Fig. 2. Pretreatment with 8-Br-cGMP reduced the alcohol-induced rise in [Ca²⁺]_i.

Panel A shows pseudocolor 340/380 ratio images of CGN cultures (representative for three replicates) before (Ai–Aii) and identical view-fields 60 seconds after adding alcohol (800 mg/dl) (Aiii–Aiv). The color scale bar maps [Ca²⁺]_i; blue and red respectively depict low and high [Ca²⁺]_i. Arrows (Aiii) highlight cells with increased [Ca²⁺]_i after alcohol exposure, shifting in color from blue (low [Ca²⁺]_i) to red/yellow (higher [Ca²⁺]_i). Fewer cells (less arrows) with raised [Ca²⁺]_i are evident in the culture (Aii, Aiv) that received 8-Br-cGMP 30 minutes before alcohol. Panel B: Lines show mean ± SEM [Ca²⁺]_i in cells (combined from six replicates) after adding alcohol (400 mg/dl) over the duration of the experiment (9 minutes). Alcohol initiated a rise in [Ca²⁺]_i which continued for the experiment duration. 8-Br-cGMP pretreatment reduced the alcohol-induced rise of [Ca²⁺]_i. Panel C: Utilizing the data from Panel B, the highest [Ca²⁺]_i attained for each cell (Peak [Ca²⁺]_i) over the duration of the experiment was derived and a mean was calculated. The dashed line indicates the baseline [Ca²⁺]_i level, measured 30 seconds after adding alcohol. Treatment with 8-Br-cGMP decreased the peak [Ca²⁺]_i level elicited by alcohol (* p ≤ 0.05). Panel D: To ascertain the cell distribution across the range of Peak [Ca²⁺]_i, a histogram was derived from the Peak [Ca²⁺]_i data in Panel C. The X-axis (Peak [Ca²⁺]_i) in Fig 3D was divided into 20 nM segments and the percent of cells in each segment was determined. A vertical dashed line is drawn at an [Ca²⁺]_i of 200 nM, and the percentage of cells with more than 200 nM [Ca²⁺]_i was determined. In the absence of 8-Br-cGMP, 27.4% of the cells attained an [Ca²⁺]_i greater than 200 nM, after alcohol addition. Treatment with 8-Br-cGMP substantially lowered this percentage to 4.1% (χ², p ≤ 0.05).