Trk 500

L. pictus sea urchins were obtained from Marinus (Long Beach, CA, USA). Dry sperm were collected after intracelomic injection of 0.5 M KCl and kept on ice until used. The fluorescent dyes 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5)), Fluo-4-AM, Quin-2 and 5-(and-6)-Carboxyfluorescein diacetate were obtained from Molecular Probes (Eugene, OR, USA). Anhydrous dimethylsulfoxide (DMSO), tolbutamide, glibenclamide, were from Sigma-Aldrich. ZnSO₄ was from Merck. Charybdotoxin and Iberiotoxin were from Alomone Labs. The Kits to measure cAMP (TRK 432) and cGMP (TRK 500) were from Amersham. Speract was synthesized in Professor Possani's Laboratory (IBT-UNAM) and fucose sulfate polymer (FSP) was prepared according to the previous report (Garbers et al., 1983 (link)). The rest of the reagents used were of the highest quality available.

Sperm diluted (1:200) in 0CaSW with 1 mM 3-isobutyl-1-methylxanthine (IBMX) were incubated with a ligand (Zn²⁺ or speract) for 1 min at 14 °C. Subsequently, the sperm suspension was transferred to a boiling bath to quench the enzymatic reaction and extract nucleotides. TRK 432 or TRK 500 Kits of Amersham were used to measure cAMP or cGMP concentration, respectively.