Proceedings of The Physiological Society
King's College London (2009) Proc Physiol Soc 14, PC43
Rate of inorganic phosphate release by permeabilised dogfish fibres
S. Park-Holohan1, M. Caremani1, T. West1, R. Woledge1, M. Ferenczi1, N. Curtin1
1. Molecular Medicine Section, Imperial College London, London, United Kingdom.
Muscle contraction depends on actomyosin interaction fuelled by ATP. The rate of consumption of ATP can be monitored by the time-resolved phosphate release detected with the fluorescent protein MDCC-PBP, N-(2[1-maleimidyl]ethyl)-7-diethylamino-coumarin-3-carboxamide phosphate binding protein. The aim of this study was to measure in permeabilised dogfish fibres the time course of inorganic phosphate (Pi) released during and after fibre shortening. Dogfish (Scyliorhinus canicula) were anaesthetised with MS-222, ethyl 3-aminobenzoate methanesulfonic acid (2g dissolved in 10 litre artificial seawater) followed by decapitation and destruction of the brain and spinal cord in accordance with Schedule I of the UK Animals (Scientific Procedures) Act 1986. Single fibres were dissected and stored as reported previously1. At 2.4µm sarcomere length the Triton X-100 permeabilised fibre was transferred through a sequence of solutions: (1) pre-rigor, (2) Ca2+-free, (3) Ca2+-rigor, (4) loading solution, (5) silicone oil. The fibre was activated from rigor state by flash photolysis of NPE-caged ATP (P3-1-(2-nitrophenyl)ethylester of adenosine 5’-triphosphate) to release 1.5mM ATP in silicone oil. Ramp release or step-ramp release was applied either early (start of release <50ms) or delayed (start of release 50 ms< t<100 ms) from the time of activation. The epifluorescence microscope and photomultiplier were used to record fluorescence time course2 for up to 3s after start of activation. Fibres were activated only once at physiological temperature of dogfish, 12C. Typical records of tension and Pi release during step-ramp fast shortening are shown with the start and end of fibre shortening indicated (-.-). Force trace: Following the photolytic release of ATP (t=0), tension increased with a biphasic profile. When tension nearly reached a plateau the fibre was allowed to shorten at a constant velocity, and tension decreased to a lower level. At the end of the shortening tension redeveloped to reach a new plateau. Pi release trace: After correction, for aci-nitro decay, and calibration, fluorescence record yielded a measure of Pi release during the fibre shortening. Saturation of the signal is shown when Pi release approached 1mM. The two important features to notice are the delay before the rate of Pi release reached a steady rate and the duration after the end of shortening for which this rate is maintained. The rate of Pi release increased during fibre shortening. This observation is in agreement with a previous publication by He and colleagues3. Further experiments are needed to explain the apparent shift in the steady Pi release rate with respect to the duration of fibre shortening.
Where applicable, experiments conform with Society ethical requirements