Evidence for Purinergic Receptors in Vestibular Dark Cell and Strial Marginal Cell Epithelia of Gerbil

Abstract

Purinergic receptors have been found to modulate ion transport in several types of epithelial cells as well as excitable cells. It was of interest to determine whether vestibular dark cells and strial marginal cells contain purinergic receptors in either the apicalor basolateral membrane which modulate transepithelial ion transport. Vestibular dark cell and strial marginal cell epithelia were mounted in a micro-Ussing chamber for the measurement of the transepithelial voltage and resistance from which the equivalent short circuit current (I(sc)) was obtained. The apical and basolateral sides were independently perfused with adenosine and adenosine 5'-triphosphate (ATP). Adenosine (10(-5) M) had no effect on I(sc) at either the apical or basolateral side of vestibular dark cells and strial marginal cells, suggesting either the absence of P(1) receptors or the absence of coupling of P(1) receptors to vectorial ion transport by these epithelia. Apical perfusion of ATP (10(-8) to 10(-4) M) caused a decrease in I(sc) of both vestibular dark cells and strial marginal cells. Apical perfusion of the nucleotides uridine 5'-triphosphate (UTP), 2-methylthioadenosine triphosphate (2-meS-ATP), adenosine 5'-O-(3-thiotriphosphate) (ATPγS) and α,β-methylene adenosine 5'-triphosphate (α,β-meth-ATP) caused qualitatively similar responses with different magnitudes of response. The sequence of the magnitude of response of each compound at 10(-6) or 10(-5) M was assessed from the fractional change of I(sc). The sequence for vestibular dark cells was UTP = ATP = ATPγS ≫ 2-meS-ATP > α,β-meth-ATP, and for strial marginal cells it was UTP = ATP ≫ 2-meS-ATP, corresponding to the sequence for the P(2U) receptor. The effect of agonist on the apical membrane was reduced by the antagonist 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) but not cibacron blue or suramin. DIDS in the absence of exogenous purinergic agonist caused a sustained increase in I(sc). The effect of ATP on the apical membrane was greater in the absence of divalent cations. Basolateral perfusion of ATP led to a biphasic response of I(sc) in vestibular dark cell and strial marginal cell epithelia, consisting of an initial rapid increase followed by a slower decrease. Perfusion of the perilymphatic surface of the stria vascularis (basal cell layer) with ATP had no acute effect on I(sc). The initial increase of I(sc) in vestibular dark cell epithelium during basolateral perfusion had a sequence of 2-meS-ATP > ATP ≫ UTP = α,β-meth-ATP = ATPγS, corresponding to the sequence for the P(2Y) receptor. Subsequently, the agonists caused a sustained decrease in I(sc) with a sequence of ATPγS > 2-meS-ATP > ATP > UTP >α,β-meth-ATP. This sequence is most simply interpreted as the result of the coexistence of P(2U) and P(2Y) receptors in the basolateral membrane. Both the increase and decrease of I(sc) by ATP at the basolateral membrane were reduced by the antagonist suramin. These findings provide evidence for the regulation of transepithelial ion transport by P(2U) receptors in the apical membrane and by coexisting P(2U) and P(2Y) receptors in the basolateral membrane of K(+)-secretory epithelial cells in the inner ear and are consistent with the hypothesis that the apical receptors are part of an autocrine negative feedback system in these cells.

Figure 1

Effects on transepithelial voltage (V_t) and resistance (R_t) of perfusion of adenosine (Ad, 10⁻⁵ M) and adenosine 5′-triphosphate (ATP, 10⁻⁵ M) on the apical membrane of vestibular dark cells (vdc-ap) and strial marginal cells (smc-ap) and on the basolateral membrane of vestibular dark cells (vdc-bl) and strial marginal cells (smc-bl). Representative traces.

Figure 2

Summary of effects (minimum and peak) on V_t, R_t and short circuit current (I_sc) from 2-minute perfusion of adenosine 5′-triphosphate (ATP) expressed as a percent of the respective values immediately prior to ATP; mean ± SEM. Top: Apical (AP) perfusion of vestibular dark cells (VDC) with ATP in the range 10⁻⁸ to 10⁻⁴ M and of strial marginal cells (SMC) with ATP in the range 10⁻⁶ to 10⁻⁴ M. Bottom: Basolateral (BL) perfusion of vestibular dark cells with ATP in the range 10⁻⁸ to 10⁻⁴ M. Number of experiments per concentration is listed next to each data point; numbers were the same for minimum and peak of V_t (basolateral side).

Figure 3

Apical (AP) and basolateral (BL) perfusion of vestibular dark cells (VDC) with 10⁻⁶ M uridine 5′-triphosphate (UTP), adenosine 5′-triphosphate (ATP), adenosine 5′-O-(3-thiotriphosphate (ATPγS), 2-methylthioadenosine triphosphate (2-meS-ATP) and α,β-methylene adenosine 5′-triphosphate (α,β-meth-ATP) and apical perfusion of strial marginal cells (SMC) with 10⁻⁵ M UTP, ATP and 2-meS-ATP. Top: increase of I_sc at peak value during basolateral perfusion of ATP expressed as a percent change from initial value;Bottom: decrease of I_sc at 2 minutes of perfusion of ATP expressed as a percent change from initial value. Bars are mean ± SEM; *Significant difference between adjacent bars; number of experiments per concentration is listed next to each bar.

Figure 4

Effect on transepithelial equivalent short circuit current (I_sc) of apical perfusion of adenosine triphosphate (ATP) (10⁻⁶ M) in the presence and absence of Ca²⁺ and Mg²⁺. Representative trace from vestibular dark cells.

Figure 5

Representative trace of effect on transepithelial equivalent short circuit current (I_sc) of apical perfusion of P_2U agonist (uridine 5′-triphosphate) [UTP] or (adenosine 5′-triphosphate) [ATP] in the presence or absence of P₂ antagonists (cibacron blue [CB] or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid [DIDS]. Top: Effects of UTP (10⁻⁶ M) and DIDS (10⁻³ M) on strial marginal cells (SMC); middle: effects of UTP (10⁻⁶ M) and DIDS (10⁻³ M) on vestibular dark cells (VDC); bottom: effects of ATP (10⁻⁵ M) and CB (10⁻⁵ M) on vestibular dark cells (VDC).

Figure 6

Effect of basolateral perfusion of 10⁻⁶ M 2-methylthioadenosine triphosphate (2-meS-ATP) on short circuit current (I_sc) of vestibular dark cells in the presence and absence of 10⁻⁶ M suramin. Top: Peak increase in I_sc during perfusion of 2-meS-ATP expressed as a percent change from the respective initial values; bottom: decrease of I_sc at 2 minutes of perfusion of 2-meS-ATP expressed as percent change from the respective initial values. Bars are mean ± SEM, n=9; *Significant difference between adjacent bars.

Figure 7

Expected responses to P₂ agonists of systems containing P_2U (left), P_2Y (middle) and coexisting P_2U (30%) and P_2Y (70%) receptor subtypes (right). The response to the most potent agonist for P_2U and P_2Y was set to 100% and the other agonists were placed in their established sequences of potency. The combined response of the coexisting receptors was derived as a linear combination from the first two sets of responses and corresponds to the sequence found for the basolateral membrane of vestibular dark cells. UTP = uridine 5′-triphosphate; ATP = adenosine 5′-triphosphate. ATPγS = adenosine 5′-O-(3-thiotriphosphate; 2-meS-ATP = 2-methylthioadenosine triphosphate; α,β-meth-ATP = α,β-methylene adenosine 5′-triphosphate.