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The vas deferens responds to a single electrical pulse with a biphasic contraction caused by cotransmitters ATP and noradrenaline. Removing Mg²⁺ (normally 1.2 mM) from the physiological salt solution (PSS) enhances the contraction. This study aimed to determine the effect of Mg²⁺ concentration on nerve cotransmitter-mediated contractions. Rat vasa deferentia were sequentially bathed in increasing (0, 1.2, 3 mM) or decreasing (3, 1.2, 0 mM) Mg²⁺ concentrations. At each concentration a single field pulse was applied, and the biphasic contraction recorded. Contractions to exogenous noradrenaline 10 μM and ATP 100 μM were also determined. The biphasic nerve-mediated contraction was elicited by ATP and noradrenaline as NF449 (10 μM) and prazosin (100 nM) completely prevented the respective peaks. Taking the contractions in normal PSS (Mg²⁺ 1.2 mM) as 100%, lowering Mg²⁺ to 0 mM …