Calcium and the contractile effect of carbachol in the depolarized guinea-pig taenia caecum.

Carbachol (10-8-10-3 M) caused a phasic contraction in the guinea-pig taenia caecum suspended in the Ca-free, K-Tyrode solution. The action of carba chol was concentration-related and was completely antagonized by atropine (10-6 g/ml). The capacity to contract in the absence of external Ca was removed by short term exposure to a high concentration of carbachol (more than 5×10-4 M), but was restored after treatment with Ca (0.1-2.0 mM). Carbachol, 10-3 M, was used throughout the following experiments. The degree of restoration increased with the duration of treatment with Ca and with the concentration of Ca. The restored capacity was not maintained but wore off again after return to the Ca-free solution. The time courses of restoration and disappearance of the capacity following addi tion and withdrawal of Ca were much slower than those of the Ca-contracture and its relaxation. The rate constant for loss of Ca component responsible for the capa city was calculated as 0.063 min-1 by assuming that there is a hyperbolic function be tween steady isometric tension and [Ca]1. These results suggest that carbachol mobi lizes Ca ions from stored Ca, which is in a dynamic equilibrium with [Ca]0, to acti vate the contractile proteins. It has been observed that excitatory drugs such as carbachol cause depolarized smooth muscles to contract and these do so for a long time even when free-Ca ions are removed from the medium (1, 2). Since there is legitimate reason to attribute contractile responses to increases in the level of intracellular free-Ca ions, the contractions in the ab sence of Ca in the medium probably result either from passage of Ca ions which are still present in the extracellular space across the cell membrane, or from liberation of Ca ions from a sequestered Ca fraction. Precisely how these two possible pools for Ca ions ac count for the mechanical response has not yet been throughly investigated. The present studies covered contractile responses of depolarized smooth muscle to an excitatory drug (carbachol) in a Ca-free environment, in the hope of throwing light on the question. The results described below support the view that Ca ions from a cellular store account for the contractile responses to carbachol, and suggest that the stored Ca may equilibrate slowly with the extracellular Ca. A preliminary account of some of these results has already been given (3). MATERIALS AND METHODS The taenia caecurn, isolated from guinea-pigs of both sexes and weighing 300 to 400 g was used throughout. A segment of taenia approx. 15 nom long was suspended in an organ bath containing 5.0 ml of Tyrode solution which had the following composition: NaCI, 137 mM; KCI, 2.7 mM; NaH2PO4, 0.4 mM; CaCI•2, 1.8 MM; MgCI2, 1.0 MM; glucose, 5.0 mM; NaHCO3, 12 mM and was bubbled with air. After equilibration in Tyrode solution for 60 min at 37'C, the muscle was then transferred to K-rich Tyrode solution in which the NaCI was replaced with equimolecular KCI, the CaCl2, NaH2PO4 and NaHCO3 were omitted and tris-maleate buffer (pH=7.4), 5.0 mM was added. The temperature of the depolarizing solution was maintained at 20_'~ __1'C. This temperature was critical in order to carry out present work. Mechanical responses to Ca and car bachol were isometrically measured using a mechano-electronic transducer and records were obtained on a potentinometric pen recorder (Hitachi, 056). At the beginning of the experiment in the K-rich solution, a resting tension of less than 0.5 g was established. To vary the Ca ion concentration and to apply carbachol, CaC12 and the drug were added to the bathing solution by rapid injection of a small volume (0.05 ml) from their concen trated solutions until the final required concentration was reached. All concentrations used refer to the final concentration in the bathing medium. Experiments to determine whether or not the capacity to contract in response to car bachol in the absence of external Ca would be restored after pre-treatment with Ca were performed using the muscles in which the capacity retaining Ca had been diminished by short-term exposure to 10-3 M of carbachol (hereafter, referred to 'Ca-deprived taeniae'). For the Ca treatment, a Ca-deprived taenia was incubated for various periods in the K-rich Tyrode solution containing Ca in different concentrations after which the muscle was washed 3 times with the K-rich Tyrode solution and was allowed to remain suspended in this solution for 10 min in order that the extracellular fluid would be depleted of Ca. To avoid a complication of deterioration of the tissue, the experiments were designed to be completed within 180 min. Drugs used were carbamylcholine chloride (carbachol, Merk) and atropine sulfate (Tanabe). RESULTS Effects of' carbachol When a segment of the taenia caecum of the guinea-pig, after equilibration in Tyrode solution at 37'C, was exposed to the K-rich Tyrode solution (Ca-free, K-Tyrode solution), a strong, rapid contraction developed, and began to decline more slowly from the maxi mum amplitude reached within 30 sec after exposure to an intermediate level, as illus trated in Fig. I a. A rapid cooling of the bathing medium to 20`C caused further relaxa tion following a small transient increase in tension. Carbachol in concentrations from 10-3 to 10-3 M contracted the muscle when applied after the relaxation had been virtu ally completed. The contractile response was also a transient one and the developed tension fell to the initial level within several min despite the fact that the bath still con tained carbachol. The action of carbachol was dose-related: The dose-response re lationship defined approximately an S-shaped Curve. Atropine (10-s g; ml) blocked the action of carbachol, indicating the contractile effect to be on the specific cholinergic re ceptors of the tissue. The contractile response to carbachol in a concentration up to 5 10-4 M was re peatable during prolonged suspension in the Ca-free, K-Tyrode solution, though the re sponse to the first dose of the stimulant was greater than subsequent ones. The latter became progressively smaller until virtually abolished. By increasing the dose of the drug, the time required for abolishment of the carbachol response was reduced. With concentrations greater than 5 x 10-4 M, the muscle contracted in response only to the first dose of carbachol. In view of the importance of Ca in activating the contraction, the abolition of the carbachol responsiveness indicates a possible depletion of Ca in some storing sites. FIG. 1. High K-induced contracture and mechanical response to carbachol. a) The muscle segment which had been bathed for 60 min in normal Tyrode solution at 37'C was exposed to Ca-free, K-Tyrode Solution (K-Tyrode). Carbachol (10' M) was applied at Carb. A period of 10 min clasped between the first and second applications. b) At Ca, Ca (1.0 mM) was added to the bathing medium. The Ca was left for 5 min and then removed by washing. Carbachol was next applied 10 min after removal of the Ca. Records of two experiments are superimposed. Restoration of tlrc carbachol responsiveness after treatment with Ca As shown in Fig. 1 b, the muscle incubated with Ca did indeed restore this capacity. The muscle was treated with 1.0 mM Ca for 5 min as described under Methods. It can be seen that a slow increase in tension of the muscle developed following the addition of Ca (Ca-contracture), and the developed tension decreased slowly following removal of the Ca and reached the initial level before measurement of the contractile response to 10-3 M carbachol was done. A series of applications of carbachol caused a series of fairly constant responses over a period of 180 min when the preparation was equally treated with Ca each time. The response then became progressively less with time. The degree of restoration produced was found to increase with the duration of in cubation with Ca and the concentration of Ca used for incubation. However, this res toration passed off gradually after return to the Ca-free Solution. When carbachol was added to the bathing solution before Ca and was present during Ca incubation, restora tion did not occur. Effects of changes of iucrrbation period with Ca on the carbachol response Seven segments of the taenia were used for the experiments. Fig. 2 a shows how the contractile responses to carbachol increased as the period of immersion of the muscle in a 1.0 mM Ca solution was prolonged. The results are summarized in Fig. 2b. In each experiment, the magnitude of the carbachol response is expressed as a per centage of the magnitude of the reference K-contracture. Each point in the graph is the mean S.E. of the percentages obtained on 7 preparations at each noted period. The curve constructed from the means showed a tendency to approach a certain value as incubation time was prolonged. This suggests that the capacity of the tissue to accumulate Ca which is responsible for the carbachol response is not unlimited, although unsaturable over the period of time observed. For the beginning of the curve, the time taken for diffusion of added Ca ions to the site where the divalent ions accumulate should be taken into account. The diffusion time is not longer than the period of time within which Ca contractures reach a maximum height (less than 8 min). Treatment with 1.0 mM Ca for 10 min was done twice. The carbachol response of the muscle was then measured and com pared to the control response after a single treatment with the same concentration of Ca. The carbachol response following two treatments was much greater than the control (on average 194.6 of the control with a range from 183 to MY,), n-8). Fr(;. 2. Time course of restoration of contrac tile effect of carbachol. a) A set of superimposed mechanical responses of a muscle segment, which has been pre-treated with a 1.0 mM Ca for various periods of time (2, 4, 8, 16 and 32 min), to carbachol applied at an in terval of 10 min after transfer to Ca-free, K-Tyrode solution. b) The graph plotting the magnitude of the carbachol response versus time. Each point of the graph is the mean S.E. of 7 experiments. Abscissa : time in min ; Ordinate : the magnitude of the carbachol responses as a percentage of the magnitude of the K-contracture in each experiment. Erects of changes in Ca-concentration for incubation on the carbachol response First, mechanical responses to carbachol were observed after the muscle had been treated with Ca by incubating in the solution with 0.5 mM Ca for various periods. The same muscle was then subjected to a second series of experiments with 1.0 mM Ca in the same manner as described above. Changes in the responses as a function of the duration of incubation are shown in Fig. 3. It is clear from the curves that the incubation with 1.0 mM Ca is more effective in restoring the carbachol responsiveness of the muscle. Another interesting finding is that the curves are approximately in parallel and never showed a tendency to fuse into one as they would if the Ca pool was a readily saturable one. Tune course of disappearance of the restored carbachol responsivveness in a Ca-free cnvi ronment FIG. 3. Comparison of the time courses of restoration of contractile effect of carbachol when two different concentrations, 0.5 (0) and 1.0 (()), of Ca were used for pre-treatment. The graphs were plotted in the same manner as in Fig. 2. The restored capacity of muscles to contract in response to carbachol after incubation with Ca was found to wear off during suspension in a Ca-free medium. In some experi ments, we attempted to follow the time course of loss of the carbachol responsiveness for 40 min. Muscles were treated with Ca by suspension in a solution containing 1.0 mM Ca for 5 min. The contractile responses to carbachol were then observed at various intervals after the removal of Ca from the medium. The response became smaller with prolongation of the interval, as illustrated in Fig. 4 a. The results obtained from 9 ex FiG. 4. Time course of disappearance of restored contractile effect of carbachol. a) A set of superimposed mechanical responses of a muscle segment, which has been pre-treated with a 1.0 mM Ca for 5 min each time, to carbachol applied at various intervals (5, 10, 20, 30 and 40 min) after transfer to Ca-free, K-Tyrode solution. b) The graph plotting the magnitude of the carbachol response versus time. Each point of the graph is the mean !_S.E. of 9 experiments. Abscissa : time in min ; Ordinate : the magnitude of the carbachol responses as a percentage of the magnitude of the reference K-contracture in each experiment. periments are summarized in Fig. 4b. I n each experiment, the magnitude of the con tractile response was expressed as a per centage of the magnitude of the reference K-contracture. Each point of the graph is the mean _ S. E. of the percentages in 9 experiments at each interval. FIG. 5. Relationship between the additional tension developed by carbachol in the presence of Ca and the time after addi tion of the Ca. Mechanical responses of a muscle seg ment to carbachol (10" Ml were obtained at various intervals (2.5, 5, 10 and 15 min) after exposure to a 0.5 mM Ca (at Ca). Contractile responses to carbachol in the presence o f Ca If loss of Ca following a fall in [Ca]o results in a decrease in the number of tissue receptors with which the drug inter acts to initiate the physiological response, the magnitude of the carbachol response could be governed not only by the level of Ca ions that are available for contraction, but also by the number of receptors. The question whether either of the two is the case can be answered only when the amount of the available Ca or the number of the receptors can be estimated accurately. A reliable method is not yet available for this determination. An approximate idea of change in the amount of available Ca was gained by the following experiments in which muscles were exposed to Ca and then to carbachol. Fig. 5 shows the additional tension deve lopments produced by carbachol applied at 4 different intervals (2.5, 5, 10 and 15 min) after exposure to Ca. The response caused by carbachol increased as the time after the addition of Ca was lengthened. There was a noticeable increase of the carbachol response after the Ca-contractures had reached their peak tension. On repetition, the Ca-contrac tures were fairly constant in magnitude and sustained the raised tension during the Ca incubation. In these experiments, Ca was present at the moment of the introduction of carbachol and the concentration of Ca in extracellular space would be expected to be con stant after the Ca-contracture had reached its equilibration. Therefore, changes in the mecha Fio. 6. Relationship between the magnitude of the response to carbachol and the interval of application of the drug in the presence of Ca. Mechanical responses to carbachol (10_3 M) applied at various intervals between two successive applications in K-Tyrode solution containing 1.0 mM Ca. Numbers over each response represent the time in min which clasped following the preceding response. nical responses produced by carbachol can be readily understood when one considers the change in available Ca. In addition, the time course of the additional tension developments was much the same as the carbachol, response in a Ca-free medium, suggesting that Ca is utilized from the same source. In some experiments, Ca was added to the K-Tyrode solution to increase the Ca concentration to 1.0 mM and con tractile responses to the same dose of carbachol (10-3 M) were measured in the sustained presence of Ca. The responses were found to increase with the passage of time as illustrated in Fig. 6. Fig. 7 re presents the graph showing the relationship of the magnitude of the carbachol responses to the interval between any successive re sponses. The magnitude of the response at the longest interval observed (40 min) was taken as 100. However, in this kind of experiment, development of tachyphylaxis should be taken into account. In similar experiments to those in Fig. 5 with short-lasting ex posures to Ca (3 min) and to carbachol (2 min), it was found that tachyphylaxis developed when the interval between successive applications of carbachol was shorter than 15 min. FIG. 7. A graph plot of the magnitude of the responses versus the interval between two successive applications of carbachol (10-3 M) in the presence of external Ca. Each point is the mean -! S.E. of 5 experiments. Abscissa : interval in min ; Ordinate : the magnitude as a percentage of the magni tude of the response at an interval of 40 min in each experiment. The estimated rate constant for movements of Ca available for the carbachol response Since the time course of disappearance of the restored carbachol responsiveness in Ca-free environment is more consistent and less variable in individual segments of the taenia, and attempt was made to calculate the rate constant for loss of Ca responsible for this responsiveness. If the contractile response of smooth muscle is a hyperbolic func tion of the concentration of Ca ions placed in the environment of the contractile elements ([Ca],), as in skeletal muscle (4, 5), and if [Ca], is directly proportional to the amount of Ca ions that are available for activation of the contractile proteins when carbachol is ap plied ([Ca],;), the mechanical response may vary as a hyperbolic function of the [Ca]" is described as follows: where Pmax is the maximum contractile response of which the smooth muscle system is capable, obtained when an infinitive amount of Ca is present, and a is a proportional constant. The loss of Ca can be described as follows : [Ca],, = [Ca]-,t _o • e'. ......................................................... (2) Hence, In [Ca], _ --alt+In[Ca],;t=o ................................................ (2t) where [Ca]rt-o is the amount of Ca at the beginning of the suspension of the muscle in the Ca-free, K-Tyrode solution, t is the time in min from the beginning of the suspension, A is the exponential rate constant (min-'). From the equation (1), the equation (2)' can be changed to where Po is the amplitude of the contractile response when t=0. The equation can be