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Am J Physiol Cell Physiol 273: C2022-C2029, 1997;
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Vol. 273, Issue 6, C2022-C2029, December 1997

P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells

Daniel C. Marcus1, Hiroshi Sunose1, Jianzhong Liu1, Zhijun Shen1, and Margaret A. Scofield2

1 Biophysics Laboratory, Boys Town National Research Hospital, Omaha 68131; and 2 Molecular Pharmacology Laboratory, Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+ (IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current (Isc) is inhibited by agonists of the apical P2U (P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). Measurements of relative K+ flux (JK) with a self-referencing K+-selective probe demonstrated a decrease in JK after apical perfusion of 100 µM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsK channel current (IIsK) by 83 ± 7% during pipette perfusion of 10 µM ATP. The magnitude of the decrease of Isc by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased IIsK by 79 ± 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4alpha -PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell IIsK . The expression of the isk gene transcript was confirmed, and the serine responsible for the species-specific response to PKC was found to be present in the gerbil IsK sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model.

P2Y2 receptor; phospholipase C; perforated-patch whole cell voltage clamp; minK channel; gerbil; self-referencing probe; slowly activating potassium channel

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

VESTIBULAR DARK CELLS (VDC) secrete K+ in the vestibular labyrinth to a luminal concentration that is unusually high for a vertebrate extracellular space (~140 mM) and the luminal K+ is used to carry the transduction current through the sensory hair cells. The constitutive ion transport mechanisms employed by VDC to take K+ across the basolateral membrane and to secrete it across the apical membrane have been established (see Ref. 15 for review). These include the Na+-K+-ATPase, Na+-Cl--K+ cotransporter, and Cl- channels in the basolateral membrane and K+-selective channels of the slowly activating K+ (IsK, or minK) type in the apical membrane. IsK channels have been shown in other systems to consist of IsK regulatory and KvLQT1 channel subunits (2, 23).

The transepithelial short-circuit current (Isc) has been shown to be accounted for by electrogenic K+ secretion (18, 19) and has recently been found to be reduced in the presence of apically perfused extracellular nucleotides (13). The mediator of this effect was determined to be a purinergic receptor of the P2U (P2Y2) subtype in the apical membrane. The present experiments were designed to test the hypothesis that activation of the apical P2U receptors results in inhibition of the K+ secretory flux (JK) by inhibition of the current through the apical IsK channels and to determine the signal pathway between these two events. In other systems, P2U receptors are coupled to phospholipase C (PLC), which activates parallel signal pathways, resulting in elevation of cytosolic Ca2+ via inositol 1,4,5-trisphosphate (InsP3) and in phosphorylation of effector proteins via protein kinase C (PKC) (4, 6). The present results are consistent with the phosphorylation of the IsK channel by PKC as the signal mechanism of the apical P2U receptor, rather than elevation of cytosolic Ca2+.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Tissue preparations. Gerbils were anesthetized with pentobarbital sodium (50 mg/kg, ip) and decapitated. The temporal bone was removed, and VDC epithelium was dissected without enzymatic treatment at 4°C in solution 2 (Table 1) as described previously (31). The tissue was either transferred to a recording chamber continuously perfused at 37°C or frozen in liquid nitrogen within 10 min of death for reverse transcription-polymerase chain reaction (RT-PCR). The heart was cut and frozen on dry ice or liquid nitrogen within 5 min of death.

Solutions and chemicals. The compositions of solutions are listed in Table 1. Nystatin (200 µg/ml; Sigma, St. Louis, MO) was dissolved with sonication in the pipette solution (solution 4, Table 1) just before use. ATP (Sigma) was dissolved directly in pipette and bath solution, whereas U-73122, U-73343 (BioMol, Plymouth Meeting, PA), A-23187, phorbol 12-myristate 13-acetate (PMA), 4alpha -phorbol 12,13-didecanoate (4alpha -PMA) (Calbiochem, La Jolla, CA), and GF109203X (Tocris Cookson, St. Louis, MO) were predissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1%. All other chemicals for the electrophysiological experiments were purchased from Sigma or Fluka (Ronkonkoma, NY).

Self-referencing K+-selective probe. The self-referencing probe techniques used were nearly identical to those previously described (14, 18). Signals were sampled from the probe amplifier with a 16-bit analog-to-digital convertor (CIO-DAS1602/16, ComputerBoards, Mansfield, MA), and the probe was moved on manipulators (Applicable Electronics, Forest Dale, MA) by a 486-based computer and specialized probe software (ASET version 1.0, Science Wares, East Falmouth, MA). Tissues were mounted in the micro-Ussing chamber, and the relative JK was monitored at 24°C with a K+-selective microelectrode moved with an excursion of 25 µm perpendicular to the plane of the tissue (10, 11). The electrode voltage was sampled at 200 Hz for 1 s at each position after a 1-s waiting period and without automatic amplifier feedback. The microelectrodes were constructed from borosilicate glass capillary (1.5 mm OD, 0.84 mm ID) pulled to a tip of ~3 µm ID and silanized with hexamethyldisilazane. The tips contained a column of K+-selective ligand (no. 60398, Fluka Chemical) ~150 µm long, and the electrode was backfilled with 100 mM KCl and 0.5% agar. The reference was Ag/AgCl with a bridge of 3 M NaCl and 3% agar. Electrodes were only used if the slope was at least 56 mV/decade in 1 and 10 mM KCl solutions. The probe was located near the thin connective tissue overlying the basal membrane of the VDC epithelium such that the signal under control conditions was >30 times the noise level. Data are expressed as voltage difference detected by the K+-selective electrode and summarized as percent of the reading under experimental conditions compared with that under control conditions.

                              
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  		Table 1.   Electrophysiological solutions

PCR amplification, subcloning, and sequencing. Genomic DNA for the mouse and rat IsK consists of a single exon encoding the entire translated region (open-reading frame) and the 3'-untranslated region and two alternate exons, which are generated from multiple transcription start sites and encode the 5'-untranslated region (12). A set of primers [sense: 5'-ACC CTG GGC ATC ATG CTG AG-3', anti-sense: 5'-GCC GCC TGG TTT TCA ATG AC-3'] was designed (A. F. Ryan, Univ. of California, La Jolla, CA) and synthesized on an Applied Biosystems 392 oligonucleotide synthesizer (Creighton Molecular Biology Core Facility). The sequence of the primers was based on the known sequences in the coding region of the rat and mouse and encompassed the consensus sequence for phosphorylation by PKC (S/T X R/K). The primers were expected to yield an RT-PCR product of 170 base pairs (bp).

Extraction of total RNA. Total RNA was extracted from samples of gerbil heart, blood, and vestibular labyrinth (mostly ampullae and utricle) by methods similar to those described previously (24). Within 5 min of death, the hearts were frozen in liquid nitrogen and pulverized in liquid nitrogen, and 100 mg of the powder were immediately transferred into 1 ml TRIzol reagent (GIBCO BRL, Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The total RNA was then extracted using TRIzol reagent according to the manufacturer's procedure. Total RNA was precipitated by isopropanol and dissolved in ribonuclease (RNase)-free water (diethyl pyrocarbonate-treated water). The nucleic acid concentration was determined spectrophotometrically, the integrity of the RNA was determined by the presence of 28S and 18S ribosomal RNA bands by horizontal agarose (1%) gel electrophoresis, and the final nucleic acid concentration was adjusted to ~1-2 µg/µl. RNA samples were stored at -70°C.

Vestibular labyrinth from gerbil was isolated by microdissection, and tissues were frozen in liquid nitrogen within 12 min of death of each animal. Tissues from eight ears were pooled in TRIzol reagent and were triturated through a 25-gauge needle. Total RNA was then extracted according to the manufacturer's procedure. Total RNA from 100 µl of blood was also isolated using 1 ml of TRIzol reagent. The final nucleotide concentration of the RNA samples from vestibular labyrinth and blood was ~0.3 and 0.25 µg/µl, respectively. Directly before the RT-PCR procedure, residual genomic DNA in RNA samples from heart, vestibular labyrinth, and blood was removed by treatment with amplification-grade RNase-free deoxyribonuclease I (GIBCO BRL, Life Technologies) for 30 min at room temperature followed by heat inactivation in the presence of EDTA, according to the protocol specified by the manufacturer.

cDNA synthesis and PCR amplification. Total RNA was reverse transcribed into cDNA in a 10-µl reaction. The reaction contained 0.1-0.5 µg total RNA, 10 units RNasin (Promega), 1 mM dNTP (GIBCO BRL, Life Technologies), 25 units Moloney murine leukemia virus (MMLV) reverse transcriptase (Perkin-Elmer), 2.5 mM MgCl2 (GIBCO BRL, Life Technologies), 25 pmol oligo(dT), 20 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, and 50 mM KCl. Tris · HCl and KCl were added from a 10× PCR buffer (GIBCO BRL, Life Technologies). The RT reaction was incubated at room temperature for 10 min, at 42°C for 50 min, at 99°C for 5 min, and at 5°C for 5 min.

The 50-µl PCR mixture contained the 10 µl RT reaction mixture in addition to 25 pmol each of antisense and sense primers for IsK and 1.25 units Taq DNA polymerase (GIBCO BRL, Life Technologies). The final concentrations of MgCl2, KCl, and Tris · HCl were adjusted to 2.5, 50, and 20 mM, respectively. The PCR mixture was incubated as follows: 1 denaturation cycle for 3 min at 95°C; 45 amplification cycles consisting of denaturation for 1 min at 95°C, annealing for 1 min at 63°C, and extension for 1 min at 72°C; and one extension cycle for 5 min at 72°C in a Perkin-Elmer thermocycler 480. PCR products were analyzed by horizontal electrophoresis in 2.5% agarose gels and visualized by ethidium bromide.

Cloning and sequencing of amplified cDNA fragments. Amplified cDNA fragments were extracted from the agarose gels using the QIA quick gel extraction kit (Qiagen) and cloned into a pCR2.1 vector with a TA cloning kit (Invitrogen). Recombinant plasmids were isolated from the colonies using the standard alkaline lysis procedure, purified by phenol/chloroform extraction, and precipitated and washed with ethanol. Insertion of the PCR product into the plasmid was confirmed by restriction endonuclease digestion with EcoR I and subsequent horizontal gel electrophoresis. The recombinant double-stranded plasmid served as a template for cycle sequencing using M13 forward and reverse primers and fluorescence-based dideoxy nucleotides (PRISM Ready Reaction dye deoxy terminator cycle sequencing kit, Perkin Elmer). The sequence was then determined using the ABI model 373 DNA sequencer (Applied Biosystems, Creighton Molecular Biology Core Facility).

Micro-Ussing chamber. The methods were described previously (16). Briefly, tissue was placed in a micro-Ussing chamber, and the seal to the aperture (80 µm diameter) between two hemichambers was made with the apical side of the VDC epithelium. The apical and basolateral sides of the tissue were perfused independently, and exchange of solution (24 or 37°C) on each side was complete within 1 s. Transepithelial voltage (Vt) was measured between calomel electrodes connected to the hemichambers via flowing 1 M KCl bridges. Transepithelial resistance (Rt) was obtained from the voltage change induced by current pulses (50 nA for 34 ms at 0.3 Hz). Sample and hold circuitry was used to obtain a signal proportional to Rt. The equivalent Isc was derived from Vt and Rt (Isc = Vt/Rt). Isc and Rt were normalized for the area defined by the aperture of the micro-Ussing chamber.

Macropatch clamp. The macropatch technique was described previously (17). Pipettes (3-6 µm ID) were made from Corning 7052 glass capillary with a two-stage puller and a microforge. Pipette tips were coated with a 2:1 mixture of alpha -tocopherol acetate and heavy mineral oil and filled with NaCl pipette solution (solution 3, Table 1). Tissues were folded into a loop with the apical membrane facing outside and mounted in the recording chamber. High-resistance seals (2-8 GOmega ) were made to the apical membrane of VDC, and currents were recorded in the cell-attached configuration. The recording technique was identical to that previously reported (17).

The patch-pipette perfusion technique was described previously (25). In brief, an internal perfusion pipette was connected to either of two solution vials via a valve and the internal pipette positioned within the macropatch pipette. Positive pressure applied to the vials drove the selected solution through the patch pipette. The arrival of new solution was delayed by ~2 min due to diffusion over ~300 µm between the outlet of the internal perfusion pipette and the membrane patch.

Perforated-patch whole cell clamp. The perforated-patch whole cell clamp technique was chosen over the conventional whole cell technique to avoid rundown of channel activity (17). Perforated-patch experiments were conducted in the absence of Cl- to reduce the contribution of the basolateral Cl- conductance to the whole cell conductance and thereby to increase the ratio between the cell membrane resistance and the access resistance. This was a necessary condition to effectively clamp the membrane potential in these cells. An unusually high Cl- conductance in the basolateral membrane of these cells is responsible for an extremely low resistance of the cell membrane, typically <20 MOmega /cell in the presence of 150 mM Cl- in the bathing medium (32). K+ secretion by the apical conductive pathway was supplied under whole cell conditions by the pipette electrolyte diffusing through the nystatin perforations.

The tissues were continuously perfused with Cl--free bath solution (solution 2, Table 1). The internal diameter of the tip of the patch pipette was 1-2 µm with a resistance of 8-12 MOmega in the Cl--free bath solution. The tip of the patch pipette was backfilled with a 1-cm column of K+-rich, Cl--free solution (solution 4, Table 1) containing 200 µg/ml nystatin. The rest of the pipette was backfilled with 15 mM Cl- solution (solution 5) to stabilize the Ag/AgCl junction. Gigaohm seals were made to the apical membrane, after which the membrane patch was perforated by insertion of the nystatin. After a stable whole cell configuration was established, the access resistance (31 ± 1 MOmega , n = 23) and the membrane capacitance (53 ± 23 pF, n = 23) were measured with the circuitry in the patch amplifier (Dagan 3900 and Axon Instruments 200A). Capacitance was nearly fully compensated and resistance compensated by typically 65%.

The seemingly large capacitance observed in the present study is consistent with the large membrane area due to extensive basolateral infoldings. Currents from individual VDC can be recorded in the native epithelium, since no gap junctions have been found among them (9).

Voltage protocols. Voltages are expressed with the usual convention of the intracellular compartment with respect to the extracellular side. In on-cell experiments, voltages are not corrected for the cell membrane potential. The membrane potential is about -18 mV in vitro with NaCl physiological saline bathing both sides of the epithelium (32).

The whole cell voltage protocol has been described in detail previously (27), and the protocol was repeated every 30 s to record the time course of changes during experimental treatments. For each instance of the protocol, the cell was first held at 0 mV; a tail-current I-V plot was produced from four pulses of 10-20 ms each. The IsK channel current (IIsK) was then deactivated by holding the cell at -50 mV for 4 s (17), and a tail-current I-V plot was produced of the "leak" current. The currents were sampled for the I-V plots after decay of capacitive transients, and the active currents were corrected for the leak currents. The interval between protocols was chosen to allow ample time for recovery of channel activity at the activating holding potential (0 mV). Measurements of currents under both control and experimental conditions in each cell allowed the application of paired statistics.

The voltage protocol used for on-cell macropatch recordings was similar, but repeated every 15 s. This interval was found to be adequate for full recovery of activation under these conditions. The deactivating voltage was -40 mV (the cell membrane voltage hyperpolarized the membrane an additional amount of ~18 mV). Currents at two voltages (-40 and 0 mV) were used to estimate the leak conductance. Parameters derived from these recordings include the current and conductance of the apical IsK channels (IIsK and gIsK, respectively), the reversal voltage without correction for leak (Vr), and the time constant of deactivation (tau off).

Data presentation and statistics. Data are expressed as means ± SE (n = number of cells). Student's t-test of paired samples was applied, and increases or decreases in parameters were considered significant at a level of P < 0.05.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Inhibition of JK by extracellular ATP. Because it was necessary to measure JK at 24°C, we controlled for the temperature dependence of the response to P2U receptor activation by first comparing the response of Isc at 24 and 37°C. Apical perfusion of ATP (100 µM) for 2 min at 24°C led to a maximal decrease in Isc by 40.8 ± 3.0% (n = 6) below the control value (Fig. 1A, Table 2). This effect was slower than that seen at 37°C (13) but similar in magnitude; it occurred with a delay of 13 ± 1 s, reaching its peak value at 148 ± 9 s. Similarly, apical perfusion of ATP (100 µM) for 3 min at 24°C led to a maximal decrease in JK by 40.9 ± 9.2% (n = 7) below the control value (Fig. 1B). The slower and delayed response of JK compared with that of Isc was most likely related to the lack of stirring in the basolateral compartment required for the probe measurement. Similar differences in response times between Isc and JK were seen for basolateral application of adenosine 3',5'-cyclic monophosphate (cAMP) (27). The results clearly show, however, that the response of VDC to apical ATP involves a decrease in transepithelial JK.


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  		Fig. 1.   Summaries of effects of apical perfusion at 24°C of ATP (100 µM) for 2 min on short-circuit current (Isc, A) and for 3 min on K+ flux (JK, B) across vestibular dark cell epithelium. Values are means ± SE (n = 6 and 7 for A and B, respectively). Error bars are shown only at intervals for clarity.

                              
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  		Table 2.   Transepithelial voltage, transepithelial resistance, and short-circuit current in the absence or presence of ATP or drug

Inhibition of IIsK by extracellular ATP. On-cell macropatch-clamp recordings were made of IIsK, gIsK, Vr, and tau off from VDC as described in METHODS. Data were obtained by averaging the last three values of the control period and of the experimental period for each experiment.

Perfusion of ATP (10 µM) for 4 min through the patch pipette led to a decrease in IIsK by 83.4 ± 7.4% and in gIsK by 48.6 ± 9.3% and to a depolarization of Vr by 24.6 ± 5.6 mV (n = 5; Fig. 2, Table 3). The tau off could not be measured for the entire duration of inhibition by ATP but did not change significantly during the first 90 s, when the current had already decreased by 58 ± 18%.


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  		Fig. 2.   Summary of on-cell macropatch-clamp recordings of slowly activating K+ (IsK) channel current (IIsK), IsK channel conductance (gIsK), and reversal voltage (Vr) during 5-min pipette perfusion of ATP. Values are means ± SE (n = 5).

                              
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  		Table 3.   IIsK, gIsK, Vr, and tau off in the absence or presence of ATP or drug

Inhibition of PLC reduces effect of apical ATP. Addition of U-73122 at 4 µM to the apical perfusate had no significant effect on Isc (n = 5). However, apical ATP (1 µM) caused less of a decrease of Isc in the presence of U-73122 than in its absence (Fig. 3A, Table 2). ATP decreased Isc by 24.1 ± 3.9% in the presence of U-73122 and by 32.7 ± 4.1% in its absence corresponding to a fractional response to ATP of 73 ± 4% in the presence of U-73122 compared with that in its absence. Raising the concentration of U-73122 to 10 µM resulted in a further reduction of the effect of ATP [by 52.0 ± 8.0% without U-73122 vs. 30.0 ± 6.4% (n = 6) in the presence of U-73122], although there was also a small decrease of Isc from the U-73122 itself at this concentration (17.6 ± 4.3%). The fractional response to ATP in the presence of 10 µM U-73122, 56 ± 7% of that in the absence of U-73122, was less than that at 4 µM U-73122. The inactive analog, U-73343 (4 µM), had no effect on Isc, and ATP (1 µM) caused the same magnitude decrease in the absence (30.3 ± 4.2%) and presence (32.0 ± 3.9%, n = 6; Fig. 3B, Table 2) of U-73343.


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  		Fig. 3.   Summary of measurements of Isc in vestibular dark cells showing the effect of apical perfusion of ATP (1 µM) in the presence and absence of an inhibitor of phospholipase C, U-73122 (4 µM, A), and an inactive analog, U-73343 (4 µM, B). Values are means ± SE (n = 5).

Elevation of cytosolic Ca2+ increases IIsK. The effect on the IIsK of raising cytosolic Ca2+ was tested in whole cell patch-clamp recordings by addition of the Ca2+ ionophore A-23187 (5 µM) to the bathing solution. Data were obtained from the average of the last two values of the control and experimental periods in each experiment.

Bath perfusion of VDC with A-23187 for 3 min led to an increase in IIsK by 43 ± 11% of the original current (n = 11) and an increase in gIsK by 34 ± 9% of the original conductance and to a hyperpolarization of Vr by 4.8 ± 2.0 mV (Fig. 4, Table 3). The tau off did not change significantly.


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  		Fig. 4.   Summary of nystatin-perforated whole cell patch-clamp recordings of IIsK, gIsK, and Vr of vestibular dark cells during 3-min bath perfusion with the Ca2+ ionophore A-23187 (5 µM). Values are means ± SE (n = 11).

Activation of PKC reduces IIsK. The effect of stimulating PKC on the IIsK was tested in whole cell patch-clamp recordings by addition of the phorbol ester PMA to the bathing solution. Data were obtained from the average of the last 2 values of the control and experimental periods in each experiment.

Bath perfusion of VDC with PMA (20 nM) for 3 min led to a decrease in IIsK by 79.1 ± 2.8% of the original current (n = 6) and in gIsK by 58.1 ± 3.5% of the original conductance and to a depolarization of Vr by 25.5 ± 3.3 mV (Fig. 5A, Table 3). There was no significant change in tau off. None of these parameters changed significantly after 3-min perfusion of 20 nM 4alpha -PMA, an inactive analog of PMA (n = 6; Fig. 5B, Table 3).


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  		Fig. 5.   A: summary of nystatin-perforated whole cell patch-clamp recordings of IIsK, gIsK, and Vr during 3-min bath perfusion of an activator of protein kinase C, phorbol 12-myristate 13-acetate (PMA, 20 nM; means ± SE, n = 6). B: similar experiments with the inactive analog, 4alpha -PMA, had no effect (n = 6).

Sequence of gerbil cDNA at putative PKC phosphorylation site. RT-PCR of total RNA from a tissue known to express the isk gene (heart) resulted in a product of the expected size based on the gene-specific primers used during amplification (Fig. 6). Furthermore, an RT-PCR product of the same expected size was also amplified from the vestibular labyrinth RNA and thus indicates the presence of an isk gene transcript in this tissue. The IsK identity of the 170-bp DNA fragments from the heart and vestibular labyrinth was confirmed by cloning and sequencing. There is no evidence of genomic DNA contamination as seen by the absence of the RT-PCR product in RNA samples subjected to only the PCR in the absence of the reverse transcriptase (MMLV). In addition, the isk gene transcript was not a result of blood contamination of the tissues, since the same RNA isolation procedure and RT-PCR of the isolated RNA from blood did not result in the amplification of a 170-bp product. The sequence is shown in Fig. 7 (GenBank no. AF029765) and is distinguished by its high homology to the same region of rat IsK cDNA (89% sequence identity between the primers) and predicted amino acid composition (91%). In particular, the PKC consensus sequence in rat is preserved in the gerbil. The serine at position 102 in the rat IsK amino acid sequence, which mediates the species-specific response to PKC, was found to be present at the analogous position in the gerbil IsK sequence.


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  		Fig. 6.   Gel electrophoresis of reverse transcription-polymerase chain reaction (RT-PCR) products from reactions with 0.5, 0.15, and 0.125 µg total RNA from gerbil heart (H), vestibular labyrinth (VL), and blood (B), respectively. +, Reactions performed in the presence of reverse transcriptase; -, reactions performed in the absence of reverse transcriptase. Position of the bands for the expected lengths of the RT-PCR products [170 base pairs (bp)] is indicated, and a 100-bp ladder marker is shown (M). Image was digitally captured and inverted.


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  		Fig. 7.   Base sequence for the 170-bp segment of gerbil IsK RT-PCR products and the corresponding amino acid sequence. A: diagrammatic map of the location of 170-bp segments within the open reading frame (TM, transmembrane segment; PKC, protein kinase C consensus sequence). B: sequence of bases and predicted amino acids from amino acid positions 58 to 114 compared between gerbil and rat. Bars indicate grouping of bases coding for amino acids; large letters next to bars indicate the corresponding amino acid; and , positions where bases differ between species. Amino acids for rat sequence are only indicated at positions that include a and  mark. Sequences corresponding to the sense and antisense primers are indicated by horizontal arrows.

Inhibition of PKC reduces response to ATP. It would be expected that if PKC is a mediating signal element in the effect of extracellular ATP on the IIsK then interference with PKC activation would reduce the effect of ATP. We tested this hypothesis by addition of the PKC inhibitor GF109203X to the apical perfusate.

Addition of GF109203X at 10 µM to the apical perfusate had no significant effect on Isc (n = 7). However, apical ATP (1 µM) caused less of a decrease of Isc in the presence of GF109203X than in its absence (Fig. 8, Table 2). ATP decreased Isc by 38.5 ± 3.2% in the absence of GF109203X and by 20.7 ± 1.6% in its presence. The fractional response to ATP in the presence of 10 µM GF109203X was 54 ± 2%.


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  		Fig. 8.   Summary of measurements of Isc showing effect of apical perfusion of ATP (1 µM) in the presence and absence of an inhibitor of protein kinase C, GF109203X (10 µM), in vestibular dark cells (values are means ± SE; n = 7).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Purinergic (P2) receptors responsive to UTP have been referred to as P2U subtypes. More recently, P2U receptors have been found to represent a family of three subtypes responsive to uridine nucleotides, which are designated P2Y2, P2Y4, and P2Y6 (22). The P2Y family of purinergic receptors is coupled via heterotrimeric G proteins in the plasma membrane to PLC (5), which leads via one branch of the pathway to activation and translocation of PKC from the cytosol to the plasma membrane (3) and via a second branch of the pathway to elevation of cytosolic Ca2+ concentration ([Ca2+]c), which occurs in response to production of InsP3 (5). Most ion transport mechanisms that are influenced by P2U receptors respond to changes in [Ca2+]c (5). In contrast, we have presented evidence that activation of apical P2U receptors in VDC leads to inhibition of K+ secretion and that this inhibition is mediated by the PKC branch of the PLC pathway. The involvement of PLC in the response to apical ATP was shown by the reduced effect of ATP in the presence of the PLC inhibitor, U-73122 (7). The involvement of PKC was demonstrated both by inhibition of the IIsK in response to activation of PKC with PMA and by the reduced effect of ATP in the presence of the PKC inhibitor, GF109203X.

PKC most likely acts by direct phosphorylation of the IsK protein. The IsK protein in rat is known to have a consensus sequence for phosphorylation by PKC, and this sequence is altered in guinea pig. The difference in sequence is correlated with different electrophysiological responses of the IIsK to stimulation of PKC (28). PKC activation in Xenopus oocytes expressing rat IsK leads to a decrease in current, as found here for gerbil, whereas wild-type guinea pig IsK (and rat IsK with the consensus sequence mutated to the corresponding sequence from guinea pig) are stimulated by PKC. Those results led to the prediction that the consensus sequence for phosphorylation by PKC in gerbil would be identical to that of rat. The result of molecular cloning and sequencing of a segment of the gerbil IsK cDNA from the vestibular labyrinth confirms that the isk gene is expressed in this tissue and that it contains the putative PKC phosphorylation site, as predicted. On the basis of these results and the presence of the same PKC phosphorylation site in human isk (21), it can be expected that the secretion of endolymph in the human vestibular system is controlled in the same way as in our animal model.

It is conceivable that extracellular nucleotides inhibit K+ secretion indirectly via cAMP, rather than via direct phosphorylation of the IsK channel. In DDT1MF-2 smooth muscle cells, activation of the P2U receptor activated PLC but also activated a distinct, pertussis toxin-sensitive G protein pathway, which caused a large sustained decrease of the level of cytosolic cAMP (26). We have recently shown that there is constitutive production of cAMP and that elevation of cytosolic cAMP stimulates the IIsK in VDC (27), so it is likely that reduction of cAMP would lead to a decrease in IIsK. If the P2U receptors in the apical membrane of VDC are coupled to Gi as well as to PLC, it is conceivable that at least part of the effect of apical perfusion of ATP was due to a reduced cAMP level, although no such coupling has yet been demonstrated in VDC.

The increase of IIsK observed in VDC under conditions expected to elevate [Ca2+]c argues against a major role of the InsP3 pathway, with its subsequent increase of [Ca2+]c, in the response of K+ secretion to activation of the apical P2U receptor. Although Ca2+ is classically known to increase PKC activity, isozymes have been identified that are Ca2+ independent (PKC-delta ) (8). PKC-delta has been identified in stria vascularis (1), a tissue in the cochlea that contains cells homologous to VDC in many other respects (29). It is not yet clear what process is directly affected by addition of A-23187, although it is most likely due to an increase in cytosolic Ca2+. Although A-23187 is a divalent cation/H+ exchanger, it is not likely that the effect was due to a change in cytosolic pH, since an influx of Ca2+ would be expected to lead to alkalinization and, by our current understanding, a concomitant decrease in K+ secretion (30).

Although the sources of agonist and the physiological functions of the apical purinergic receptors are not yet clear, several hypotheses can be advanced. The constitutive level of ATP in the luminal fluid of the cochlea has been estimated at 13 nM (20), a concentration that causes a small but significant decrease in Isc of VDC (13). If one assumes a similar level of ATP in the vestibular labyrinth, the receptor would be poised to be either stimulated further by additional agonists or inhibited by antagonists. Functionally, it may be extremely important for these epithelial cells to receive signals from neighboring VDC, since they are not coupled to each other by gap junctions. In addition, there are many other epithelial cell types lining the vestibular lumen that may communicate via apical release of ATP. Conditions stimulating this hypothetical release of ATP remain to be discovered.

    ACKNOWLEDGEMENTS

This work was supported by the National Institute on Deafness and Other Communication Disorders research grant 5R01-DC-00212 to D. C. Marcus.

    FOOTNOTES

Address for reprint requests: D. C. Marcus, Biophysics Laboratory, Boys Town National Research Hospital, 555 No. 30th St., Omaha, NE 68131.

Received 21 January 1997; accepted in final form 20 August 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C2022-C2029
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society


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