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Hypotonicity modulates tetrodotoxin-sensitive sodium current in trigeminal ganglion neurons.

Li L, Liu C, Chen L, Chen L - Mol Pain (2011)

Bottom Line: Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability.Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers.We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Nanjing Medical University, Nanjing, PR China.

ABSTRACT
Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability. We previously reported that the excitability of nociceptor was increased by hypotonic stimulation. The present study tested the effect of hypotonicity on tetrodotoxin-sensitive sodium current (TTX-S current) in cultured trigeminal ganglion (TG) neurons. Our data show that after hypotonic treatment, TTX-S current was increased. In the presence of hypotonicity, voltage-dependent activation curve shifted to the hyperpolarizing direction, while the voltage-dependent inactivation curve was not affected. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers. We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response. We conclude that hypotonic stimulation enhances TTX-S current, which contributes to hypotonicity-induced nociception. TRPV4 receptor and PKC intracellular pathway are involved in the increase of TTX-S current by hypotonicity.

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Effect of hypotonicity on TTX-S current in TG neurons. A. Plot of current densities as function of hypotonic stimuli shows that the increase of TTX-S current was conspicuous at the largest osmotic gradient. B. The typical recordings show that TTX-S current was increased by hypotonicity. Upper: total sodium current, middle: TTX-R current obtained during application of 300nM TTX, below: TTX-S current obtained by subtracting TTX-R current from total. C. Comparison of voltage-current relationship (I-V curve) for TTX-S current in isotonic and hypotonic solution. D. TTX-S current was converted to a conductance and fitted to a Boltzman function. The mid-point of activation (V0.5) was significantly more negative in hypotonic than in isotonic solution (-41.17 ± 1.09 mV vs. -34.41 ± 2.16 mV, n = 9, pared t-test, P < 0.05). However, the slope factor (k) was not significantly different between isotonic and hypotonic solution (3.54 ± 0.23 vs. 3.29 ± 0.89, n = 9, pared t-test, P > 0.05). E. Unlike G-V curve, the inactivation-voltage curve did not shift before and during hypotonic treatment. V0.5 were -69.66 ± 2.17 mV and -71.55 ± 3.21 mV (n = 10, paired t-test, P > 0.05), k were -9.86 ± 1.81 and -11.25 ± 1.03 (n = 10, paired t-test, P > 0.05) for 300mOsm and 260mOsm respectively.
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Figure 1: Effect of hypotonicity on TTX-S current in TG neurons. A. Plot of current densities as function of hypotonic stimuli shows that the increase of TTX-S current was conspicuous at the largest osmotic gradient. B. The typical recordings show that TTX-S current was increased by hypotonicity. Upper: total sodium current, middle: TTX-R current obtained during application of 300nM TTX, below: TTX-S current obtained by subtracting TTX-R current from total. C. Comparison of voltage-current relationship (I-V curve) for TTX-S current in isotonic and hypotonic solution. D. TTX-S current was converted to a conductance and fitted to a Boltzman function. The mid-point of activation (V0.5) was significantly more negative in hypotonic than in isotonic solution (-41.17 ± 1.09 mV vs. -34.41 ± 2.16 mV, n = 9, pared t-test, P < 0.05). However, the slope factor (k) was not significantly different between isotonic and hypotonic solution (3.54 ± 0.23 vs. 3.29 ± 0.89, n = 9, pared t-test, P > 0.05). E. Unlike G-V curve, the inactivation-voltage curve did not shift before and during hypotonic treatment. V0.5 were -69.66 ± 2.17 mV and -71.55 ± 3.21 mV (n = 10, paired t-test, P > 0.05), k were -9.86 ± 1.81 and -11.25 ± 1.03 (n = 10, paired t-test, P > 0.05) for 300mOsm and 260mOsm respectively.

Mentions: Osmotic balance is of great significance for maintaining the internal environment homeostasis. Many pathological processes, in accompany with the changes in osmolality (such as the facial or intraoral edema which is not contained within a rigid physical restraint), are painful. Both in vitro and in vivo experiments have proved that hypotonic stimuli can induce nociception or pain-related behavior [1,2]. We previously reported that hypotonic stimulation caused an increase of action potential (AP) generation in small to medium-sized trigeminal ganglion (TG) neurons that are likely to be nociceptive in nature, resulting in the hyperexcitability of nociceptors [3]. Voltage-gated sodium channels (VGSCs), providing an inward current that underlies the upswing of an AP, contribute to the control of membrane excitability and underlie AP generation [4]. In nociceptors, VGSCs are pharmacologically separated into tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) channels [5]. Our recent study found that TTX-R current was decreased by hypotonic stimulation [6] and this result seemingly can not explain hypotonicity-induced hyperexcitability of TG neurons. However, the modulation of VGSCs varies between laboratories and models. The selective up-regulation of TTX-S channel is detected in the pain caused by nerve injury or in inflammatory pain [7,8]. This phenomenon indicates that TTX-S channel may also play an important role in the pain sensation. Therefore, we tested the effect of hypotonic stimulation on TTX-S current in cultured small- to medium-sized TG neurons which have characteristics of nociceptors. Voltage-gated sodium current was measured first in the absence of TTX to get the total sodium current and then in the presence of TTX to obtain the TTX-R current. TTX-S current was obtained by subtracting the latter from the former. We found that TTX-S current was increased by 37.74 ± 2.12% from -160.89 ± 14.11 pA/pF to -220.18 ± 18.57 pA/pF (n = 16, paired t-test, P < 0.05) when the external solution was changed from isotonicity (300mOsm) to hypotonicity (260mOsm) (Figure 1A and 1B). Hypotonicity-induced increase was largely reversible and TTX-S current recovered to -163.75 ± 12.13 pA/pF after hypotonicity was washed out for 3 min. We also found that the voltage-dependent activation curve (G-V curve) markedly shifted to the hyperpolarizing direction in the presence of hypotonic stimulation (paired t-test, P < 0.05) (Figure 1D). Unlike the activation function, the voltage-dependent inactivation curve (inactivation-voltage curve) did not markedly shift before and during hypotonic treatment (paired t-test, P > 0.05) (Figure 1E).


Hypotonicity modulates tetrodotoxin-sensitive sodium current in trigeminal ganglion neurons.

Li L, Liu C, Chen L, Chen L - Mol Pain (2011)

Effect of hypotonicity on TTX-S current in TG neurons. A. Plot of current densities as function of hypotonic stimuli shows that the increase of TTX-S current was conspicuous at the largest osmotic gradient. B. The typical recordings show that TTX-S current was increased by hypotonicity. Upper: total sodium current, middle: TTX-R current obtained during application of 300nM TTX, below: TTX-S current obtained by subtracting TTX-R current from total. C. Comparison of voltage-current relationship (I-V curve) for TTX-S current in isotonic and hypotonic solution. D. TTX-S current was converted to a conductance and fitted to a Boltzman function. The mid-point of activation (V0.5) was significantly more negative in hypotonic than in isotonic solution (-41.17 ± 1.09 mV vs. -34.41 ± 2.16 mV, n = 9, pared t-test, P < 0.05). However, the slope factor (k) was not significantly different between isotonic and hypotonic solution (3.54 ± 0.23 vs. 3.29 ± 0.89, n = 9, pared t-test, P > 0.05). E. Unlike G-V curve, the inactivation-voltage curve did not shift before and during hypotonic treatment. V0.5 were -69.66 ± 2.17 mV and -71.55 ± 3.21 mV (n = 10, paired t-test, P > 0.05), k were -9.86 ± 1.81 and -11.25 ± 1.03 (n = 10, paired t-test, P > 0.05) for 300mOsm and 260mOsm respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: Effect of hypotonicity on TTX-S current in TG neurons. A. Plot of current densities as function of hypotonic stimuli shows that the increase of TTX-S current was conspicuous at the largest osmotic gradient. B. The typical recordings show that TTX-S current was increased by hypotonicity. Upper: total sodium current, middle: TTX-R current obtained during application of 300nM TTX, below: TTX-S current obtained by subtracting TTX-R current from total. C. Comparison of voltage-current relationship (I-V curve) for TTX-S current in isotonic and hypotonic solution. D. TTX-S current was converted to a conductance and fitted to a Boltzman function. The mid-point of activation (V0.5) was significantly more negative in hypotonic than in isotonic solution (-41.17 ± 1.09 mV vs. -34.41 ± 2.16 mV, n = 9, pared t-test, P < 0.05). However, the slope factor (k) was not significantly different between isotonic and hypotonic solution (3.54 ± 0.23 vs. 3.29 ± 0.89, n = 9, pared t-test, P > 0.05). E. Unlike G-V curve, the inactivation-voltage curve did not shift before and during hypotonic treatment. V0.5 were -69.66 ± 2.17 mV and -71.55 ± 3.21 mV (n = 10, paired t-test, P > 0.05), k were -9.86 ± 1.81 and -11.25 ± 1.03 (n = 10, paired t-test, P > 0.05) for 300mOsm and 260mOsm respectively.
Mentions: Osmotic balance is of great significance for maintaining the internal environment homeostasis. Many pathological processes, in accompany with the changes in osmolality (such as the facial or intraoral edema which is not contained within a rigid physical restraint), are painful. Both in vitro and in vivo experiments have proved that hypotonic stimuli can induce nociception or pain-related behavior [1,2]. We previously reported that hypotonic stimulation caused an increase of action potential (AP) generation in small to medium-sized trigeminal ganglion (TG) neurons that are likely to be nociceptive in nature, resulting in the hyperexcitability of nociceptors [3]. Voltage-gated sodium channels (VGSCs), providing an inward current that underlies the upswing of an AP, contribute to the control of membrane excitability and underlie AP generation [4]. In nociceptors, VGSCs are pharmacologically separated into tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) channels [5]. Our recent study found that TTX-R current was decreased by hypotonic stimulation [6] and this result seemingly can not explain hypotonicity-induced hyperexcitability of TG neurons. However, the modulation of VGSCs varies between laboratories and models. The selective up-regulation of TTX-S channel is detected in the pain caused by nerve injury or in inflammatory pain [7,8]. This phenomenon indicates that TTX-S channel may also play an important role in the pain sensation. Therefore, we tested the effect of hypotonic stimulation on TTX-S current in cultured small- to medium-sized TG neurons which have characteristics of nociceptors. Voltage-gated sodium current was measured first in the absence of TTX to get the total sodium current and then in the presence of TTX to obtain the TTX-R current. TTX-S current was obtained by subtracting the latter from the former. We found that TTX-S current was increased by 37.74 ± 2.12% from -160.89 ± 14.11 pA/pF to -220.18 ± 18.57 pA/pF (n = 16, paired t-test, P < 0.05) when the external solution was changed from isotonicity (300mOsm) to hypotonicity (260mOsm) (Figure 1A and 1B). Hypotonicity-induced increase was largely reversible and TTX-S current recovered to -163.75 ± 12.13 pA/pF after hypotonicity was washed out for 3 min. We also found that the voltage-dependent activation curve (G-V curve) markedly shifted to the hyperpolarizing direction in the presence of hypotonic stimulation (paired t-test, P < 0.05) (Figure 1D). Unlike the activation function, the voltage-dependent inactivation curve (inactivation-voltage curve) did not markedly shift before and during hypotonic treatment (paired t-test, P > 0.05) (Figure 1E).

Bottom Line: Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability.Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers.We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Nanjing Medical University, Nanjing, PR China.

ABSTRACT
Voltage-gated sodium channels (VGSCs) play an important role in the control of membrane excitability. We previously reported that the excitability of nociceptor was increased by hypotonic stimulation. The present study tested the effect of hypotonicity on tetrodotoxin-sensitive sodium current (TTX-S current) in cultured trigeminal ganglion (TG) neurons. Our data show that after hypotonic treatment, TTX-S current was increased. In the presence of hypotonicity, voltage-dependent activation curve shifted to the hyperpolarizing direction, while the voltage-dependent inactivation curve was not affected. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased TTX-S current and hypotonicity-induced increase was markedly attenuated by TRPV4 receptor blockers. We also demonstrate that inhibition of PKC attenuated hypotonicity-induced inhibition, whereas PKA system was not involved in hypotonic-response. We conclude that hypotonic stimulation enhances TTX-S current, which contributes to hypotonicity-induced nociception. TRPV4 receptor and PKC intracellular pathway are involved in the increase of TTX-S current by hypotonicity.

Show MeSH
Related in: MedlinePlus