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K W Snowdowne

Publications and source records attributed to K W Snowdowne.

25 records · Page 2Linked to original sources

Ruthenium red: differential effects on excitation and excitation-contraction coupling in frog skeletal muscle.

The effects of purified ruthenium red (RR6+) on action potentials, resting potentials, twitches, tetani and potassium contractures in fibre bundles dissected from frog leg muscles were compared to the effects of supranormal concentrations of Ca2+. The effects of RR6+ include a shift of action potential threshold and reduction in rates of depolarization, repolarization and conduction velocity. Resting potentials and action potential overshoots are unaffected by RR6+ in concentrations of 10 microM or less. Higher RR6+ concentrations cause membrane depolarization. Low concentrations of RR6+ cause twitch potentiation but do not affect tetanus tension. Unlike Ca2+, RR6+ has no effect on the mechanical activation curve. However, crude preparations of RR6+ contain an unidentified contaminant which does shift the mechanical activation curve to the right, i.e. it increases mechanical threshold. RR6+ itself appears to be largely excluded from the t-system.

Action Potentials↗

Measurement of cytosolic free calcium in mammalian cells with aequorin.

Measurement of cytosolic free calcium (Cai) in small mammalian cells has been achieved by incorporating into cells the photoprotein aequorin by hypoosmotic shock treatment (HOST). The method and the instrumentation necessary for monitoring Cai are described in detail. We present evidence that the aequorin-Ca light signal originates in the cytosol and that the cells subjected to the HOST procedure have a normal viability and functional integrity with respect to growth, respiration, membrane transport calcium metabolism, and hormone responsiveness. From 36 measurements made at 25 degrees C, the cytosolic free calcium of cultured kidney cells is estimated to be 60 nM assuming an intracellular free magnesium of 1 mM. Anoxia and metabolic inhibitors (10 mM cyanide) dramatically increase the cytosolic free calcium, and both effects are fully reversible.

Aequorin↗

Changes in cytosolic ionized calcium induced by activators of secretion in GH3 cells.

Cytosolic ionized calcium (Cai) was measured in GH3 cells with aequorin incorporated by hypoosmotic shock treatment. Cai rose from 0.4 to 1.3 microM after the administration of 10(-7) M thyrotropin-releasing hormone (TRH) and from 0.3 to 1.1 microM after depolarization caused by high-K concentration (60 mM). Repeated TRH stimulations decreased the magnitude of the Cai response. This magnitude was directly related to the interval between stimuli. Incubations in low-calcium media (0.13 mM Ca) for 0.5 h did not alter the Cai response to TRH, but incubations in Ca-free media reduced the response. These results suggest that TRH mobilized calcium from an endogenous pool of Ca that can be depleted and replenished. The rise in Cai induced by K depolarization was slower than that evoked by TRH (time constant, t = 9 s for TRH and t = 111 s for K depolarization). Even when Cai was elevated by K depolarization, it could still be further increased by TRH, suggesting that these two stimuli mobilize calcium from two different pools and by different mechanisms.

Aequorin↗

Measurement of intracellular free calcium in monkey kidney cells with aequorin.

A method has been developed for the measurement of intracellular free calcium in mammalian cells. The calcium-sensitive photoprotein aequorin can be incorporated into isolated cells by hypo-osmotic treatment without altering the cell viability, permeability, or metabolism. Intracellular calcium activity (Cai2+) was monitored in a perfusion system. In monkey kidney cells (LLC-MK2), Cai2+ is approximately 57 nanomoles per liter. Changes in Cai2+ with time can also be followed: exposure of the cells to anaerobiosis or the calcium ionophore A23187 reversibly increases Cai2+. The method has also been successfully tested in rat hepatocytes.

Aequorin↗

Inhibition of tetanus tension by elevated extracellular calcium concentration.

Extracellular [Ca2+] in the range of 5-20 mM produces a concentration-dependent reversible reduction in tetanus tension in single frog skeletal muscle fibers. Both peak tension and ability to sustain tension during tetanus is reduced. The effect is unrelated to osmotic effects and independent of stimulation frequency in the range 100-200 Hz. The effect occurs both at 8 and 24 degrees C. Tetanus tension is most strongly inhibited by elevated extracellular [Ca2+] at short muscle lengths, but the effect can be seen at all lengths. Microelectrode recordings during tetanus indicate that action potentials remain undiminished in amplitude and duration throughout the tetanus. The evidence suggests that the inhibition results from a failure of action potentials propagation within the transverse tubular system.

Action Potentials↗