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

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 Ca2+ 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 Ca2+ was present. This stimulation is not affected when either 50 microM valinomycin, or 50 mM K+ is present. The activity of this bidirectional Ca2+/Na+ exchanger could be inhibited by 100 microM bepridil or 200 microM La3+. 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 microM valinomycin or 50 mM K+. However, the transporter is completely inhibited by 50 microM furosemide.