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The effect of ATP on the response of intact and toxin-permeabilized isolated rat anococcygeus muscle to noradrenaline and acetylcholine
J Mol
pw1
Cell
Cardiol
22 (Supplement
III)
(1990)
THE EFFECT OF ATP ON THE RESPONSE OF INTACT AND TOXIN-PERMEALIILIZED ANOCOCCYGEUS MUSCLE TO NORADRENALINE AND ACEIYLCHOLINE.
ISOLATED
RAT
Catherine A Crichton, Godfrey L. Smith, David J. Miller, John C. McGrath, Institute of Physiology, University (ii Glasgow, Glasgow G12 8QQ. ATP(SmM), noradrenaline (30 PM) and acetylcholine (3 mM) individually cause maintained contractions m l(X) pm diameter strips of rat anococcygeus muscle bathed in Tyrodes solution. Bathing the muscle in 120 mM KCl, 40 mM NaCI, 2 mM MgCl*and 0.12 PM Ca*+ (0.2 mM EGTA pH 7.1), all three agonists caused a large transient contracture, indicating the release of Ca2+ from internal stores. However, in the continued presence of 5 mM ATP the response to acetylcholine was almost completely abolished, while the response fo noradrenaline remained unaffected. The ability of ATF’ to block the cholinergic response was not affected by the addition of a-p-methylene ATP (100 PM) or'flurbiprofen:lOO nM). Treatment of strips of smooth muscle with the a-toxin from staphylococcus aureus creates holes in the membranes that allow free diffusion of small molecules while the surface membrane receptors remain functional. After this treatment the muscle was bathed in a mock intracellular solution containing 5 mM ATP to prevent a rigor contracture. Under these conditions noradrenaline caused a transient contracture, bur the preparation did not respond to acetylcholine. This effect persisted after Ihe addition of 100 PM GTP. a
manoeuvre which has previously been shown to enhance the responseto acetylcholne in toxin permeabilized smooth muscle (Kitazawa et al, 1989). In conclusion ATP appears to block the ability of acetylcholine to release Ca*+ from the SR in intact muscle. This result may explain the absence of a cholnergic response in toxin permeahili& preparations.
Kirazawa, T, Kobayashi, S. Horiutu, K., Somlyo. A.V., Somlyo. A.P. (1989) J. Biol. Chem. 212, 5339.5342,
pW2PROTEIN KINASES CHANGE Ca SENSITIVITY OF CARDIAC MYOFILAMENTS IN SKINNED SINGLE RAT CELLS. 0. Clbment, M. Puceat, J-M. Pelosin, W. Landgraf, M. Walsh, G. Vassort. INSERM U-241, Univ. Paris-Sud ORSAY. Cardiac contraction could be regulated by changes in the properties of the contractile proteins as well as by changes in cytosolic calcium during action potential. Applications of p-adrenergic agonists to single myocytes before skinning induce a decrease of myofilament sensitivity to Ca++ ions, or muscarinic agonists while stimulations by aI- adrenergic lead to a leftward shift of the tension/pCa relationships. We have applied three protein kinases (PK-A, PK-G and PK-C) to skinned single rat cells and measured the force they developed at different [Ca++]. PKA reproduced the rightward shift of the tension-pCa relationship induced by j3-adrenergic agonists. PK-G which phosphorylates cardiac TN1 at the same sites as PK-A also decreased Ca sensitivity of the myofilaments. The addition of PK-C (whole brain extract) in the presence of phosphatidylserine and DOG was without effect. Hovewer. avolication of 'the complex Cat+-calmodulin-MLCK, known to phosphorylate t-h-e MLC2, led to a leftward shift (pCa,, = 5.92 instead of 5.78) and might be involved in the pathway inducing the increase in Ca++ sensitivity observed after a,-adrenergic and muscarinic stimulations.
PW3COORDINATED POSTNATAL BINDING OF CREATINE KINASE TO MYOFIBRILLS AND MITOCHONDRIA IN RABBIT HEART. R. Ventura-Clanier. J.A. Hoerter. A. Kuznetsov. Laboratoire de Physiologie celluiaire Cardiaque, i-24; INSERM, Universit6 Paris Sud, 91405-Orsay, France Perinatal development of functional activity of myofibrillar creatine kinase (MM-CK bound to triton X-100 skinned fibers) and of mitochondrial CK (mito-CK bound to treated fib&s) days before birthwtz followed in LV of rabbit ranging in aglafPFzr?2 adult. MM-CK is cytosolic before birth and mito-CK is not expressed (Carlsson et al., 1982 ; Ingwall et al., 1990). Total CK activity/mg protein was unchanged. Myofibrillar fixation and functional activity, absent at birth, - reached adult level within 3 weeks. Creatin& stimulated respiration was absent at birth and followed the same pattern of increase than myofibrillar bound CK. Thus compartmentation of CK isozymes occurs during postnatai cardiac development. Coordinate fixation at both ends of the creatine shuttle, in mitochondria and myofibrille, is evident by parallel development oi functional activity. S.1
pw1
Cell
Cardiol
22 (Supplement
III)
(1990)
THE EFFECT OF ATP ON THE RESPONSE OF INTACT AND TOXIN-PERMEALIILIZED ANOCOCCYGEUS MUSCLE TO NORADRENALINE AND ACEIYLCHOLINE.
ISOLATED
RAT
Catherine A Crichton, Godfrey L. Smith, David J. Miller, John C. McGrath, Institute of Physiology, University (ii Glasgow, Glasgow G12 8QQ. ATP(SmM), noradrenaline (30 PM) and acetylcholine (3 mM) individually cause maintained contractions m l(X) pm diameter strips of rat anococcygeus muscle bathed in Tyrodes solution. Bathing the muscle in 120 mM KCl, 40 mM NaCI, 2 mM MgCl*and 0.12 PM Ca*+ (0.2 mM EGTA pH 7.1), all three agonists caused a large transient contracture, indicating the release of Ca2+ from internal stores. However, in the continued presence of 5 mM ATP the response to acetylcholine was almost completely abolished, while the response fo noradrenaline remained unaffected. The ability of ATF’ to block the cholinergic response was not affected by the addition of a-p-methylene ATP (100 PM) or'flurbiprofen:lOO nM). Treatment of strips of smooth muscle with the a-toxin from staphylococcus aureus creates holes in the membranes that allow free diffusion of small molecules while the surface membrane receptors remain functional. After this treatment the muscle was bathed in a mock intracellular solution containing 5 mM ATP to prevent a rigor contracture. Under these conditions noradrenaline caused a transient contracture, bur the preparation did not respond to acetylcholine. This effect persisted after Ihe addition of 100 PM GTP. a
manoeuvre which has previously been shown to enhance the responseto acetylcholne in toxin permeabilized smooth muscle (Kitazawa et al, 1989). In conclusion ATP appears to block the ability of acetylcholine to release Ca*+ from the SR in intact muscle. This result may explain the absence of a cholnergic response in toxin permeahili& preparations.
Kirazawa, T, Kobayashi, S. Horiutu, K., Somlyo. A.V., Somlyo. A.P. (1989) J. Biol. Chem. 212, 5339.5342,
pW2PROTEIN KINASES CHANGE Ca SENSITIVITY OF CARDIAC MYOFILAMENTS IN SKINNED SINGLE RAT CELLS. 0. Clbment, M. Puceat, J-M. Pelosin, W. Landgraf, M. Walsh, G. Vassort. INSERM U-241, Univ. Paris-Sud ORSAY. Cardiac contraction could be regulated by changes in the properties of the contractile proteins as well as by changes in cytosolic calcium during action potential. Applications of p-adrenergic agonists to single myocytes before skinning induce a decrease of myofilament sensitivity to Ca++ ions, or muscarinic agonists while stimulations by aI- adrenergic lead to a leftward shift of the tension/pCa relationships. We have applied three protein kinases (PK-A, PK-G and PK-C) to skinned single rat cells and measured the force they developed at different [Ca++]. PKA reproduced the rightward shift of the tension-pCa relationship induced by j3-adrenergic agonists. PK-G which phosphorylates cardiac TN1 at the same sites as PK-A also decreased Ca sensitivity of the myofilaments. The addition of PK-C (whole brain extract) in the presence of phosphatidylserine and DOG was without effect. Hovewer. avolication of 'the complex Cat+-calmodulin-MLCK, known to phosphorylate t-h-e MLC2, led to a leftward shift (pCa,, = 5.92 instead of 5.78) and might be involved in the pathway inducing the increase in Ca++ sensitivity observed after a,-adrenergic and muscarinic stimulations.
PW3COORDINATED POSTNATAL BINDING OF CREATINE KINASE TO MYOFIBRILLS AND MITOCHONDRIA IN RABBIT HEART. R. Ventura-Clanier. J.A. Hoerter. A. Kuznetsov. Laboratoire de Physiologie celluiaire Cardiaque, i-24; INSERM, Universit6 Paris Sud, 91405-Orsay, France Perinatal development of functional activity of myofibrillar creatine kinase (MM-CK bound to triton X-100 skinned fibers) and of mitochondrial CK (mito-CK bound to treated fib&s) days before birthwtz followed in LV of rabbit ranging in aglafPFzr?2 adult. MM-CK is cytosolic before birth and mito-CK is not expressed (Carlsson et al., 1982 ; Ingwall et al., 1990). Total CK activity/mg protein was unchanged. Myofibrillar fixation and functional activity, absent at birth, - reached adult level within 3 weeks. Creatin& stimulated respiration was absent at birth and followed the same pattern of increase than myofibrillar bound CK. Thus compartmentation of CK isozymes occurs during postnatai cardiac development. Coordinate fixation at both ends of the creatine shuttle, in mitochondria and myofibrille, is evident by parallel development oi functional activity. S.1