Figure 3. AVP stimulates Mn²⁺ entry: no rebound after AVP removal.

(A) Unidirectional Mn²⁺ entry was measured at three time points (labelled a, b and c) in fura 2-loaded A7r5 cell monolayers based on the rate of quench of fura 2 fluorescence (λ_ex=358 nm). Three sets of experiments were performed, represented by the three labelled traces in the top panel: the time course of Mn²⁺ entry in Ca²⁺-free medium without any treatment (Control); the time course of Mn²⁺ entry before, during and after treatment with 100 nM AVP; the time course of Mn²⁺ entry before and during 100 nM AVP and after a brief introduction of 10 mM CaCl₂ to allow partial refilling of the intracellular Ca²⁺ stores. (B) The rates of fluorescence quench were measured (linear regression) at each of the three time points (black bars superimposed on traces on A) for each set of experiments (n=3–4) and are summarized in the bar graph. The results indicate that capacitative Mn²⁺ entry is enhanced while AVP is present and is not further increased after AVP removal. (D) Changes in [Ca²⁺]_i that occur during the treatments for the third set of Mn²⁺ quench experiments (AVP followed by Ca²⁺). [Ca²⁺]_i was estimated from the 340:380 fura 2 fluorescence ratios recorded from a different coverslip treated with the same solutions. In this case, the cells were exposed a second time to AVP to demonstrate that transient exposure to extracellular Ca²⁺ enabled the subsequent additional release of intracellular Ca²⁺. The representative Mn²⁺-entry recording is aligned temporally on (C) above the [Ca²⁺]_i recording, demonstrating that the rates of quench were determined at those time points when cytosolic [Ca²⁺] was low and would not interfere with the Mn²⁺-entry measurements.