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Mechanism of phospholamban-induced stimulation of cardiac sarcoplasmic reticulum Ca-pumping ATPase: The role of the surface membrane potential
J Mol Cell Cardiol20
P-22
(Supplement
IV) (1988)
NECUAUISH OF PHOSPEOLAURAN-INDUCRD STIUULATION OF CARDIAC SARCOPLASNIC RRTICULUR Ca-PUUPING ATPase: TUR ROLE OF TUR SURPACB UEUBRANR POTRNTIAL. M.Chiesi and R.Schwaller, Deptm. Research, Pharmaceuticals Division, CIBA-GEIGY Ltd., Basel. The function of biomembranes can be modified by changes in the electrostatic surface potential. This latter influences the local concentration of relevant ionic species near the membrane and thus can modulate, for instance, the apparent affinity and rates of pumps and ion channels. The activity of the Ca-pump in cardiac SR is controlled by the phosphorylation level of the intrinsic regulatory protein phospholamban (PLB) which affects both the V,., and the apparent K,(Ca) of the transport process. Since PLB is abundant and up to 10 Pi/PLB complex can be incorporated via the specific action of various kinases, it is plausible that its phosphorylation could modify the membrane surface potential. The partition of the anionic probe AN% into the SR membrane was found to be clearly dependent on the phosphorylation level of PLB. From the change in the association constant of ANS- to cardiac SR, it can be calculated that the process of PLB phosphorylation modifies the electrostatic surface potential by about 7 mV. High ionic strength reduces the surface potential and all the processes which depend on it. The decrease in the apparent K,(Ca) of the Ca-pump observed upon phosphorylation of PLB could be clearly reduced at high ionic strength. The results show that the modulation of the ATPase by PLB can, in part, be explained by purely electrostatic phenomena.
P-23cGMP-DEPENDENT PROTEIN KINASE PHOSPHORYLATES PHOSPHOLAMBAN Q VITRO Tessa J. Mattinsley & John P. Huggins, Dept. of Cellular Pharms'gy, SK&F Research Ltd., The Frythe. Welwyn. Herts. AL6 9AR, UK. Phospholamban (plb) is a protein of the cardiac sarcoplasmic reticulum (SRI which, when phosphorylated by CAMP-dependent or a Ca2+/calmodulin-dependent protein kinase enhances the activity of the SR Ca*+ATPase pump. We report here that purified plb (method: Jones & Chem. 260, 7721) is phosphorylated by purified al., 1985, J. Biol. cGMP-dependent protein kinase (method: Lincoln, 1983, Meths. Enzymol. 99, This reaction is not prevented by the inhibitor protein of 62). CAMP-dependent protein kinase and so is not the result of any The kinetics of cGMP-dependent plb contamination by this kinase. phosphorylation have been studied using guinea pig cardiac SR where it is certain that plb is not denatured nor proteolysed. Plb is a good substrate for cGMP-dependent protein kinase. compared to the activity of the C-subunit of CAMP-dependent protein kinase using plb as a substrate. We have confirmed the presence of small amounts of plb in smooth muscle SR and it is possible that cGMP-dependent phosphorylation of this plb may mediate the relaxant effects of agents which increase cGMP in smooth muscle.
P-24
HETEROGENOUS RESPONSE IN TROPONIN-I AND PHOSPHOLAMBAN PHOSPHORYLATION IN RAT HEART AFTER ISOPRENALINE EXPOSURE. P. Karczewski, S. Bartel and E.-G. Krause. Central Institute for Cardiovascular Research, Academy of Sciences of GDR, Berlin-Buch, GDR The l3-adrenergic stimulation of cardiac contractility seems to be essentially connected with the O-adrenergic CAMP-mediated acceleration of cardiac relaxation. The latter process is thought to be related to enhanced phosphorylation of troponin-I (TN-I) and phospholamban (Plb). Phosphorylation oc,Tf+l an-+Plb was studied in perfused rat hearts freeze-clamped 30 s after 10 -10 M isoprenaline (ISO) using the indirect back-phosphorylation technique with I32P IS0 caused a dose-dependent enhance-ATP and exogenous C-subunit of PKA. ment in p+hosphate incorpqration into both proteins reaching maximal increases pmoles/mg of cardiac fraction protein for TN-I of il.8 - 1.6 and 72.0 - 2.6 respectively. The obtained dose-response curves differed significantly and Plb, indicating a higher sensitivity of TN-I phosphorylation towards IS0 or a compartmentalization of CAMP within the cytosol. On the other hand, the different sensitivity of TN-I and Plb phosphorylation in response to IS0 points to an additive or sequential cooperation in terminating the calcium signal and realizing the relaxation of cardiac muscle over a wide concentration range of ISO. 344
P-22
(Supplement
IV) (1988)
NECUAUISH OF PHOSPEOLAURAN-INDUCRD STIUULATION OF CARDIAC SARCOPLASNIC RRTICULUR Ca-PUUPING ATPase: TUR ROLE OF TUR SURPACB UEUBRANR POTRNTIAL. M.Chiesi and R.Schwaller, Deptm. Research, Pharmaceuticals Division, CIBA-GEIGY Ltd., Basel. The function of biomembranes can be modified by changes in the electrostatic surface potential. This latter influences the local concentration of relevant ionic species near the membrane and thus can modulate, for instance, the apparent affinity and rates of pumps and ion channels. The activity of the Ca-pump in cardiac SR is controlled by the phosphorylation level of the intrinsic regulatory protein phospholamban (PLB) which affects both the V,., and the apparent K,(Ca) of the transport process. Since PLB is abundant and up to 10 Pi/PLB complex can be incorporated via the specific action of various kinases, it is plausible that its phosphorylation could modify the membrane surface potential. The partition of the anionic probe AN% into the SR membrane was found to be clearly dependent on the phosphorylation level of PLB. From the change in the association constant of ANS- to cardiac SR, it can be calculated that the process of PLB phosphorylation modifies the electrostatic surface potential by about 7 mV. High ionic strength reduces the surface potential and all the processes which depend on it. The decrease in the apparent K,(Ca) of the Ca-pump observed upon phosphorylation of PLB could be clearly reduced at high ionic strength. The results show that the modulation of the ATPase by PLB can, in part, be explained by purely electrostatic phenomena.
P-23cGMP-DEPENDENT PROTEIN KINASE PHOSPHORYLATES PHOSPHOLAMBAN Q VITRO Tessa J. Mattinsley & John P. Huggins, Dept. of Cellular Pharms'gy, SK&F Research Ltd., The Frythe. Welwyn. Herts. AL6 9AR, UK. Phospholamban (plb) is a protein of the cardiac sarcoplasmic reticulum (SRI which, when phosphorylated by CAMP-dependent or a Ca2+/calmodulin-dependent protein kinase enhances the activity of the SR Ca*+ATPase pump. We report here that purified plb (method: Jones & Chem. 260, 7721) is phosphorylated by purified al., 1985, J. Biol. cGMP-dependent protein kinase (method: Lincoln, 1983, Meths. Enzymol. 99, This reaction is not prevented by the inhibitor protein of 62). CAMP-dependent protein kinase and so is not the result of any The kinetics of cGMP-dependent plb contamination by this kinase. phosphorylation have been studied using guinea pig cardiac SR where it is certain that plb is not denatured nor proteolysed. Plb is a good substrate for cGMP-dependent protein kinase. compared to the activity of the C-subunit of CAMP-dependent protein kinase using plb as a substrate. We have confirmed the presence of small amounts of plb in smooth muscle SR and it is possible that cGMP-dependent phosphorylation of this plb may mediate the relaxant effects of agents which increase cGMP in smooth muscle.
P-24
HETEROGENOUS RESPONSE IN TROPONIN-I AND PHOSPHOLAMBAN PHOSPHORYLATION IN RAT HEART AFTER ISOPRENALINE EXPOSURE. P. Karczewski, S. Bartel and E.-G. Krause. Central Institute for Cardiovascular Research, Academy of Sciences of GDR, Berlin-Buch, GDR The l3-adrenergic stimulation of cardiac contractility seems to be essentially connected with the O-adrenergic CAMP-mediated acceleration of cardiac relaxation. The latter process is thought to be related to enhanced phosphorylation of troponin-I (TN-I) and phospholamban (Plb). Phosphorylation oc,Tf+l an-+Plb was studied in perfused rat hearts freeze-clamped 30 s after 10 -10 M isoprenaline (ISO) using the indirect back-phosphorylation technique with I32P IS0 caused a dose-dependent enhance-ATP and exogenous C-subunit of PKA. ment in p+hosphate incorpqration into both proteins reaching maximal increases pmoles/mg of cardiac fraction protein for TN-I of il.8 - 1.6 and 72.0 - 2.6 respectively. The obtained dose-response curves differed significantly and Plb, indicating a higher sensitivity of TN-I phosphorylation towards IS0 or a compartmentalization of CAMP within the cytosol. On the other hand, the different sensitivity of TN-I and Plb phosphorylation in response to IS0 points to an additive or sequential cooperation in terminating the calcium signal and realizing the relaxation of cardiac muscle over a wide concentration range of ISO. 344