Endothelial Ca2+ signals exhibit oscillatory behavior. The occurrence of these oscillations was identified automatically using the discrete (first) derivative (d(F/F0)/dt) of the Ca2+ signal (F/F0). Discrete derivate signals were calculated by convolving F/F0 traces with the first derivative of a Gaussian kernel in the programming language, Python (8, 51). An increase in F/F0 corresponds to a positive deflection in the discrete derivative, and a decrease in F/F0 corresponds to a negative deflection in the discrete derivative. At the peak of an F/F0 Ca2+ spike, the derivative changes sign from positive to negative. A “zero-crossing detector” was used on the discrete derivative to identify the time at which peaks occurred in the F/F0 data, i.e., when the sign of the rate changed from positive to negative and the rate of change is zero. The zero-crossing detector provided a list of times of “zero crossing,” i.e., the times at which a peak occurs in the data. Therefore, the zero-crossing detector enabled the times of all peaks, with a magnitude greater than 10 times the SD of baseline noise (considered to be the threshold), in the derivate signal to be extracted. Those times were then used to extract conventional measurements (e.g., amplitude) from the corresponding F/F0 data. The detection of a peak from the discrete derivative trace (above threshold) was used to determine whether a cell responded. If a peak was detected in a cell, then the cell was defined as being “active.” Oscillatory Ca2+ signals evoked by the agonists were measured in terms of peak amplitude of the initial Ca2+ rise, the response averaged over 60 s after this peak, and percentage of the total number of cells which responded. The times of occurrence of the first peak in each Ca2+ response were used to generate latency profiles of cellular Ca2+ responses. These latency profiles are presented as histograms with time, t = 0, corresponding to the time of the first detected peak.

Ca2+ responses were recorded over a 5-min period. This allowed for the recording of basal Ca2+ activity (no agonist; ~1 to 2 min) and the response to stimuli (~3 to 4 min).

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