release of calcium bound to superficial sites which will carry the current for the spike. This explains spike suppression, absence of hyperpolarization, absence of an effect of strontium, and increase in effect caused by omission of sodium. Omission of sodium may in itself increase the attraction between calcium and anionic membrane sites. Since the initial spike inhibiting effect of bradykinin depends on the amount of calcium, possibly bound at the superficial anionic sites, it is tempting to speculate that bradykinin might have some affinity for this calcium complex which would suppress release of calcium from its binding site. This supposed mechanism of bradykinin in its initial phase of action does not explain the second excitatory phase of bradykinin action. It is reasonable to suppose that this second phase is caused by an action of bradykinin at a different site.
ACKNOWLEDGEMENT It is a pleasure to thank Prof.Dr. C.Van Der Meer for his advise and helpful criticism during the investigation and during the preparation of this manuscript.
REFERENCES Antonio, A., 1968, The relaxing effect of bradykinin on intestinal smooth muscle, Brit. J. Pharmacol. 32, 78-86. Brading, A.F. and T.Tomita, 1968, The action potential of the guinea-pig taenia coli in low sodium solution, J. Physiol. 197, 30-31P. Brading, A.F., E.Biilbting and T.Tomita, 1969a, The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coil, J. Physiol. 200, 637-654. Brading, A.F., E.Biilbring and T.Tomita, 1969b, The effect of temperature on the membrane conductance of the smooth muscle of the guinea-pig taenia coil, J. Physiol. 200, 621-635. Bueding, E. and E.Biilbring, 1964, The inhibitory action of adrenaline. Biochemical and biophysical observations, in: Pharmacology of Smooth Muscle, ed. E.Biilbring (Czechoslovak Medical Press, Prague) pp. 37-56.
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Biilbring, E., 1957, Changes of configuration of spontaneously discharged spike potentials from smooth muscle of the guinea-pig's taenia coli. The effect of electrotonic currents and of adrenaline, acetylcholine and histamine, J. Physiol. 135,412-425. Biilbring, E. and G.Burnstock, 1960, Membrane potential changes associated with tachyphylaxis and potentiation of the response to stimulating drugs in smooth muscle, Brit. J. Pharmacol. 15, 6 1 1 - 6 2 4 . Biilbring, E. and H.Kuriyama, 1963a, Effects of changes in the external sodium and calcium concentrations on spontaneous electrical activity in smooth muscle of guinea-pig taenia coli, J. Physiol. 166, 2 9 - 5 8 . Biilbring, E. and H.Kuriyama, 1963b, Effects of changes in ionic environment on the action of acetylcholine and adrenaline on the smooth muscle cells of guinea-pig taenia coli, J. Physiol. 166, 5 9 - 7 4 . Biilbring, E., P.J.Goodford and J.Setekleiv, 1966, The action of adrenaline on the ionic content and on sodium and potassium movements in the smooth muscle of the guinea-pig taenia coli, Brit. J. Pharmacol. 28, 2 9 6 - 3 0 7 . Biilbring, E. and T.Tomita, 1969, Effect of calcium, barium and manganese on the action of adrenaline in the smooth muscle of the guinea-pig taenia coli, Proc. Roy. Soc. London, B172, 121-136. Elliot, D.F., E.W.Horton and G.P.Lewis, 1960, Actions of pure bradykinin, J. Physiol. 1 5 3 , 4 7 3 - 4 8 0 .
Goodford, P.J., 1967, The calcium content of smooth muscle of the guinea-pig taenia coli, J. Physiol. 192, 145 147. Hotta, Y. and R.Tsukui, 1968, Effect on the guinea-pig taenia coli of the substitution of strontium or barium ions for calcium ions, Nature 2 1 7 , 8 6 7 - 8 6 9 . Khairallah, P.A. and I.H.Page, 1963, Effects of bradykinin and angiotensin on smooth muscle, Ann. N.Y. Acad. Sci. 104, art. 1 , 2 1 2 - 2 2 1 . Nonomura, Y., Y.Hotta and H.Ohashi, 1966, Tetrodotoxin and manganese ions: Effects on electrical activity and tension in taenia coli of guinea-pig, Science 152, 97- 99. Ohashi, H., Y.Nonomura and A.Ohga, 1967, Effects of angiotensin, bradykinin and oxytocin on electrical and mechanical activities in the taenia coli of the guinea-pig, Japan J. Pharmacol. 17,247 257. Schnieden, H. and A.H. Weston, 1969, Investigation of the spasmogenic effect of manganese on the guinea-pig isolated ileum preparation, Brit. J. Pharmacol. 3 6 , 4 9 6 - 5 0 9 . St~npfli, R., 1954, A new method for measuring membrane potentials with external electrodes, Experienta 10, 508. Stiirmer, E. and B.Berde, 1963, A comparative pharmacological study of synthetic eledoisin and synthetic bradykinin, J. Pharmacol. Exptl. Therap. 140, 349 355. Umbreit, W.W., R.H.Burris and J.F.Stauffer, 1951, Manometric Techniques and Tissue Metabolism (Burgers, Minneapolis) p. 119.
EFFECT
OF CHANGES
IN IONIC ENVIRONMENT
ON THE ACTION OF
BRADYKININ ON T H E G U I N E A - P I G T A E N I A C O L I P. N. A A R S E N and M. VAN CASPEL-DE BRUYN Pharmacological Laboratory of the University of A msterdam, PoMerweg 104, Amsterdam, The Netherlands
Received 25 March 1970
Accepted 25 June 1970
P.N.AARSEN and M.VAN CASPEL-DE BRUYN, Effect of changes in ionic environment on the action of bradykinin on the guinea-pig taenia coli, European J. Pharmacol. 12 (1970) 348-358. The effect of changes in the concentration of sodium and calcium in the medium and the effect of adrenaline on the mechanical and electrical responses of the guinea-pig taenia coli to bradykinin were investigated using an organ bath and a sucrose-gap method. In normal Krebs solution the response to bradykinin was mostly biphasic: an acute initial phase charaterized by spike inhibition and decreased isometric tension, followed by a second phase with an increased frequency and amplitude of the spikes and membrane depolarization, accompanied by an increase in isometric tension. Omission of sodium at normal or twice the normal calcium concentration strengthened and lengthened the initial phase and suppressed the second phase. Lowering the calcium concentration abolished the initial phase of bradykinin, while the second phase, especially the increased electrical activity, was enhanced. After lowering both the calcium and the sodium concentration the spontaneous spike activity ceased almost immediately. Under these conditions bradykinin only caused slight hyperpolarization. The effect of substituting strontium for calcium was similar to that of lowering the calcium concentration. However, after a long period of exposure to strontium the initial phase of the response to bradykinin reappeared and was enhanced by doubling the strontium concentration. After a preceding dose of adrenaline, 5 #g, the initial phase of the bradykinin effect was lengthened, while the second phase was not significantly affected. It is suggested that bradykinin acts on more than one site at the cell membrane. The spike inhibition in the initial phase may be explained if it is assumed that bradykinin prevents the release of calcium superficially bound to the cell membrane and involved in the spike generation. Spike stimulation accompanied by depolarization in the second phase is thought to be due to an action of bradykinin at another site in the membrane. Bradykinin Adrenaline
Guinea-pig taenia coli
1. I N T R O D U C T I O N Bradykinin is known to act directly on smooth muscle cells (Khairallah and Page, 1963; Ohashi, Nonomura and Ohga, 1967; Schnieden and Weston, 1969). According to the smooth muscle preparation used, bradykinin can induce contraction, relaxation or a biphasic effect, i.e. an initial relaxation followed by a contraction (Elliott, Horton and Lewis, 1960). Since, in most pharmacological organ preparations bradykinin causes contraction (StiJrmer and Berde,
Sodium Calcium
1963), the question arises as to whether the bradykinin induced relaxation e.g. in the rat duodenum, is due to a different mode o f action or to a difference in the condition o f the cell membrane. Ohashi, Nonomura and Ohga (1967) showed that changes in the biphasic response of the guinea-pig taenia coli to bradykinin appeared to be dependent in some cases on the electrical membrane activity at the moment o f addition of bradykinin, whereas in several other cases no such relation was found. They suggested that bradykinin might have a dual action on the smooth
P.N,4arsen, M.van Caspel-deBruyn, The action o[ bradykinin muscle membrane. Antonio (1968) showed that lowering the external c~cium concentration changed the bradykinin induced relaxation of the rat duodenum into a biph~sic response. In |he im~ent imm~ig~tion the various responses of smooth muscle cells to bradykinin were investigated after changing their ionic environment and hence their electrical and mechanical activities. The guineapig taenia coli was chosen since: l)this preparation usually shows a biphasic reaction to bradykinin in Krebs solution with a normal calcium concentration; 2) both electrical and mechanical activities can be recorded in this preparation by using the sucrose-gap technique; 3)the taenia coli has been investigated very extensively.
2. METHODS Female guinea-pigs of about 500 g body weight were killed by a blow on the head. Strips of 15 to 20 × 0.5 mm and about 0.5 mm thick, were torn out of the superficial layers of the taenia coli. The strips were suspended either in a small organ bath or in a sucrose-gap. One side of the strip was connected by means of a thin nylon thread to an RCA5734 transducer. In all experiments, the tension of the strips was adjusted to 0.5 g. The organ bath and the active side of the sucrose-gap were continuously perfused with salt solution at 37°C with a rate of about 2 ml per min; the perfusion fluid could be changed by means of a multiway stopcock. During the control periods the salt solution used was a modified Krebs-Ringer solution (further indicated as normal Krebs), containing (mM): NaCI 118.4; KC1 4.7; CaC12 2.5; MgSO4 1.2; KH2PO4 1.2; NaHCOs 15.5; glucose 11.1. This solution was prepared from stock solutions according to the method described by Umbreit, Burris and Staufer (1951). The following test solutions were used: Krebs solutions with 0.25 or 5 mM CaCI2 instead of 2.5 mM; a Krebs solution in which CaC12 was replaced by 2.5 mM SrCI2 and 0.1 mM EGTA (ethylenglycol-2-(2-aminoethyl)-tetracetic acid); a sodium-free and a sodium-deficient Krebs solution. In the case of the Krebs solution with double the Ca2+-ion concentration, 10 ml tris-HCl buffer (pH 7.3) was added per 1.3 1. The sodium-free Krebs solution, in which sod-
349
ium was substituted by tris-HCl buffer, was prepared according to Biilbring and Kuriyama (1963a). In the sodium-deficient Krebs solution containing 15.5 mM Na÷ instead of 133.9 mM, NaCI was replaced by an equivalent amount of tris-HCl buffer (pH 7.3). Unless stated otherwise 0.25 ml of a solution containing 1 jag of synthetic bradykinin (Sandoz BRS/640) per ml was added during 30 sec to the supply of perfusion fluid by a continuous infusion apparatus. Bradykinin reached the taenia coli strip within one second after the beginning of the injection period. 2.1. Organ bath method The strip of taenia coli was suspended in the cylindrical hole, 20 X 2 mm, of a perspex organ bath by means of nylon threads. The thread at the upper end of the strip was connected to the anode pin of the transducer RCA 5734. The changes of the isometric tension were recorded by a Kipp pen-recorder. In all experiments a constant dose of bradykinin (0.25/ag) was added to the organ preparation at 4 min intervals. The perfusion fluid under test was always changed immediately after the effect of the preceding dose of bradykinin had been recorded. 2.2. Sucrose-gap method Changes of the membrane potential and the electrical activity as well as the isometric tension were recorded using the sucrose-gap of St~impfli (1954), as modified by Biilbring and Burnstock (1960). In all experiments the inactive side of the gap was perfused with normal Krebs at 5 to IO°C. Changes in the membrane potential and in the electrical activity were measured differentially by using two unity gain preamplifiers and a differential oscilloscope. The changes in the isometric tension were recorded on the same oscilloscope.
3. RESULTS 3.1. The effect of varying the calcium concentration, in the presence and absence o f sodium, on the mechanical responses to bradykinin In three series of organ bath experiments the effects of the following changes in the perfusion fluid were studied: 1) absence of sodium (8 exp.); 2) double the normal calcium concentration (5 mM) in the
350
P.N.Aarsen, M.van Caspel~e Bruyn, The action o f bradykinin
presence and absence of sodium (8 exp.); 3)one tenth of the normal calcium concentration in the presence and absence of sodium (6 exp.). Each experiment was started with three successive additions of 0.25/ag of bradykinin to the taenia coli perfused with normal Krebs. Thereafter, the perfusion was changed either to a sodium-free solution (first series) or to a Krebs solution having an altered calcium concentration first in the presence, and second in the absence of sodium (2nd and 3rd series). The results of these three series of experiments are plotted in figs. la, lb and 2. The points on the drawn lines represent the mean isometric tension ( t o n e ) a t the moment bradykinin was added. The vertical broken and drawn lines represent respectively the mean initial relaxation and the following contraction induced by bradykinin. These figures show that usually bradykinin produces a biphasic effect in normal Krebs (2.5 mM Ca). In sodium-free solution, muscle tone and the relaxing activity of bradykinin increased considerably in the presence of either normal or twice normal calcium concentration (see figs. l a and b). However, the increased relaxation induced by bradykinin was not related to the increased tone; figs. la and lb show that at the end of the experiments relaxation decreased progressively despite increased tone. Fig. 1b further shows that twice normal calcium concentration in the presence of a normal concentration of sodium did not affect the mechanical response to bradykinin. At a low calcium concentration (0.25 mM) both the tone and the reaction to bradykinin were decreased considerably (see fig. 2). The relaxing activity of bradykinin almost completely disappeared within eight min after changing the perfusion fluid and bradykinin only produced contraction; the height of the contraction was decreased. In the absence of sodium the effect of lowering the calcium concentration on the response to bradykinin appeared earlier; the initial relaxation was almost completely abolished within four min. The inhibition of the second contractile phase also occurred more rapidly and was more pronounced. These findings demonstrate that the reaction to bradykinin can be changed by changing the external medium. Omission of sodium enhanced the initial, relaxing, phase, whereas the second, contractile, phase was suppressed. The predominant effect of reducing calcium to one tenth of the normal concen.
Isometric tension(g) 5 Ca (raM) 2.5 i 2.5.Na-free 4
i
i--l,~
i./[ 2
t\
\l ;d .
i
, ii i
i I
12
24
36
i2.5 .l _1/'\, r[
5 Ca (raM) 2.5 i5
4 3 2 1
15, Na-free
0 12
24
36
48 time (rain)
Fig. 1. The effect of the calcium concentration, in the presence and absence of sodium, on the mean isometric tension (tone, o) of taenia coli and changes induced by a constant dose of bradykinin (0.25 #g) added at 4 min intervals. The vertical broken and drawn lines indicate respectively the mean initial decrease (relaxation) and increase (contraction) in tension caused by bradykinin added at the moments indicated on the abscissae. a) Tone and response to bradykinin of the taenia coli (8 exp.) perfused first with normal Krebs (2.5 mM Ca), and second with a sodium-free Krebs solution (2.5 mM Ca, Na-free). Omission of sodium causes an increase in tone, while the relaxation response to bradykinin increases. Eight min after changing perfusion the bradykinin-induced relaxation decreases progressively in spite of the fact that the tone remains
high. b) Tone and response to bradykinin of the taenia coli (8 exp.) perfused successivelywith normal Krebs, Krebs with twice the normal calcium concentration (5 mM), normal Krebs and sodium-free Krebs containing 5 mM calcium. Double the calcium concentration in the presence of sodium does not considerably affect both tone and response to bradykinin, whereas omission of sodium has about the same influence as at a normal calcium concentration.
P.N.Aarsen, M.van CaspelwleBruyn, The action o f bradykinin Isometric tension(g) 5-C,a (mM)2.5 0.25
Z5
4-
i0.25 i Na-f ree I
32-
,i
[
i
i 1 ~ 1
;i
:i 1
0
,
,
,I
12
,
,
,
24
?
,
48 time(rain)
Fig. 2. Effect of calcium concentration, in the presence and absence of sodium, on the mean isometric tension (tone, s) of taenia coil and changes induced by a constant dose of bradykinin (0.25 ~g) added at 4 rain intervals. In 6 experiments the taenia coli was superfused with normal Krebs (2.5 mM Ca), Krebs with one tenth the normal calcium concentration (0.25 raM), normal Krebs and sodinm-free Krebs containing 0.25 mM calcium. Lowering the calcium concentration causes both a decrease in tone and in response to bradykinin. The relaxing activity of bradykinin almost completely disappeared within eight rain o f changing the perfusion fluid. In the absence of sodium the effect of lowering the calcium concentration on the response to bradykinin appeared earlier: the mean initial relaxation was almost completely abolished within four min. For further description see fig. 1.
tration was suppression of the relaxing phase. In a low calcium and sodium-free medium both phases of the response to bradykinin were suppressed. 3.2. The effect o f various calcium concentrations, in the presence and absence o f sodium, on the electrical response to bradykinin and its relation to the mechanical response The organ bath experiments were repeated in a similar way using the sucrose-gap method; each experiment was repeated at least five times. The response to bradykinin in normal Krebs was, in most cases, biphasic: an initial phase characterized by acute spike suppression and decreased isometric tension was followed by a second phase which often started with irregular rlring of spikes and an increase in isometric tension. Later, firing became more regular; frequency and amplitude of the spikes were increased. Depolari-
351
zation ensued and isometric tension increased further. In our experiments, the membrane potential did not always increase during the initial phase as stated by Ohashi et al. (1967). Apart from a slight increase observed in a few cases, no change or even a decrease in membrane potential was usually found. However, the effect of bradykinin depended on the conditions prevailing before treatment. In preparations with a fluctuating, unstable membrane potential (see control in fig. 4), the predominant response to bradykinin was contraction (second phase). In preparations with a stable membrane potential, coupled with regular spike activity, the initial relaxing phase of the response to bradykinin was dominant. Such a pattern is shown by the first control in fig. 3. Furthermore this figure shows that on repeated administration of bradykinin the initial phase decreased; in the second control the initial phase was interrupted by a few spikes, resulting in fluctuations of the isometric tension. 3.2.1. Sodium-free solution Fig. 3 shows an experiment in which the influence of sodium-free Krebs on the response to bradykinin was investigated. The most characteristic effect of omission of sodium was an increase and prolongation of the initial phase within four min after changing the sodium content. The duration of the spike inhibition, which was 53 sec during the first control experiment, increased to more than 135 sec within four min after the omission of sodium. At 12, 16 and 20 min after starting the perfusion with sodium-free Krebs, the spike inhibition lasted 70, I01 and 105 sec respectively and was accompanied by a concurrent increase in duration of the relaxation. It must be stressed that during this phase no hyperpolarization occurred. The second phase of the response to bradykinin was abolished in sodium-free medium: the spike frequency did not increase, and in most cases it remained lower than before the addition of bradykinin. The tone usually returned to its resting value. Furthermore, usually depolarization of the membrane potential was not observed. An exception is shown in fig. 3 where a slight depolarization occurred 12 min after the perfusion with sodium-free solution was started, accompanied by a slight increase in tone over the resting value.
P.N.Aarsen, M.van Caspel-de Bruyn, The action o f bradykinin
352
|
Control
I
5 mV
lg B
B
!
Na -free
!
12'
1
5 mV
lg B
B
20'
16'
5mV
lg !
B
B
!
33sec
Fig. 3. The effect of bradykinin on spontaenous membrane activity recorded by means of the sucrose-gap method (upper traces), and on the isometric tension (lower traces) of the guinea-pig taenia coli. Horizontal bars indicate periods when 0.25 pg bradykinin (B) was added to the perfusion fluid at the active side of the gap, being either normal Krebs during the control period (control), or sodium-free Krebs (Na-free). Records were taken at 4, 12, 16 and 20 min respectively after changing from normal Krebs to sodium-free Krebs.
P.N.Aarsen, M. van Caspel-de Bruyn, The action o f bradykinin
3.2.2. The effect of varying the calcium concentration in the presence and absence of sodium Doubling the calcium concentration to 5 mM without changing the sodium Concentration had only a small effect on the electrical response to bradykinin. Fig. 4 shows that within four min after exposure to this concentration a short-lasting initial phase (inhibition of spikes) was observed, whereas in the preceding control experiment this initial phase was absent. After twelve min, however, the response was not different from that in the control period. In the second control period the response to bradykinin had become biphasic. Exposure to twice the normal calcium concentration, in the absence of sodium in this case, had a stabilizing effect on the spike firing mechanism. The response to bradykinin changed in a characteristic
353
way: a pronounced initial phase occurred, whereas' the second phase was suppressed. Exposure to a tenth of the normal calcium concentration (0.25 mM) had the following effect. The initial phase was suppressed and the second phase, especially its electrical phenomena, was activated (fig. 5). In spite of the increase in frequency and amplitude of the spikes during the second phase, the increase in isometric tension was smaller than in the control situation. After a second control period, in which the active side of the sucrose-gap was again perfused with normal Krebs, the muscle was exposed to a solution containing 0.25 mM calcium and 15.5 mM sodium. In spite of the fact that approximately 10% of the sodium remained, spontaneous activity ceased within four min and the tone was considerably decreased.
Control
5mM Ca
5mV[
E
,g[
¢ ,7
B
4'
B
Control
12'
B
5mM Ca, Na_free
5mY[
Ig[ !
B
4'
B
12'
B
J
33 sec
Fig. 4. The effect of 0.25 tag bradykinin (B) on the spontaneous membrane activity recorded with the sucrose-gap method (upper traces), and on the isometric tension (lower traces) of the guinea-pig taenia coll. The effects were investigated using the same preparation superfused in succession with normal Krebs (control), Krebs with twice the normal calcium concentration (5 mM Ca) (at 4 rain and 12 rain after starting the perfusion), normal Krebs (control), and sodium-free Krebs with twice the normal calcium concentration (5 mM Ca, Na-free) (at 4 rain and 12 rain after starting the perfusion).
P.N.Aarsen, M.van Caspei-deBruyn, The action of bradykinin
354
Con t rol
0.25 mM Ca !
5 mV B
4'
B
Control
12'
B
Q25mM Ca,15.5 mM Na
!
B
4'
B
12'
B
!
33sec
Fig. 5. The effects of 0.25 pg hradykinin (B) on the spontaneous membrane activity recorded with the sucrose-gap method and on the isometric tension of the guinea-pig taenia eoli superfused successively with normal Krebs (control), Krebs with one tenth the normal calcium concentration (0.25 mM Ca), normal Krebs (control), and sodium-deficient Krebs with one tenth the normal calcium concentration (0.25 mM Ca, 15.5 mM Na). For further description see figs. 3 and 4. Under these conditions bradykinin only produced hyperpolarization, which was increased after a longer exposure to the low calcium, low sodium solution (see fig. 5). In this case no change in the tension occurred; in some o f the other experiments a small increase in tension occurred after hyperpolarization.
3.4. The effect o f substituting strontium for calcium Replacement o f calcium by an equivalent amount o f strontium in the presence o f 0.1 mM EGTA produced a characteristic pattern o f changes in the firing o f spikes, i.e. the spike amplitude was increased and the spike frequency was increased initially but declined after some time (see fig. 6). Moreover, the spikes became more regular. At low frequencies (about 0.1/sec), a pronounced positive after-potential
could be demonstrated. Replacement of calcium by strontium changed response to bradykinin as follows: in the beginning, when the spike frequency was still high, bradykinin caused only stimulation, an increase in spike frequency and amplitude accompanied by a small depolarization. Later on this stimulation was preceded by a short4asting initial phase, consisting mainly o f a decrease in spike frequency. After exposure to twice the strontium concentration (5 mM) a pronounced initial phase appeared within four min (see fig. 6). During the second phase of the response to bradykinin a contraction still occurred, even after two hours exposure to the calcium-free medium. After lowering the sodium concentration (to 15.5 mM) o f the strontium containing medium, the electrical activity stopped almost immediately.
355
P.N.Aarsen, M.van Caspel-deBruyn, The action ol'bradykinin
Control
2.5mM Sr +0.1mM EGTA
5 mV I. lg t B
12'
B
96'
B
5 m M Sr +0.1mM E G T A I
,11.
!
J
.
,tufallfyI ,
5 mV [
# I
4'
B
12'
B
ZS'
B
!
33 sec
Fig. 6. The effect of 0.25 #g bradykinin (B) on the spontaneous membrane activity recorded with the sucrose-gap method (upper traces), and on the isometric tension (lower traces) of the guinea-pig taenia eo5 superfused successively with normal Krebs (control), calcium-free, strontium containing Krebs (2.5 mM Sr + 0.1 mM EGTA), and calcium-free Krebs with twice the concentration of strontium (5 mM Sr + 0.1 mM EGTA). For further description see figs. 3 and 4. 3.5. The effect o f halving the calcium concentration and the effect o f exposure to adrenaline In these experiments the effect of 30 min exposure to half the normal calcium concentration on the electrical responses of the taenia coli to bradykinin was investigated and compared with that of a preceding dose of adrenaline in normal Krebs. Adrenaline was used as a pharmacological tool, because its action resembles that of excess calcium (Biilbring, 1957; BiJlbring and Kuriyama, 1963b). In these experiments a constant volume o f 0.1 ml of bradykinin solution (10/ag/ml) was injected into the drop counter placed in the supply tube at the active side of the sucrosegap. The adrenaline (5/ag) was added by means of a continuous infusion apparatus as follows: 0.2 ml per min o f an adrenaline solution (10/ag/ml) during 2.5 min. Four min after the end o f this addition, the
injection of bradykinin was started. In each experiment the electrical response to bradykinin was investigated successively in Krebs solutions with 2.5, 1.25 and 2.5 mM calcium. Thereafter the effect of a preceding exposure to adrenaline was determined. The mean (-+ S.E.M.) o f the electrical responses, i.e. the duration o f the initial spike inhibition, the secondary increase in spike frequency and the depolarization, are plotted in fig. 7. This figure shows that halving the calcium concentration reduced the mean duration of the initial spike inhibition from 30 -+ 6.2 (S.E.M.) sec to 2 -+ 1.6 (S.E.M.) sec; in nine out of ten determinations, this initial response disappeared with half the normal calcium concentration. After exposure to adrenaline the initial spike inhibiting effect o f bradykinin increased from 31 -+ 6.2 (S.E.M.) see to 78 + 21.5 (S.E.M.) sec. However, the
356
P.N.Aarsen, M.van Caspel-de Bruyn, The action of bradykinin
number of
sec 100
spikes/1OOsec
mV 100
Plh [ ] period of inhibition(sec) / r ] i n c r e a s e m number
oh / of spikes/lO0sec 80 2 /~] depolarization(mV)
80
60
60
(10)
(10)
(6)
(6)
40
40
20 ~
20
0
2.5
1.25
2.5
0 2.5 mM Ca
after exposure to adrenaline
Fig. 7. The effects of halving the calcium concentration and of preceding exposure to adrenaline on the first (ph 1) and the second phase (ph 2) of electrical responses of the guinea-pig taenia coli induced by 1 pg of bradykinin. In each experiment the active side of the sucrose-gap was perfused in succession with Krebs containing 2.5, 1.25 and 2.5 mM calcium. In the last solution the effect of a preceding dose of 5 pg adrenaline on the response to bradykinin was studied. The heights of the columns indicate the means of the in brackets indicated number of determinations; the vertical bars indicate the S.E.M.
electrical phenomena of the second phase, increase in spike frequency and depolarization, were not affected by both halving the calcium concentration and preceding exposure to adrenaline.
4. DISCUSSION In normal Krebs solution, the response of the taenia coli to bradykinin was usually biphasic. An initial phase characterized by spike inhibition and a decrease in isometric tension was followed by a second phase with an increased frequency and amplitude of the spikes and membrane depolarization, accompanied by an increase in isometric tension. Spike inhibition and a decrease in isometric tension occurred simultaneously during the initial phase as did stimulation of spike activity and increase in isometric tension during the second phase. Less correlation was found between the mechanical response
and the change in membrane potential. In some cases the initial relaxation seemed to be accompanied by a small hyperpolarization, probably due to suppression of the superimposed spikes of numerous firing cells, ~ausing a slight decrease in the base-potential. In other cases either a depolarization or no change of the membrane potential was found during the relaxation. The maximum increase in isometric tension, observed in the second phase, occurred before membrane depolarization. Thus, bradykinin-induced changes in isometric tension must be a direct consequence o f changes in the spontaneous spike activity, which according to Bueding and Bi]lbring (1964) maintains tone. However, in some cases either predominantly inhibitory or predominantly stimulatory responses to bradykinin were f o u n d in normal Krebs, probably depending on the membrane activity before the addition of bradykinin. If the spontaneous spikes were regular and the resting membrane potential constant, clear-cut inhibition of the spikes commenced within 15 sec of adding bradykinin. This reaction was frequently followed by a period of increased spike activity. When the spontaneous spikes were irregular. and the resting membrane potential fluctuated, the response to bradykinin was predominantly a stimulation. Since Biilbring and Kuriyama (1963a) demonstrated that a solution containing excess calcium stabilizes the membrane activity, regardless of whether sodium is present or not, it was assumed that the differences in response of the taenia coli to bradykinin might depend on differences in the amount of calcium bound to the cell membrane. The more calcium is bound to the cell membrane, the stronger the initial response to bradykinin. This hypothesis is supported by the following findings. Lowering of the calcium concentration abolished the initial phase of spike inhibition, whereas the second phase of the electrical response started sooner and was more pronounced. A predominance of the initial inhibitory phase was also found after omission of sodium. This may be explained by an increase in the amount of calcium bound to the superficial anionic sites caused by a reduction in the concentration o f competing sodium, as postulated by Goodford (1967). The fact that doubling the calcium concentration at a normal sodium concentration had only a slight effect, is not at variance with this explanation, since the concentra-
P.N.Aarsen, M.van Caspel-cleBruyn, The action of bradykinin
tion of competing sodium ions is many times higher than that of calcium ions. After pretreatment with adrenaline the initial phase of the bradykinin effect was also lengthened, but the second phase was not suppressed. According to Bueding and Biilbring (1964), Biilbring, Goodford and Setekleiv (1966) and BiJlbring and Tomita (1969) adrenaline may cause an increase in the amount of calcium bound to the cell membrane. Therefore, the observed effect of adrenaline may also support the view that the occurrence of the initial phase of the response to bradykinin is somehow related to the amount of calcium bound to the cell membrane. However, adrenaline did not affect the second phase whereas sodium deficiency caused a reduction of the second phase in addition to its effect on the initial phase. This shows that a reduction of the second phase is not necessarily coupled with a predominance of the initial phase. The suppression of the second phase during sodium deficiency might therefore be caused by an additional effect not related to an increase in the amount of calcium bound at the anionic sites. The calcium ion is very important for spike activity in taenia smooth muscle, and under normal conditions probably carries most of the current during the action potential (Biilbring and Kuriyama, 1963a; Nonomura, Hotta and Ohashi, 1966; Brading and Tomita, 1968; Brading, BLilbring and Tomita, 1969a). According to Biilbring (personal communication) the results described by Brading, Biilbring and Tomita (1969a,b) favour superficially bound calcium as the source from which ions can be released to carry the inward current of the spike. Strontium and barium are supposed to replace calcium in its function of generating the spike (Hotta and Tsukui, 1968). The initial phase of the bradykinin action resembled the effect of adrenaline in so far as both compounds suppressed the spikes in normal Krebs solution and both effects were enhanced in excess calcium. However, the initial phase of bradykinin differs from the effect of adrenaline in the following respects: 1) in contrast to adrenaline bradykinin does not usually cause hyperpolarization; 2) in sodium-free solution, the effect of bradykinin is enhanced but the effect of adrenaline is decreased (Biilbring and Kuriyama, 1963b); 3)according to Biilbring and Tomita (1969) substitution of barium for calcium suppresses
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the effect of adrenaline, whereas substitution of strontium for calcium never suppresses the initial phase of bradykinin action completely. The most likely explanation for the initial effect of bradykinin is that bradykinin prevents the release of calcium bound to superficial sites which will carry the current for the spike. This explains spike suppression, absence of hyperpolarization, absence of an effect of strontium, and increase in effect caused by omission of sodium. Omission of sodium may in itself increase the attraction between calcium and anionic membrane sites. Since the initial spike inhibiting effect of bradykinin depends on the amount of calcium, possibly bound at the superficial anionic sites, it is tempting to speculate that bradykinin might have some affinity for this calcium complex which would suppress release of calcium from its binding site. This supposed mechanism of bradykinin in its initial phase of action does not explain the second excitatory phase of bradykinin action. It is reasonable to suppose that this second phase is caused by an action of bradykinin at a different site.
ACKNOWLEDGEMENT It is a pleasure to thank Prof.Dr. C.Van Der Meer for his advise and helpful criticism during the investigation and during the preparation of this manuscript.
REFERENCES Antonio, A., 1968, The relaxing effect of bradykinin on intestinal smooth muscle, Brit. J. Pharmacol. 32, 78-86. Brading, A.F. and T.Tomita, 1968, The action potential of the guinea-pig taenia coli in low sodium solution, J. Physiol. 197, 30-31P. Brading, A.F., E.Biilbting and T.Tomita, 1969a, The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coil, J. Physiol. 200, 637-654. Brading, A.F., E.Biilbring and T.Tomita, 1969b, The effect of temperature on the membrane conductance of the smooth muscle of the guinea-pig taenia coil, J. Physiol. 200, 621-635. Bueding, E. and E.Biilbring, 1964, The inhibitory action of adrenaline. Biochemical and biophysical observations, in: Pharmacology of Smooth Muscle, ed. E.Biilbring (Czechoslovak Medical Press, Prague) pp. 37-56.
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Biilbring, E., 1957, Changes of configuration of spontaneously discharged spike potentials from smooth muscle of the guinea-pig's taenia coli. The effect of electrotonic currents and of adrenaline, acetylcholine and histamine, J. Physiol. 135,412-425. Biilbring, E. and G.Burnstock, 1960, Membrane potential changes associated with tachyphylaxis and potentiation of the response to stimulating drugs in smooth muscle, Brit. J. Pharmacol. 15, 6 1 1 - 6 2 4 . Biilbring, E. and H.Kuriyama, 1963a, Effects of changes in the external sodium and calcium concentrations on spontaneous electrical activity in smooth muscle of guinea-pig taenia coli, J. Physiol. 166, 2 9 - 5 8 . Biilbring, E. and H.Kuriyama, 1963b, Effects of changes in ionic environment on the action of acetylcholine and adrenaline on the smooth muscle cells of guinea-pig taenia coli, J. Physiol. 166, 5 9 - 7 4 . Biilbring, E., P.J.Goodford and J.Setekleiv, 1966, The action of adrenaline on the ionic content and on sodium and potassium movements in the smooth muscle of the guinea-pig taenia coli, Brit. J. Pharmacol. 28, 2 9 6 - 3 0 7 . Biilbring, E. and T.Tomita, 1969, Effect of calcium, barium and manganese on the action of adrenaline in the smooth muscle of the guinea-pig taenia coli, Proc. Roy. Soc. London, B172, 121-136. Elliot, D.F., E.W.Horton and G.P.Lewis, 1960, Actions of pure bradykinin, J. Physiol. 1 5 3 , 4 7 3 - 4 8 0 .
Goodford, P.J., 1967, The calcium content of smooth muscle of the guinea-pig taenia coli, J. Physiol. 192, 145 147. Hotta, Y. and R.Tsukui, 1968, Effect on the guinea-pig taenia coli of the substitution of strontium or barium ions for calcium ions, Nature 2 1 7 , 8 6 7 - 8 6 9 . Khairallah, P.A. and I.H.Page, 1963, Effects of bradykinin and angiotensin on smooth muscle, Ann. N.Y. Acad. Sci. 104, art. 1 , 2 1 2 - 2 2 1 . Nonomura, Y., Y.Hotta and H.Ohashi, 1966, Tetrodotoxin and manganese ions: Effects on electrical activity and tension in taenia coli of guinea-pig, Science 152, 97- 99. Ohashi, H., Y.Nonomura and A.Ohga, 1967, Effects of angiotensin, bradykinin and oxytocin on electrical and mechanical activities in the taenia coli of the guinea-pig, Japan J. Pharmacol. 17,247 257. Schnieden, H. and A.H. Weston, 1969, Investigation of the spasmogenic effect of manganese on the guinea-pig isolated ileum preparation, Brit. J. Pharmacol. 3 6 , 4 9 6 - 5 0 9 . St~npfli, R., 1954, A new method for measuring membrane potentials with external electrodes, Experienta 10, 508. Stiirmer, E. and B.Berde, 1963, A comparative pharmacological study of synthetic eledoisin and synthetic bradykinin, J. Pharmacol. Exptl. Therap. 140, 349 355. Umbreit, W.W., R.H.Burris and J.F.Stauffer, 1951, Manometric Techniques and Tissue Metabolism (Burgers, Minneapolis) p. 119.