Progressive irreversible hearing loss is caused by stria vascularis degeneration in an Slc26a4-insufficient mouse model of large vestibular aqueduct syndrome.

Hearing loss of patients with enlargement of the vestibular aqueduct (EVA) can fluctuate or progress, with overall downward progression. The most common detectable cause of EVA is mutations of SLC26A4. We previously described a transgenic Slc26a4-insufficient mouse model of EVA in which Slc26a4 expression is controlled by doxycycline administration. Mice that received doxycycline from conception until embryonic day 17.5 (DE17.5; doxycycline discontinued at embryonic day 17.5) had fluctuating hearing loss between 1 and 6 months of age with an overall downward progression after 6 months of age. In this study, we characterized the cochlear functional and structural changes underlying irreversible hearing loss in DE17.5 mice at 12 months of age. The endocochlear potential was decreased and inversely correlated with auditory brainstem response thresholds. The stria vascularis was thickened and edematous in ears with less severe hearing loss, and thinned and atrophic in ears with more severe hearing loss. There were pathologic changes in marginal cell morphology and gene expression that were not observed at 3 months. We conclude that strial dysfunction and degeneration are the primary causes of irreversible progressive hearing loss in our Slc26a4-insufficient mouse model of EVA. This model of primary strial atrophy may be used to explore the mechanisms of progressive hearing loss due to strial dysfunction.

Localization of beta1-adrenergic receptors in the cochlea and the vestibular labyrinth.

Sympathetic activation in a "fight or flight reaction" may put the sensory systems for hearing and balance into a state of heightened alert via beta1-adrenergic receptors (beta1-AR). The aim of the present study was to localize beta1-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize beta1-AR by Western immunoblots, and to identify beta1-AR pharmacologically by measurements of cAMP production. Staining for beta1-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated beta1-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, beta1-AR are present and functional in inner ear epithelial cells that are involved in K+ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.

CGRP receptors in the gerbil spiral modiolar artery mediate a sustained vasodilation via a transient cAMP-mediated Ca2+-decrease.

Alteration of cochlear blood flow may be involved in the etiology of inner ear disorders like sudden hearing loss, fluctuating hearing loss and tinnitus. The aim of the present study was to localize the vasodilator calcitonin gene-related peptide (CGRP) and to identify CGRP receptors and their signaling pathways in the gerbil spiral modiolar artery (SMA) that provides the main blood supply of the cochlea. CGRP was localized in perivascular nerves by immunocytochemistry. The vascular diameter and cytosolic Ca2+ concentration [Ca2+]i in the smooth muscle cells were measured simultaneously with videomicroscopy and fluo-4-microfluorometry. Calcitonin receptor-like receptor (CRLR) mRNA was identified by RT-PCR as a specific 288 bp fragment in total RNA isolated from the vascular wall. The SMA was preconstricted by a 2-min application of 1 nM endothelin-1 (ET1). CGRP, forskolin, and dibutyryl-cAMP caused a vasodilation (EC50 = 0.1 nM, 0.3 mM, and 20 mM). CGRP and forskolin caused an increase in cAMP production and a transient decrease in the [Ca2+]i. The CGRP-induced vasodilation was antagonized by CGRP8-37 (KDB = 2 mM). The K+-channel blockers iberiotoxin and glibenclamide partially prevented the CGRP- or forskolin-induced vasodilations but failed to reverse these vasodilations. These results demonstrate that CGRP is present in perivascular nerves and causes a vasodilation of the ET1-preconstricted SMA. The data suggest that this vasodilation is mediated by an increase in the cytosolic cAMP concentration, a transient activation of iberiotoxin-sensitive BK and glibenclamide-sensitive KATP K+ channels, a transient decrease in the [Ca2+]i and a long-lasting Ca2+ desensitization.

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.

Differential desensitization of Ca2+ mobilization and vasoconstriction by ET(A) receptors in the gerbil spiral modiolar artery.

Endothelins are known to be among the most potent endogenous vasoconstrictors. Vasoconstriction of the spiral modiolar artery, which supplies the cochlea, may be implicated in hearing loss and tinnitus. The purpose of the present study was to determine whether the spiral modiolar artery responds to endothelin, whether a change in the cytosolic Ca2+ concentration ([Ca2+]i) mediates the response and which endothelin receptors are present. The vascular diameter and [Ca2+]i were measured simultaneously by videomicroscopy and microfluorometry in the isolated spiral modiolar artery from the gerbil. ET-1 induced a transient [Ca2+]i increase and a strong and long-lasting vasoconstriction. The transient [Ca2+]i increase underwent rapid desensitization, was independent of extracellular Ca2+ and inhibited by the IP3-receptor blocker (75 microm) 2-aminoethoxydiphenyl borate (2-APB) and by depletion of Ca2+ stores with 10(-6) m thapsigargin. In contrast, the vasoconstriction displayed no comparable desensitization. The initial vasoconstriction was independent of extracellular Ca2+ but maintenance of the constriction depended on the presence of extracellular Ca2+. The half-maximal concentration values (EC50) for the agonists ET-1, ET-3 and sarafotoxin S6c were 0.8 nm, >10 nm and >100 nm, respectively. Affinity constants for the antagonists BQ-123 and BQ-788 were 24 nm and 77 nm, respectively. These observations demonstrate that ET-1 mediates a vasoconstriction of the gerbil spiral modiolar artery via ETA receptors and an IP3 receptor-mediated release of Ca2+ from thapsigargin-sensitive Ca2+ stores. The marked difference in desensitization between Ca2+ mobilization and vasoconstriction suggests that Ca2+ mobilization is not solely responsible for the vasoconstriction and that other signaling mechanisms must be present.

Functional beta2-adrenergic receptors are present in nonstrial tissues of the lateral wall in the gerbil cochlea.

Recently, we have demonstrated that functional beta1-adrenergic receptors are the dominant beta-adrenergic receptor subtype in the stria vascularis and that beta1-adrenergic receptors stimulate K+ secretion in strial marginal cells. The goal of the present study was to determine whether nonstrial tissues in the cochlear lateral wall contain beta-adrenergic receptors and if so which subtype is present. Pharmacological tools were used to identify receptors in functional studies where cAMP production was measured. Further, receptors were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed on gerbil nonstrial lateral wall tissues. Tissues stimulated with 10(-5) M isoproterenol produced 0.42 +/- 0.22 pmol cAMP per ear within 12 min (n = 14). The selective beta-adrenergic receptor agonist isoproterenol stimulated cAMP production with an EC50 of (2 +/- 3) x 10(-7) M (n = 7). Isoproterenol-stimulated cAMP production was inhibited by the beta2-adrenergic receptor antagonist ICI 118551 with an IC50 of (7 +/- 7) x 10(-6) M, which corresponds to an affinity constant of 1 x 10(-7) M (pK(DB) = 6.89 +/- 0.23, n = 3). Isoproterenol-stimulated cAMP production was not inhibited by the highly selective beta1-adrenergic receptor antagonist CGP 20712A. The IC50 and the affinity constant for CGP 20712A were estimated to be >3 x 10(-4) and >6 x 10(-6) M, respectively. RT-PCR of total RNA isolated from nonstrial lateral wall tissues using primers specific for beta1-, beta2- and beta3-adrenergic receptors revealed products of the predicted sizes for the beta1- and beta2- but not for the beta3-subtype. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that nonstrial lateral wall tissues contain transcripts for beta1- and beta2- but not for beta3-adrenergic receptors and that the beta2-adrenergic receptor is the dominant functional receptor subtype. The cellular localization and function of the beta2-adrenergic receptors remains to be determined.

K+ secretion in strial marginal cells is stimulated via beta 1-adrenergic receptors but not via beta 2-adrenergic or vasopressin receptors.

Pharmacologic tools were used to identify receptors in functional studies by measuring either transepithelial current (I(sc)) in strial marginal cells (SMC) or cAMP production in stria vascularis (SV). Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Experiments were performed using tissues isolated from gerbils unless specified otherwise. I(sc) under control conditions was 1090 +/- 21 microA/cm(2) (n = 213) in gerbil SMC and 2001 +/- 95 microA/cm(2) (n = 6) in murine SMC. Direct stimulation of adenylate cyclase with 10(-5) m forskolin but not with 10(-5) m 1,9-dideoxy-forskolin resulted in an increase in the I(sc) by a factor of 1.14 +/- 0.01 (n = 6). The vasopressin-receptor agonist 10(-8) m Arg(8)-vasopressin had no significant effect on I(sc) in gerbil and murine SMC. The beta-adrenergic agonists isoproterenol, norepinephrine and epinephrine stimulated I(sc) with an EC(50) of (6 +/- 2) x 10(-7) m (n = 28), (3 +/- 1) x 10(-6) m (n = 40) and (7 +/- 2) x 10(-6) m (n = 38), respectively. Isoproterenol stimulated cAMP production in SV with an EC(50) of (5 +/- 2) x 10(-7) m (n = 8). The beta-antagonist 10(-4) m propanolol completely inhibited 2 x 10(-5) m isoproterenol-induced stimulation of I(sc). The beta-antagonists atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of I(sc) with a K(DB) of 1 x 10(-7) m (pK(DB) = 6.96 +/- 0.15, n = 14), 1 x 10(-7) m (pK(DB) = 7. 01 +/- 0.14, n = 15), 2 x 10(-9) m (pK(DB) = 8.73 +/- 0.13, n = 19), respectively. CGP20712A inhibited isoproterenol-induced cAMP production with a K(DB) of 1 x 10(-10) m (pK(DB) = 9.94 +/- 0.55, n = 9). RT-PCR of total RNA isolated from SV using primers specific for the beta(1)-, beta(2)- and beta(3)-adrenergic receptors revealed products of the predicted sizes for the beta(1)- and beta(2)- but not the beta(3)-adrenergic receptor. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results demonstrate that K(+) secretion in SMC is under the control of beta(1)-adrenergic receptors but not beta(2)-adrenergic or vasopressin-receptors and that the beta(1)-subtype is the primary beta-adrenergic receptor in SV although SV contains transcripts for both beta(1)- and beta(2)-adrenergic receptors.

Evidence for a calcium-sensing receptor in the vascular smooth muscle cells of the spiral modiolar artery.

The vascular diameter of the gerbilline spiral modiolar artery has been shown to depend on the presence of extracellular Ca(2+) but it remained unknown whether the smooth muscle cells of this arteriole contain a Ca(2+) sensing receptor (CaSR). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) was monitored as fluo 3 fluorescence and the vascular diameter was measured by video-microscopy in isolated in vitro superfused spiral modiolar arteries. RT-PCR was used to probe for the presence of CaSR transcripts. Increasing the extracellular Ca(2+) concentration ([Ca(2+)](o)) from 1 to 10 mm caused a biphasic increase in [Ca(2+)](i) that was paralleled by a vasoconstriction. The initial rate of this vasoconstriction, 2.01 +/- 0.07 microm/sec (n = 131), was inhibited when cytosolic Ca(2+) stores were presumably depleted with thapsigargin (IC(50) = 3 x 10(-9) m, n = 26) or ryanodine (IC(50) = 4 x 10(-8) m, n = 25) or when PLC was inhibited by 10(-6) m U73122 (n = 8). The initial rate of this constriction was not affected by the L-type Ca(2+) channel blocker 10(-6) m nifedipine (n = 5), by 10(-6) m U73343 (n = 6), which is the inactive analogue of U73122, by the T-type Ca(2+) channel blocker 10(-6) Gd(3+) (n = 6) or the Na(+)/Ca(2+) exchanger blocker 10(-4) m Ni(2+) (n = 5). The agonist rank potency order was Gd(3+) > Ni(2+) > Ca(2+) >> neomycin = Mg(2+). Analysis of RNA isolated from the SMA revealed a RT-PCR product of the appropriate size for the CaSR (448 bp). Sequence analysis of the amplified cDNA fragment revealed a 94-96% amino acid identity compared to other CaSRs. These results demonstrate that the spiral modiolar artery contains a CaSR, which is most likely located in the vascular smooth muscle cells.

Beta1-adrenergic receptors but not beta2-adrenergic or vasopressin receptors regulate K+ secretion in vestibular dark cells of the inner ear.

Receptors were identified pharmacologically in functional studies where K+ secretion was monitored as transepithelial current (Isc). Further, receptors were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Isc under control conditions was 796 +/- 15 microA/cm2 (n = 329) in gerbilline VDC and 900 +/- 75 microA/cm2 (n = 6) in murine VDC. Forskolin (10(-5) m) but not 1, 9-dideoxy-forskolin increased Isc by a factor of 1.42 +/- 0.05 (n = 7). 10(-9) m Arg8-vasopressin and 10(-9) m desmopressin had no significant effect in gerbilline and murine VDC. Isoproterenol, norepinephrine, epinephrine and prenalterol stimulated Isc maximally by a factor of 1.38 +/- 0.04 (n = 7), 1.59 +/- 0.06 (n = 6), 1.64 +/- 0.03 (n = 8) and 1.37 +/- 0.03 (n = 6), respectively. The EC50 values were (1.4 +/- 0.7) x 10(-8) m (n = 36), (2.5 +/- 1.0) x 10(-8) m (n = 31), (1.7 +/- 0.7) x 10(-7) m (n = 36) and (5 +/- 4) x 10(-7) m (n = 32), respectively. Propanolol inhibited isoproterenol-induced stimulation of Isc. Atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of Isc with a pKDB of 5.0 x 10(-8) m (pKDB = 7.30 +/- 0.07, n = 38), 4.4 x 10(-8) m (pKDB = 7.36 +/- 0.14, n = 37) and 6.8 x 10(-12) m (pKDB = 11.17 +/- 0.12, n = 37), respectively. RT-PCR of total RNA isolated from microdissected vestibular labyrinth tissue using specific primers revealed products of the predicted sizes for beta1- and beta2-adrenergic receptors but not for beta3-adrenergic receptors. Sequence analysis of the amplified cDNA fragments from gerbilline tissues revealed a 96.4%, 91.5% and 89.6% identity compared to rat beta1-, beta2- and beta3-adrenergic receptors, respectively. These results demonstrate that K+ secretion in VDC is under the control of beta1- but not beta2- or beta3-adrenergic receptors or vasopressin-receptors.

Alpha1A-adrenergic receptors mediate vasoconstriction of the isolated spiral modiolar artery in vitro.

Several lines of evidence suggest that cochlear blood flow is under the control of the sympathetic nervous system and that this control is mediated via alpha-adrenergic receptors. The goal of the present study was to determine whether alpha-adrenergic receptors mediate vasoconstriction of the spiral modiolar artery and, if so, to determine which subtype dominates this response. Vascular diameter was measured with video microscopy in the isolated superfused spiral modiolar artery in vitro. The diameter of the spiral modiolar artery under control conditions was 61 +/- 2 microm (n = 60). Spontaneous vasomotion was observed in most specimens. Addition of norepinephrine to the superfusate caused a phasic vasoconstriction and an increase in the amplitude of vasomotion. These effects were limited to the vicinity of arteriolar branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction occurred with EC50 of (1.9 +/- 0.4) x 10(-5) M (n = 44) and the vascular diameter was maximally reduced by a factor of 0.87 +/- 0.01 (n = 29). Neither the phasic nature nor the EC50 of the norepinephrine-induced vasoconstrictions was altered in the presence of the beta2-adrenergic receptor antagonist 10(-5) M ICI118551 or the nitric oxide synthase inhibitor 10(-4) M NOARG. In contrast, the alpha2-adrenergic receptor antagonist 10(-7) M yohimbine and the alpha1-adrenergic receptor antagonist 10(-9) and 10(-8) M prazosin caused a significant shift in the dose-response curve. The affinity constants (K(DB)) for yohimbine and prazosin were (5+/-2) x 10(-8) M (n=4) and (2.0+/-0.7) x 10(-10) M (n=18), respectively. The alpha1A-adrenergic receptor antagonist 10(-8) M 5-methyl urapidil and the alpha1D-adrenergic receptors antagonist 5 x 10(-6) M BMY7378 caused a significant shift in the dose-response curve. The K(DB) values for 5-methyl urapidil and for BMY7378 were (2.7 +/- 0.7) x 10(-10) M (n = 8) and (4.4 +/- 2.7) x 10(-7) M (n = 8), respectively. Further, total RNA was isolated from microdissected spiral modiolar arteries and the presence of transcripts for alpha1-adrenergic receptor subtypes was determined by reverse transcription polymerase chain reaction (RT-PCR). Primers specific for gerbil alpha1-adrenergic receptor subtypes were developed using RNA from rat and gerbil brain. Analysis of RNA extracted from the spiral modiolar artery revealed RT-PCR products of the appropriate size for the alpha1A-adrenergic receptor, however, no evidence for the alpha1B- and alpha1D-adrenergic receptor was found. Further, analysis of RNA extracted from blood, which was a contaminant of the microdissected spiral modiolar arteries, revealed no RT-PCR products. Sequence analysis of the RT-PCR product of the alpha1A-adrenergic receptor from the spiral modiolar artery confirmed its identity. Identity between the 175 nt gerbil sequence fragment and the known rat, mouse and human alpha1A-adrenergic receptor sequences was 90.9, 92.0 and 85.2%, respectively. These observations demonstrate that the spiral modiolar artery contains alpha1A-adrenergic receptors which mediate vasoconstriction at branch points.

Ca2+-dependence and nifedipine-sensitivity of vascular tone and contractility in the isolated superfused spiral modiolar artery in vitro.

The regulation of the vascular diameter of the spiral modiolar artery may play a major role in the regulation of cochlear blood flow and tissue oxygenation since the spiral modiolar artery provides the main blood supply to the cochlea. The goal of the present study was to determine whether vascular tone and contractility of the spiral modiolar artery depend on the presence of extracellular Ca2+ and involves nifedipine-sensitive Ca2+ channels. The spiral modiolar artery was isolated and superfused in vitro and the diameter was measured continuously by video microscopy. Isolated segments of the spiral modiolar artery had an outer diameter of 61 +/- 3 microm (n = 59) and displayed vasomotion characterized by 5-15 clearly distinguishable constrictions per min. Removal of Ca2+ from the superfusion medium caused a reversible relaxation and cessation of vasomotion and was used to determine the magnitude of basal vascular tone. The basal vascular tone consisted of a sustained reduction of the vascular diameter to 95.1 +/- 0.3% (n = 51) of the maximal diameter in Ca2+-free medium. Nifedipine reduced the basal vascular tone with an IC50 of (1.1 +/- 0.3) x 10(-9)) M although 22% of the basal vascular tone was insensitive to nifedipine. Elevation of the K+ concentration from 3.6 to 150 mM caused a transient vasoconstriction which was dependent on the presence of extracellular Ca2+. Nifedipine fully inhibited K+-induced vasoconstriction with an IC50 of (2.0 +/- 0.7) x 10(-9) M. Norepinephrine (10(-4) M) caused a transient vasoconstriction and an increase of vasomotion at branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction was fully inhibited in the absence of Ca2+ and partially inhibited by 10(-7) M nifedipine. These observations suggest that the spiral modiolar artery contains voltage-dependent nifedipine-sensitive Ca2+ channels which are involved in the maintenance of basal vascular tone as well as in the mediation of K+- and norepinephrine-induced contractility. Further, the data suggest that cytosolic Ca2+ stores, if present in the spiral modiolar artery, are of limited capacity compared to other vessels.

Vestibular dark cells contain an H+/monocarboxylate- cotransporter in their apical and basolateral membrane.

The transport of lactate and pyruvate across membranes of vestibular dark cells (VDC) may be important under aerobic, ischemic or hypoxic conditions. This study addresses the questions whether VDC from the gerbil contain an H+/monocarboxylate- cotransporter (MCT) and in which membrane, apical or basolateral, MCT is located. Uptake of monocarboxylates into VDC was monitored in functional studies by measuring the cytosolic pH (pHi) and by measuring the pH-sensitive equivalent short circuit current (Isc). Subtypes of the functionally identified MCT which are present in vestibular labyrinth tissues were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Monocarboxylates but not dicarboxylates induced a transient acidification of pHi which was inhibited by 5 mM alpha-cyano-4-hydroxycinnamate (CHC) but not by 1 microM DIDS or 500 microM pCMBS. The initial rate of acidification induced by monocarboxylates was dose-dependent in the range between 1 and 20 mM. K(m) values were for pyruvate 1.3, acetate 3.7, L-lactate 3.8 and D-lactate 7.3 mM. Both apical and basolateral application of monocarboxylates caused a transient increase of Isc which was sensitive to 5 mM CHC. RT-PCR revealed the presence of transcripts for the MCT subtypes MCT1 and MCT2. The identity of transcripts was confirmed by sequence analysis. These observations suggest that VDC contain an MCT in their apical and basolateral membrane and that the vestibular labyrinth contains transcripts for the subtypes MCT1 and MCT2.

The isolated in vitro perfused spiral modiolar artery: pressure dependence of vasoconstriction.

We developed a new technique, the isolated in vitro perfused spiral modiolar artery, which allowed the continuous measurement of the vascular diameter and control of the intravascular pressure. An isolated section of the spiral modiolar artery from the gerbil was perfused on one end with a set of concentric pipettes and occluded on the other end in order to apply a defined intravascular pressure in the range between 10 and 230 cm H2O. The preparation was continuously superfused with a NaCl solution. The diameter of the spiral modiolar artery in NaCl solution displayed little dependence on the applied intravascular pressure. The diameter was 73 +/- 10 microm (n = 5) at 10 cm H2O and increased with pressure to 85 +/- 7 microm (n = 5) at the highest applied pressure (220 or 230 cm H2O). Elevation of the K+ concentration from 3.6 to 150 mM in the superfusate caused a transient vasoconstriction. The amplitude of the K+-induced vasoconstriction depended strongly on the applied intravascular pressure. At 10 cm H2O the amplitude was maximal and the outer diameter decreased transiently by 49 +/- 9% (from 73 +/- 10 to 38 +/- 9 microm; n = 5). The amplitude of K -induced vasoconstriction was nearly maximal at pressures lower than 30 cm H2O, declined at higher pressures, and was not significantly different from zero at pressures larger than 100 cm H2O. These observations in conjunction with an estimation of the intravascular pressures in vivo suggest that cochlear blood flow can be regulated on two levels: (1) cochlear blood flow can be regulated by controlling the vascular diameter of the spiral modiolar artery (intracochlear blood flow regulation) and (2) intracochlear blood flow regulation can be modulated by altering the perfusion pressure which is controlled by the vasculature upstream of the cochlea.

Endothelin-A receptors mediate vasoconstriction of capillaries in the spiral ligament.

The purpose of this study was to determine whether endothelin-1 (ET-1), endothelin-2 (ET-2) or endothelin-3 (ET-3) alter the vascular diameter of capillaries in the spiral ligament. Changes in vascular tone were measured in capillaries from the isolated spiral ligament in vitro. Capillaries were occluded on one end and opened on the other end. Red blood cells trapped in the capillaries served as markers for a luminal volume defined by the red cell itself, the capillary wall and the occluder. Movement of the red cell toward the open end was taken as evidence for vasoconstriction and movement of the red cell toward the occluder was taken as evidence for vasodilation. The inner diameter of the capillaries was 7.0 microm and decreased maximally by a factor of 0.8 in response to ET-1 and ET-2 (both 10(-8) M). Vasoconstriction induced by ET-1 and ET-2 was concentration-dependent in the range between 10(-12) and 10(-8) M whereas ET-3 (10(-8) M) had no effect. The EC50s for ET-1 and ET-2 were 1.2 x 10(-10) M and 1.4 x 10(-9) M, respectively. Thus, the potency order was ET-1 > ET-2 >> ET-3. Vasoconstriction induced by ET-1 and ET-2 was completely inhibited by the competitive antagonist 10(-6) M BQ-123 (cyclic D-Asp-L-Pro-D-Val-L-Leu-D-Trp). Vasoconstriction induced by ET-1 or ET-2 continued for more than 1 min after removal of agonist from the perfusate. Rapid vasodilation of capillaries preconstricted by ET-1 was observed in response to 10(-3) M sodium nitroprusside. Sodium nitroprusside, however, had no significant effect on the vascular diameter of resting capillaries. These results demonstrate that capillaries in the spiral ligament can constrict and the endothelin-mediated vasoconstriction occurs via ET(A) receptors.

[Potassium ion secretion and generation of the endocochlear potential in the stria vascularis]. / Kaliumionensekretion und Entstehung des endokochlearen Potentials in der Stria vascularis.

Central to inner ear research are questions regarding the homeostasis of the high endolymphatic potassium concentration (approximately 150 mmol/l) and the high endocochlear potential (approximately +80 mV). Disturbances of the endocochlear potential can lead to the immediate loss of hearing which may be irreversible. The molecular mechanism leading to the generation of the endocochlear potential has not yet been discovered in spite of its clinical relevance. It is long known, however, that the stria vascularis is responsible for both the generation of the endocochlear potential as well as the secretion of potassium into endolymph. Recent investigations have clarified the mechanisms leading to the secretion of potassium and have led to the formulation of a now widely accepted model. This model explaining potassium secretion as well as the generation of the endocochlear potential is discussed in the present article.

Functional evidence for a monocarboxylate transporter (MCT) in strial marginal cells and molecular evidence for MCT1 and MCT2 in stria vascularis.

The transport of lactate, pyruvate and other monocarboxylates across plasma membranes of metabolically active cells such as strial marginal cells (SMC) may be important under aerobic conditions as well as under ischemic and hypoxic conditions. This study addresses the question whether SMC from the gerbil contain a membrane transport mechanism for monocarboxylates. The type of transporter was identified in functional studies by monitoring uptake of monocarboxylates into SMC through measurement of the cytosolic pH (pHi) with the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Further, subtypes of the functionally identified transporter which are present in stria vascularis were identified as transcripts by cloning and sequencing the reverse-transcription polymerase chain reaction (RT-PCR) products. All functional experiments were performed under nominally HCO3--free conditions. The monocarboxylates acetate and pyruvate (each 20 mM) induced an acidification of pHi. In contrast, the dicarboxylate malonate (20 mM) had no significant effect on pHi. Alpha-cyano-4-hydroxycinnamate (CHC; 5 mM), a blocker of H+/monocarboxylate cotransporter (MCT), reduced reversibly the acidification induced by 5 mM pyruvate. In contrast, 1 microM DIDS, a blocker of band-3 protein, had no significant effect on the acidification induced by 20 mM acetate. The presence of the transcripts for each of the MCT subtypes, MCT1 and MCT2, was determined by RT-PCR of stria vascularis from gerbil. RT-PCR performed with primers for the MCT1 and MCT2 subtypes on total RNA from stria vascularis revealed PCR products of the predicted sizes. Sequence analysis confirmed that amplified MCT1 and MCT2 cDNA fragments encoded a nucleotide sequence of MCT1 and MCT2, respectively. These observations suggest that SMC contain a MCT and that stria vascularis contains RNA for the subtypes MCT1 and MCT2 subtypes.

Inner ear defects induced by null mutation of the isk gene.

The isk gene is expressed in many tissues. Pharmacological evidence from the inner ear suggests that isk mediates potassium secretion into the endolymph. To examine the consequences of IsK null mutation on inner ear function, and to produce a system useful for examining the role(s) IsK plays elsewhere, we have produced a mouse strain that carries a disrupted isk locus. Knockout mice exhibit classic shaker/waltzer behavior. Hair cells degenerate, but those of different inner ear organs degenerate at different times. Functionally, we show that in mice lacking isk, the strial marginal cells and the vestibular dark cells of the inner ear are unable to generate an equivalent short circuit current in vitro, indicating a lack of transepithelial potassium secretion.

K(+)-induced stimulation of K+ secretion involves activation of the IsK channel in vestibular dark cells.

Vestibular dark cells in the inner ear secrete K+ from perilymph containing 4 mM K+ to endolymph containing 145 mM K+. Sensory transduction causes K+ to flow from endolymph to perilymph, thus threatening the homeostasis of the perilymphatic K+ concentration which is crucial for maintaining sensory transduction since the basolateral membranes of the sensory cells and adjacent neuronal elements need to be protected from K(+)-induced depolarization. The present study addresses the questions (1) whether increases in the perilymphatic K+ concentration by as little as 1 mM are sufficient to stimulate KCl uptake across the basolateral membrane of vestibular dark cells, (2) whether K(+)-induced stimulation of KCl uptake causes stimulation of the IsK channel in the apical membrane, and (3) whether the rate of transepithelial K+ secretion depends on the perilymphatic (basolateral) K+ concentration when the apical side of the epithelium is bathed with a solution containing 145 mM K+, as in vivo. Uptake of KCl was monitored by measuring cell height as an indicator for cell volume. The current (IIsK), conductance (gIsK) and inactivation time constant (tau IsK) of the IsK channel as well as the apparent reversal potential of the apical membrane (Vr) were obtained with the cell-attached macro-patch technique. Vr was corrected for the membrane voltage previously measured with microelectrodes. The rate of transepithelial K+ secretion JK was obtained as equivalent short circuit current from measurements of the transepithelial voltage (Vt) and resistance (Rt) measured in the micro-Ussing chamber. Cell height of vestibular dark cells was 7.2 microns (average). Elevations of the extracellular K+ concentration from 3.5 to 4.5 mM caused cell swelling with an initial rate of cell height change of 11 nm/s. With 3.6 mM K+ in the pipette IIsK was outwardly directed and elevation of the extracellular K+ concentration from 3.6 to 25 mM caused an increase of IIsK from 12 to 65 pA, gIsK from 152 to 950 pS and tau IsK from 278 to 583 ms as well as a hyperpolarization of Vr from -50 to -60 mV. With 150 mM K+ in the pipette IIsK was inwardly directed and the elevation of the extracellular K+ concentration caused an increase of IIsK from -1 to -143 pA, gIsK from 141 to 1833 pS and tau IsK from 248 to 729 ms. Vr remained within +/- 10 mV from zero. JK was 4.8 nmol x cm-2 x s-1 when the both the apical side and the basolateral side of the epithelium were perfused with a solution containing 3.5 mM K+. Elevation of the basolateral K+ concentration by 1 mM caused JK to increase by 1.1 nmol x cm-2 x s-1 or 23%. When the basolateral side of the epithelium was perfused with a solution containing 3.5 mM K+ and the apical side with a solution containing 145 mM K+, as in vivo, JK was 0.8 nmol x cm-2 x s-1 and elevation of the basolateral K+ concentration by 1 mM caused JK to increase by 0.8 nmol x cm-2 x s-1 or 100%. These data suggest that physiologically relevant increases in the perilymphatic K+ concentration increase JK by increasing KCl uptake across the basolateral membrane and activation of K+ release via the IsK channel in the apical membrane. Thus, the data demonstrate that vestibular dark cells adjust the rate of K+ secretion into endolymph according to the perilymphatic K+ concentration.

Osmotic water permeability of capillaries from the isolated spiral ligament: new in-vitro techniques for the study of vascular permeability and diameter.

Perilymph is separated from blood by a barrier called the blood-labyrinth or blood-perilymph barrier in analogy to the blood-brain or blood-cerebrospinal fluid barrier. These barriers consist mainly of vascular endothelial cells. To characterize the blood-labyrinth barrier we developed in vitro techniques for the quantitative determination of the osmotic water permeability and for the determination of changes in the diameter of isolated inner ear capillaries. Both techniques rely on measurement of the velocity of marker red cells trapped in the lumen of capillaries. The velocity of marker red cells is a measure for the capillary permeability when a water flux across the capillary wall is induced by an osmotic gradient or a measure for a change in the capillary diameter. With these techniques the osmotic water permeability coefficient (Pf) and the pH sensitivity of isolated capillaries from the spiral ligament of the inner ear was determined. Pf at 23 degrees C was (1.49 +/- 0.17) 10(-3) cm/s at pH 7.4 and (1.61 +/- 0.23) 10(-3) cm/s at pH 6.8 (n = 12: mean +/- SEM: n = number of tissues). Pf at 37 degrees C was (2.26 +/- 0.23) 10(-3) cm/s at pH 7.4 and (2.35 +/- 0.17) 10(-3) cm/s at pH 6.8 (n = 13). No change in capillary diameter was observed when the pH of the interstitial fluid was lowered from pH 7.4 to 6.8. These data demonstrate that Pf and the capillary diameter of spiral ligament capillaries are pH independent and suggest that water crosses the blood-labyrinth barrier via an aqueous pathway. Further, these data suggest that the relatively low Pf is another characteristic shared by the blood-labyrinth and the blood-brain barrier.

Vestibular dark cells contain the Na+/H+ exchanger NHE-1 in the basolateral membrane.

Vestibular dark cells and strial marginal cells transport K+ by similar mechanisms. We have shown that K+ transport in vestibular dark cells is sensitive to the cytosolic pH (pHi) (Wangemann et al. (1995a): J. Membrane Biol. 147: 255-262). The present study addresses pharmacologically the questions whether vestibular dark cells and strial marginal cells from the gerbil contain a Na+/H+ exchanger (NHE) and in which membrane, apical or basolateral, NHE is located. Further, the study addresses the question which NHE subtype is present in vestibular dark cells. pHi was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF), cell volume which is a measure of the net balance between ion influx and efflux was monitored as cell height (CH) and the equivalent short circuit current (Isc) which is a measure of transepithelial K+ secretion was calculated from measurements of the transepithelial voltage (Vt) and the transepithelial resistance (Rt). Changes in pHi were induced by 20 or 40 mM propionate. In the presence of propionate a transient acidification of pHi was observed in vestibular dark cells as well as a subsequent alkalinization to pHi values exceeding those under control conditions. The alkalinization of pHi in the presence of propionate was inhibited by the NHE blockers amiloride and EIPA. Propionate-induced swelling of vestibular dark cells was inhibited by amiloride. The NHE blocker amiloride caused in vestibular dark cells an acidification of pHi and a decrease in CH. Amiloride caused in both vestibular dark cells and strial marginal cells a transient stimulation of Isc when added to the basolateral side but not added to the apical side. Similar effects and pHi were observed in vestibular dark cells with the amiloride analog ethyl-isopropyl-amiloride (EIPA) and similar effects on Isc were observed with EIPA and the NHE blocker HOE694 when applied to the basolateral side of vestibular dark cell epithelium. The IC50 for these basolateral effects of EIPA, HOE694 and amiloride on Isc in vestibular dark cells were 2 x 10(-7) M, 8 x 10(-7) M and 4 x 10(-5) M. These observation suggest that vestibular dark cells and strial marginal cells contain NHE in their basolateral membrane, that K+ transport in strial marginal cells in pHi sensitive similar to K+ transport in vestibular dark cells and that NHE in vestibular dark cells consists of the subtype NHE-1.