The small numbers of neurons with clear pharmacological evidence of TRPV4 channels is probably due to toxicity of 4α PDD on up to 34% of the tested neurons. The activation of TRPV4 is thought to be Alpelisib cell line mediated by phospholipase A2 (PLA2) and can thus be inhibited by the PLA2 inhibitor 3N-(p-amyl-cinnamoyl)anthranilic acid (ACA) ( Vriens et al., 2004). However, hypo-osmotically induced Ca2+ increases in thoracic DRG neurons were not significantly altered by 20 μm ACA ( Figure 3A). This lack of inhibition by ACA has not been observed for recombinantly expressed TRPV4 channels ( Vriens et al., 2004)

and thus it may well be that the mode of activation of TRPV4 in the physiological click here context of osmoreceptors is distinctive.

These results strongly suggested that the Ca2+-response was mediated by a Ca2+ influx through a TRP-like ion channel, most probably TRPV4. Hence osmosensitive neurons, as determined using Ca2+-imaging, should also exhibit an inward current in response to hypo-osmotic stimulation. To test this hypothesis we combined simultaneous Ca2+-imaging with whole-cell patch-clamp recordings. Strikingly, in 12 from 12 tested osmosensitive thoracic neurons, increases in [Ca2+]i were accompanied by a fast activating inward current (Figure 4A). To test whether this osmosensitive current is carried, at least in part, by calcium ions and thus directly mediates the calcium signal we next examined its current-voltage relationship. Therefore, neurons were step depolarized from −60 mV to +20 mV for 200 ms (to inactivate voltage-gated sodium channels) followed by a 200 ms ramp depolarization from −100 mV to +100 mV (Figure 4B, inset). The osmosensitive currents reversed at membrane potentials around 0 mV (−6.7 ± 3.3 mV, n = 5), which is characteristic

for nonselective cation channels. To rule ablukast out a possible contribution of swelling-activated chloride channels because such currents reverse at similar potentials under the recording conditions employed ( Nilius et al., 2001), we substituted extracellular chloride with gluconate. However, changing the driving force for chloride did not affect the osmosensitive current ( Figure 4D). We next applied a series of osmotic stimuli of decreasing osmolalities (260–290 mOsm) to determine the osmolality dependence of the inward current ( Figure 4C). This experiment showed that the current was half-maximally activated with a stimulus of just 278.9 ± 0.6 mOsm (n = 17), which was well within the range of physiological changes in blood osmolality following water intake ( Figure 1A). Hence the osmosensitive current found in thoracic DRG neurons is an excellent candidate as the primary detector of rapid and physiological changes in osmolality in hepatic blood vessels.