Styrene alters potassium endolymphatic concentration in a model of cultured utricle explants.

Despite well-documented neurotoxic and ototoxic properties, styrene remains commonly used in industry. Its effects on the cochlea have been extensively studied in animals, and epidemiological and animal evidence indicates an impact on balance. However, its influence on the peripheral vestibular receptor has yet to be investigated. Here, we assessed the vestibulotoxicity of styrene using an in vitro model, consisting of three-dimensional cultured newborn rat utricles filled with a high­potassium (K+) endolymph-like fluid, called "cysts". K+ entry in the cyst ("influx") and its exit ("efflux") are controlled by secretory cells and hair cells, respectively. The vestibular epithelium's functionality is thus linked to K+ concentration, measured using a microelectrode. Known inhibitors of K+ efflux and influx validated the model. Cysts were subsequently exposed to styrene (0.25; 0.5; 0.75 and 1 mM) for 2 h or 72 h. The decrease in K+ concentration measured after both exposure durations was dose-dependent, and significant from 0.75 mM styrene. Vacuoles were visible in the cytoplasm of epithelial cells from 0.5 mM after 2 h and from 0.25 mM after 72 h. The results presented here are the first evidence that styrene may deregulate K+ homeostasis in the endolymphatic space, thereby altering the functionality of the vestibular receptor.

In Vitro Survival of Human Mesenchymal Stem Cells is Enhanced in Artificial Endolymph with Moderately High Concentrations of Potassium.

While mesenchymal stem cells (MSCs) are promising candidates for inner ear hair cell regeneration, to date, there have been no convincing reports indicating whether MSCs can survive in the cochlea for more than a few weeks, as the high levels of potassium (K+) in the endolymph (EL) are thought to be toxic to transplanted stem cells. For conditioning the EL for MSC transplantation, we conducted this in vitro study to examine the effects of artificial EL with altered K+ concentration levels, in the range of 5-153.8 mM, on proliferation, apoptosis, and morphological changes in MSCs derived from various human tissues. Our findings demonstrate that altering the K+ concentration in artificial EL could significantly influence the survival of MSCs in vitro. We discovered that K+ concentrations of 55-130 mM in artificial EL could enhance the survival of MSCs in vitro. However, MSCs exhibited reduced proliferation regardless of K+ concentration.

Glucocorticoids stimulate endolymphatic water reabsorption in inner ear through aquaporin 3 regulation.

Menière's disease, clinically characterized by fluctuating, recurrent, and invalidating vertigo, hearing loss, and tinnitus, is linked to an increase in endolymph volume, the so-called endolymphatic hydrops. Since dysregulation of water transport could account for the generation of this hydrops, we investigated the role of aquaporin 3 (AQP3) in water transport into endolymph, the K-rich, hyperosmotic fluid that bathes the apical ciliated membrane of sensory cells, and we studied the regulatory effect of dexamethasone upon AQP3 expression and water fluxes. The different AQP subtypes were identified in inner ear by RT-PCR. AQP3 was localized in human utricle and mouse inner ear by immunohistochemistry and confocal microscopy. Unidirectional transepithelial water fluxes were studied by means of (3)H2O transport in murine EC5v vestibular cells cultured on filters, treated or not with dexamethasone (10(-7) M). The stimulatory effect of dexamethasone upon AQP3 expression was assessed in EC5v cells and in vivo in mice. AQP3 was unambiguously detected in human utricle and was highly expressed in both endolymph secretory structures of the mouse inner ear, and EC5v cells. We demonstrated that water reabsorption, from the apical (endolymphatic) to the basolateral (perilymphatic) compartments, was stimulated by dexamethasone in EC5v cells. This was accompanied by a glucocorticoid-dependent increase in AQP3 expression at both messenger RNA (mRNA) and protein level, presumably through glucocorticoid receptor-mediated AQP3 transcriptional activation. We show that glucocorticoids enhance AQP3 expression in human inner ear and stimulate endolymphatic water reabsorption. These findings should encourage further clinical trials evaluating glucocorticoids efficacy in Menière's disease.

P2Y4-mediated regulation of Na+ absorption in the Reissner's membrane of the cochlea.

The epithelial cells of Reissner's membrane (RM) are capable of transporting Na(+) out of endolymph via epithelial Na(+) channel (ENaC). However, much remains to be known as to mechanism of regulation of Na(+) absorption in RM. We investigated P2Y signaling as a possible regulatory mechanism of ENaC in gerbil RM using voltage-sensitive vibrating probe technique and immunohistochemistry. Results showed that UTP induced partial inhibition of the amiloride-sensitive short-circuit current but did not change short-circuit current when applied in the presence of amiloride. The inhibitory effect of UTP was not completely reversible in minutes. The response to UTP was inhibited by reactive blue-2 and 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate but not by suramin or pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid, which indicates this P2Y receptor as the P2Y(4) subtype. The phospholipase C (PLC) inhibitors 1-[6[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine markedly inhibited the effect of UTP on ENaC. In contrast, neither modulation of protein kinase C nor application of 2-aminoehoxydiphenyl borate affected P2Y(4)-mediated inhibition of ENaC. Immunoreactive staining for P2Y(4) was observed in the RM, apical membrane of stria vascularis, spiral ligament, and organ of Corti, including outer hair cell, inner hair cell, outer pillar cell, Deiters' cell, and Hensen cell. These results suggest that the physiological role of P2Y(4) receptor in RM is likely to regulate Na(+) homeostasis in the endolymph. The acute inhibition of ENaC activity by activation of P2Y(4) receptor is possibly mediated by decrease of phosphatidylinositol 4,5-biphosphate in the plasma membrane through PLC activation.

Endolymphatic sac is involved in the regulation of hydrostatic pressure of cochlear endolymph.

To clarify the role of the endolymphatic sac (ES) in the regulation of endolymphatic pressure, the effects of isoproterenol, a beta-adrenergic receptor agonist, and acetazolamide, a potent carbonic anhydrase inhibitor, both of which decrease ES direct current potential on cochlear hydrostatic pressure, were examined in guinea pigs. When isoproterenol was applied intravenously, hydrostatic pressures of cochlear endolymph and perilymph were significantly increased with no change in endocochlear potential or the hydrostatic pressure of cerebrospinal fluid. Acetazolamide produced no marked change in the hydrostatic pressure of cochlear endolymph. In ears with an obstructed ES, the action of isoproterenol on the hydrostatic pressure of cochlear endolymph and perilymph was suppressed. These results suggest that the ES may regulate the hydrostatic pressure of the endolymphatic system via the action of the agents such as catecholamines on the ES.

Endolymphatic hydrops and therapeutic effects are visualized in 'atypical' Meniere's disease.

A 53-year-old male with fluctuating low frequency sensorineural hearing loss and tinnitus, but without vertigo, was evaluated by MRI obtained by intratympanic injection of a gadolinium-based contrast agent (GBCA) before and after the administration of isosorbide. The endolymphatic hydrops was semi-quantitatively evaluated by a 3.0-T MR scanner. For quantification, the affected side/contralateral side ratios were calculated. A gadodiamide (a kind of GBCA)-enhanced space surrounding the endolymph in the affected side with a 0.50 ratio (which may have represented endolymphatic hydrops) improved after isosorbide therapy to a 0.98 ratio. Thus, endolymphatic hydrops was demonstrated in a patient with 'atypical' Meniere's disease (MD), suggesting that at least some atypical MD may share similar etiology with, and therefore be a continuum of, MD. Also, therapeutic effects could be visualized by using MRI. Therefore, MRI-based diagnosis of MD-related disease will be a powerful tool not only because of its precision but also its usefulness for therapeutic evaluation.

Ca(2+) regulation of endocochlear potential in marginal cells.

We examined the effect of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca(2+) chelator or inhibitors of Ca(2+)-permeable channels or Ca(2+)-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca(2+) channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca(2+)-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the gamma subunit of epithelial Na(+) channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 muM amiloride or 10 muM phenamil. Taken together, these findings suggest that [Ca(2+)](c) regulated by endoplasmic Ca(2+)-pump and Ca(2+)-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na(+) across the basolateral membrane in marginal cells.

TRPV4 enhances the cellular uptake of aminoglycoside antibiotics.

The cochlea and kidney are susceptible to aminoglycoside-induced toxicity. The non-selective cation channel TRPV4 is expressed in kidney distal tubule cells, and hair cells and the stria vascularis in the inner ear. To determine whether TRPV4 is involved in aminoglycoside trafficking, we generated a murine proximal-tubule cell line (KPT2) and a distal-tubule cell line (KDT3). TRPV4 expression was confirmed in KDT3 cells but not in KPT2 cells. Removal of extracellular Ca(2+) significantly enhanced gentamicin-Texas-Red (GTTR) uptake by KDT3, indicative of permeation through non-selective cation channels. To determine whether TRPV4 is permeable to GTTR, stable cell lines were generated that express TRPV4 in KPT2 (KPT2-TRPV4). KPT2-TRPV4 cells took up more GTTR than control cell lines (KPT2-pBabe) in the absence of extracellular Ca(2+). TRPV4-dependent GTTR uptake was abolished by a point mutation within the crucial pore region of the channel, suggesting that GTTR permeates the TRPV4 channel. In an endolymph-like extracellular environment, clearance of GTTR was attenuated from KPT2-TRPV4 cells in a TRPV4-dependent fashion. We propose that TRPV4 has a role in aminoglycoside uptake and retention in the cochlea.

Simulation of cupulolithiasis and canalolithiasis by an animal model.

The physical mechanisms responsible for cupulolithiasis and canalolithiasis have been investigated by two groups of experiments in isolated posterior semicircular canal (SCC) of frog (Rana esculenta L.). First, clouds of 10-30 isolated otoconia were let to fall (snowfall of otoconia) either through the ampulla onto the cupula, or inside the long arm of the canal, opposite to the cupula. Second, microspheres ranging 30 to 350 microm in diameter were gently moved to and fro inside the long arm of the canal by a micromanipulator. The effects were evaluated by recording the firing rate (Nfr) of the ampullary nerve. Snowfall of otoconia produced detectable changes of Nfr only when otoconia got in contact with the cupula, but not when falling through the endolymph. Movement of the microspheres in the canal long arm induced Nfr changes only if the microsphere diameter exceeded about 50 microm. Although the exact microsphere size needed for receptor stimulation may depend on the experimental conditions, these results strongly suggest that debris moving inside a SCC (canalolithiasis) can produce transcupular pressures able to stimulate ampullar receptors only if they have suitable size, whereas isolated otoconia cannot, except when lying on the cupula (cupulolithiasis).

Does BAPTA leave outer hair cell transduction channels closed?

The calcium chelator BAPTA was iontophoresed into the scala media of the second turn of the guinea pig cochlea. This produced a reduction in low frequency cochlear microphonic (CM) measured in scala media and an elevation of the cochlear action potential (CAP) threshold that lasted for the duration of the experiment. Using two pipettes, one filled with KCl and the other KCl and BAPTA (50, 20 and 5 mM) it was possible to observe the effect of passing current through one electrode while measuring the endolymphatic potential (EP) with the other. The results demonstrated that current passed via the BAPTA pipette caused a sustained increase in EP of 8.2, 12.9 and 7.8 mV in the three animals used. This increase coincided with the decrease in low frequency CM that indicated a causal connection between the two. In a second series of experiments, pipettes with larger tips were inserted into scala media in the first cochlear turn and BAPTA was allowed to diffuse from the pipette. The results confirmed the relationship between EP increase and the fall of scala media CM. One interpretation of these results is that lowering the Ca2+ concentration of endolymph with BAPTA inhibits mechano-electrical transduction in outer hair cells (OHCs) and leaves the hair cell transduction channels in a closed state, thus increasing the resistance across OHCs and increasing the EP. These findings are consistent with a model of hair cell transduction in which tension on stereo cilia opens the transduction channels.

Effects of CO2/HCO3- in perilymph on the endocochlear potential in guinea pigs.

The effect of CO(2)/HCO(3)(-) on the endocochlear potential (EP) was examined by using both ion-selective and conventional microelectrodes and the endolymphatic or perilymphatic perfusion technique. The main findings were as follows: (i) A decrease in the EP from approximately +75 to approximately +35 mV was produced by perilymphatic perfusion with CO(2)/HCO(3)(-)-free solution, which decrease was accompanied by an increase in the endolymphatic pH (DeltapH(e), approximately 0.4). (ii) Perilymphatic perfusion with a solution containing 20 mM NH(4)Cl produced a decrease in the EP (DeltaEP, approximately 20 mV) with an increase in the pH(e) (DeltapH(e), approximately 0.2), whereas switching the perfusion solution from the NH(4)Cl solution to a 5% CO(2)/25 mM HCO(3)(-) solution produced a gradual increase in the EP to the control level with the concomitant recovery of the pH(e). (iii) The perfusion with a solution of high or low HCO(3)(-) with a constant CO(2) level within 10 min produced no significant changes in the EP. (iv) Perfusion of the perilymph with 10 microg/ml nifedipine suppressed the transient asphyxia-induced decrease in EP slightly, but not significantly. (v) By contrast, the administration of 1 microg/ml nifedipine via the endolymph inhibited significantly the reduction in the EP induced by transient asphyxia or perilymphatic perfusion with CO(2)/HCO(3)(-)-free or 20 mM NH(4)Cl solution. These findings suggest that the effect of CO(2) removal from perilymphatic perfusion solution on the EP may be mediated by an increase in cytosolic Ca(2+) concentration induced by an elevation of cytosolic pH in endolymphatic surface cells.

Effects of lithium on endolymph homeostasis and experimentally induced endolymphatic hydrops.

There is evidence to suggest that water homeostasis in the inner ear is regulated via the vasopressin (VP)-aquaporin 2 (AQP2) system in the same fashion as in the kidney. The VP-AQP2 system in the kidney is well known to be inhibited by lithium, resulting in polyuria due to a decrease in reabsorption of water in the collecting duct of the kidney. Therefore, lithium is also likely to inhibit the VP-AQP2 system in the inner ear, and consequently exert some influence on inner ear fluid homeostasis. In this study, we investigated the effects of lithium on AQP2 expression in the rat inner ear, and on the cochlear fluid volume in hydropic ears of guinea pigs. A quantitative PCR study revealed that lithium reduced AQP2 mRNA expression in the cochlea and endolymphatic sac. Lithium application also decreased the immunoreactivity of AQP2 in the cochlea and endolymphatic sac. In a morphological study, lithium intake significantly reduced endolymphatic hydrops dose-dependently. These results indicate that lithium acts on the VP-AQP2 system in the inner ear, consequently producing a dehydratic effect on the endolymphatic compartment.

[Effect of adriamycin on expression of mdr1 gene in guinea pig cochlea lateral wall].

OBJECTIVE: To study whether ADM affect the expression of mdr1 gene in guinea pig cochlea lateral wall. METHOD: Sixty white guinea pigs with red eyes were divided into three groups: control group, NS group and ADM group. Guinea pigs were administered an intravenous injection of 30 mg/kg ADM. Four hoars later,ten animals were then immediately sacrificed, and the bony labyrinths were removed. The remaining ten animals were sacrificed after 24 h. The effect of ADM, an ototoxicity drug, on expression of mdrl gene in guinea pigs cochlea lateral walls were observed with RT-PCR. Furthermore, the ADM concentration in endolymph were measured by high performance liquid chromatography. RESULT: For ADM group, the density ratio of mdr1 to beta-action of the 24 h was significantly greater compared to that of the 4 h or controls; In contrast, the ADM concentration of 4 h was significantly higher than that of the 24 h. CONCLUSION: mdr1 gene in normal guinea pig cochlea lateral wall is a stress gene. It can be induced by ADM; it may be not only related to reduce the accumulation of ototoxicity drug in endolymph, but also associated with the protective function of inner ear.

BAPTA induces frequency shifts in vivo of spontaneous otoacoustic emissions of the bobtail lizard.

Spontaneous otoacoustic emissions (SOAEs) are indicators of active processes in the inner ear and are found in all classes of land vertebrates. In the Australian bobtail lizard, earlier work showed that otoacoustic emissions are generated by an active motility process in the hair-cell bundle. This is likely to be driven by calcium-sensitive mechanisms implicated in other non-mammalian hair cell systems. If so, it should be fundamentally influenced by the extracellular calcium concentration. In in vitro studies, the rate of force generation in hair cell stereovilli is linked to the extracellular calcium concentration. In such preparations, low-calcium solutions, buffered by the calcium chelator BAPTA, were reported to change the frequency of hair cell bundle oscillations. In the present study, BAPTA was iontophoresed into the endolymph of the bobtail skink in vivo, and SOAEs were monitored. Application of BAPTA resulted in a prolonged downward shift in the frequency of individual SOAE spectral peaks. Recovery took more than 1 h, consistent with a slow clearance of BAPTA from endolymph. SOAE peak amplitudes were most often enhanced, suggesting there was no functional disruption of tip links. The direction and degree of frequency shifts were consistent with in vitro and in vivo data showing the effects of changing calcium concentrations in the endolymph directly.

Endolymphatic perfusion with EGTA-acetoxymethyl ester inhibits asphyxia- and furosemide-induced decrease in endocochlear potential in guinea pigs.

We examined the effect of the Ca(2+) concentration in the endolymph ([Ca](e)) or in the endolymphatic surface cells ([Ca](i)) on the endocochlear potential (EP) by using an endolymphatic or perilymphatic perfusion technique, respectively. (i) A large increase in [Ca](e) up to approximately 10(-3) M with a fall in the EP was induced by transient asphyxia ( approximately 2 min) or by the intravenous administration of furosemide (60 mg/kg), and a significant correlation was obtained between the EP and p[Ca](e) (= -log [Ca](e), r = 0.998). (ii) Perfusion of the endolymph with 10 mM EGTA for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP (DeltaEP(asp)), suggesting that neither [Ca](e) nor the Ca(2+) concentration gradient across the stria vascularis contributed directly to the generation of the EP in the condition of low [Ca](e). In contrast, endolymphatic perfusion with high Ca(2+) (more than 10 mM) produced a decrease in EP and a significant correlation was obtained between the EP and the Ca(2+) concentration of perfusion solution (r = 0.982), suggesting that Ca(2+) permeability may exist across the stria vascularis. (iii) The administration of a Ca(2+) chelator, EGTA-acetoxymethyl ester (AM, 0.3 mM), to the endolymph, which produced a gradual increase in EP, suppressed significantly, by 60-80%, DeltaEP(asp) or furosemide-induced changes in EP. In contrast, perilymphatic administration of 0.5 mM EGTA-AM caused no significant suppression of the DeltaEP(asp). These findings suggest that [Ca](i) plays an important role in generating/maintaining a large positive EP.

Ca2+ current-driven nonlinear amplification by the mammalian cochlea in vitro.

An active process in the inner ear expends energy to enhance the sensitivity and frequency selectivity of hearing. Two mechanisms have been proposed to underlie this process in the mammalian cochlea: receptor potential-based electromotility and Ca(2+)-driven active hair-bundle motility. To link the phenomenology of the cochlear amplifier with these cellular mechanisms, we developed an in vitro cochlear preparation from Meriones unguiculatus that affords optical access to the sensory epithelium while mimicking its in vivo environment. Acoustic and electrical stimulation elicited microphonic potentials and electrically evoked hair-bundle movement, demonstrating intact forward and reverse mechanotransduction. The mechanical responses of hair bundles from inner hair cells revealed a characteristic resonance and a compressive nonlinearity diagnostic of the active process. Blocking transduction with amiloride abolished nonlinear amplification, whereas eliminating all but the Ca(2+) component of the transduction current did not. These results suggest that the Ca(2+) current drives the cochlear active process, and they support the hypothesis that active hair-bundle motility underlies cochlear amplification.

Time course of dehydrating effects of isosorbide on experimentally induced endolymphatic hydrops in guinea pigs.

Osmotic diuretics are therapeutic agents used to reduce endolymphatic hydrops. However, glycerol-induced change in endolymph volume is followed by a rebound phenomenon. In this study, we investigated the rebound phenomenon occurring with isosorbide, an osmotic diuretic used as a therapeutic agent for Ménière's disease in Japan. Forty guinea pigs underwent surgical obliteration of the endolymphatic sac. Thirty received isosorbide orally 1 month after surgery. These animals were sacrificed 3, 6, or 12 h after isosorbide intake. The remaining 10 animals served as controls. Quantitative assessment of changes in the endolymphatic space was performed light-microscopically. Isosorbide reduced cochlear endolymph volume, with a peak reduction 6 h after intake. Thereafter, no prominent rebound phenomenon was noted. Clinically, since isosorbide is orally administered every 8 h, rebound phenomenon need not be considered in the treatment with isosorbide.

Endolymphatic sodium homeostasis by Reissner's membrane.

Cochlear sensory transduction depends on active extrusion of sodium ion (Na(+)) from the luminal fluid, endolymph. Reissner's membrane epithelium forms much of the barrier between cochlear endolymph and perilymph and we hypothesized that Reissner's membrane might be responsible for this function. We found that Reissner's membrane isolated from gerbil produced a short circuit current (I(sc)) directed into the apical side, consistent with cation absorption and/or anion secretion. I(sc) was inhibited by amiloride analogs in the potency sequence benzamil>amiloride>>ethylisopropylamiloride, consistent with Na(+) absorption through an epithelial sodium channel in the apical cell membrane. I(sc) was also inhibited by an inhibitor of Na(+),K(+)-ATPase, ouabain, and by the K(+) channel blockers Ba(2+), 4-aminopyridine and quinine but not tetraethylammonium nor glibenclamide, consistent with the presence of a voltage-activated K(+) channel. Bumetanide, an inhibitor of the Na(+),2Cl(-),K(+)-cotransporter, had no effect on I(sc). Contrary to previous hypotheses, no evidence was found for electrogenic secretion of Cl(-) under control of cAMP since neither forskolin nor genistein affected I(sc) when Na(+) absorption was blocked. These results provide the first direct evidence that Reissner's membrane contributes to normal cochlear function by absorption of Na(+) from endolymph.

Asphyxia and diuretic-induced changes in the Ca2+ concentration of endolymph.

Using Ca2+ -selective microelectrodes based on the neutral carrier, ETH-1001 with polyvinyl chloride (PVC), we have measured changes in the free Ca2+ concentration of guinea pig cochlear endolymph ([Ca](e)) after transient asphyxia or intravenous administration of diuretics. Under the control conditions, the endocochlear potential (EP) was +80 mV, and the [Ca](e) was in the range 1.4 x 10(-7)-2.4 x 10(-6) M (n = 16). Transient asphyxia (1-1.5 min) produced an increase in the [Ca](e) with a fall in the EP, whereas the cessation of the asphyxia led to a quick recovery of both [Ca](e) and EP to their control levels. Intravenous administration of furosemide (60 mg/kg) or bumetanide (30 mg/kg) also caused an increase in the [Ca](e) with a fall in the EP, followed by a gradual recovery of both [Ca](e) and EP. From these results, we obtained a significant correlation between EP and p[Ca](e) (= -log[Ca](e)), and conclude that (1) the [Ca](e) is extremely low, around 10(-6) M or less, under normal conditions and (2) the [Ca](e) is directly correlated with EP under physiological conditions.

Otolith growth in trout Oncorhynchus mykiss: supply of Ca2+ and Sr2+ to the saccular endolymph.

Kinetic and pharmacological characteristics of Ca2+ fluxes across the saccular epithelium of trout were studied using a perfused isolated inner ear. 45Ca2+influx from the Ringer solution to the endolymph was 3-4 nmoles h(-1)microl(-1) endolymph, which corresponds to a global turnover rate of the endolymph calcium of 200 % h(-1). Ca2+ entry into the proximal endolymph was faster than into the distal fluid. Net Ca2+ movement across the saccular epithelium depended on the direction and intensity of the chemical gradient of calcium between the Ringer solution and the endolymph. Increasing the calcium concentration in the Ringer solution up to 4.4 mmol l(-1) provoked an accumulation of Ca2+ in both proximal and distal endolymphs, and equilibrium was reached about 30 min after the beginning of perfusion. Perfusion with calcium-free Ringer partially emptied the proximal compartment of calcium, whereas the calcium levels in the distal endolymph did not vary during 70 min of perfusion. Verapamil (10(-5) mol l(-1)) and cyanide (CN, 10(-3) mol l(-1)) did not modify the accumulation of Ca2+ within the endolymph in the presence of a favourable calcium chemical gradient. Furthermore the relationship between Ca2+ net fluxes and the chemical calcium gradient across the saccular epithelium was linear, indicating a passive diffusional mechanism via a paracellular pathway. Similar relationships were found for Sr2+ fluxes across the saccular epithelium in the presence of positive chemical gradients (1, 2 and 4 mmol l(-1) Sr2+). In vivo experiments in which trout were intraperitoneously injected with CaCl2 solution confirmed the tight relationship between the calcium levels in plasma and endolymph (both proximal and distal). Sampling proximal and distal endolymphs in trout and turbot saccules revealed a decreasing proximo-distal calcium gradient in endolymph of both fish species. The present results strongly suggest that the endolymph is supplied with Ca2+ and Sr2+ via a paracellular pathway located in the proximal area of the saccular epithelium.