Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum

1 To understand whether the molecular mechanism of Tetrandrine (Tet)'s phar macological effects is concerned with sarcoplasmic reticulum calcium transp ort so as to be involved in myocardial contractility. we observed the effec ts of Tet on calcium transport and membrane structure of rabbit skeletal mu scle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reti culum vesicles (CSR). 2 Calcium uptake was monitored with a dual-wavelength spectrophotometer. Pr otein conformation and fluorescence polarization were measured by fluospect rophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 3 128 mu mol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca2+Mg2+-A TPase activity, causing the stoichiometric ratio of SR Ca2+/ATP to decrease to 1.43 from 2.0 of control. 4 Inhibitory rates on SR Ca2+, Mo2+-ATPasr by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced fr om 92% in I mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. 5 Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)modified protein fluorescence of SR labelled with N -(3-pyrene)-maleimide. 6 Tet slightly increased fluorescence polarization in the middle and deep l ayers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1,6-di phenyl-1,3,5-hexatrine (DPH). 7 These results revealed that prevention of SR calcium uptake by Tet was du e to inhibition of the SK calcium pump Ca2+, Mg2+-ATPase, changes in spatia l conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca2 +](i) and myocardial contractility.