Voltage-Dependent Calcium Channels in Merkel Cells of Hamster Oral Mucosa

Masakazu Tazaki 1, Yuki Tazaki 1, Takashi Inoue2 and Masaki Shimono3

1 Department of Physiology, Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihamaku, Chiba 261-8502, Japan

2Department of Clinical Pathophysiology, Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihamaku, Chiba 261-8502, Japan

3Department of Pathology, Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihamaku, Chiba 261-8502, Japan

Summary

Previous studies have demonstrated the existence of voltage-dependent Ca2+ channels in the membrane of Merkel cells. In order to investigate which categories

of ca2+ channels are involved, we examined the changes in the fluorescence ratio of single isolated Merkel cells loaded with fura-2 during 334/380 nm excitation. Cells were depolarized with a solution of high KCl concentration (100 mM),

known to open voltage-dependent ca2+ channels. A transient increase in the fura-2 334/380 nm excitation ratio which occurred in Merkel cells was induced by 100 mM KCI. The results suggest that voltage-dependent L-type, N-type and P/Q-

type ca2+ channels exist in most Merkel cells. However, the number of voltage­

dependent N-type ca2+ channels in Merkel cells appears to be smaller or variable

in comparison with the other types of ca2+ channels.

Introduction

The Merkel cells-neurite complex that exists in the hamster cheek touch domes, are similar to those observed in the touch dome from primate hairy skin (lggo and Muir 1969), thus they are considered to behave as slowly adapting type I mech­anoreceptors (Tazaki and lggo 1995). It has been hypothesized that Merkel cells from the Merkel cells-neurite complex are mechano-electric transducers (lggo and

Findlater 1984), based on the fact that voltage-dependent ca2+ channels (VDCCs)

K. I. Baumann et al. (eds.), The Merkel Cell© Springer-Verlag Berlin Heidelberg 2003

144 Tazaki et al.

exist on the Merkel cell membrane (Yamashita et al. 1992; Chan et al. 1996). However, in previous studies concerning the classification of VDCCs, there are still some aspects that need to be discussed (Yamashita et al. 1992; Chan et al. 1996). In the present study, the categories ofVDCCs were investigated with spe­cific blockers using microfluorimetric techniques in isolated single Merkel cells from hamster cheek touch domes.

Materials and Methods

In order to investigate the categories of VDCCs, isotonic highly concentrated potassium chloride solution (100 mM KCl; depolarizing stimulation) was applied to single Merkel cells using the Y-tube method and changes in fura-2AM 334/380 nm excitation ratio of single Merkel cells were observed. The excitation ratio for 100 mM KCl after applying each channel blocker was compared with the excitation ratio (control ratio) for 100 mM KCl before application of the channel blocker.

Results and Discussion

L-Type ca2+ Channels The effects of (+)-Bay K 8644 (50 J.!M) which is an L-type VDCCs blocker

(Chan et al. 1996) were tested and are shown in Fig. 1. A transient increase in the fura-2 334/380 nm excitation ratio which occurred in Merkel cells was induced by 100 mM KCl (first black bar). Superfusion of (+)-Bay K 8644 (clear bar) showed a decrease in the fura2-AM 334/380 nm excitation ratio for the control ratio. The decrease in fura2-AM 334/380 nm excitation ratio was 39.2±6.4% (mean ± SE, range 12.3-68.1%, n=8). After washing out the (+)-Bay K 8644, a recovery of the fura2-AM 334/380 nm excitation ratio to 100 mM KCl (third black bar) was ob­served. These results suggested that the L-type VDCCs exist in the Merkel cells membrane.

N-Type ca2+ Channels The effects of ro-ConotoxinGVIA (1 J.!M) which is an N-type VDCCs blocker

(Chan et al. 1996) were tested and shown in Fig. 2. Superfusion of ro­ConotoxinGVIA (clear bar) showed a decrease in the fura2-AM 334/380 nm exci­tation ratio for control ratio. The decrease in fura2-AM 334/380 nm excitation ra­tio was 13.9±0.02% (mean±SE, range 3.0-28.4%, n=18). These results suggested that the N-type VDCCs exist in the Merkel cells membrane. However, only 18 of 25 Merkel cells (72%) examined showed a decrease in the calcium signal as men­tioned above, while in 28% of the Merkel cells (7 out of25 cells) application of ro­ConotoxinGVIA had no effect. Thus, it appears that the number ofN-type VDCCs

Voltage dependent Ca-channels in Merkel cells 145

2. 74,,--------------------,-----------,----------.

2. 25 ~ -- -- - -----------1

l. 76 ----,--lt · --- --- ---- -- ----

Fig. 1. Effect of (+)-Bay K 8644 (50 f..!M) in the intracellular influx of extracellular

ca2+. Clear bars indicate the superfusion of (+)-Bay K 8644, black bars KCl

I. 17 l r------~---r--~-----~----~

I 0. 997 1------~------~-----'----·------·-+

I :00 ~ o. 823 -n-----~1 -'-----t \-----.---Jc------H ...... 0

0.619 -

553 1656 2207

Fig. 2. Effect of (J}-ConotoxinGVIA (l f..!M) in the intracellular influx of extracellular

Ca2+. Clear bars indicate the superfusion of oo-ConotoxinGVIA, black bars KCl

1. 007

:00 ~0. 820 ...... 0

175 959 Timetsec. )

1121 1899

Fig. 3. Effect of oo-AgatoxiniV A ( l f..!M) in the intracellular influx of extracellular Ca2+. Clear bars indicate the superfusion of oo-AgatoxiniV A, black bars KCl

146 Tazaki et al.

in Merkel cells is smaller or variable in comparison with the other types of ca2+ channels. Further studies regarding theN-type VDCCs will be required.

P/Q-Type ca2+ Channels The effects of ro-AgatoxiniVA {1J.1M) which is a P/Q-type VDCCs blocker

(Cruz et al. 1987) were tested and shown in Fig. 3. Superfusion of ro­AgatoxiniVA (clear bar) showed a decrease in the fura2-AM 334/380 nm excita­tion ratio for control ratio. The decrease in fura2-AM 334/380 nm excitation ratio was 27.5±0.04% (mean± SE, range 8.7-46.7%, n=12). These results suggested that P/Q-type VDCCs exist in the Merkel cell membranes.

Although some studies have mentioned that L- or N-type VDCCs do not exist in Merkel cell membrane (Chan et al. 1996), in the present study based on there­sults of several investigations, it is suggested that L-type VDCCs actually exist in Merkel cell membrane (Yamashita et al. 1992). This study is also the first report regarding the existence ofP/Q-type VDCCs in Merkel cell membranes.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research Nos. 00085771 and 40155065 toM. Shimono and M.Tazaki from the Ministry of Edu­cation, Science and Culture, Japan, and by a grant for Scientific Research No. HRC5A08 to T. Inoue from the Oral Health Science Center of Tokyo Dental Col­lege.

References

Chan E, Yung W-H, Baumann KI (1996) Cytoplasmic ca2+ concentrations in intact Merkel cells of an isolated, functioning rat sinus hair preparation. Exp Brain Res 108:357-366

Cruz LJ, Johnson DS, Olivera BM (1987) Characterization of the ro-conotoxin target. Evi­dence for tissue-specific heterogeneity in calcium channel types. Biochemistry 26:820-824

Iggo A, Findlater GS (1984) A review of Merkel cell mechanisms. In: Hamann W, lggo A (eds) Sensory receptor mechanisms. World Scientific, Singapore, pp 117-131

lggo A, Muir AR (1969) The structure and function of a slowly adapting touch corpuscle in hairy skin. J Physiol200:763-796

Tazaki M, lggo A (1995) SAl-type mechanoreceptors in the buccal pouch of the golden hamster (Mesocricetus auratus ). Prim Sensory Neuron 1: 157-169

Yamashita Y, Akaike N, Wakamori M, Ikeda I, Ogawa H (1992) Voltage-dependent cur­rents in isolated single Merkel cells of rats. J Physiol 450:143-162