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  • 1
    Electronic Resource
    Electronic Resource
    Vestibular Dark Cells Contain an H+/Monocarboxylate− Cotransporter in Their Apical and Basolateral Membrane (1998)
    Springer
    The journal of membrane biology 163 (1998), S. 37-46 
    Details
    ISSN: 1432-1424
    Keywords: Key words: H+/monocarboxylate− cotransporter — BCECF — Vestibular labyrinth — RT-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. 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 (pH i ) and by measuring the pH-sensitive equivalent short circuit current (I sc ). 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 pH i which was inhibited by 5 mmα-cyano-4-hydroxycinnamate (CHC) but not by 1 μm DIDS or 500 μm 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 I sc 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900368
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  • 2
    Electronic Resource
    Electronic Resource
    The pH-sensitivity of transepithelial K+ transport in vestibular dark cells (1995)
    Springer
    The journal of membrane biology 147 (1995), S. 255-262 
    Details
    ISSN: 1432-1424
    Keywords: Labyrinth ; Slowly activating K+ channel ; IsK channel ; MinK channel ; pH
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The pH-sensitivity of transepithelial K+ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pH iwas measured microfluorometrically with the pH-sensitive dye 2′,7′-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (I sc), which is a measure for transepithelial K+ secretion, was calculated from measurements of the transepithelial voltage (V t)and the transepithelial resistance (R t) in a micro-Ussing chamber. All experiments were conducted in virtually HCO 3 − -free solutions. Under control conditions, pH iwas 7.01±0.04 (n=18), V twas 9.1±0.5 mV, R t16.7±0.09 Ωcm2, and I sc was 587±30 μA/cm2 (n=49). Addition of 20 mm propionate− caused a biphasic effect involving an initial acidification of pH i, increase in V tand I sc and decrease in R tand a subsequent alkalinization of pH i, decrease of V tand increase of R t. Removal of propionate− caused a transient effect involving an alkalinization of pH i, a decrease of V tand I sc and an increase in R t. pH iin the presence of propionate− exceeded pH iunder control conditions. Effects of propionate − on V t, R tand I sc were significantly larger when propionate− was applied to the basolateral side rather than to the apical side of the epithelium. The pH i-sensitivityof I sc between pH 6.8 and 7.5 was −1089 μA/(cm2 · pH-unit) suggesting that K+ secretion ceases at about pH i7.6. Acidification of the extracellular pH (pH o)caused an increase of V tand I sc and a decrease of R tmost likely due to acidification of pH i. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pH osensitivity of I sc between pH 7.4 and 6.4 was −155 μA/(cm2 · pH unit). These results demonstrate that transepithelial K+ transport is sensitive to pH iand pH oand that vestibular dark cells contain propionate− uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K+ channel (I sK)in the apical membrane. Whether the effect of pH ion the I sK channel is a direct or indirect effect remains to be determined.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234523
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  • 3
    Electronic Resource
    Electronic Resource
    β1-Adrenergic Receptors but not β2-Adrenergic or Vasopressin Receptors Regulate K+ Secretion in Vestibular Dark Cells of the Inner Ear (1999)
    Springer
    The journal of membrane biology 170 (1999), S. 67-77 
    Details
    ISSN: 1432-1424
    Keywords: Key words: Beta1-adrenergic receptors — Vestibular labyrinth — Ussing chamber — RT-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Receptors were identified pharmacologically in functional studies where K+ secretion was monitored as transepithelial current (I sc ). Further, receptors were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. I sc under control conditions was 796 ± 15 μA/cm2 (n= 329) in gerbilline VDC and 900 ± 75 μA/cm2 (n= 6) in murine VDC. Forskolin (10−5 m) but not 1,9-dideoxy-forskolin increased I sc 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 I sc 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 EC 50 values were (1.4 ± 0.7) × 10−8 m (n= 36), (2.5 ± 1.0) × 10−8 m (n= 31), (1.7 ± 0.7) × 10−7 m (n= 36) and (5 ± 4) × 10−7 m (n= 32), respectively. Propanolol inhibited isoproterenol-induced stimulation of I sc . Atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of I sc with a pKDB of 5.0 × 10−8 m (pK DB = 7.30 ± 0.07, n= 38), 4.4 × 10−8 m (pK DB = 7.36 ± 0.14, n= 37) and 6.8 × 10−12 m (pK DB = 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 β1- and β2-adrenergic receptors but not for β3-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 β1-, β2- and β3-adrenergic receptors, respectively. These results demonstrate that K+ secretion in VDC is under the control of β1- but not β2- or β3-adrenergic receptors or vasopressin-receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002329900538
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  • 4
    Electronic Resource
    Electronic Resource
    DIDS increases K+ secretion through an I sKchannel in apical membrane of vestibular dark cell epithelium of gerbil (1995)
    Springer
    The journal of membrane biology 146 (1995), S. 283-291 
    Details
    ISSN: 1432-1424
    Keywords: I sKpotassium channel ; Min K channel ; Disulfonic stilbene ; Inner ear ; Ion-selective vibrating probe ; Macropatch clamp technique
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Vestibular dark cell epithelium secretes K+ via I sKchannels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (I sc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in I scwas associated with an increase in K+ flux and if the effect was directly on the I sKchannel or indirectly via a cytosolic intermediary. Measurements of transepithelial K+ flux with the K+-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) increased K+ flux by a factor of 1.96±0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (I sK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K+. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K+ secretion through I sKchannels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233948
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  • 5
    Electronic Resource
    Electronic Resource
    Hypo-osmotic challenge stimulates transepithelial k+ secretion and activates apical i sK channel in vestibular dark cells (1995)
    Springer
    The journal of membrane biology 147 (1995), S. 263-273 
    Details
    ISSN: 1432-1424
    Keywords: Regulatory volume decrease ; Slowly activating K+ channel ; Vestibular Labyrinth ; Vibrating probe ; Micro-Ussing chamber ; Patch clamp
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Volume regulation of vestibular dark cells from the gerbilline inner ear in response to a hypoosmotic challenge depends on the presence of cytosolic K+ and Cl−. The present study addresses the questions: (i) whether and by what mechanism K+ is released during volume regulation, (ii) whether the osmolarity of the basolateral medium has an effect on the steady-state rate of transepithelial K+ transport and (iii) whether there is cross-talk between the basolateral membrane responsible for K+ uptake and the apical membrane responsible for K+ release. K+ secretion $$(J_{{\text{K}}^{\text{ + }} ,{\text{probe}}} )$$ and current density (I sc,probe) were measured with vibrating probes in the vicinity of the apical membrane and the transepithelial potential (V t) and resistance (R t) were measured in a micro-Ussing chamber. The equivalent short-circuit current (I sc) was calculated. The current (I IsK), conductance (g IsK) and inactivation time constant (τIsK) of the I sK channel and the apparent reversal potential of the apical membrane (V r) were obtained with the cell-attached macropatch technique. V rwas corrected (V rc) for the membrane voltage (V m) measured separately with microelectrodes. A hypo-osmotic challenge (294 to 154 mosm by removal of 150 mm mannitol) on the basolateral side of the epithelium increased $$J_{K^{\text{ + }} ,{\text{probe}}} $$ and I sc,probe by a factor of 2.7 and 1.6. When this hypo-osmotic challenge was applied to both sides of the epithelium V tand I sc increased from 5 to 14 mV and from 189 to 824 μA/cm2 whereas R tdecreased from 27 to 19 Ω-cm2. With 3.6 mm K+ in the pipette I IsK was outwardly directed, τIsK was 267 msec and the hypo-osmotic challenge caused I IsK and g IsK to increase from 14 to 37 pA and from 292 to 732 pS. V rc hyperpolarized from −44 to −76 mV. With 150 mm K+ in the pipette I IsK was inwardly directed, τIsK was 208 msec and the hypo-osmotic challenge caused I IsK and g IsK to increase in magnitude from 0 to −21 pA and from 107 to 1101 pS. V rc remained unchanged (−2 vs. 1 mV). These data demonstrate that a hypo-osmotic challenge stimulates transepithelial K+ secretion and activates the apical I IsK channel. The hypoosmotically-induced increase in K+ secretion exceeded the estimated amount of K+ release necessary for the maintenance of constant cell volume, suggesting that the rate of basolateral K+ uptake was upregulated in the presence of the hypo-osmotic challenge and that cross-talk exists between the apical membrane and the basolateral membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00234524
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