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Eugenol Inhibits Calcium Currents in Dental Afferent Neurons

M.H. Lee, K.-Y. Yeon, C.-K. Park, H.-Y. Li, Z. Fang, M.S. Kim, S.-Y. Choi, S.J. Lee, S. Lee², K. Park, J.-H. Lee, J.S. Kim, and S.B. Oh^1,*

¹ Department of Physiology, and
² Department of Craniomaxillofacial Cell and Developmental Biology, College of Dentistry and Dental Research Institute, Seoul National University, 28-2 Yeongeon-Dong Chongno-Ku Seoul, Seoul 110-749, Korea;

View larger version (32K): [in a new window]   Figure 1. Inhibition of HVACC currents by eugenol in dental primary afferent neurons. (Aa) Trigeminal neurons were acutely isolated from adult rats 3 wks after placement of a fluorescent dye, DiI, into maxillary molars. Arrows indicate the 2 labeled cells when visualized under fluorescence microscopy (Ab). Scale bar, 50 µm. (B) The effect of 1 mM eugenol on membrane currents and HVACC barium currents (IBa). B shows continuous chart records of membrane current with eugenol applied at the time indicated by the horizontal bar (1, before; 2, during; 3, after the application of eugenol). Eugenol produced inhibitory effects on IBa without the change in membrane-holding currents (n = 13). (C) Superimposed IBa evoked by test pulse at the points indicated in (B). Individual currents are numbered on the chart record for ease of identification and superimposed at higher time resolution in (B). Calcium currents were recorded during 200-ms voltage clamp steps from a holding potential of –80 mV to a test potential of 0 mV every 10 sec.

View larger version (22K): [in a new window]   Figure 2. Effect of eugenol on IBa in capsaicin-sensitive and capsaicin-insensitive neurons. (A) Time course of the effect of capsaicin (1 µM) and eugenol (1 mM) on IBa. The IBa inhibition by eugenol (1 mM) was observed in capsaicin-sensitive dental primary afferent neurons (n = 20). (B) In dental primary afferent neurons in which IBa was not inhibited by capsaicin (1 mM), eugenol (1 mM) also inhibited IBa, indicating that the inhibitory effects of eugenol on IBa can be induced without the involvement of TRPV1 activation. (C) Dose-response relationship of eugenol-induced IBa inhibition in capsaicin-sensitive dental primary afferent neurons (mean ± SEM). The numbers in parentheses represent the numbers of cells studied. (D) The summary of IBa inhibition in dental primary afferent neurons. 1 mM eugenol (Eug)-induced IBa inhibition in capsaicin-insensitive neurons (Cap-Ins) was similar to that obtained in capsaicin-sensitive neurons (Cap-S). Capsazepine (CZP, 10 µM) failed to block eugenol (1 mM)-induced IBa inhibition completely. The IBa inhibition by the combined application of eugenol and CZP was not significantly different from that of eugenol (mean ± SEM, p > 0.05).

View larger version (23K): [in a new window]   Figure 3. Inhibition of N-type Ca currents by eugenol in C2D7 cells. (A) RT-PCR analysis shows no endogenous expression of TRPV1 in C2D7 cells (lane -). TRPV1 is clearly expressed in C2D7 cells following transient transfection (lane +), and in both DRG and TG neurons. The expected size of PCR product was 536 bp. Lane C indicates no PCR products with H2O. (B) Time course of the effect of eugenol (1 mM) on IBa in C2D7 cells which have no endogenous TRPV1 expression (n = 5). (C) Superimposed IBa evoked by a test pulse at the points indicated in (B). (D) The summary of IBa inhibition in TG neurons and C2D7 cells. The IBa inhibition by eugenol in C2D7 cells was significantly greater than that in TG neurons (mean ± SEM, *p < 0.01). The number in parentheses represents the number of cells studied.