Ca2+/Na+ exchanger and Na+,K+ 2Cl- cotransporter in lens fiber plasma membrane vesicles.

Abstract When the Ca 2+ -sensitive fluorescent probe, Fura-2, or the Na + -sensitive probe, SBFI, in their cell permeable forms or the Cl − -specific probe, SPQ, were incubated with plasma membrane vesicles prepared from dogfish and bovine lenses fibers, there was a selective accumulation of the ion-specific probes within the vesicles. The SBFI and Fura-2 fluorescent excitation ratios of 340 nm to 380 nm (em: 505 nm) in the presence of an outwardly-directed Na + gradient across the vesicles membrane, indicate that the influx of Ca 2+ is increased by 152·5% and 147·4% for dogfish and bovine vesicles, respectively. The Na + influx into the vesicles is also enhanced by 154·1% for dogfish and 149·1% for bovine lens when an outwardly-directed Ca 2+ was present. This stimulation is not affected when either 50 μ m valinomycin, or 50 m m K + is present. The activity of this bidirectional Ca 2+ Na + exchanger could be inhibited by 100 μ m bepridil or 200 μ m La 3+ . The entrance behaviour of Cl − as monitored by the SPQ fluorescent signal indicates that, the Cl − influx is Na + -dependent. The Cl − influx is stimulated 152·8% and 187·6% for dogfish and bovine lens, respectively, when an inwardly-directed Na + gradient is present, and is further enhanced when a K + gradient is also present. The stoichiometry of Na + to Cl − entering the vesicles was 1:2. This Na + , K + 2Cl − cotransporter is not affected by 20 μ m valinomycin or 50 m m K + . However, the transporter is completely inhibited by 50 μ m furosemide. H 2 O 2 , 20 μ m diamide or oxidizing DTT, significantly activates the Ca 2+ Na + exchanger, whereas the Na + ,K + 2Cl − cotransporter totally lost its activity in the presence of 200 μ m H 2 O 2 .