Muscarinic receptors control K+ secretion in inner ear strial marginal cells.
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.
Wangemann
P
P
Cell Physiology Laboratory, Anatomy & Physiology Department Kansas State University, 1600 Dension Ave., Manhattan KS 66506, USA.
Liu
J
J
Scherer
E Q
EQ
Herzog
M
M
Shimozono
M
M
Scofield
M A
MA
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D003959:Diamines / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005849:Gerbillinae; D007545:Isoproterenol / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine; D011188:Potassium / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013316:Stria Vascularis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D018679:Cholinergic Agonists; D003959:Diamines; D018727:Muscarinic Antagonists; D010276:Parasympatholytics; D010880:Piperidines; D011976:Receptors, Muscarinic; C058297:4-fluorohexahydrosiladifenidol; D010890:Pirenzepine; D001285:Atropine; D002217:Carbachol; D000242:Cyclic AMP; D007545:Isoproterenol; C054938:methoctramine; D011188:Potassium; D002118:Calcium