Excitation-Contraction Coupling in Rabbit Aorta Studied by the Lanthanum Method for Measuring Cellular Calcium Influx

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Abstract
Lanthanum inhibits 45Ca efflux from internally injected squid axons. Evidence for La3+ blockade of Ca2+ fluxes across the cell membrane was also obtained for aortic smooth muscle: 1mM La3+ completely inhibited contractions which otherwise resulted from adding Ca2+ to calcium-free depolarizing solutions, and 2 mM La3+ caused a 50% inhibition of the late 45Ca efflux. Ca2+ bound extracellularly could be displaced by La3+, and the binding sites preferred La3+ over Ca2+. We could thus use La3+ to eliminate extracellular bound Ca2+ from our calcium-influx measurements as follows: after exposure of the aortic strips to experimental solutions labeled with 45Ca, the extracellular Ca2+ label was displaced by putting the strips in a calcium-free solution containing 2 mM La3+ for 60 minutes. The loss of intracellular Ca2+ was minimized during this hour by the La3+ blockade of Ca2+ membrane fluxes. Tissue weight, 45Ca, and total Ca2+ were then measured using standard techniques. Studies employing this new method showed that depolarization by high K+, Na+ replacement, and high pH activate smooth muscle contraction by stimulating Ca2+ influx. Low pH and La3+ block Ca2+ influx. Norepinephrine initiates aortic contractions by release of intracellularly sequestered Ca2+. Angiotensin and histamine appear to release Ca2+ from this same fraction.