Calcium source for contractile response of guinea pig taenia caecum to carbachol in a calcium deficient, potassium rich solution.

The Ca storing site for the carbachol-induced contraction in a high-K medium without adding CaCl2 was further investigated in the guinea pig taenia caecum. La3+ (0.05-0.5 mM) caused an increase in peak tension and relaxation rate in the carbachol contraction but reduced or abolished the response to CaCl2. Simultaneous application of carbachol and CaCl2 produced tension development the trace of which was compa rable to the curve obtained by adding graphically the respective traces of the carbacholinduced and Ca-induced tensions. La3+ (0.5 mM) abolished the late, sustained com ponent without appreciable change in the initial, transient contraction. The carbachol contraction persisted after 5 min perfusion of solution containing 0.25 mM EGTA (estimated free Ca2+ concentration less than 10-8 M). The intracellular Ca content was little changed before and after exposure to carbachol. Histamine induced a transient contraction with a dose-dependent amplitude and resulted in a decrease in tension development produced by subsequently applied carbachol. There was an inversely proportional relationship in amplitude between the histamine contraction and the following carbachol contraction, while the sum of tensions raised by both drugs showed little variation. These results favour an intracellular site for the car bachol-sensitive Ca store. Histamine appears to release Ca2+ from the carbacholsensitive store. The guinea pig taenia caecum incubated in a high K-medium retains its capacity to contract in response to carbachol in the virtual absence of external Ca2+ (1-3). The con tractile capacity disappears gradually in such a Ca-deficient environment and is restored during incubation with Ca. The rates of disappearance and restoration are much slower than the rate of diffusion of Ca ions across the cell membrane which is inferred from the time course of tension development and relaxation following application and withdrawal of Ca (3). Calcium-antagonists such as La", Mn2+, verapamil and D-600, have no direct effect on the carbachol-sensitive Ca store but they do interfere with Ca entry into the store during incubation with Ca, causing depletion of Ca2+ (4). From these results, it has been suggested that the carbachol-sensitive Ca store may be located in more internal sites than the Ca-channels where the Ca-antagonists act. The present study was undertaken to provide further support to the hypothesis. Lantha num ions were used to block Ca entry into the carbachol-sensitive store, since these ions have been shown to be most potent in this effect (4). MATERIALS AND METHODS A segment of the taenia caecum (15 mm in length), isolated from guinea pigs of either sex which weighed 300 to 400 g, was suspended in an organ bath containing 5.0 ml of Tyrode solution bubbled with air at 37°C. After equilibration with the solution for about one hour, the muscle was transferred to a K-rich solution without adding CaC12 (hereafter referred to the Ca-free, K-Tyrode solution) which was bubbled with air and kept at 20'C. Isometric tension was measured using a mechano-electronic transducer (Nihon Kohden, SB-1T) and recorded by a potentinometric pen recorder (Hitachi, 056). The drugs and CaC12 were added to the bathing solution by rapid injection in a small volume (0.05 ml) of their concentrated solutions and the final required concentration was obtained. All con centrations in the present paper refer to the final concentration in the bathing medium. Concentrations of CaC12 and carbachol were 0.5 mM and 1 mM, respectively throughout the experiments, thus their description has been omitted. The effect of each drug was examined in more than six preparations. Each preparation was deprived of the carbachol-sensitive Ca-component by exposing 1 mM carbachol for 3 min in the Ca-free, K-Tyrode solution and then suspended in this solution for 10 min. The Ca-deprived muscle was incubated in the Ca-containing, K Tyrode solution for 10 min during which a tonic contraction developed as shown in Fig. 1. The tonic contraction hereafter refers to the Ca-contracture. The muscle was rinsed three times with the Ca-free, K-Tyrode solution and was left in this solution for 10 min. During this period, the muscle completely relaxed but restored its capacity to contract in response to carbachol (Ca-loaded taenia). The Ca-loaded taenia responded by a transient and rapid contraction on exposure to carbachol (see Fig. 1) (hereafter referred to as the carbachol contraction). When the carbachol contraction fell to the initial level (about three min after the carbachol addition) the muscle was again rinsed with the Ca-free, K-Tyrode solution and left in this solution for 10 min. By repeating the sequence of pro cedures, the amplitude of the Ca con tracture and of the carbachol contraction virtually became constant. In this study, effects of the drugs on the fifth Ca contracture or carbachol contraction were observed and the fourth respective res ponses were taken as the control. FIG. I. Traces of mechanical responses to CaCl2 and carbachol in the K-rich Tyrode solution without adding CaCI2. Left, a contracture evoked by addition of 0.5 mM CaC12 (Ca) to the bathing medium in the taenia which had exposed to the solution containing carbachol (1 mM) for 3 min and then suspended in the drug-free solution for 10 min; right, a contraction in duced by I mM carbachol (Carb) applied 10 min after removal of the Ca. Calcium cfeterrnination: To estimate the cellular Ca content, the tissue was treated in a manner similar to the lanthanum method (5, 6). After blotting on filter paper (Toyo Roshi, No. 7) the tissue was weighed on a torsion balance to determine the fresh wet weight. The tissue in a crucible was dried at 90'C for 18 hours and then ashed in a mufle furnace at 600°C for 3 hours. This ash was dissolved in 5 ml of 0.5 N HCl solution containing I % LaC13. The Ca content of this solution was measured by an atomic absorption spectrophotometer (Hitachi, 208) and expressed as mM/kg wet weight of the tissue. To determine the Ca content of the medium, the bathing medium was transferred into a crucible and evaporated to dryness by heating at 90'C for 18 hours, before being ashed in a mufle furnace at 600°C for 3 hours. The amount of Ca in the ash was estimated. Solutions: Tyrode solution (mM); NaCl 137.0, KCI 2.7, NaH2PO4 0.4, CaC12 1.8, MgCl2 1.0, NaHCO3 12.0 and glucose 5.0. The Ca-free, K-Tyrode solution; KCI 137.0, MgC12 1.0, tris-maleate buffer (pH=7.4) 5.0 and glucose 5.0. Drugs: Carbamylcholine hydrochloride (carbachol) (Merck), histamine dihydro chloride (Ishizu), mepyramine maleate (May & Baker), Lanthanum chloride and EGTA (Wako). FIG. 2. Tension development produced by simultaneous application of CaC12 and carbachol. a and b, Ca-contractures; a' carbachol-induced contraction following the Ca-contracture (a); b', contraction evoked by simultaneous application of CaCl2 and carbachol (Carb-F Ca) following the Ca-contracture (b). RESULTS Effects of La" on the carbachol contraction Previous exposure of a Ca-loaded taenia to the K-rich solution containing La3+ (0.05 0.5 mM) resulted in an appreciable increase in the amplitude and the rate of falling phase of the carbachol contraction. This increase occurred in a dose-dependent manner. The averaged amplitude of the carbachol contractions in the presence of 0.5 mM La3+ was found to be 120.3=1-3.6`x„ (--S.E., n=6) of the control. When a Ca-deprived taenia was exposed to La3+ in varied concentrations before its Ca loading, it showed reduced tension develop ment during the Ca loading and by subsequently-applied carbachol. La3+ also slows the rates of tension development and of relaxation following application and withdrawal of Ca by diminishing Ca2+ membrane flux. These inhibitory effects of La3+ on the Ca-contracture were dose-dependent and virtually irreversible. Some of these observations confirm our previous findings (4). The marked difference in the La3+ sensitively between the Ca contracture and the carbachol contraction suggests that such are induced by Ca2+ from different sources. If such is the case, both contractile responses should not fuse but rather remain independent and additive (2). Fig. 2 shows tension development produced in a Ca-loaded muscle by simultaneous application of CaC12 and carbachol. The response consists of an initial, transient component and a late, sustained component. The trace of this response was compared with the curve obtained by adding graphically the respective traces of the control Ca-contracture and carbachol contraction, as shown in Fig. 3. It can be seen that the two curves are almost superimposable, which suggests that the Ca and carbachol induced con tractions are independent and additive. The peak tension of the contraction evoked by simultaneous application of CaC12 and carbachol was compared with that of the contraction elicited by carbachol alone after normalization by expressing as a percentage of the peak tension of the control carbachol contraction (the fourth carbachol response, see Methods). The mean percentage of the former in six preparations was 108.9±1.7% (±S.E.) and that of the latter in another six preparations was 103.8±2.4%. The difference between the two means was statistically significant (p<0.05). The level of the sustained tension varied in a dependent manner to Ca concentrations added, as shown in Fig. 4. Furthermore, when a Ca-loaded muscle had been exposed to 0.5 mM La3+, it responded to simultaneous ap plication of CaCl2 and carbachol by a monophasic, transient contraction which was quite similar to that elicited by carbachol alone (Fig. 5). Thus it is apparent that the initial, transient component of the response is due to intracellular Ca2+ release by carbachol and that the late, sustained component is due to Ca2+ influx. FIG. 3. Comparison between the trace of contraction (d) evoked by simultaneous application of CaC2 and carbachol and the curve (c, broken line) constructed by adding graphically tensions of the control Ca-contracture (b) and car bachol contraction (a). The respective traces were photographically superim posed. FIG. 4. Traces of the contractions evoked by simultaneous exposure to CaCl2 in varied concentrations (a, 0.5 mM; b, 0.25 mM; c, 0.125 mM; d, 0 mM) and carbachol in a concentration of 1 mM. The traces show that the strength of the sustained component of the con tractions depends on the concentration of CaCl2. In all preparations, the peak tension and the line of the falling phase of the transient component and of the early part of the sustained component were distorted somewhat above from the graphically-constructed curve. It was found that the peak tension and the maximum rate of tension development of Ca-contractures were appreciably increased in the presence of carbachol, as illustrated in Fig. 6. This indicates that carbachol increases Ca perme ability of the depolarized smooth muscle cell membrane as in the polarized membrane (7) and accelerates Ca2+ transport across the membrane. The increased tension development observed on addition of CaCl2 in the presence of carbachol was still abolished by La3+ of 0.5 mM. The slight distortion from the constructed curve, therefore, may be due to an increased Ca2+ entry during exposure to carbachol. FIG. 5. Effects of La3+ on contraction evoked by simultaneous application of CaC12 and carbachol. a and a', Control Ca-contracture and contraction evoked by simultaneous application of CaCJ2 and carbachol; b, Ca-contracture, followed by contraction (b') on exposure to CaCl2 and carbachol at the same time (Carb+ Ca) in the presence of 0.5 mM La3` (La), added 5 min prior to the simultaneous exposure. See the transient contraction (b'), just like that elicited by carbachol alone. FIG. 6. Effects of carbachol on Ca-contrac ture. a, Control carbachol contraction and Ca-contracture; b, carbachol-con traction, followed by contraction evoked on exposure to CaCl2 in the presence of 1 mM carbachol, added at the mark (") 2 min prior to the Ca exposure. FIG. 7. Effects of EGTA on carbachol con traction. a and a', Control Ca-contrac ture and carbachol-contraction; b, Ca contracture, followed by contraction induced by carbachol in the presence of 0.25 mM EGTA, added 5 min prior to the carbachol exposure. Effects of EGTA on the carbachol contraction To decrease free Ca2+ of the solution in the bath EGTA was added. Calcium con centration in the bathing solution was determined to be 0.024+0.001 mM (mean+S.E., n=6) and the free Ca2+ concentration was reduced to less than 10-8 M by adding 0.25 mM EGTA. This free Ca2+ concentration was far lower than the concentrations required to activate the contractile elements in the skeletal muscle (8). After 5 min perfusion by the solution containing EGTA, carbachol contractions were still evoked and their amplitude was reduced only to 85 % or so of the control (Fig. 7). Effects of carbachol on intracellular Ca content Twenty-four Ca-loaded preparations were divided into two groups and were used to determine the intracellular Ca content before and after exposing same to carbachol. The mean Ca contents were determined to be 1.1110.12 mM/kg wet weight tissue before the exposition and 1.18 X0.12 mM/kg wet weight tissue after the exposition. There was no statistically significant difference between the values (P>0.05). FIG. 8. Effects of histamine on carbachol contraction. a, Ca-contracture, followed by contraction induced by 5 x 10-6 M histamine (Hist) and that by subsequently applied carbachol (a'); b and b', control Ca-contracture and carbachol contraction. FIG. 9. Relationship between the peak tension of contractions evoked by histamine in varied concentrations and that of the fol lowing carbachol contractions. The graph plotting the peak tension of the histamine induced contractions the peak tension of the following carbachol con tractions (C) ~) and the total tension obtained by adding the former two versus concentration of hista mine. Ordinate: the respective peak ten sions expressed as a percentage of that of the control carbachol contraction; ab scissa: concentration (M). Each point of the graph is the mean L_S.E. of 11-14 ex periments. Effects of histamine on carbachol contraction Histamine was applied to determine whether or not the carbachol-sensitive Ca store is also sensitive to other smooth muscle stimulants. Exposure to a Ca-loaded muscle to histamine (2.5X 10-6-2.5 x 10-5 M) resulted in a contraction. The contraction was transient and completely subsided even when perfusion by the solution containing histamine was continued. The peak tension developed during exposure to histamine was increased in a dose-dependent way. The effect of histamine could be specifically blocked by mepyramine. The amplitude of the contraction evoked by carbachol applied at 2 min after the histamine exposure was reduced (Fig. 8). Fig. 9 shows the relationship between the peak tension of contractions evoked by histamine in varied concentrations and that of the following carbachol contractions. It can be seen that the amplitude of the carbachol contractions decreased in an inversely proportional manner to that of the preceding histamine contractions, while the total tensions raised by both drugs showed little variation over the range of the histamine concentrations used. To exclude a possible incomplete recovery of the contractile elements, the interval between their applications was prolonged up to 8 min. This manner of the inhibitory effect of histamine on the carbachol contraction was little affected by varying the interval. These results are what would be expected if histamine can in fact mobilize Ca2+ from the carbachol-sensitive site and reduce the quantity of Ca2+ available for inducing the subsequent carbachol contraction.