Ion transport regulation by P2Y receptors, protein kinase C and phosphatidylinositol 3-kinase within the semicircular canal duct epithelium

Abstract

Background: The ionic composition of the luminal fluid in the vestibular labyrinth is maintained within tight limits by the many types of epithelial cells bounding the lumen. Regulatory mechanisms include systemic, paracrine and autocrine hormones along with their associated intracellular signal pathways. The epithelium lining the semicircular canal duct (SCCD) is a tissue that is known to absorb sodium and calcium and to secrete chloride.

Findings: Transport function was assessed by measurements of short circuit current (Isc) and gene transcript expression was evaluated by microarray. Neither ATP nor UTP (100 microM) on the apical side of the epithelium had any effect on Isc. By contrast, basolateral ATP transiently increased Isc and transepithelial resistance dropped significantly after basolateral ATP and UTP. P2Y2 was the sole UTP-sensitive purinergic receptor expressed. Isc was reduced by 42%, 50% and 63% after knockdown of alpha-ENaC, stimulation of PKC and inhibition of PI3-K, while the latter two increased the transepithelial resistance. PKCdelta, PKCgamma and PI3-K were found to be expressed.

Conclusions: These observations demonstrate that ion transport by the SCCD is regulated by P2Y2 purinergic receptors on the basolateral membrane that may respond to systemic or local agonists, such as ATP and/or UTP. The sodium absorption from endolymph mediated by ENaC in SCCD is regulated by signal pathways that include the kinases PKC and PI3-K. These three newly-identified regulatory components may prove to be valuable drug targets in the control of pathologic vestibular conditions involving dysfunction of transport homeostasis in the ear, such as Meniere's disease.

Figure 1

Effects of purinergic agonists on short circuit current of SCCD epithelium. Representative recordings of apical (AP) and basolateral (BL) addition of purinergic agonists to SCCD epithelia. The continuous line is the open-circuit transepithelial voltage (V_T) and the pulses are the voltage response to current pulses. A) ATP was added to the apical or basolateral chamber at the times indicated by the bars at the top of the figure. A single pulse is shown with an expanded time scale in the insert for clarity and shows no time-dependent response. Vertical arrows indicate where readings were taken for the initial (I) value, change after apical (ΔA) agonist, peak basolateral (B_p) and change after basolateral (ΔB) agonist for tabulation in Table 2. B) UTP was added to the apical or basolateral chamber at the times indicated by the bars at the top of the figure; example of recording with transient increase in V_T after basolateral addition of UTP. C) Similar to recording in B, but an example of a recording without transient increase in V_T after basolateral addition of UTP.

Figure 2

Effect of RNA interference on short circuit current of SCCD epithelium. Effect of α-ENaC siRNA on dexamethasone stimulated Na⁺ transport: Epithelia were incubated with dexamethasone (100 nM) for 24 hr concurrently with transfection. Amiloride-sensitive short circuit current, AMIL-I_sc, was measured from both transfected and non-transfected monolayers as a function of net Na⁺ transport. α-ENaC siRNA (anti-sense) but not non-silencing siRNA (sense) inhibits dexamethasone-stimulated Na⁺ transport. "Control" epithelia were not incubated with dexamethasone. Data are mean ± SEM, n = 3 - 10. P < 0.05 (*) compared to AMIL-I_sc with dexamethasone alone. Note the break in the vertical axis.

Figure 3

Proposed cell model for P2Y, PKC and PI3-K regulation of ion transport by SCCD epithelium. Na⁺ is absorbed from endolymph by entry into the cytosol across the apical membrane via the epithelial Na⁺ channel (ENaC). Na⁺ is removed from the cytosol across the basolateral membrane by the Na⁺, K⁺-ATPase (Na⁺-pump); K⁺ entering the cell on the Na⁺, K⁺-ATPase recycles across the basolateral membrane via K⁺ channels. Activity of this Na⁺ absorptive mechanism is stimulated by activation of the glucocorticoid receptor (GR), by activity of phosphoinositide-3 kinase (PI3-K) and inhibited by activity of protein kinase C (PKC). Activation of the P2Y2 receptor (P2Y2-R) in the basolateral membrane reduces the barrier to ions of the tight junctions (TJ) and stimulates Cl^- secretion.