Muscarinic Receptors Control K⁺ Secretion in Inner Ear Strial Marginal Cells

Abstract.

K⁺ secretion in strial marginal cells (SMC) of stria vascularis (SV) is stimulated by β₁-adrenergic receptors. The aim of the present study was to determine, whether SMC from the gerbil inner ear contain muscarinic receptors that inhibit K⁺ secretion. Receptors were identified with pharmacological tools in functional studies where K⁺ secretion was monitored as transepithelial current (I _sc ). The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured as fluo-4 fluorescence and cAMP production with a colorimetric immunoassay. Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. The cholinergic receptor agonist carbachol (CCh) caused a transient increase in [Ca²⁺]_i with a half-maximal concentration value (EC ₅₀) of (5 ± 6) × 10⁻⁶ m (n= 29) and a decrease in basal and stimulated cAMP production. Apical CCh had no effect on I _sc but basolateral CCh caused a transient increase in I _sc with an EC ₅₀ of (3 ± 1) × 10⁻⁶ m and a sustained decrease of I _sc with an EC ₅₀ of (1.2 ± 0.2) × 10⁻⁵ m (n= 129). The effects of CCh on I _sc and [Ca²⁺]_i were inhibited in the presence of muscarinic antagonist 10⁻⁶ m atropine. Further, the muscarinic antagonists pirenzipine, methoctramine and para-fluoro-hexahydo-sila-defenidol (pFHHSiD) inhibited the CCh-induced transient increase of I _sc with affinity constants (K _DB ) of 3 × 10⁻⁸ m (pK _DB = 7.54 ± 0.19, n= 17), 2 × 10⁻⁶ m (pK _DB = 5.71 ± 0.26, n= 19) and 2 × 10⁻⁸ m (pK _DB = 7.65 ± 0.28, n= 19) and the sustained decrease of I _sc with K _DB of 7 × 10⁻⁸ m (pK _DB = 7.05 ± 0.09, n= 33), 6 × 10⁻⁶ m (pK _DB = 5.21 ± 0.13, n= 23), 5 × 10⁻⁸ m (pK _DB = 7.34 ± 0.13, n= 31), respectively. RT-PCR of total RNA isolated from SV using primers specific for the M1–M5 muscarinic receptors revealed products of the predicted sizes for the M3- and M4- but not the M1-, M2- and M5-muscarinic receptor subtypes. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results suggest that K⁺ secretion in SMC is under the control of M3- and M4-muscarinic receptors that may be located in the basolateral membrane of strial marginal cells.