Purinergic modulation of cochlear partition resistance and its effect on the endocochlear potential in the Guinea pig

Abstract

Introduction of adenosine 5'-triphosphate (ATP) into the endolymphatic compartment of the guinea-pig cochlea decreases the endocochlear potential (EP). To determine if this is due to an ATP-induced change in compartment resistance, the cochlear partition resistance (CoPR) was measured using constant current injections into scala media before, during, and after microinjection of ATP into the same compartment. The CoPR (mean = 3.13 +/- 0.13 kOmega) decreased with ATP in a dose-dependent manner (25.1 +/- 3.0% decrease in relation to baseline values) and this was linearly correlated ( R(2) = 0.91) to the magnitude of the ATP-induced decline in EP (41.6 +/- 7.0% decline in relation to the baseline). Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, a P2X receptor antagonist) injected prior to ATP application blocked this ATP-induced reduction in EP and CoPR. This indicates that ATP-gated ion channels (P2X receptors) provide a latent shunt capable of regulating the majority of the electrical potential across the luminal surface of the sensory hair cells, which is necessary for sound transduction. The results suggest a novel sound transduction regulatory mechanism, which, via extracellular ATP, has the capability of adjusting hearing sensitivity.

Figure 1

A. Diagram showing the arrangement for microinjection of ATP into scala media and measurement of EP and CoPR. SV = scala vestibuli, SM = scala media, ST = scala tympani. B. An example of ATP-induced reduction in CoPR. ATP (500 nl, 100 µM, applied over 1 min) reduced EP from 83 to 36 mV concomitant with a reduction in the magnitude of the 1 µA current-induced voltage shift (3 s) from 3.8 mV (arrow a) to 2.5 mV (arrow b), corresponding to a decrease of CoPR from 3.8 kΩ to 2.5 kΩ, respectively. After ATP injection, CoPR returned to baseline levels (arrow c). The inset shows the voltage shifts increased in size for better illustration. ATP injection produced a decrease in the EP (C) and CoPR (D) in every experiment. Open symbols in C and D represent the mean EP and CoPR values, respectively. E. The reduction in the magnitude of the EP was concomitant with a reduction in the CoPR in every experiment. Each paired data point (open symbol, before the ATP pulse; filled symbol, during the ATP pulse) represents one experiment. Dotted line represents the best-fit intercept.

Figure 2

Relationship between the magnitude of the ATP-induced reduction (A) or percentage reduction (B) in EP and CoPR (filled circles). For comparison, open circles in A and B represent the relationship of the EP and CoPR changes induced by ATP when applied after PPADS (from the PPADS subset of experiments). Crosses in A and B represent the data from kanamycin-treated animals from which the dotted linear regression lines were produced for comparison (see text). Solid linear regression lines and equations apply only to data represented by the filled circles.

Figure 3

Histograms showing the effect of artificial endolymph (EL), PPADS, ATP, and ATP following PPADS on the EP (A) and CoPR (B). Endolymph (mean of 250–1000 nl pulses) and PPADS (1 mM, 50 nl) control injections had a small effect on EP and CoPR. In contrast, ATP (100 µM in 500 nl) caused a significant decline in both measures (*, p= 0.0014 and p < 0.001, Student’s paired t-tests for EP and CoPR, respectively), which was substantially reduced by the pre-application of PPADS (†, p < 0.001, Student’s paired t-tests for both EP and CoPR). C. Example showing that PPADS blocks the ATP-induced reduction in EP and CoPR. The initial 26 mV fall in EP observed during ATP pulse injection (from 67 to 41 mV) was reduced by prior application of PPADS to only a 3 mV fall (from 70 to 67 mV). Concomitantly, the initial 0.7 kΩ fall in CoPR (calculated from 2.9 and 2.2 mV, the current-induced voltage shifts produced before and during ATP injection, respectively) induced by ATP was reduced to a 0.25 kΩ fall by PPADS pre-application (from 2.8 to 2.55 kΩ). Both, EP and CoPR returned to baseline levels after the ATP pulse. Only one current-induced voltage shift is shown for each experimental stage for clarity. Data records were digitally reconstructed.

Figure 4

Light microscopy of the organ of Corti from the first turn of normal (A) and kanamycin-treated (B) guinea pigs. The normal organ of Corti shows the three rows of outer sensory hair cells (OHC), supported at their base by the Deiters’ supporting cells (D) and separated by the supporting pillar cells (arrow) from a single row of inner sensory hair cells (IHC). The upper surface of the organ of Corti is bathed by endolymph of the scala media (SM) and the basal surface is bathed by perilymph in the scala tympani (ST). In contrast, after kanamycin treatment, both types of sensory cell are missing and the space is now occupied by expanded supporting cells (stars). The remaining pillar cells (arrow) define the original architecture. Scale bar = 20 µm.