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Localisation of intracellular calcium stores in the striated muscles of the jellyfish Polyorchis penicillatus: possible involvement in excitation–contraction coupling

Y.-C. James Lin and Andrew N. Spencer^*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 and Bamfield Marine Station, Bamfield, British Columbia, Canada V1R 1B

View larger version (42K): [in a new window]   Fig. 1. Force–frequency relationship of the swimming muscle in the medusa of P. penicillatus and the effect of caffeine. (A) A typical force trace of muscle contraction at stimulation frequencies of 0.1, 0.2, 0.4 and 0.8 Hz using 30 ms square pulses that were just suprathreshold. As stimulation frequency increased there was a stepwise increase in contractile tension. (B) Quantitative data for the force–frequency relationship. All maximal contraction forces were normalized to the mean force at 0.1 Hz in ASW. Increasing frequency of stimulation significantly increases the amplitude of contractile force (ANOVA test, P<0.05). The underline between 0.4 Hz and 0.8 Hz at the upper right corner marks the results of the Student–Neuman–Keuls multiple comparison test, indicating no significant difference between amplitudes of contractile force when muscles were stimulated at the rate of 0.4 Hz and 0.8 Hz. Data were collected from nine muscle strips. Values are means ± S.E.M. (C) A typical trace of the effect of 10 mmol l-1 caffeine on the force–frequency relationship. Arrow indicates the beginning of 10 mmol l-1 caffeine perifusion, which produced a transient increase in tension. Caffeine perifusion was continued to the end of the trace. After caffeine treatment there was no facilitation of tension at increased frequencies. (D) Quantitative data for the force–frequency relationship when 10 mmol l-1 caffeine was present in the perifusate. The data for the ‘washed’ group were obtained at least 10 min after the removal of caffeine perifusate with ASW and after the contractile force had stabilised. All maximal contraction forces were normalised to the mean force at 0.1 Hz in ASW. Data were collected from three muscle strips. Caffeine significantly inhibited the maximal tension developed at all frequencies (ANOVA test, P<0.05, indicated by asterisks). Values are means ± S.E.M.

View larger version (171K): [in a new window]   Fig. 2. Ultrastructure of the swimming muscle of the medusa of Polyorchis penicillatus. Each muscle cell is separated into a somal region and attached muscle feet containing the myofibrils. The somal region contains the nucleus (Nu), vacuoles, Golgi complex, and mitochondria (Mi). Note the larger number of mitochondria (Mi) lying along the striated myofibrils. The myofibrils are closely applied to the basal mesoglea (Me). Arrows indicate vesicles in the subsarcolemmal region that are thought to be part of a sarcoplasmic reticular system. Scale bar, 0.5 µm.

View larger version (204K): [in a new window]   Fig. 3. Ca2+-ATPase activity in the swimming muscle of Polyorchis penicillatus. The reaction product (lead phosphate precipitate) of Ca2+-ATPase activity is located in (A) the nucleus; (B) Golgi stacks; (C) the outer leaflet of the lateral plasma membrane (arrowhead) and vesicles of the sarcoplasmic reticulum (arrow). The apical region, at the top of panel, shows little precipitation and there is an absence of reaction products in the mitochondria. (D) Precipitates were found along both sides of the sarcolemma surrounding myofibres and at their basal attachment to the mesoglea, but none were seen in the mitochondria. Arrows point to subsarcolemmal vesicles showing Ca2+-ATPase activity. The arrowhead indicates a tubular structure that does not have any precipitate. (E) Negative control for the Ca2+-ATPase activity in swimming muscle. A section through the nuclear region showed no lead phosphate precipitate when tissues were incubated in a medium without ATP. (F) Negative control for the Ca2+-ATPase activity in swimming muscle. Tangential section through myofibrils showing that there was no lead phosphate precipitation when tissues were incubated in a calcium-free medium with 10 mmol l-1 EGTA. The arrow indicates one of the vesicles and the arrowhead the sarcolemmal membrane, both of which are free of precipitate. Mi, mitochondria; Me, mesoglea. Scale bars, 1 µm (A,E); 50 nm (B); 100 nm (C); 0.5 µm (D,F).

View larger version (175K): [in a new window]   Fig. 4. Localisation of calcium in the swimming muscle of Polyorchis penicillatus by potassium dichromate precipitation. (A) Within the nucleus, grainy electron-dense precipitates (EDPs) can be seen. (B) Vesicles in the somal region show a high calcium content. The arrowhead indicates the plasma membrane, showing EDPs located on the inner side of the membrane. (C) Vesicles (arrow) in the subsarcolemma (arrowhead) contain EDPs. (D) Large vesicles between mitochondria and myofilaments in the subsarcolemma contain EDPs (arrow), which are located on the inner side of the membrane. The arrowhead indicates the sarcolemma membrane. Mi, mitochondria; Me, mesoglea. All scale bars are 1 µm.

View larger version (109K): [in a new window]   Fig. 5. Electron energy loss spectroscopy of calcium localisation in Polyorchis penicillatus swimming muscle. (A) Net calcium distribution image. This image is generated by computer subtraction of a background image taken at dE=330 eV from the image taken at dE=355 eV, which is above the edge of electron absorption specific for calcium (CaL2,3 edge 346 eV). The bright spots indicate the calcium signal. (B) Corresponding image taken using conventional TEM (zero energy loss). In both images the arrowhead indicates a desmosome and the arrow a subsarcolemmal vesicle. Scale bar, 0.5 µm. Mi, mitochondria; Me, mesoglea.