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1、 Received 1998206208 Revised 19982092073This study was supported by a grant from National Natural Science Foundation of China (No139670242) .33T o whom correspondence should be addressed. Tel : 021264370080 , ext1147; Fax: 86221264332445; E2mail : phzhu server1shcnc1ac1cnActa Physiologica Sinica 153
2、 Apr. 1999 , 51 (2) , 153160POTENTIATION OF CAFFEINE2INDUCED CONTRACTURE BY RAISING EXTRACELLULARPOTASSIUM IN FROG SKELETAL MUSCLE3CHENKE2YING,ZHUPEI2HONG33( Unit of Cell Signal Transduction , Shanghai Institute of Physiology ,Chinese Academy of Sciences , Shanghai200031)ABSTRACT The effect of raisi
3、ng extracellular potassium ( K+O) on caffeine contracture was inves2tigated , using small bundles dissectedfromfrog anterior tibialis muscle. Elevating K+Ofrom the control of 2 mmol/L to 10 or 25 mmol/L significantly potentiated the contracture induced by 3 mmol/L caffeine. The po2 tentiation repres
4、ented by PKC/ PC, where PKCand PCare the peak tension of the caffeine contracture evoked inhigh and normal K+Orespectively , was dependent on K+Oand the duration of conditioning high K+ex2posure. With 10 mmol/L K+O, the potentiation was gradually increased by prolonging conditioning exposureup to 10
5、 min. On the contrary , with 25 mmol/L K+Othe potentiation reached a maximum within only 1 min , and then subsided to the control. These different time courses of PKC/ PCcould not be accounted for byhigh K+induced depolarization , but were in general consistence with the time courses of the change i
6、n my2 oplasmic free calcium induced by corresponding high K+O10. It is suggested that , at least in frog skeletalmuscle , the high K+Oinduced potentiation of caffeine contracture is mainly due to an increase of myoplas2 mic free calcium.Key words : skeletal muscle ; ryanodine receptor; caffeine ; hi
7、gh potassium; contractureCaffeine can directly act on ryanodine receptors/ calcium release channels (RyRs) to release cal2 cium ions from the sarcoplasmic reticulum of skeletal muscle fibres and to produce contracture1. Re2 cently , it has been shown in mammalian skeletal muscle that the caffeine co
8、ntracture can be potentiated by raising extracellular potassium concentration ( K+O)2 ,3.It is known that the potentiation of caffeine contracture resultsfrom increased release of Ca2 +from the intracellular calcium stores , rather than change of calcium sensitivity of contractile proteins3. But , t
9、he mechanism underlying the increased release of Ca2 +is still unclear. It is well established that the gating of RyRs in skeletal muscle fibres is regulated by the potentials across the transverse tubule membrane as well as by calcium ions4. Several studies suggested that calcium release through ca
10、ffeine sensitive pathways is controlled by the membrane potential3 ,5. On the other hand , the elevated myo2 plasmic free calcium (Ca2 +i)produced by high K+Ois thought to be responsible for this 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.potentiation2.The predominant iso
11、form of RyRs in mammalian skeletal muscle is RyR1 (skeletal muscle iso2form) , while other (13) % belongs to RyR3 (brain isoform) . However , the skeletal muscle in non2mammalian vertebrate , including amphibian , contains other two isoforms: RyRand RyR. They havebeen recognized being homologous to
12、RyR1 and RyR3 , respectively. However , RyRand RyRinnonmammalian vertebrate skeletal muscle are present in approximately equal amounts6. At present , itis unclear whether or not the diversity of the RyR isoforms serves different functions. Considering thatthe composition of RyR isoforms in mammalian
13、 and nonmammalian vertebrate skeletal muscle is so dif2ferent , it would be interesting to investigate if caffeine contracture can be potentiated by raising K+Oin frog skeletal muscle. After finding its presence , it was attempted to see whether or not the change ofmembrane potential or Ca2 +iis res
14、ponsible for the potentiation of caffeine contracture.1 MATERIALS AND METHODS111 Preparation and solution The experiments were performed on small bundles of anterior tib2ialis muscle dissected out from pithed frogRana nigromaculataat a temperature of 15. Due to thelimited sensitivity of transducer ,
15、 the bundle used in this study comprised about 1020 fibres.The composition of Ringers solution was (in mmol/L) : NaCl 120 , KCl 2 , CaCl2118 , sucrose10 , and HEPES 4. The solution was titrated to pH 712 with NaOH. In high K+medium , potassiumwas equivalently substituted for sodium. In order to keep
16、 the product of K+ and Cl- constant ,Cl-was partially replaced by CH3HSO23. Caffeine was dissolved in the Ringers solution or in the highK+medium.112 Contractile assessment and experimental protocols The dissected muscle bundle was put in aperfusion chamber with one end fixed by a clamp and the other end connected to the lever of a trans2ducer. The preparation was then perfused with Ringers solution at a rate of about 2
