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Effects of low ionic strength and calmodulin on the decline in maximal calcium-activated force in permeabilized
1Institute of Physiology, University of Glasgow, Scotland.
Abstract:
Maximal calcium-activated tension (Tmax) was measured from saponin-and alpha-toxin permeabilized strips of rat anococcygeus muscle stimulated repetitively. 50 mins after saponin treatment, Tmax had declined to 20% of the initial value. Calmodulin (1 microM) only partially reduced this decline. Lowering the ionic strength of the bathing solution from 0.2M to 0.07M markedly reduced the rate of decline of tension. Addition of calmodulin to the bathing medium of low ionic strength prevented the decline of force still further; 50 mins after permeabilization, Tmax declined to 80% of the original level. Loss of endogenous calmodulin should be prevented by permeabilization with alpha-toxin from Staphylococcus aureus. Nevertheless, Tmax still declined to approximately 50% of the original value after 50mins. However, negligible force reduction was observed if the bathing solution had an ionic strength of 0.07M.
Insights
Maintaining muscle tension requires careful control of ionic strength and calmodulin levels during permeabilization. Low ionic strength solutions and calmodulin supplementation significantly preserve maximal calcium-activated tension (Tmax) in muscle tissue.
Area of Science:
- Muscle physiology
- Biochemistry
Background:
- Permeabilization of muscle tissue is crucial for studying calcium-activated tension.
- Saponin and alpha-toxin are common agents used for muscle permeabilization.
- Endogenous calmodulin plays a role in maintaining muscle contractility.
Purpose of the Study:
- To investigate the effects of permeabilization methods and solution conditions on maximal calcium-activated tension (Tmax).
- To determine the role of calmodulin and ionic strength in preserving muscle force during experimental procedures.
Main Methods:
- Maximal calcium-activated tension (Tmax) was measured in saponin- and alpha-toxin permeabilized rat anococcygeus muscle strips.
- Experiments varied ionic strength of the bathing solution (0.2M vs. 0.07M) and included calmodulin (1 microM) supplementation.
- Tension decline was monitored over 50 minutes post-permeabilization.
Main Results:
- Saponin permeabilization led to a rapid decline in Tmax to 20% of the initial value within 50 minutes.
- Lowering ionic strength to 0.07M significantly reduced the rate of tension decline.
- Calmodulin supplementation, especially in low ionic strength solutions, largely prevented force reduction, preserving Tmax at 80% of the original level.
- Alpha-toxin permeabilization also showed Tmax decline to ~50%, but this was negligible at 0.07M ionic strength.
Conclusions:
- Both ionic strength and calmodulin availability are critical for maintaining muscle tension after permeabilization.
- Low ionic strength solutions (0.07M) are effective in preserving Tmax, regardless of permeabilization agent.
- Optimizing experimental conditions, including ionic strength and calmodulin levels, is essential for accurate measurements of muscle contractility.