Changes in osmolality modulate voltage-gated calcium channels in trigeminal ganglion neurons

Abstract

Voltage-gated calcium channels (VGCCs) participate in many important physiological functions. However whether VGCCs are modulated by changes of osmolarity and involved in anisotonicity-induced nociception is still unknown. For this reason by using whole-cell patch clamp techniques in rat and mouse trigeminal ganglion (TG) neurons we tested the effects of hypo- and hypertonicity on VGCCs. We found that high-voltage-gated calcium current (I(HVA)) was inhibited by both hypo- and hypertonicity. In rat TG neurons, the inhibition by hypotonicity was mimicked by Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator but hypotonicity did not exhibit inhibition in TRPV4(-/-) mice TG neurons. Concerning the downstream signaling pathways, antagonism of PKG pathway selectively reduced the hypotonicity-induced inhibition, whereas inhibition of PLC- and PI3K-mediated pathways selectively reduced the inhibition produced by hypertonicity. In summary, although the effects of hypo- and hypertonicity show similar phenotype, receptor and intracellular signaling pathways were selective for hypo- versus hypertonicity-induced inhibition of I(HVA).

Figure 1. Hypotonicity reduces I_HVA in rat TG neurons

A The typical recordings show that I_HVA was reduced from −1.4 nA to −0.8 nA when the external solution was changed from 300mOsm to 260mOsm for 3 min and the current recovered to −1.2 nA after washout. The time–course curve was shown before, during and after hypotonicity treatment. B. The voltage–current relationship was shown in the I–V curve before and during application of hypotonicity. C. In the presence of hypotonicity, G–V curve did not significantly shift. V_0.5 were −17.3±6.8 mV and −19.6±2.8 mV (n=10, paired t test, P>0.05), k were 6.5±5.9 and 6.8±3.3 (n=10, paired t test, P>0.05) for 300mOsm and 260mOsm respectively. Data were transformed from the I–V data shown in B. D. In the presence of hypotonicity, inactivation–voltage curve significantly shifted to hyperpolarizing direction. V_0.5 were −31.3±1.9 mV and −46.3±2.2 mV (n=10, paired t test, P<0.05), k were −8.5±2.2 and −10.8±3.8 (n=10, paired t test, P<0.05) for 300mOsm and 260mOsm respectively.

Figure 2. Hypertonicity reduces I_HVA in rat TG neurons

A. The typical recordings show that I_HVA was reduced from −1.1 nA to −0.7 nA when the external solution was changed from 300mOsm to 322mOsm for 3min but the current did not recover (−0.8 nA) after washout. The time–course curve was shown before, during and after hypertonicity treatment. B. I–V curve was shown before and during application of hypertonicity. C. In the presence of hypertonicity G–V curve did not shift, in which V_0.5 were −17.8±3.8 mV and −16.0±4.8 mV (n=6, paired t test, P>0.05), k were 6.8±4.1 and 6.0±1.9, (n=6, paired t test, P>0.05) for 300mOsm and 322mOsm respectively. Data were transformed from the I–V data shown in B. D. The typical recordings show that inactivation–voltage curve did not significantly shift with Boltzmann parameters V₀^.5 being −28.6±2.3 mV and −31.5±3.4 mV (n=6, paired t test, P>0.05), k being −7.4±0.9 and −8.8±1.0 for 300mOsm and 322mOsm respectively (n=6, paired t test, P>0.05). E. I_HVA was inhibited by anisotonic stimuli with “V” shape dose–response curve.

Figure 3. 4α-PDD reduces I_HVA in rat TG neurons

A. The typical recordings show that I_HVA was reduced from −1.2 nA to −0.8 nA in the presence of 1 μM 4α-PDD for 3 min and the current recovered to −1.0 nA after washout. The time–course curve was shown before, during and after 4α-PDD application. B. I–V curve was shown before and during 4α-PDD treatment. C. There was no change in the G–V curve with V_0.5 being −18.3±5.3 mV and −16.1±5.0 mV (n=8, paired t test, P>0.05), k being 6.1±3.6 and 6.4±8.5 (n=8, paired t test, P>0.05) respectively before and during 4α-PDD treatment. Data were transformed from the I–V data shown in B. D. In the presence of 4α-PDD inactivation–voltage curve significantly shifted to hyperpolarizing direction. V_0.5 were −29.7±1.2 mV and −36.2±4.9 mV (n=8, paired t test, P<0.05), k were 2−8.5±3.1 and −8.9±3.1 (n=8, paired t test, P<0.05) before and during 4α-PDD treatment respectively. E. The plot showed the percentage inhibition of 4α-PDD at different concentrations of 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30 and 100 μM. The dose–response curve fits to Hill equation with IC50 being 1.9 μM and n being 0.7.

Figure 4. Effects of hypo- and hypertonicity on I_HVA in TRPV4^+/+ and TRPV4−/− mice TG neurons

A. The typical recordings show the effect of hypotonicity on I_HVA. In TG neurons from TRPV4^+/+ mice, after exposure to hypotonicity (260mOsm), I_HVA was reduced from −1.6 nA to −1.0 nA and recovered to −1.4 nA after a 3 min wash. In TG neurons from TRPV4−/− mice, I_HVA were −1.37 nA, −1.29 nA and −1.35 nA before, during and after hypotonicity was applied. The inhibition by hypotonicity in TRPV4−/− mice (4.9±3.6%, n=18) is significantly different from that in TRPV4^+/+ mice TG neurons (43.6±9.1%, n=18) (unpaired t test, P<0.01). B. The typical recordings show the effect of hypertonicity on I_HVA. In TRPV4^+/+ mice TG neurons, after exposure to hypertonicity (322mOsm), I_HVA was reduced from −1.1 nA to −0.6 nA and recovered to −0.8 nA after washout. In TRPV4^−/− mice TG neurons, after exposure to hypertonicity, I_HVA was reduced from −1.1 nA to −0.6 nA and recovered to −0.7 nA after washout. On the average, I_HVA was reduced by 44.7±8.9% (n=14) and 48.0±9.8% (n=14) in TRPV4^+/+ and TRPV4^−/− mice TG neurons respectively (unpaired t test, P>0.05).

Figure 5. The role of G-protein, PKA and PKC system in the inhibition of anisotonicity on I_HVA

A. For G-protein system, neither GTP-γs nor GDP-βs had significant effect on the inhibitions induced by anisotonic stimuli. In hypotonic solution (260mOsm) I_HVA was reduced by 37.2±8.9% (n=15) (unpaired t test, P>0.05) and 39.6±8.6% (n=16) (unpaired t test, P>0.05) with GTP-γs and GDP-βs in the pipette solution, respectively, while in hypertonic solution (322mOsm) I_HVA was reduced by 36.8±9.4% (n=19) (unpaired t test, P>0.05) and 32.8±7.6% (n=18) (unpaired t test, P>0.05), respectively. B. For PKA system, pre-incubation with H-89 10 min did not statistically alter the inhibitions of I_HVA by hypo or hypertonicity, with I_HVA being reduced by 33.5±8.1% (n=14) (unpaired t test, P>0.05) and 35.5±7.2% (n=16) (unpaired t test, P>0.05), respectively. C. For PKC system, application of BIM did not significantly affect the inhibition induced by hypo or hypertonicity. I_HVA was reduced by 45.0±7.0% (n=13) (unpaired t test, P>0.05) and 31.3±6.6% (n=23) (unpaired t test, P>0.05) with 1μM BIM in hypotonic and hypertonic solution, respectively.

Figure 6. Inhibition of PKG system selectively reverses the inhibition of hypotonicity on I_HVA

A. In the presence of PKG inhibitors KT5823 (10 μM) or Rp-8-Br-PET-cGMPs (1 μM), the inhibition of I_HVA by hypotonicity was significantly reduced to 13.8±5.4% (n=12) (unpaired t test, P<0.01) and 11.2±3.1% (n=7) (unpaired t test, P<0.01) respectively. B. Application of KT5823 and Rp-8-Br-PET-cGMPs had no significant effect on the inhibition by hypertonicity and I_HVA was reduced by 38.8±9.5% (n=22) (unpaired t test, P>0.05) and 37.4±7.0% (n=8) (unpaired t test, P>0.05), respectively.

Figure 7. Lipids system is selectively involved in the inhibition of hypertonicity on I_HVA

A I_HVA was reduced by 43.2±9.0% (n=15), 41.2±6.1% (n=16) and 42.0±7.9% (n=18) in hypotonic solution with Wortmannin, LY294002 and U73122 in the pipette solution respectively. Compared with the inhibition by hypotonicity in normal pipette solution (38.3±10.6%, n=33), none of them was significantly different (unpaired t test, P>0.05). B. Pre-incubation with Wortmannin, LY294002 and U73122 significantly reversed the inhibition of I_HVA by hypertonicity from 37.1±9.0%, (n=22) to 22.5±10.3% (n=11) (unpaired t test, P<0.05), 18.9±6.5% (n=16) (unpaired t test, P<0.01) and 23.0±8.2% (n=16) (unpaired t test, P<0.05), respectively.