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. 2012 Jul 1;590(13):2995-3007.
doi: 10.1113/jphysiol.2012.229153. Epub 2012 May 8.

Extracellular matrix proteoglycan plays a pivotal role in sensitization by low pH of mechanosensitive currents in nociceptive sensory neurones

Asako Kubo  1 , Kimiaki Katanosaka, Kazue Mizumura
Affiliations

Extracellular matrix proteoglycan plays a pivotal role in sensitization by low pH of mechanosensitive currents in nociceptive sensory neurones

Asako Kubo et al. J Physiol. .

Abstract

Ischaemia, inflammation, and exercise lead to tissue acidosis, which induces pain and mechanical hyperalgesia. Corresponding to this, enhanced thin-fibre afferent responses to mechanical stimulation have been recorded in vitro at low pH. However, knowledge about how this sensitization by low pH occurs is lacking. In this study, we found that all three types (rapidly adapting (RA), intermediately adapting and slowly adapting) of mechanically activated currents recorded with the whole cell patch-clamp method were sensitized by low pH in rat cultured dorsal root ganglion neurones. This sensitization was mainly observed in neurones positively labelled with isolectin B4 (IB4), which binds to versican, a chondroitin sulfate proteoglycan. Inhibitors of acid-sensitive channels (amiloride and capsazepine) did not block sensitization by low pH except in RA neurones, and extracellular calcium was not involved even in the sensitization of this type of neurone. A broad spectrum kinase inhibitor and a phospholipase C inhibitor (staurosporine and U73122) failed to block pH-induced sensitization in IB4-positive neurones, suggesting that these intracellular signalling pathways are not involved. Notably, both excess chondroitin sulfate in the extracellular solution and pretreatment of the neurone culture with chondroitinase ABC attenuated this low pH-induced sensitization in IB4-positive neurones. These findings suggest that a change in interaction between mechanosensitive channels and/or their auxiliary molecules and the side chain of versican on the cell surface causes this sensitization, at least in IB4-positive neurones. This report proposes a novel mechanism for sensitization that involves extracellular proteoglycans (versican).

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Figures

Figure 1
Figure 1. Low pH application potentiates all types of MA currents in nociceptive DRG neurones (sample recordings)
A, mechanical stimulation evoked currents and action potentials (APs). From top to bottom: representation of mechanical stimulation (stimulation steps were 5 for RA and 6 for SA), current recording in voltage clamp mode and voltage recording in current clamp mode. Many APs were observed in an SA neurone in current clamp mode (right bottom), while only one AP was evoked in an RA neurone (bottom left) with almost the same peak current amplitude as SA. Inset: an AP with a hump (two minima in first dV/dt) evoked with the current injection method in the same RA neurone. B, continuous sample recording of MA currents and effects of low pH application. Each low pH solution (pH 6.2, 6.6 and 7.0) was applied for 30 s and washed out with extracellular solution (pH 7.4) for 30 s. The application order of the low pH solution was randomized. Mechanical stimulation was applied every 30 s at the end of low pH application (about 25 s from application start) and was held for 500 ms. The low pH application period is marked with black bars and the markers of the mechanical stimuli are shown just above the trace. Stimulation steps were 4. The current type of this neurone was RA (see recording in inset with an expanded time scale, τ: 1.8 ms). IA (C) and SA (D) types of MA currents were also potentiated by low pH (6.2). Stimulation steps were 4 for IA and 6 for SA.
Figure 2
Figure 2. Summary of MA current potentiation by low pH
A, percentage of neurones potentiated by different pH applications. The criterion for potentiation was > 20% increase of the peak MA current amplitude during low pH application compared with the mean peak MA current amplitude at pH 7.4 before and 30 s after the low pH application. Each neurone was challenged by all levels of low pH in random order. n= 45. B, peak MA current amplitude (% of control) of potentiated neurones by pH 6.2 increased in a pH-dependent manner. Error bars represent SEM. **P < 0.01 (repeated measures ANOVA followed by Dunnett's multiple comparison test). n= 17. C, difference in the percentage of neurones potentiated by pH 6.6 at different postnatal days. The number of examined neurones is in parentheses under each bar. Note: the percentage of potentiated neurones at postnatal day 13 (though by pH 6.2) was almost the same (namely 47%) as that by pH 6.6 at postnatal day 10–11 (not shown in the figure).
Figure 3
Figure 3. IB4-positive DRG neurones are more frequently potentiated by low pH application
A, percentage of neurones potentiated by pH 6.2 application. Potentiation percentage was significantly greater in IB4-positive neurones than in -negative ones in every MA current type. *P < 0.05 (χ2 test). The number of examined neurones is in parentheses on each bar. B, proportion of current types in IB4-positive and -negative neurones. There was no significant difference between groups (IB4-positive: n= 73, IB4-negative: n= 57). C, sample recordings showing total charge transfer change. Upper panel shows an RA-type response (grey trace with pH 6.2 application, black trace with pH 7.4) and lower panel shows an SA-type response. The total charge transfer increased as a result of inactivation time constant prolongation. D, increase in the total charge transfer of MA current in neurones potentiated by low pH application. IB4-positive: n= 14, IB4-negative: n= 9. Error bars represent SEM; **P < 0.01, ***P < 0.001 (Student's paired t test).
Figure 4
Figure 4. Effects of acid sensitive channel blockers on mechanical sensitization by low pH
A, sample recordings of the RA current. The mixture of 10 μm capsazepine (CPZ) and 200 μm amiloride (AMI) was applied during the time period marked with an open arrow. This RA current was potentiated with pH 6.2 (left) and the blocker mixture attenuated the potentiation (right). Stimulation steps were 4. Inset shows MA current form with an expanded time scale (τ: 1.7 ms). B, comparison of the peak current amplitude (% of control) with and without blocker mixture (10 μm CPZ + 200 μm AMI) in each type of MA current. White bars, without blockers; grey bars, with blockers. Only RA current potentiation was blocked by the mixture. RA, n= 4; IA, n= 7; SA, n= 7. C, the effect of each blocker on sensitization of RA current. White bars, without blocker; grey bars, with CPZ or AMI. Ten micromolar CPZ was effective on RA current potentiation with pH 6.2 (n= 6), while 200 μm AMI had no effect (n= 6). Error bars represent SEM **P < 0.01, *P < 0.05, n.s., no significant difference (Student's paired t test).
Figure 5
Figure 5. No contribution of extracellular Ca2+ to RA current potentiation by low pH
Sample recording showing the effect of Ca2+-free extracellular solution. RA current potentiation was also observed under Ca2+-free conditions. Stimulation steps were 6. Inset: MA current form with an expanded time scale (τ: 2.9 ms). This neurone responded to 1 μm capsaicin (CAP), a TRPV1 agonist (white arrow).
Figure 6
Figure 6. Effects of intracellular signalling enzyme inhibitors on mechanical sensitization by low pH in IB4-positive neurones
Percentages of potentiated IB4-positive neurones were compared in the presence and absence of inhibitors. White bars, percentage of potentiated neurones without inhibitors (same as IB4-positive neurones in Fig. 3A); pale grey bars, with a mixture of 100 nm staurosporine + 1 μm U73122; and dark grey bars, 500 nm staurosporine in patch pipette solution. The number of examined neurones is in parentheses on each bar. Percentage of sensitization in every MA current type was not significantly different with or without inhibitors (χ2 test). Note: among SA neurones, the percentage of neurones sensitized in the 500 nm staurosporine group tended to be lower than that in the non-treated group. Theoretically, however, the difference would not be significant even if the number of staurosporine-treated neurones was increased to 21 (the same number as the non-treated group) and none of them showed sensitization.
Figure 7
Figure 7. Chondroitin sulfate contribution to low pH-induced sensitization of IB4-positive neurones
A, sample recordings from an IB4-positive neurone. This IA current was potentiated with pH 6.2 (left) and 0.1% CS attenuated this potentiation (right). The white arrow shows CS application and the black bars show pH 6.2 application. Inset shows the MA current form of this neurone with an expanded time scale (τ: 5.6 ms). Stimulation steps were 4. B, summary of the effect of CS on the IB4-positive neurones. White bars, the peak current amplitude before CS application; grey bars, after CS. The number of examined neurones is in parentheses under the graph. Error bars represent SEM. **P < 0.01, *P < 0.05, n.s., no significant difference (Student's paired t test). Note: CS application reversed the sensitization of MA current of IB4-positive neurones with pH 6.2, but it was not effective in IB4-negative neurones sensitized by low pH (RA, n= 6; IA, n= 5; SA, n= 3; see the text). C, effect of chondroitinase ABC treatment on the sensitization with pH 6.2. After pretreatment with 2 U ml−1 chondroitinase ABC (37°C, 30 min), the percentage of sensitized neurones was significantly decreased. *P < 0.05 (χ2 test). Non-treated group, n= 73; chondroitinase ABC-treated group, n= 54.
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