P2X2 receptor mediates stimulation of parasensory cation absorption by cochlear outer sulcus cells and vestibular transitional cells

Abstract

Cochlear outer sulcus cells (OSC) and vestibular transitional cells (VTC) are part of the parasensory epithelium in the inner ear and are located in homologous positions between the sensory hair cells and the cation secretory epithelial cells in the cochlea and the vestibular labyrinth. OSC are known to sustain a reabsorptive transepithelial current and to contain an immunoreactivity for P2X(2) purinergic receptors. This study addresses whether OSC and VTC share functional similarities and extends this hypothesis to the question of whether both cell types contain functional P2X(2) receptors. The current density (I(sc)) was recorded with the vibrating probe technique and was found to be similar in VTC and OSC. Both gadolinium and flufenamic acid reduced I(sc) in VTC, as reported previously for OSC. I(sc) was stimulated by extracellular ATP but not by selective agonists of P2Y receptors. Purinergic receptor agonists increased I(sc) with a potency order of ATP > 2'- and 3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate alpha,beta-methyleneadenosine 5'-triphosphate in both OSC and VTC. In the presence of suramin (100 micrometer) or gadolinium (100 micrometer), the responses of ATP were inhibited significantly in both OSC and VTC. This pharmacological profile is consistent with that of the P2X(2) receptor. These results demonstrate that VTC participate in vestibular parasensory cation absorption and that both OSC and VTC regulate their parasensory cation flux via P2X(2) receptors, which would regulate the endolymphatic concentration of the current-carrying ion species in auditory and vestibular transduction.

Fig. 1.

Tissue preparation for OSC and VTC.A, C, Schematic illustration showing the location of OSC and VTC in the cochlea and semicircular canal ampulla, respectively.B, D, Prepared tissue for the measurement ofI_sc in OSC and VTC, respectively.HC, Vestibular hair cell (damaged); SL, spiral ligament; SMC, strial marginal cell;SV, stria vascularis; VP, vibrating probe. Scale bar, 50 μm. A andC adapted from graphics by P. Wangemann (Wangemann, 1997).

Fig. 2.

Effects of Gd³⁺ and flufenamic acid on I_sc in VTC. A, Gd³⁺ (1 mm, n = 6).B, Flufenamic acid (FFA) (0.1 mm, n = 9). Bumetanide (10 μm) was added to the bath solution to exclude influence from vestibular dark cells. The SEM bars are plotted only at intervals for clarity.

Fig. 3.

Comparison of effects of ATP, UTP, UDP, and ADP in OSC and VTC. A, OSC; B, VTC. Each drug was perfused at 100 μm. For the experiments in VTC, bumetanide (10 μm) was added to the bath solution to exclude influence from vestibular dark cells. UDP and ADP were preincubated with hexokinase (1 U/ml) in the presence of glucose for at least 1.5 hr (see Materials and Methods).

Fig. 4.

Dose–response relationships of ATP analogs in OSC and VTC. A, B, C, OSC; D, E, F, VTC. There is a discontinuity in B that indicates recordings from two different tissues. Numbersin C and F indicate numbers of observations at each concentration. In the dose–response curves (C, F), the data were normalized based on the response to 100 μm ATP. In the experiments on VTC, bumetanide (10 μm) was added to the bath solution to exclude influence from vestibular dark cells. αβ, αβmeATP; Bz, BzATP.

Fig. 5.

Effect of ATP in the presence of suramin or gadolinium on OSC and VTC. A, B, C, OSC; D, E, F, VTC. In the experiments on VTC, bumetanide (10 μm) was added to the bath solution to exclude influence from vestibular dark cells. Significance was tested based on the 100 μm ATP response (C, F).SUR, Suramin; Gd, gadolinium. *p < 0.05.