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On the influence of low extracellular calcium level on the contractilityand ultrastructure of smooth muscle cells of ileum and colon
Exp. Path., Bd. 14, S. 180-188 (1977) Academy of Sciences of the GDR, Research Center for Molecular Biology and Medicine, Institute of Drug Research, Berlin (Director: Prof. Dr. P. OEHME)
On the influence of low extracellular calcium level on the contractility and ultrastructure of smooth muscle cells of ileum and colon By H. LowE and J. BERGMANN With 6 figures (Received March 28, 1977) Key-words: calcium level: extracellular; ileum; colon; contractility: smooth muscle cells; acetylcholine; myosin like filaments; verapamil; sarcoplasm; activator calcium; myosin; actin; ultrastructure; guinea pig
Summary After decrease of calcium level in organ bath to one tenth of the normal concentration the isolated ileum of guinea pigs shows decrease of acetylcholine induced contraction to 50 per cent. The same effect is shown after addition of 5 . 10-7 M verapamil to normal bath fluid (Tyrode solution). If isolated tissue strips of ileum have been incubated in a calcium reduced bath for 20 minutes (37°C), there will occur formation of myosin-like filaments in the smooth muscle cells of the tunica muscularis. 'l'he same changes in ultrastructure can be demonstrated under the influence of 2 . 10-5 M verapamil. In contrast to this the taenia coli of guinea pigs shows no changes in contractility when the calcium level in the organ bath is lowered. In parallel with this there are no formations of myosin-like filaments in the smooth muscle cells of this tissue. After addition of verapamil to the incubation solution, the contractility of the taenia coli decreases, but not in the same degree as in the ileum. Under these conditions ultrastructural changes cannot be demonstrated in the sarcoplasm of the smooth muscle cells. It is discussed that the contraction of the smooth muscle cells in the ileum is mainly caused by the influx of calcium from the extracellular space, in the taenia coli, however, by the efflux of calcium from storage sites in the sarcoplasm. The formation of thick myofilaments is discussed as a result of a change in myosin structure following a changed calcium concentration and therefore a further indication for the myosin-linked calcium regulation of the smooth muscle contraction. The increase of the free calicum level in sarcoplasm from 10-7 M to 10-5 M in smooth muscle cells is responsible for the contraction like in the skeletal muscle. The free calcium inducing contraction (activator calcium) can originate from intracellular storage sites or from the extracellular space. The different smooth muscle tissues are distinguished among other things, by the fact that the activator calcium originates from one of these two sites. In contrast to the cells of skeletal muscle the contraction of smooth muscle cells is not essentially dependent on the linkage of calcium to the actin-tropomyosin-troponin-system, put on the linkage of calcium to myosin (BREMEL 1974, SOBIESZEK and BREMEL 1975, SOBIESZEK and SMALL 1976). Thereby myosin undergoes a structural change during the calcium linkage, followed by an interaction of myosin with actin to actomyosin, and so the contraction is introduced. This has been shown by MORIMOTO and HARRINGTON (1974) by means of viscosimetric measurements and by BREMEL (1974) in the change in activity of myosin-ATPase. There are relations between the myosin structure, which is different under the influence of a changed cation milieu, and the different myosin filament formations seen by electron microscopy (SOBIESZEK and SMALL 1976, ASHTON et al. 1975). It is only possible to demonstrate reproducibly such myosin filaments under constant conditions of preparation in connection with a constant cation mileu (COOKE and FAY 1972, ASHTON et al. 1975, CAMPBELL and CHAMLEY 1975).
180
A systematical change of the calcium level in the cell following a change of the extracellular calcium concentration or after blockade of the calcium influx by calcium antagonists (verapamil) can result in a change of myosin formation, visible in the electron microscope. For this reason it is of interest to compare the smooth muscle cells of taenia coli and of ileum, if there is a difference in the dependence of contraction on the extracellular calcium level between these two tissues. In previous studies we could demonstrate such a difference between guinea pig ileum and rat colon (BERGMANN 1975). Here we want to present our studies in this field.
Material and methods 1. Pharmacology: The dose response curves of acetylcholine in buffered Tyrode solution (37°C, pH 7.4, gassed with 02) were determined on the isolated terminal ileum and on the taenia coli of guinea pigs to study the different contraction behaviour. Contents of the Tyrode solution: 9 gil NaCI, 0.2 gil KCI, 0.2 gil CaCI2 , 0.1 gil MgCI 2 , 1.0 gil glucose, 5 mIll 0.2 mol Tris-HCl-Buffer. After maximal concentration the bath solution was substituted by a solution with 1/1c calcium concentration. After 20 min incubation and washing for many times we registered a second dose-response curve. The maximal contraction in normal conditions was defined as 100 %. 2. Morphology: Tissue strips of taenia coli and terminal ileum were incubated for 20 min in a CaCl 2-free Tyrode solution under 37°C, after that the tissue was fixed for 60 min in a 2.5 % buffered glutaraldehyde solution (pH 7.3; 4°C). As a control the tissue probes were incubated for the same time in a Tyrode solution with normal calcium concentration and also fixed in glutaraldehyde solution. In some cases there are microprecipitations after the interaction of glutaraldehyde with tris-buffer, which could not be abolished during washing. Therefore the Tyrode solution was buffered with equimolar cacodylate (0.1 mol). After satisfactory washing the tissues were dissected and postfixed for 90 min in a buffered 1 %osmium tetroxide solution (pH 7.3), dehydrated by ascending alcohol concentrations and embedded in Epon 812. After polymerization sections were made by glass knives on an ultrotom I (LKB Sweden), after that they were contrasted by lead citrate and studied in the electron microscope. In some cases the native tissue probes were incubated in Tyrode solution with addition of 2 . 10-5 mol verapamil and then prepared in parallel with the others.
Results 1. Behaviour of ileu m in low calcium concentrations Figure 1 shows a decrease of 50 % in acetylcholine induced contractility, if the calcium concentration in bath solution is decreased to lito of the norm. The same effect can be shown, if 5 . 10-7 mol verapamil is added to a normal Tyrode solution (with normal calcium concentration). As described in the introduction, too, there is in all experiments a change in the contractility of ileum during a change in extracellular calcium level; in our special case a decrease of contractility after a decrease in the extracellular calcium.
hJl wi/hout
with
Veropamil
Fig. 1. Dose response curves of acetylcholine on guinea pig ileum. • - - - . normal conditions; 1/10 of normal calcium concentration; x---x verapamil (5· 10-7 M). The inserted scheme shows a single contraction without and with verapamil.
0---0
181
182
Fig. 3. Longitudinal sectioned smooth muscle cells of the tunica muscularis of guinea pig ileum after incubation in normal Tyrode solution for 20 min. C = caveolae intercellularis; db = dense bodies; M = mitochondria; N = nucleus. Fig. 2. Smooth muscle cells of the tunica muscularis of guinea pig ileum after incubation in calcium diminished Tyrode solution for 20 min (see methods). a = Longitudinal sections through smooth muscle cells. b = Cross sections through smooth muscle cells. The sectioned thick myofilaments are visible. M = mitochondria.
183
Fig. 4. Longitudinal sectioned smooth muscle cells of the tunica muscularis of the guinea pig ileum after incubation in normal Tyrode solution with addition of verapamil for 20 min. See the thick myofilaments in the sarcoplasma (see methods). M = mitochondria.
184
without
with
Yerapamil
16
Fig. 5. Dose response curves of aeetylcholine on taenia coli of guinea pig.• - - . normal conditions; 0 - - 0 1/10 of normal calcium concentration; x - - x verapamil (5.10- 7 M). The inserted scheme shows a single contraction without and with verapamil.
If a native, fresh strip of ileum is incubated for 10-20 min in Tyrode solution without calcium (37°C) thick, myosin-like myofilaments, as shown in fig. 2, can be demonstrated under total relaxation in the smooth muscle cells of the tunica muscularis. These thick myofilaments have an average diameter of 110 A; the assemblence of thicker filaments is possible. If the tissue strips are incubated under the same conditions in a normal Tyrode solution with calcium no thick myofilaments comparable with those in calcium-free Tyrode solution can be demonstrated. But there are partial contractions in ileum strips during incubation in normal Tyrode solution and therefore in some muscle cells actomyosin complexes with myosin filaments are visible (fig. 3). As we have seen in preliminary experiments, similar myosin-like filaments can be shown in relaxed smooth muscle cells of the ileum after an incubation of tissue strips in Tyrode solution with calcium and addition of verapamil (fig. 4). The described formation of myosin-like filaments at low calcium level was only partly reversible during a postincubation in normal Tyrode solution with calcium. But these studies were not significant, because there are already distinct destructions in the tissue. 2. Behaviour of the taenia coli in low calcium concentrations Fig. 5 shows that there is no decrease in acetylcholine induced contractility of taenia coli following a decrease of extracellular calcium level to 1/10 of the norm in contrast to ileum. But under the influence of 5 . 10-7 mol verapamil there is a decrease in maximum contractility to 60 % compared with the control tissue. But in contrast to the taenia coli the contraction of the ileum under the influence of verapamil is very short with a duration of 10 sec; the decrease in contractility of taenia coli is slow, so that after 30 sec a resting contraction can be demonstrated (see inserted parts of fig. 1 and fig. 5). If a tissue strip of taenia coli is incubated under the same conditions as the ileum strip in calcium-free Tyrode solution for 10-20 min (37°C), there is no change in structure of myofilaments in the smooth muscle cells compared with control tissues, which were incubated in normal Tyrode solution with calcium. Here is a difference between taenia coli and ileum. Also after incubation with Tyrode solution with added verapamil there are no distinct differences in the myofilament structures compared with control tissues (fig. 6).
Discussion Our experiments have demonstrated that the contractility as well, as the formation of myofilaments in the smooth muscle cells of the tunica muscularis of the ileum is influenced directly by the extracellular calcium concentration. This is in contrast to the behaviour
185
Fig. 6. Longitudinal sectioned smooth muscle cells of the taenia coli of guinea pig after incubation in calcium diminished Tyrode solution for 20 min (see methods). Normal formation of the myofilaments is visible. C = caveolae intercellulares; db = dense bodies; M = mitochondria; n = nucleus.
of taenia coli. Here is no change in the contractility in vitro after decrease of calcium concentration to lito of the norm in the bath fluid, and there are no formations of myosinlike filaments in the smooth muscle cells, as is shown in isolated ileum strips. Such solutions to which calcium was not added are not free from calcium, but calcium diminished; SOMLYO (1968) could demonstrate in solutions without addition of chelating agents a calcium concentration of about 10-5 mol. The activator calcium, which is necessary for the contraction, is caused by an increased calcium influx from the extracellular space, or by a liberation of membrane-bound calcium, or of intracellular storage sites (mainly the sarcoplasmatic reticulum). It has been demonstratrated, that the acetylcholine-induced contraction of ileum and of the taenia coli of the guinea pig is dependent on the extracellular calcium concentration (OEHME et al. 1969, OHASHI et al. 1973). The contraction of the smooth muscles in ileum is mainly induced by an influx of calcium from the extracellular space, as we could 186
show. In contrast to this the smooth muscles of taenia coli are mainly contracted during liberation of activator calcium from intracellular storage sites (HURWITZ et al. 1973); a decrease of calcium concentration to 1/10 of the norm is not paralleled by a decrease of contractility. This agrees with the studies of SOMLYO (1968), who could find contraction of taenia coli in calcium-free organ bath during an increase of potassium concentration, but not in ileum preparation. We do not know, why there is a decreased contractility of taenia coli by verapamil. On the one side verapamil blocks as a "calcium inhibitor" the influx of calcium from the extracellular space into the cell (FLECKENSTEIN 1970), on the other side verapamil has its own effects, for instance on the membrane permeability of the other cations, which can explain the findings contrary to our conclusions. Our findings in the morphological experiments with the smooth muscle cells of ileum and taenia coli are in agreement with our hypothesis. In the smooth muscle cells of the ileum formation of thick myofilaments occurs at low extracellular calcium concentration. We want to discuss these filaments as myosin filaments. This would be in agreement with the studies referred to myosin-linked calcium regulation of the contraction process in smooth muscle cells. A change of calcium concentration in sarcoplasm of the smooth muscle cells of ileum following a change of extracellular calcium concentration induced a change in myosin structure. This caused the formation of thick myofilaments visible by electron microscopy. The same situation is reached, if the calcium influx is totally blocked by a high dosis of verapamil during an unchanged calcium efflux, as we concluded. In contrast to this it is not possible to influence the actual intracellular calcium concentration during a decrease of extracellular calcium content in taenia coli. Therefore the myosin structure is not directly changed. As we could demonstrate earlier, the formation of myosin-like filaments in the muscle cells of ileum is bound to the contractile proteins. If they are extracted with 0.2 mol solution of Kel in presence of ATP, there are no formations of such myofilaments after incubation in calcium diminished Tyrode solution (Low E1975). It is not possible to conclude from our experiments whether these myosin-like filaments playa role in the contraction process. Our studies show, too, if our hypothesis is correct, that a change in calcium concentration caused a change in myosin structure and this is in agreement with the studies of MORIMOTO and HARRINGTON (1974) and of BREMEL (1974).
Acknowledgements We thank Mrs. G. Grochalski, Mrs. M. Eichstadt and Mrs. M. Rudet for skilful technical assistance.
Literature ASHTON, F. T., A. V. SOMLYO and A. P. SOMLYO, The contractile apparatus of vascular smooth muscle: Intermediate high voltage stereo electron microscopy. J. Mol. BioI. 98, 17 (1975). BERGMANN, J., P. OEHME, M. BIENERT, H. NIEDRICH und C. ALBRECHT, Untersuchungen zum Wirkungsmechanismus von Tachykininsequenzen an glattmuskularen isolierten Organen. 17. Jahrestagung der Gesellschaft fur Pharmakologie und Toxikologie der DDR, Berlin, 17.-19. September 1975. BREMEL, R. D., Myosin linked calcium regulation in vertebrate smooth muscle. Nature 252, 405 (1974). CAMPELL, C. R., and J. H. CHAMLEY, Thick filaments in vertebrate smooth muscle. Cell Tiss. Res. 156, 201 (1975). COOKE, P. H., and F. S. FAY, Thick myofilaments in contracted and relaxed mammalian smooth muscle cells. Exptl. Cell. Res. 71, 265 (a972). FLECKENSTEIN, A., Die Zugelung des Myocardstoffwechsels durch Verapamil, Angriffspunkte und Anwendungsmoglichkeiten. Arzneimittelforsch. 20, 1317 (1970). HURWITZ, L., D. F. FITZPATRICK, G. DEBBAS and E. J. LANDON, Localization of calcium pump activity in smooth muscle. Science 179, 384 (1973). LOWE, H., Anderung der Strukturen kontraktiler Elemente in glatten Muskelzellen unter Calciummangel in vitro. 17. Jahrestagung der Gesellschaft fUr Pharmakologie und Toxikologie der DDR, Berlin, 17.-19. September 1975. MORIMOTO, K., and W. F. HARRINGTON, Evidence for structural changes in vertebrate thick filaments induced by calcium. J. Mol. BioI. 88, 693 (1974).
187
OEHME, P., J. BERGMANN, M. RUDEL, M. EICHStADt und F. JUNG, Calcium-Abhiingigkeit der Wirkung von Pharmaka am Meerschweinchen-I1eum und Beziehung zu ihrem Wirkungsmechanismus. Arch. into Pharmacodyn. 179, 6 (1969). OHASHI, H., A. OHGA and K. SAITO, Enhancement of Ca-contractions by catecholamines and temperature dependency in the depolarized taenia coli of the guinea-pig. Japan. J. Pharmacol. 23, 467 (1973). SOBIESZEK, A., and R. D. BREMEL, Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin. Eur. J. Biochem. 1i5, 49 (1975). - and J. V. SMALL, Myosin-linked calcium regulation in vertebrate smooth muscle. J. Mol. BioI. 101, 75 (1976). SOMLYO, A. P., and A. V. SOMLYO, Vascular smooth muscle. I. Normal structure, pathology, biochemistry and biophysics. Pharmacol. Rev. 20, 197 (1968). Author's address: Prof. Dr. H. LOWE, Akademie der Wissenschaften der DDR, Institut fur Wirkstofforschung, Alfred-Kowalke-StraBe 4, DDR - 1136 Berlin.
188
On the influence of low extracellular calcium level on the contractility and ultrastructure of smooth muscle cells of ileum and colon By H. LowE and J. BERGMANN With 6 figures (Received March 28, 1977) Key-words: calcium level: extracellular; ileum; colon; contractility: smooth muscle cells; acetylcholine; myosin like filaments; verapamil; sarcoplasm; activator calcium; myosin; actin; ultrastructure; guinea pig
Summary After decrease of calcium level in organ bath to one tenth of the normal concentration the isolated ileum of guinea pigs shows decrease of acetylcholine induced contraction to 50 per cent. The same effect is shown after addition of 5 . 10-7 M verapamil to normal bath fluid (Tyrode solution). If isolated tissue strips of ileum have been incubated in a calcium reduced bath for 20 minutes (37°C), there will occur formation of myosin-like filaments in the smooth muscle cells of the tunica muscularis. 'l'he same changes in ultrastructure can be demonstrated under the influence of 2 . 10-5 M verapamil. In contrast to this the taenia coli of guinea pigs shows no changes in contractility when the calcium level in the organ bath is lowered. In parallel with this there are no formations of myosin-like filaments in the smooth muscle cells of this tissue. After addition of verapamil to the incubation solution, the contractility of the taenia coli decreases, but not in the same degree as in the ileum. Under these conditions ultrastructural changes cannot be demonstrated in the sarcoplasm of the smooth muscle cells. It is discussed that the contraction of the smooth muscle cells in the ileum is mainly caused by the influx of calcium from the extracellular space, in the taenia coli, however, by the efflux of calcium from storage sites in the sarcoplasm. The formation of thick myofilaments is discussed as a result of a change in myosin structure following a changed calcium concentration and therefore a further indication for the myosin-linked calcium regulation of the smooth muscle contraction. The increase of the free calicum level in sarcoplasm from 10-7 M to 10-5 M in smooth muscle cells is responsible for the contraction like in the skeletal muscle. The free calcium inducing contraction (activator calcium) can originate from intracellular storage sites or from the extracellular space. The different smooth muscle tissues are distinguished among other things, by the fact that the activator calcium originates from one of these two sites. In contrast to the cells of skeletal muscle the contraction of smooth muscle cells is not essentially dependent on the linkage of calcium to the actin-tropomyosin-troponin-system, put on the linkage of calcium to myosin (BREMEL 1974, SOBIESZEK and BREMEL 1975, SOBIESZEK and SMALL 1976). Thereby myosin undergoes a structural change during the calcium linkage, followed by an interaction of myosin with actin to actomyosin, and so the contraction is introduced. This has been shown by MORIMOTO and HARRINGTON (1974) by means of viscosimetric measurements and by BREMEL (1974) in the change in activity of myosin-ATPase. There are relations between the myosin structure, which is different under the influence of a changed cation milieu, and the different myosin filament formations seen by electron microscopy (SOBIESZEK and SMALL 1976, ASHTON et al. 1975). It is only possible to demonstrate reproducibly such myosin filaments under constant conditions of preparation in connection with a constant cation mileu (COOKE and FAY 1972, ASHTON et al. 1975, CAMPBELL and CHAMLEY 1975).
180
A systematical change of the calcium level in the cell following a change of the extracellular calcium concentration or after blockade of the calcium influx by calcium antagonists (verapamil) can result in a change of myosin formation, visible in the electron microscope. For this reason it is of interest to compare the smooth muscle cells of taenia coli and of ileum, if there is a difference in the dependence of contraction on the extracellular calcium level between these two tissues. In previous studies we could demonstrate such a difference between guinea pig ileum and rat colon (BERGMANN 1975). Here we want to present our studies in this field.
Material and methods 1. Pharmacology: The dose response curves of acetylcholine in buffered Tyrode solution (37°C, pH 7.4, gassed with 02) were determined on the isolated terminal ileum and on the taenia coli of guinea pigs to study the different contraction behaviour. Contents of the Tyrode solution: 9 gil NaCI, 0.2 gil KCI, 0.2 gil CaCI2 , 0.1 gil MgCI 2 , 1.0 gil glucose, 5 mIll 0.2 mol Tris-HCl-Buffer. After maximal concentration the bath solution was substituted by a solution with 1/1c calcium concentration. After 20 min incubation and washing for many times we registered a second dose-response curve. The maximal contraction in normal conditions was defined as 100 %. 2. Morphology: Tissue strips of taenia coli and terminal ileum were incubated for 20 min in a CaCl 2-free Tyrode solution under 37°C, after that the tissue was fixed for 60 min in a 2.5 % buffered glutaraldehyde solution (pH 7.3; 4°C). As a control the tissue probes were incubated for the same time in a Tyrode solution with normal calcium concentration and also fixed in glutaraldehyde solution. In some cases there are microprecipitations after the interaction of glutaraldehyde with tris-buffer, which could not be abolished during washing. Therefore the Tyrode solution was buffered with equimolar cacodylate (0.1 mol). After satisfactory washing the tissues were dissected and postfixed for 90 min in a buffered 1 %osmium tetroxide solution (pH 7.3), dehydrated by ascending alcohol concentrations and embedded in Epon 812. After polymerization sections were made by glass knives on an ultrotom I (LKB Sweden), after that they were contrasted by lead citrate and studied in the electron microscope. In some cases the native tissue probes were incubated in Tyrode solution with addition of 2 . 10-5 mol verapamil and then prepared in parallel with the others.
Results 1. Behaviour of ileu m in low calcium concentrations Figure 1 shows a decrease of 50 % in acetylcholine induced contractility, if the calcium concentration in bath solution is decreased to lito of the norm. The same effect can be shown, if 5 . 10-7 mol verapamil is added to a normal Tyrode solution (with normal calcium concentration). As described in the introduction, too, there is in all experiments a change in the contractility of ileum during a change in extracellular calcium level; in our special case a decrease of contractility after a decrease in the extracellular calcium.
hJl wi/hout
with
Veropamil
Fig. 1. Dose response curves of acetylcholine on guinea pig ileum. • - - - . normal conditions; 1/10 of normal calcium concentration; x---x verapamil (5· 10-7 M). The inserted scheme shows a single contraction without and with verapamil.
0---0
181
182
Fig. 3. Longitudinal sectioned smooth muscle cells of the tunica muscularis of guinea pig ileum after incubation in normal Tyrode solution for 20 min. C = caveolae intercellularis; db = dense bodies; M = mitochondria; N = nucleus. Fig. 2. Smooth muscle cells of the tunica muscularis of guinea pig ileum after incubation in calcium diminished Tyrode solution for 20 min (see methods). a = Longitudinal sections through smooth muscle cells. b = Cross sections through smooth muscle cells. The sectioned thick myofilaments are visible. M = mitochondria.
183
Fig. 4. Longitudinal sectioned smooth muscle cells of the tunica muscularis of the guinea pig ileum after incubation in normal Tyrode solution with addition of verapamil for 20 min. See the thick myofilaments in the sarcoplasma (see methods). M = mitochondria.
184
without
with
Yerapamil
16
Fig. 5. Dose response curves of aeetylcholine on taenia coli of guinea pig.• - - . normal conditions; 0 - - 0 1/10 of normal calcium concentration; x - - x verapamil (5.10- 7 M). The inserted scheme shows a single contraction without and with verapamil.
If a native, fresh strip of ileum is incubated for 10-20 min in Tyrode solution without calcium (37°C) thick, myosin-like myofilaments, as shown in fig. 2, can be demonstrated under total relaxation in the smooth muscle cells of the tunica muscularis. These thick myofilaments have an average diameter of 110 A; the assemblence of thicker filaments is possible. If the tissue strips are incubated under the same conditions in a normal Tyrode solution with calcium no thick myofilaments comparable with those in calcium-free Tyrode solution can be demonstrated. But there are partial contractions in ileum strips during incubation in normal Tyrode solution and therefore in some muscle cells actomyosin complexes with myosin filaments are visible (fig. 3). As we have seen in preliminary experiments, similar myosin-like filaments can be shown in relaxed smooth muscle cells of the ileum after an incubation of tissue strips in Tyrode solution with calcium and addition of verapamil (fig. 4). The described formation of myosin-like filaments at low calcium level was only partly reversible during a postincubation in normal Tyrode solution with calcium. But these studies were not significant, because there are already distinct destructions in the tissue. 2. Behaviour of the taenia coli in low calcium concentrations Fig. 5 shows that there is no decrease in acetylcholine induced contractility of taenia coli following a decrease of extracellular calcium level to 1/10 of the norm in contrast to ileum. But under the influence of 5 . 10-7 mol verapamil there is a decrease in maximum contractility to 60 % compared with the control tissue. But in contrast to the taenia coli the contraction of the ileum under the influence of verapamil is very short with a duration of 10 sec; the decrease in contractility of taenia coli is slow, so that after 30 sec a resting contraction can be demonstrated (see inserted parts of fig. 1 and fig. 5). If a tissue strip of taenia coli is incubated under the same conditions as the ileum strip in calcium-free Tyrode solution for 10-20 min (37°C), there is no change in structure of myofilaments in the smooth muscle cells compared with control tissues, which were incubated in normal Tyrode solution with calcium. Here is a difference between taenia coli and ileum. Also after incubation with Tyrode solution with added verapamil there are no distinct differences in the myofilament structures compared with control tissues (fig. 6).
Discussion Our experiments have demonstrated that the contractility as well, as the formation of myofilaments in the smooth muscle cells of the tunica muscularis of the ileum is influenced directly by the extracellular calcium concentration. This is in contrast to the behaviour
185
Fig. 6. Longitudinal sectioned smooth muscle cells of the taenia coli of guinea pig after incubation in calcium diminished Tyrode solution for 20 min (see methods). Normal formation of the myofilaments is visible. C = caveolae intercellulares; db = dense bodies; M = mitochondria; n = nucleus.
of taenia coli. Here is no change in the contractility in vitro after decrease of calcium concentration to lito of the norm in the bath fluid, and there are no formations of myosinlike filaments in the smooth muscle cells, as is shown in isolated ileum strips. Such solutions to which calcium was not added are not free from calcium, but calcium diminished; SOMLYO (1968) could demonstrate in solutions without addition of chelating agents a calcium concentration of about 10-5 mol. The activator calcium, which is necessary for the contraction, is caused by an increased calcium influx from the extracellular space, or by a liberation of membrane-bound calcium, or of intracellular storage sites (mainly the sarcoplasmatic reticulum). It has been demonstratrated, that the acetylcholine-induced contraction of ileum and of the taenia coli of the guinea pig is dependent on the extracellular calcium concentration (OEHME et al. 1969, OHASHI et al. 1973). The contraction of the smooth muscles in ileum is mainly induced by an influx of calcium from the extracellular space, as we could 186
show. In contrast to this the smooth muscles of taenia coli are mainly contracted during liberation of activator calcium from intracellular storage sites (HURWITZ et al. 1973); a decrease of calcium concentration to 1/10 of the norm is not paralleled by a decrease of contractility. This agrees with the studies of SOMLYO (1968), who could find contraction of taenia coli in calcium-free organ bath during an increase of potassium concentration, but not in ileum preparation. We do not know, why there is a decreased contractility of taenia coli by verapamil. On the one side verapamil blocks as a "calcium inhibitor" the influx of calcium from the extracellular space into the cell (FLECKENSTEIN 1970), on the other side verapamil has its own effects, for instance on the membrane permeability of the other cations, which can explain the findings contrary to our conclusions. Our findings in the morphological experiments with the smooth muscle cells of ileum and taenia coli are in agreement with our hypothesis. In the smooth muscle cells of the ileum formation of thick myofilaments occurs at low extracellular calcium concentration. We want to discuss these filaments as myosin filaments. This would be in agreement with the studies referred to myosin-linked calcium regulation of the contraction process in smooth muscle cells. A change of calcium concentration in sarcoplasm of the smooth muscle cells of ileum following a change of extracellular calcium concentration induced a change in myosin structure. This caused the formation of thick myofilaments visible by electron microscopy. The same situation is reached, if the calcium influx is totally blocked by a high dosis of verapamil during an unchanged calcium efflux, as we concluded. In contrast to this it is not possible to influence the actual intracellular calcium concentration during a decrease of extracellular calcium content in taenia coli. Therefore the myosin structure is not directly changed. As we could demonstrate earlier, the formation of myosin-like filaments in the muscle cells of ileum is bound to the contractile proteins. If they are extracted with 0.2 mol solution of Kel in presence of ATP, there are no formations of such myofilaments after incubation in calcium diminished Tyrode solution (Low E1975). It is not possible to conclude from our experiments whether these myosin-like filaments playa role in the contraction process. Our studies show, too, if our hypothesis is correct, that a change in calcium concentration caused a change in myosin structure and this is in agreement with the studies of MORIMOTO and HARRINGTON (1974) and of BREMEL (1974).
Acknowledgements We thank Mrs. G. Grochalski, Mrs. M. Eichstadt and Mrs. M. Rudet for skilful technical assistance.
Literature ASHTON, F. T., A. V. SOMLYO and A. P. SOMLYO, The contractile apparatus of vascular smooth muscle: Intermediate high voltage stereo electron microscopy. J. Mol. BioI. 98, 17 (1975). BERGMANN, J., P. OEHME, M. BIENERT, H. NIEDRICH und C. ALBRECHT, Untersuchungen zum Wirkungsmechanismus von Tachykininsequenzen an glattmuskularen isolierten Organen. 17. Jahrestagung der Gesellschaft fur Pharmakologie und Toxikologie der DDR, Berlin, 17.-19. September 1975. BREMEL, R. D., Myosin linked calcium regulation in vertebrate smooth muscle. Nature 252, 405 (1974). CAMPELL, C. R., and J. H. CHAMLEY, Thick filaments in vertebrate smooth muscle. Cell Tiss. Res. 156, 201 (1975). COOKE, P. H., and F. S. FAY, Thick myofilaments in contracted and relaxed mammalian smooth muscle cells. Exptl. Cell. Res. 71, 265 (a972). FLECKENSTEIN, A., Die Zugelung des Myocardstoffwechsels durch Verapamil, Angriffspunkte und Anwendungsmoglichkeiten. Arzneimittelforsch. 20, 1317 (1970). HURWITZ, L., D. F. FITZPATRICK, G. DEBBAS and E. J. LANDON, Localization of calcium pump activity in smooth muscle. Science 179, 384 (1973). LOWE, H., Anderung der Strukturen kontraktiler Elemente in glatten Muskelzellen unter Calciummangel in vitro. 17. Jahrestagung der Gesellschaft fUr Pharmakologie und Toxikologie der DDR, Berlin, 17.-19. September 1975. MORIMOTO, K., and W. F. HARRINGTON, Evidence for structural changes in vertebrate thick filaments induced by calcium. J. Mol. BioI. 88, 693 (1974).
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