Effects of temperature on muscular contraction in isolated skeletal muscle of frog and rat
[한글]냉혈동물인 개구리는 동면하는 물속의 온도가 1∼2℃이지만 운동이 가능하며 이런 온도에서도 움직일 수 있다는 것은 세포막을 비롯한 각종 생체막에서 Ca**++ 을 비롯한 여러가지 물질의 이동이 가능함을 시사한다. 그러나 온혈동물인 쥐에서는 이런 낮은 온도에서는 근육수축이 이루어질 수 없으므로 온혈동물과 냉혈동물에 있어서 수축기구(contractile components)의 차이점이 있으리라 사료되어 본 실험을 하였다.
개구리와 쥐의 gastrocnemius근육을 적출하여 온도에 따른 근장력의 변화를 측정하였으며 Fiehn 및 Peter(1971) 방법에 의하여 개구리와 쥐의 gastrocnemius 근육으로부터 sarcoplasmic reticulum 을 분러하고 millipore filter로 여과하여 Ca**45 -uptake rate를 구
하였으며 개구리와 쥐에서 각 온도에 대한 영향을 비교하였다.
Cha등(1971)의 방법으로 sarcoplasmic reticulum의 Ca**++ -activated ATPase의 활성도를 측정하여 이를 μM Pi/mg protein/hr로 나타냈으며 개구리와 쥐에서 각 근도에 따른 영향을 관찰하였다.
Levy 및 Fleisher(1965) 방법에 의하여 개구리와 쥐의 gastrocnemius근육으로부터 actomyosin을 분리하여 actomyosin의 산광도(light scattering)에 의한 superprecipitation을 spectrophotometer로 측정하였고, 또한 actomyosin의 superprecipitation이 최고로 될때 까지의 시간을 각각 측정하여 비교하였다.
이상의 방법들에 의하여 다음과 같은 결과를 얻었다.
1. 온도에 따른 근장력이 최고치를 나타내는 것은 쥐에서는 16℃, 개구리에서는 8℃였으며 자극에 대하여 반응할 수 있는 최저온도가 쥐에서는 7℃였고 개구리에서는 0℃였다.
2. Sarcoplasmic reticulum에 의한 Ca**45 -uptake rate는 양군 모두 온도에 비례하여 증가하였으며 쥐에서의 uptake rate가 각 온도에서 개구리의 것보다 의의있게 높았다.
3. Sarcoplasmic reticulum의 Ca**++ -activated ATPase 활성도는 양군 모두 온도에 비례하여 증가하였으며 각 온도에서 쥐에서의 활성도가 개구리의 것보다 높았으며 10℃이상에서 의의있는 차를 보였다(P<0.05).
4. Actomyosin의 superprecipitation형성은 온도상승에 따라 증가하였으며 20℃이하에서는 쥐에서보다 개구리에서 훨씬 높았고 superprecipitation이 최고치에 달하는 시간도 개구리에서 의의있게 짧았다.
이상의 성적으로 미루어 보아 개구리의 골격근이 쥐에서와는 달리 낮은 온도에서도 수축반응을 일으킬 수 있는 것은 sarcoplasmic reticulum에 의한 Ca**45 -uptake rate나 Ca**++ -activated ATPase 활성도는 낮다 하더라도 수축기구의 하나인 Ca**++에 의한 개구
리의 actomyosin의 superprecipitation이 낮은 온도에서도 쥐에 비하여 용이하게 이루어지기 때문이라고 사료된다.
[영문]It has been well known that the frog in hibernation responds to stimulation. Thus a coldblooded animal, the frog in hibernation is able to move even at a temperature as low as 1-2℃., which suggests that transport of Ca++ and numerous materials across the cell and various biological membrane is likely to occur.
On the contrary, in a warm-blooded animal, the rat, skeletal muscular contraction is not demonstrated at this lower temperature.
The present study was undertaken to investigate some defferences in constractile component between a cold-blooded and a warm-blooded animal.
Materials and methods
1. Effect of temperature on the isometric tension in the isolated gastrocnemius muscle of frog and rat:
A strip of gastrocnemius muscle extracted from frog and rat was fixed in a thermostatically controlled water bath and stimulated with an electric stimulator (Grass SD5) for 10-20msec with 20-50 volts, as temperature of water bath was changed from 1 to 36℃. The iso metric tension on muscular contraction was recorded
in a polygraph (Grass Model 7) connecting to a force-transducer (FTO 3C). The incubation medium used was Krebs-Henseleit Ringer (Krebs and Henseleit, 1932) in the rat, and Clark-frog Ringer (Gaddum, 1953) in the frog.
2. Effect of temperature on the Ca^^45 -uptake rate by sarcoplasmic reticulum:
Sarcoplasmic reticulum was isolated from the gastrocnemius muscle of the frog and rat by the method of Fiehn and Peter (Fiehn and Peter, 1971). The Ca^^45 -uptake rate was measured with a millipore filter (pore size 0.45μ) at temperatures of 20,
10, 5, and 1℃ of incubation medium.
3. Effect of temperature on the Ca++ -activated ATPase activity:
It was measured by the method of Cha et al (Cha et al., 1971) at temperatures of 20, 10, 5, and 1℃ and represented as μM Pi/mg protein/hr.
4. Effect of temperature on the superprecipitation of actomysin:
Actomyosin was isolated from the gastrocnemius muscle of the frog and rat by the method of Levy and Fleisher (Levy and Fleisher, 1965).
Superprecipitation of actomyosin was measured with a spectrometer (Bausch and Lomb-88) at temperatures of 30, 20, 10, 5, and 1℃ by light scattering of actomyosin and represented as change in optical density (O.D.).
Also, the maximum rate of superprecipitation in relation to time was measured at temperatures of 20, 15, 10, and 5℃
Results and conclusions:
1. The maximum value of the muscular tension was 16℃ in rats and 8℃ in frogs. The lowest temperature with response to stimulation was 7℃ in rats and 0℃ in frogs.
2. The Ca^^45 -uptake rate by sarcoplasmic reticulum was increased in proportion to the rise of temperature in both frogs and rats. The value in rats was higher than that is frogs at various temperatures studied.
3. The Ca++ -activated ATPase activity by sarcoplasmic reticulum was increased in proportion to the rise of temperature in both frogs and rats. The value in rats was higher than that in frogs at various temperature studied, which was significant
difference above a temperature of 10℃ (P<0.05).
4. As the temperature was raised, the degree of superprecipitation of actomyosin was increased in both frogs and rats. Below a temperature of 20℃, the value in frog was statistically significantly higher than that in rats (P<0.05). The maximum rate of superprecitation was remarkably faster in frog than in rats, as the time passed. From these results, although the Ca^^45 -uptake rate and the Ca++ -activated ATPase activity in the frog is lower than that of rat, it is suggested
that in contrast to the rat, the contraction of frog's skeletal muscle at the lower temperature was due to the easier induction of superprecipitation of actomyosin by Ca++.