The contractile vacuole (CV) is an osmoregulatory organelle found in amoebae that live in hypotonic environments. Calmodulin, alkaline phosphatase, and a proton pump are known to be associated with the CV and its spongiome of tubules. However, the ion gradients generated across the CV membrane and their role in the water-pumping mechanism remain unknown. An improved understanding of CV water regulation may be relevant to the physiology of other systems, such as the vertebrate kidney. The techniques of rapid freezing, cryosectioning, scanning transmission electron microscopy, x-ray spectroscopy, and electron energy-loss spectroscopy have been adapted to measure CV ion concentrations in cryosections of rapidly frozen Acanthamoeba castellanii. Low-dose dark-field STEM has provided an estimation of the ion concentrations in dilute subcellular compartments for the first time. For cells in standard growth medium, preliminary results indicate a [Na+]:[K+] ratio of approximately 6:1; low [Cl-], suggesting HCO3- as the major anion; and a total osmolality of approximately 21 mosmoles. This new approach allows us to explore how external hypotonic and hypertonic ionic environments affect contracile vacuole ion concentrations.
