Biomedical subjects

D C Marcus

Publications and source records attributed to D C Marcus.

At least 37 records · Page 2Linked to original sources

PubMed1996

Inhibitory effect of erythromycin on ion transport by stria vascularis and vestibular dark cells.

A previous study showed that systemic administration of erythromycin caused a reversible decline in the endocochlear and cochlear microphonic potentials. Those data were thought to suggest that erythromycin caused hearing loss by interference with ion transport processes in the stria vascularis. The present study was undertaken to test this hypothesis by measuring the effects of erythromycin perfused on either the apical or basolateral side on the transepithelial short circuit current (Isc), a measure of the K+ secretion rate. Isc was measured from preparations of strial marginal cells and the homologous vestibular dark cells in vitro with a micro-Ussing chamber. Erythromycin was found to have no effect when perfused on the apical side but to cause a reversible decrease in Isc when perfused on the basolateral side for both epithelia. These data are consistent with the notion that at least one ototoxic effect of erythromycin is the inhibition of K+ secretion in the inner ear.

Animals↗

PubMed1995

DIDS increases K+ secretion through an IsK channel in apical membrane of vestibular dark cell epithelium of gerbil.

Vestibular dark cell epithelium secretes K+ via IsK channels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (Isc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in Isc was associated with an increase in K+ flux and if the effect was directly on the IsK channel 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 (IsK) 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 IsK channels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Hypo-osmotic challenge stimulates transepithelial K+ secretion and activates apical IsK channel in vestibular dark cells.

Volume regulation of vestibular dark cells from the gerbilline inner ear in response to a hypo-osmotic 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 (JK+,probe) and current density (Isc,probe) were measured with vibrating probes in the vicinity of the apical membrane and the transepithelial potential (Vt) and resistance (Rt) were measured in a micro-Ussing chamber. The equivalent short-circuit current (Isc) was calculated. The current (IIsK), conductance (gIsK) and inactivation time constant (tau IsK) of the IsK channel and the apparent reversal potential of the apical membrane (Vr) were obtained with the cell-attached macropatch technique. Vr was corrected (Vrc) for the membrane voltage (Vm) 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 JK+,probe and Isc,probe by a factor of 2.7 and 1.6. When this hypo-osmotic challenge was applied to both sides of the epithelium Vt and Isc increased from 5 to 14 mV and from 189 to 824 microA/cm2 whereas Rt decreased from 27 to 19 omega-cm2. With 3.6 mM K+ in the pipette IIsK was outwardly directed, tau IsK was 267 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase from 14 to 37 pA and from 292 to 732 pS. Vrc hyperpolarized from -44 to -76 mV. With 150 mM K+ in the pipette IIsK was inwardly directed, tau IsK was 208 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase in magnitude from 0 to -21 pA and from 107 to 1101 pS. Vrc remained unchanged (-2 vs. 1 mV). These data demonstrate that a hypo-osmotic challenge stimulates transepithelial K+ secretion and activates the apical IsK channel. The hypo-osmotically-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.

Animals↗

PubMed1995

Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro.

It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K+. The present study was designed to provide evidence for this hypothesis and for a cell model proposed to explain K+ secretion and the generation of the endocochlear potential. Stria vascularis from the cochlea of the gerbil was isolated and mounted into a micro-Ussing chamber such that the apical and basolateral membrane of marginal cells could be perfused independently. In this preparation, the transepithelial voltage (Vt) and resistance (Rt) were measured across marginal cells and the resulting equivalent short circuit current (Isc) was calculated (Isc = Vt/Rt). Further, K+ secretion (JK+,probe) was measured with a K(+)-selective vibrating probe in the vicinity of the apical membrane. In the absence of extrinsic chemical driving forces, when both sides of the marginal cell epithelium were bathed with a perilymph-like solution, Vt was 8 mV (apical side positive), Rt was 10 ohm-cm2 and Isc was 850 microA/cm2 (N = 27). JK+,probe was outwardly directed from the apical membrane and reversibly inhibited by basolateral bumetanide, a blocker of the Na+/Cl-/K+ cotransporter. On the basolateral but not apical side, oubain and bumetanide each caused a decline of Vt and an increase of Rt suggesting the presence of the Na,K-ATPase and the Na+/Cl-/K+ cotransporter in the basolateral membrane. The responses to [Cl-] steps demonstrated a significant Cl- conductance in the basolateral membrane and a small Cl- conductance in the paracellular pathway or the apical membrane. The responses to [Na+] steps demonstrated no significant Na+ conductance in the basolateral membrane and a small Na+ or nonselective cation conductance in the apical membrane or paracellular pathway. The responses to [K+] steps demonstrated a large K+ conductance in the apical membrane. Apical application of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and basolateral elevation of K+ caused an increase in Vt and a decrease in Rt consistent with stimulation of the apical K+ conductance. Similar observations have been made in vestibular dark cells, which suggest that strial marginal cells and vestibular dark cells are homologous and transport ions by the same pathways. Taken together, these observations are incompatible with a model for the generation of the endocochlear potential which ascribes the entire potential to the strial marginal cells [Offner et al. (1987) Hear. Res. 29, 117-124].(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Transepithelial voltage and resistance of vestibular dark cell epithelium from the gerbil ampulla.

Transepithelial voltage (Vt) and resistance (Rt) were measured across the dark cell epithelium of the gerbil ampulla using a micro Ussing chamber of improved design in order to test the view that the histologically similar epithelia in the utricle and in the ampullae exhibit similar electrophysiologic functions. Vt was found to be 8.0 +/- 0.3 mV and Rt was 11.6 +/- 0.4 ohm-cm2 (N = 179) when both sides of the tissue were perfused with symmetric perilymph-like solution. The equivalent short circuit current (Isc = Vt/Rt) was 712 +/- 18 microA/cm2 (N = 179). Isc was reduced from 638 +/- 60 to 48 +/- 16 microA/cm2 (N = 14) by basolateral perfusion of 10(-3) M ouabain and from 538 +/- 27 to 27 +/- 4 microA/cm2 (N = 15) by basolateral perfusion of 5 x 10(-5) M bumetanide. Basolateral K+ steps (Na+ substitution) from 3.6 to 25 mM increased Vt from 6.5 +/- 0.5 to 12.2 +/- 0.6 mV and reduced Rt from 9.7 +/- 0.7 to 7.4 +/- 0.5 ohm-cm2 (N = 43). Apical K+ steps from 3.6 to 25, to 100 mM or to 145 mM led to a decrease in both Vt and Rt. The steady state Vt during apical perfusion of 145 mM K+ was near zero. Upon return to 3.6 mM K+, Vt transiently overshot its original level. Apical Cl- steps from 150 to 50 mM (gluconate substitution) monophasically decreased Vt from 5.9 +/- 0.7 to 4.1 +/- 0.8 mV (N = 15) and increased Rt from 9.6 +/- 1.3 to 12.0 +/- 1.5 ohm-cm2 (N = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe.

Detection of motion and position by the vestibular labyrinth depends on the accumulation of potassium within a central compartment of the inner ear as a source of energy to drive the transduction process. Much circumstantial evidence points to the vestibular dark cell (VDC) epithelium as being responsible for concentrating K+ within the lumen. We have used the vibrating probe technique to directly observe voltage and ion gradients produced by this tissue to put this assumption on a solid experimental footing. Relative current density (Isc,probe) over the apical membrane of VDC epithelium was measured with the vibrating voltage-sensitive probe, and this technique was validated by performing maneuvers known to either stimulate or inhibit the transepithelial equivalent short circuit current. Basolateral bumetanide (5 x 10(-5) M) and ouabain (1 x 10(-3) M) caused a decrease in Isc,probe by 55 +/- 6% and 39 +/- 3%, respectively while raising the basolateral K+ concentration from 4 to 25 mM caused an increase by 35 +/- 8%. A K+ gradient directed toward the apical membrane was detected with the vibrating K(+)-selective electrode, demonstrating that, indeed, the VDC epithelium secretes K+ under control conditions. This secretion was inhibited by bumetanide (by 94 +/- 7%) and ouabain (by 52 +/- 8%). The results substantiate the supposition that dark cells produce a K+ flux and qualitatively support the correlation between this flux and the transepithelial current.

Animals↗

PubMed1994

Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells.

Dark cell epithelium secretes K+ into the lumen of the vestibular labyrinth by a previously unidentified apical transport mechanism. Previous single-channel patch-clamp studies demonstrated nonselective cation channels and maxi-K+ channels in the apical membrane, but in too low a density to account for transepithelial K+ transport. In this report, we demonstrated with the cell-attached macro-patch-clamp technique an outward apical membrane current at 0-mV pipette voltage, which was stimulated by elevating bath K+ concentration from 3.6 to 25 mM and inhibited by 10 microM bumetanide, similar to their known effects on transepithelial short-circuit current and K+ secretion. Furthermore, the patch current was activated over several seconds by a sustained depolarization and deactivated over several hundred milliseconds by a hyperpolarization. Current-voltage relationships from tail currents were obtained with either NaCl or KCl in the pipette. Depolarization from -40 to +40 mV led to an increased conductance by a factor of 7.3 +/- 1.7 (n = 7) and 19.2 +/- 7.6 (n = 6) for NaCl and KCl, respectively, and to a reversal voltage near the presumed equilibrium potential for K+. The results demonstrate that dark cell K+ secretion occurs via K(+)-selective channels with characteristics similar to those associated with the IsK protein.

Animals↗

PubMed1993

The Na+/H+ exchanger in transitional cells of the inner ear.

The cytosolic pH (pHi) of transitional cells from the ampulla of the gerbil was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) to assess the possible contribution of a Na+/H+ exchanger to the regulation of pHi. All experiments were conducted in virtually HCO3(-)-free solutions. Under control conditions, pHi was 7.19 and addition of 10(-5) M ethylisopropylamiloride (EIPA), a blocker of Na+/H+ exchange, caused a small but significant acidification of pHi. A transient exposure to 21.4 mM NH4Cl caused a rapid cytosolic alkalinization followed by a brisk acidification and prompt recovery of pHi to its control value. The cytosolic buffer capacity (Bi) was determined in the absence of Na+ from changes in pHi which were elicited by [NH4+] steps. Bi was 4.7 mM/pH at pHi 7.19 and varied with pHi. The initial net proton flux JH, representative of Na+/H+ exchange activity, was calculated from the product of the initial rate of alkalinization after an NH4(+)-prepulse and Bi at the corresponding pHi. JH was dependent on the extracellular Na+ with an apparent Km of 64 mM, sensitive to the cytosolic [H+] with an apparent Km of 2.7 * 10(-7) M (i.e. pH 6.6), and inhibited by EIPA with an IC50 of 5 * 10(-7) M. These data suggest that transitional cells contain in the basolateral membrane a Na+/H+ exchanger of the amiloride-sensitive subtype.

Amiloride↗

PubMed1993

Two types of chloride channel in the basolateral membrane of vestibular dark cells.

Transepithelial and cell membrane potential measurements have suggested that the basolateral membrane of gerbil vestibular dark cells contains Cl- conductive pathways. We used the patch clamp technique to search this membrane for Cl- conductive channels which could account for the macroscopic observations. Two types of Cl- channel were found in both cell-attached and excised membrane patches. One type was found with an incidence of 19% and had a single-channel conductance of 95 +/- 1 pS (N = 20) in symmetrical Cl- solutions. The other type was found with an incidence of 3% and had a large single-channel conductance of 360 +/- 11 pS (N = 12) in symmetrical Cl- solutions (LC-type Cl- channel). Both types of Cl- channel had linear current-voltage relations and at least 2 substates. In asymmetrical Cl- solutions (gluconate substitution) the current-voltage relations fit the Goldman-Hodgkin-Katz current equation for Cl-. Neither channel was blocked by Zn2+, NPPB, DIDS, DNDS or quinine. The 95 pS channel exhibited a spontaneous 'rundown' of its activity within 1 to 10 min after being excised. This rundown was not reversed by the catalytic subunit of protein kinase A. Channel activity was not dependent on the presence of cytosolic Ca2+ nor markedly altered by variations in cytosolic pH between 6.5 and 8.0. The two Cl- channels were distinguished by the membrane voltage ranges in which they were active and by their anion selectivity. The open probability of the 95 pS channel was insensitive to voltage and the anions NO3-, I- and Br- were only half as permeable as Cl-. By contrast, the LC-type Cl- channel was mostly active between about +/- 30 mV and equally permeable to NO3-, I-, Br- and Cl-. The 95 pS Cl- channel may account for the observed transepithelial and intracellular voltage responses to Cl- concentration steps and provide the path for the recirculation of Cl- across the basolateral membrane. The LC-type Cl- channel shows the same lack of anion discrimination as the anion pathway activated during hyposmotic challenge.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Ca(2+)-activated nonselective cation, maxi K+ and Cl- channels in apical membrane of marginal cells of stria vascularis.

Patch clamp recordings on the apical membrane of marginal cells of the stria vascularis of the gerbil were made in the cell-attached and excised configuration. Marginal cells are thought to secrete K+ into and absorb Na+ from endolymph. Four types of channel were identified; the most frequently observed channel was a small, nonselective cation channel which was highly similar to that found in the apical membrane of vestibular dark cells (Marcus et al., (1992) Am. J. Physiol. 262, C1423-C1429). The small nonselective cation channel was equally conductive (26.7 +/- 0.3 pS; N = 49) for K+, Na+, Rb+, Li+ and Cs+, 1.6 times more permeable to NH4+, but not permeable to Cl-, Ca2+, Ba2+ or N-methyl-D-glucamine. This channel yielded linear current-voltage relations which passed nearly through the origin (intercept: -2.2 +/- 0.4 mV, N = 49) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face but not the extracellular (endolymphatic) face; there was essentially no activity for cytosolic Ca2+ less than or equal to 10(-7) M Ca2+ and full activity for greater than or equal to 10(-5) M. Cell-attached recordings had a conductance of 28.6 +/- 2.2 pS (N = 6) and a reversal voltage of -2.2 +/- 5.2 mV (N = 3) which was interpreted to reflect the intracellular potential of marginal cells under the present conditions. The three other types of channel were a Cl- channel (approximately 50 pS; N = 2), a maxi-K+ channel (approximately 230 pS; N = 1), and another large channel, probably cation nonselective (approximately 170 pS; N = 1). The 27 pS nonselective cation channel may be involved in K+ secretion and Na+ absorption under stimulated conditions which produce an elevated intracellular Ca2+; however, consideration of the apparent channel density in relation to the total transepithelial K+ flux suggests that these channels are not sufficient to account for K+ secretion.

Animals↗

PubMed1992

The membrane potential of vestibular dark cells is controlled by a large Cl- conductance.

The K+ secretory epithelium of the vestibular labyrinth (dark cells) was impaled with glass microelectrodes in order to test the hypothesis that it contains a large Cl- conductance. In the first series of experiments, the short-circuited epithelium was perfused on both sides by a solution containing 150 mmol/l Cl-. The membrane voltage (PD) was -18 +/- 1 mV (N = 101), showed a Gaussian distribution, and the estimated input resistance of the cell (R 'cell') was 17 +/- 3 M omega. The PD responded to 10(-4) mol/l ouabain with a depolarization, suggesting the presence of a (Na(+) + K+)-ATPase. The PD responses to Cl- steps yielded an apparent transference number tCl = 0.34 +/- 0.03 (N = 65) and those to K+ steps yielded a tK = 0.16 +/- 0.01 (N = 48). In the second series of experiments, cells presumed to be Cl(-)-depleted were impaled in Cl(-)-free solutions. The distribution of the PD was not Gaussian; PDs as negative as -90 mV were observed. Cells with a highly negative PD also had a high R 'cell'. With the addition of Cl- the PD collapsed to -19 +/- 1 mV and R collapsed to 16 +/- 3 M omega (N = 145) which are not significantly different from values obtained in the first series of experiments when cells were impaled in a solution containing 150 mmol/l Cl-. Alternating the bath perfusate between Cl(-)-free and Cl(-)-containing solutions led to large PD transients.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1992

Ca(2+)-activated nonselective cation channel in apical membrane of vestibular dark cells.

Patch-clamp recordings were made on cell-attached and excised apical membrane from dark cells of the semicircular canal of the gerbil. These cells are thought to secrete K+ and absorb Na+ from the luminal fluid (endolymph). Single-channel events were identified as being equally conductive (27.6 +/- 0.4 pS; n = 48) for K+, Na+, Rb+, Li+, and Cs+ and 1.4 times more permeable to NH4+ but not permeable to Cl-, Ca2+, Ba2+, nor to N-methyl-D-glucamine. The channels displayed linear current-voltage relations that passed nearly through the origin (intercept: -2.6 +/- 0.5 mV; n = 48) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face; there was no activity at less than or equal to 10(-7) M Ca2+ and full activity at greater than or equal to 10(-5) M Ca2+. Cell-attached recordings had a mean reversal voltage of -36.4 +/- 7.9 mV (n = 7), which was interpreted to reflect the intracellular potential of dark cells under the present conditions. We have identified a nonselective cation channel in the apical membrane of vestibular dark cells that might participate in K+ secretion or Na+ absorption under stimulated conditions, but the density appears to be insufficient to fully account for the transepithelial K+ flux.

Animals↗

PubMed1992

Maxi K+ channel in apical membrane of vestibular dark cells.

Recordings were made on excised apical membrane patches from vestibular dark cells from the semicircular canal of gerbils to determine if ion channels could be involved in the process of K+ secretion. Both nonselective cation channels [Am. J. Physiol. 262 (Cell Physiol. 31): C1430-C1436, 1992] and K(+)-selective channels were found. The K+ channels occurred in only 0.7% of the patches. In symmetrical 145 mM KCl solutions, the current-voltage (I-V) relation of the K(+)-selective channel was linear, indicating the absence of rectification, and the conductance was 240 +/- 8 pS (n = 8). The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relation in asymmetrical K+ and Na+ solutions and yielded a K+ permeability of 5.78 x 10(-13) cm3/s (n = 12). The channel was shown to be impermeable to Li+, NH4+, N-methyl-D-glucamine, and Cl-. Channel activity increased with depolarization and with increasing free [Ca2+]; for voltages between +40 and -60 mV, the strongest regulation occurred in the range 10(-6) to 10(-5) M Ca2+. Tetraethylammonium (2 x 10(-2) M) had from the cytosolic side no effect on the open probability (Po) but completely inhibited activity from the extracellular side. Po was reduced by Ba2+ (5 x 10(-3) M), verapamil (10(-4) M), quinine (10(-4) M), and quinidine (10(-4) and 10(-3) M), while lidocaine (5 x 10(-3) M) had no measurable effect on Po but decreased the amplitude. Rb+ and Cs+ were either poorly permeable or partially blocked the channel in a voltage-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Evidence for the involvement of a K+ channel in isosmotic cell shrinking in vestibular dark cells.

Cell volume changes were measured in dark cells. Isosmotic addition of 21.4 mM K+, Rb+, Cs+, or NH4+ to a control solution containing 3.6 mM K+ caused piretanide-sensitive cell swelling (initial rate for K+, 0.100 +/- 0.005 microns/s; n = 119), suggesting dependence on the Na(+)-Cl(-)-K+ cotransporter. Subsequent isosmotic removal of 21.4 mM K+ caused piretanide-insensitive cell shrinking (initial rate, -0.104 +/- 0.005 microns/s; n = 119), which was inhibited by barium, lidocaine, quinidine, quinine, verapamil, and 4-aminopyridine but not tetraethylammonium (TEA) or glibenclamide, suggesting the involvement of K+ channel(s). Barium, lidocaine, quinine, quinidine, and 4-aminopyridine caused cell swelling in control solution (initial rate for barium, 0.011 +/- 0.004 microns/s; n = 6), suggesting that the K+ channel is also involved in efflux under control conditions. Cell shrinking was slowed by 21.4 mM extracellular K+, Rb+, or Cs+ but unaffected by Na+, Li+, TEA+, or NH4+ (all in the presence of piretanide and compared with N-methyl-D-glucamine), supporting the notion that the efflux mechanism is permeable to and/or inhibited by K+, Rb+, and Cs+. Cell shrinking was slowed by the presumed replacement of intracellular K+ by Cs+ but not by Rb+. Circumstantial evidence suggests that this putative K+ channel is present in the basolateral membrane. The physiological relevance of such a K+ channel might encompass regulatory volume decrease during K+ secretion.

Animals↗

PubMed1990

K(+)-induced swelling of vestibular dark cells is dependent on Na+ and Cl- and inhibited by piretanide.

Epithelial cell height was measured in order to estimate the cell volume of dark cells from the ampullae of the semicircular canal of the gerbil. Under control conditions, addition of 10(-4) mol/l piretanide, 10(-5) mol/l 5-nitro-2(3-phenylpropylamino)-benzoic acid (NPPB), 5 mmol/l barium or 10(-3) mol/l quinidine had no significant effect on cell height. Addition of 10(-4) mol/l NPPB or 10(-3) mol/l ouabain led to a small significant decrease in cell height which was not reversible. Substitution of Na+ by N-methyl-D-glucamine or of Cl- by gluconate led to a significant and reversible reduction in cell height. Isotonic elevation of [K+] from 3.6 to 25 mmol/l in a PO4-buffered, HCO3-free solution led to an increase in cell height from 5.8 +/- 0.1 (SEM) to 8.7 +/- 0.2 microns (n = 62) during the first 40 s. During prolonged exposure to elevated [K+] (3-5 min; n = 19), some tissue samples underwent a regulatory volume decrease. K(+)-induced swelling was absent in both isotonic Cl(-)-free and isotonic Na(+)-free solutions and was inhibited by the loop diuretic piretanide (10(-5) and 10(-4) mol/l) or by the (Na+ + K+) ATPase inhibitor ouabain (10(-3) mol/l) or by 10(-4) mol/l NPPB. After the removal of ouabain or 10(-4) mol/l NPPB, K(+)-induced swelling under control conditions was enhanced and was less reversible as compared to control conditions before the experiment. K(+)-induced swelling was not altered by NPPB (10(-5) mol/l) or barium (5 mmol/l); however, barium slowed shrinking upon return of [K+] to control level. In the presence of 10(-3) mol/l quinidine, K(+)-induced swelling was enhanced and not reversible. These data suggest that dark cells from the semicircular canal possess an Na+2Cl-K+ cotransporter as a solute uptake mechanism and a solute efflux mechanism which is sensitive to barium and inhibited by quinidine.

Animals↗

PubMed1990

Transepithelial electrical responses to sodium and potassium of nonsensory region of gerbil utricle.

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber. Step changes in [Na] and [K] on each side of the epithelium led to changes in transepithelial potential difference (Vt) and resistance (Rt) which were consistent with the presence of ionic conductances for these two ions. Responses of Vt to 20 mM Rb steps were similar to those to 20 mM K steps. Several common Na and K channel blockers were applied: No evidence for the presence of amiloride-sensitive Na channels was found. At 10(-6) M, amiloride caused no significant changes in Vt but at 10(-3) M it initially decreased or increased Vt when applied from the apical or the basolateral side, respectively. Ba and quinidine each reduced Vt, quinidine more strongly from the basolateral than from the apical side. Tetraethylammonium, another K channel blocker, had no significant effect from either side. These data suggest that in spite of the low value of Rt, the cellular pathway contributes significantly to the voltage response to cation concentration steps in this epithelium. The mode of action of 10(-3) M amiloride and the K channel blockers remains uncertain.

Animals↗

PubMed1989

Transepithelial electrical responses to Cl- of nonsensory region of gerbil utricle.

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber in order to identify and localize chloride conductances. The [Cl-] was rapidly reduced (substituted with isethionate) in the apical or basolateral perfusate and the transepithelial potential difference (Vt) and transepithelial resistance (Rt) were monitored continuously. In addition, agents known to inhibit anion transport in other epithelia were applied. The direction of all initial changes in Vt and Rt due to Cl- substitutions were consistent with the presence of ionic conductances for Cl- on both sides of the epithelium. The time-courses and magnitudes of the fall in Vt and increase in Rt during apical [Cl-] steps in the presence and absence of basolateral bumetanide were monophasic and identical in the two cases. The response of Vt to basolateral [Cl-] steps was biphasic and the initial response was greatly attenuated by bumetanide. These findings demonstrate that the largest conductance for Cl- is in the basolateral cell membrane, but that the paracellular and/or apical pathway also possess a finite Cl- conductance. All three agents tested, 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), caused an increase in Vt. NPPB and DIDS were more effective from the apical side. DCDPC and DIDS administered from the apical side led to a decrease in Rt. These results suggest that these agents act in this tissue by enhancing a conductive pathway on the apical membrane rather than blocking the basolateral Cl- conductance.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

Membrane potential measurements of transitional cells from the crista ampullaris of the gerbil. Effects of barium, quinidine, quinine, tetraethylammonium, cesium, ammonium, thallium and ouabain.

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was -87 +/- 1 mV (n = 103). This value is not significantly different from -83 +/- 2 mV (n = 24) measured in Cl- free solution. [Cl-] steps from 150 to 15 mmol/l (n = 24) depolarized the membrane by about 2 mV, indicating a minor Cl- conductance. The transference number for K+ was 0.75 +/- 0.01 (n = 79) obtained from the PD responses to K+ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K+] steps was reduced by Ba2+ (2.10(-6) to 10(-3) mol/l), quinidine (10(-3) mol/l), quinine (10(-3) mol/l), Rb+ (20 mmol/l), Cs+ (20 mmol/l), NH4+ (20 mmol/l) and Tl+ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba2+ in the presence of 3.6 mmol/l K+ was shifted to the right by approximately three decades in the presence of 25 mmol/l K+ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl-. Transitional cells were depolarized by ouabain, suggesting the presence of (Na+ + K+)-ATPase.

Animals↗