Ca(2+) regulation of endocochlear potential in marginal cells

Abstract

We examined the effect of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca(2+) chelator or inhibitors of Ca(2+)-permeable channels or Ca(2+)-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca(2+) channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca(2+)-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the gamma subunit of epithelial Na(+) channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 muM amiloride or 10 muM phenamil. Taken together, these findings suggest that [Ca(2+)](c) regulated by endoplasmic Ca(2+)-pump and Ca(2+)-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na(+) across the basolateral membrane in marginal cells.

Fig. 1

Typical recording of the EP after transient asphyxia or intravenous injection of 60 mg/kg furosemide with or without endolymphatic administration of 300 μM EGTA-AM. a Transient asphyxia for about 100 s or the intravenous administration of furosemide produces a rapid decrease in the EP. b After administration of EGTA-AM to the endolymph, the asphyxia- or the furosemide-induced decrease in the EP is significantly suppressed

Fig. 2

Recording of the EP after transient asphyxia or intravenous injection of 60 mg/kg furosemide with endolymphatic administration of 1 μg/ml nifedipine or 100 μM SKF96365. a Administration of nifedipine into the endolymph significantly suppresses the TAID, but only slightly reduces the FUID. b Administration of SKF96365 into the endolymph significantly suppresses both the TAID and FUID

Fig. 3

Summarized data on the changes in the decrease in EP induced by transient asphyxia with or without the endolymphatic administration of drugs. Administration of EGTA-AM, nifedipine, or SKF96365 into the endolymph significantly suppresses the asphyxia-induced decrease in EP. *P > 0.05, **P > 0.01, ***P > 0.005 indicate significant differences from the corresponding data for the control experiment

Fig. 4

Summarized data on the changes in the decrease in EP induced by furosemide infusion with or without the endolymphatic administration of EGTA-AM, nifedipine, or SKF96365. Administration of EGTA-AM or SKF96365 into the endolymph significantly suppressed the FUID, but nifedipine did not. Moreover, in the presence of nifedipine, the recovery rate from the FUID was significantly retarded. *P > 0.05, **P > 0.01, ***P > 0.005 indicate significant differences from the corresponding data for the control experiment

Fig. 5

Summarized data on the maximum change in FUID and the recovery from it. The maximum change in FUID at 2.7 min after the furosemide injection was not significantly different from the recovery change in the FUID at 30 min after the maximum change in FUID. The recovery from FUID was significantly suppressed by the endolymphatic perfusion with EGTA-AM, nifedipine, or SKF96365, indicating that all of these drugs directly or indirectly suppress the Ca²⁺-pump in the ESC

Fig. 6

Effect of endolymphatic application of 100 μM BTP2 on TAID and FUID. Endolymphatic administration of BTP2 suppresses both the TAID and FUID and also suppresses the recovery from FUID

Fig. 7

Effect of endolymphatic application of 1 μM thapsigargin on the TAID and FUID. Endolymphatic administration of thapsigargin suppresses both the TAID and FUID and also suppresses the recovery from FUID

Fig. 8

Expression of TRPC3/6/7 channels in the stria vascularis of cochlea. a Control. b Strong and significant immunoreactivity indicating TRPC3/6/7 channels (green) is seen in the luminal side in the stria vascularis

Fig. 9

Expression of TRPC3/6/7 channels in the stria vascularis of cochlea. a Strong and significant immunoreactivity for TRPC3/6/7 channels (green) is seen in the marginal cells (MC) of the stria vascularis. b High magnification of the stria vascularis. Significant immunoreactivity from TRPC3/6/7 channels is evident in the luminal and basolateral membrane of marginal cells, but not in the intermediate or basal cells

Fig. 10

Expression of γENaC in the stria vascularis of the cochlea. High magnification of the stria vascularis. Significant and strong immunoreactivity indicating γENaC (green) is evident in the marginal cells, especially in their luminal and basolateral membranes

Fig. 11

Effect of endolymphatic administration of 10 μM phenamil or 10 μM amiloride on the EP. a Effect of endolymphatic administration of phenamil on the EP. A few minutes after the administration of phenamil, the EP decreases to ~0 mV. b Effect of endolymphatic administration of amiloride on the EP. A few minutes after the administration of amiloride the EP drops to ~+20 mV. In both experiments, an initial biphasic change in the EP was observed, but this change cannot be explained at the present time

Fig. 12

a Possible mechanisms of the regulation in ENaCs and tight junctions (TJs) by increase in [Ca²⁺]_c in marginal cells. b Electrical circuit model in the stria vascularis. In this model, we propose the electrical shunt pathway through marginal cells, and we do not need to consider the negative potential of hair cells, as was proposed previously [9]. Negative EP should be analyzed under the consideration of shunt resistance in the ESC in the future. EP was almost determined by the shunt resistance, because EP ~ E_M if the R ₂ is much larger than R ₁ or R ₃. We considered that E_M is mainly produced by Na⁺ diffusion potential across the basolateral membrane of marginal cells