No measurable currents were activated within 30 min after achieving the whole-cell configuration, and subsequent stimulation with 12.5 or 25% hypotonic solutions activated and (data not shown). Potentiation of 1993) on = 12) and was completely reversible. and dephostatin were purchased from Calbiochem. Mibefradil was kindly provided by Dr J.-P. Clozel, Hoffmann-La Roche, Basel, Switzerland. Genistein, daidzein, the tyrphostins and dephostatin were added to the appropriate extracellular solution from 50 mm stocks in DMSO just before Xphos each experiment. It was found that the effects of these compounds were already strongly diminished after 1 h at room temperature (22C25C) (see also Shuba, Asai, Pelzer & McDonald, 1996), which may explain some of the previous negative results (Szcs 1996is the drug concentration, IC50 is the drug concentration for 50% inhibition and is the Faraday constant, the gas constant and absolute temperature. Pooled data are given as means s.e.m. from cells. Significance was tested using Student’s paired or unpaired tests. Differences were considered significant at the level of 0.05. RESULTS Inhibition of 1994= 65) and 12.4 3.2 pA pF?1 at ?80 mV and 40.8 4.2 pA pF?1 at +100 mV for the 12.5% hypotonic solution (= 48). Figure 1shows an experiment in which tyrphostin B46 (10 m), a potent inhibitor of PTK activity (Gazit 1991), was applied to the bath 5 min before superfusion Xphos of the cell with the 25% hypotonic solution. Tyrphostin B46 partially inhibited indicates that both inhibition and Xphos recovery from inhibition were rather slow when compared with the effect of presumably direct channel blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate (Nilius 1994show that and = 7) and 50.6 4.2% (= 6), respectively. To examine whether direct activation of a tyrosine kinase could induce activation of and (Liu 1993). JNKK1 These peptides stimulate Src tyrosine kinase activity by binding to the SH2-domain and have been used by others Xphos to show regulation of NMDA channels by Src (Yu, Askalan, Keil & Salter, 1997). No measurable currents were activated within 30 min after achieving the whole-cell configuration, and subsequent stimulation with 12.5 or 25% hypotonic solutions activated and (data not shown). Potentiation of 1993) on = 12) Xphos and was completely reversible. The current-voltage relations presented in Fig. 3show that Na3VO4 had in fact two distinct effects on with = 7), without an initial blocking effect (Fig. 31996). In CPAE cells, intracellular perfusion with GTPS (100 m), a GTP analogue known to activate G proteins (Barritt & Gregory, 1997), induced the transient activation of an inward current at ?80 mV (Fig. 4= 23). This activation was followed by a decay phase, as 5 min after reaching this maximum the current decreased to 18 5% of the peak value. Current-voltage relations show that the GTPS-activated current was outwardly rectifying and reversed close to the reversal potential for Cl? ions (Fig. 419941996; Nilius 1997= 6) = 5). The GTPS-activated current is volume sensitive Although we did not observe volume changes during the intracellular perfusion with GTPS, the concomitant activation of the Cl? current was clearly volume sensitive. Pre-incubation of the cells in hypertonic solution almost completely prevented the activation of the current (Fig. 7= 10) to 23.4 2.9 pA pF?1 (= 10). In addition, in the presence of intracellular GDPS the GTPS-activated current decreased at a much slower rate than in its absence. Open in a separate window Figure 8 Effect of GDPS on the GTPS-activated Cl? currentAverage time course for the activation of a whole-cell current after breaking into the cell with a pipette solution containing 100 m GTPS (; = 10) or 100 m GTPS + 1 mm GDPS (?; = 10, cells from the same coverslips). Intracellular GDPS also caused a time-dependent inhibition of shows the effect of repetitive stimulation with the 25% hypotonic solution on a cell which was intracellularly perfused with GDPS (1 mm). The amplitude of.