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 beta1-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 (Isc). 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 (EC50) of (5 +/- 6) x 10(-6) m (n = 29) and a decrease in basal and stimulated cAMP production. Apical CCh had no effect on Isc but basolateral CCh caused a transient increase in Isc with an EC50 of (3 +/- 1) x 10(-6) m and a sustained decrease of Isc with an EC50 of (1.2 +/- 0.2) x 10(-5) m (n = 129). The effects of CCh on Isc 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 Isc with affinity constants (KDB) of 3 x 10(-8) m (pKDB = 7.54 +/- 0.19, n = 17), 2 x 10(-6) m (pKDB = 5.71 +/- 0.26, n = 19) and 2 x 10(-8) m (pKDB = 7.65 +/- 0.28, n = 19) and the sustained decrease of Isc with KDB of 7 x 10(-8) m (pKDB = 7.05 +/- 0.09, n = 33), 6 x 10(-6) m (pKDB = 5.21 +/- 0.13, n = 23), 5 x 10(-8) m (pKDB = 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.

Muscarinic Signaling in the Cochlea: Presynaptic and Postsynaptic Effects on Efferent Feedback and Afferent Excitability

“…1 C ) or when the nerve trunk was included with the spiral ganglion (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), an M 3 band was present: M 3 receptors have been detected, by RT-PCR, in microdissected stria vascularis (Wangemann et al, 2001) and may also be expressed in the nerve-root nucleus and/or parts of the cochlear nucleus that are present when the modiolus is included (Merchan et al, 1988). All PCR bands were sequenced to verify the identity of the amplicons.…”

“…Previous RT-PCR study in gerbil reported M 3 expression in microdissected stria vascularis (Wangemann et al, 2001). Transcripts for M 1 , M 3 , and M 5 (but not M 2 or M 4 ) were amplified from whole mouse cochleas (Drescher et al, 1992), and, in rats and guinea pigs, M 3 transcripts were found in microdissected organ of Corti and spiral ganglion (Safiedine et al, 1996).…”

“…Reverse transcription (RT)-PCR, in situ hybridization, and immunohistochemistry suggest that all five mAChR subtypes may be expressed in the mammalian cochlea, (1) in the postsynaptic targets of the cholinergic olivocochlear system, i.e., sensory hair cells (Drescher et al, 1992) or sensory neurons (Safieddine et al, 1996), (2) as autoreceptors in the olivocochlear neurons themselves (Safieddine et al, 1996), or (3) in the stria vascularis, an epithelium involved in ionic regulation of cochlear fluids with no known cholinergic innervation (Wangemann et al, 2001). Pharmacological studies of isolated hair cells (Shigemoto and Ohmori, 1991; Sziklai et al, 1996) and cochlear sensory neurons (Rome et al, 1999; Ito and Dulon, 2002) also suggest functional mAChRs.…”

Functional significance of channels and transporters expressed in the inner ear and kidney

“…Furthermore, the rate of K ϩ secretion is increased by ␤ 1 -adrenergic receptors via cAMP-dependent stimulation of the KCNQ1/KCNE1 K ϩ channel (258,259,309,310). Conversely, muscarinic and purinergic receptors suppress K ϩ secretion (307). Purinergic P2Y 4 receptors decrease currents through the KCNQ1/KCNE1 K ϩ channel via protein kinase C (160,166).…”

Reactive blue 2, an antagonist of rat P2Y4, increases K+ secretion in rat cochlea strial marginal cells

“…P2X 2 receptors are widely distributed in the inner ear, but not in the stria vascularis (Housley et al, 1998;Housley et al, 1999), and it has been shown that K + secretion is regulated via P2Y 4 receptors in the apical membrane of the marginal cells residing in the stria vascularis (Marcus et al, 2005). Moreover, P2Y 2 (Sage and Marcus, 2002), 1-adrenergic, and muscarinic receptors (Wangemann et al, 2001) have been reported at the basolateral membrane, and it is believed that the sources of agonists for these receptors are the strial marginal cells (White et al, 1995) and the organ of Corti (Wangemann, 1996).…”