1 Department of Physiology, University of Bristol, Bristol BS8 1TD, United Kingdom; and Departments of 2 Otolaryngology and 3 Physiology and Biophysics, University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1063
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In amniotes,
there are two types of hair cells, designated I and II, that differ in
their morphology, innervation pattern, and ionic membrane properties.
Type I cells are unique among hair cells in that their basolateral
surfaces are almost completely enclosed by an afferent calyceal nerve
terminal. Recently, several lines of evidence have ascribed a motile
function to type I hair cells. To investigate this, elevated external
K+, which had been used previously
to induce hair cell shortening, was used to induce shape changes in
dissociated mammalian type I vestibular hair cells. Morphologically
identified type I cells shortened and widened when the external
K+ concentration was raised
isotonically from 2 to 125 mM. The shortening did not require external
Ca2+ but was abolished when
external Cl was replaced
with gluconate or sulfate and when external
Na+ was replaced with
N-methyl-D-glucamine.
Bumetanide (10-100 µM), a specific blocker of the
Na+-K+-Cl
cotransporter,
significantly reduced K+-induced
shortening. Hyposmotic solution resulted in type I cell shape changes
similar to those seen with high
K+, i.e., shortening and widening.
Type I cells became more spherical in hyposmotic solution, presumably
as a result of a volume increase due to water influx. In hypertonic
solution, cells became narrower and increased in length. These results
suggest that shape changes in type I hair cells induced by high
K+ are due, at least in part, to
ion and solute entry via an
Na+-K+-Cl
cotransporter, which
results in cell swelling. A scheme is proposed whereby the type I hair
cell depolarizes and K+ leaves the
cell via voltage-dependent K+
channels and accumulates in the synaptic space between the type I hair
cell and calyx. Excess K+ could
then be removed from the intercellular space by uptake via the
cotransporter.
crista ampullaris; utricle; bumetanide; guinea pig; sodium-potassium-adenosinetriphosphatase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TWO MORPHOLOGICALLY DISTINCT types of hair cells occur in the vestibular epithelia of mammals, birds, and certain reptiles. Type I cells are flask shaped, having a broad base, narrow neck region, and flared apex. The basolateral regions of the type I cell are encased by an afferent nerve calyx. Type II cells, present in all vertebrates, are generally more cylindrical in shape and receive bouton-type innervation at their basal pole. Displacement of cilia, projecting from the apical surface of the hair cells into the K+-rich endolymph, modulates the flow of mechanoelectric transduction current into the cells. Vestibular dark cells, located adjacent to the neuroepithelium, secrete K+ into the endolymph through apical K+ channels (17). The basolateral electrical membrane properties of type I and type II cells have been found to vary significantly, suggesting that these two cell types play different roles in the processing of mechanoelectric transduction signals (reviewed in Ref. 4). Vestibular hair cells maintain their characteristic morphology after isolation (26) but, under appropriate stimulation, may undergo somatic shape changes (6, 12, 16, 25, 36). Mammalian type I vestibular hair cells are reported to shorten by 0.5-1.0 µm in response to a variety of stimuli, including depolarizing steps in voltage clamp (12, 25), cell cooling (36), and external perfusion of isosmotic solutions with a raised K+ concentration (6, 16). The physiological significance of these changes is not known. However, because the membrane properties of type I hair cells are dominated by a large resting K+ conductance, it is likely that K+ levels in the restricted space between hair cell and calyx could rise substantially during hair cell stimulation (10, 11, 24).
Motile behavior has been described in outer hair cells of the mammalian cochlea (for review see Ref. 5). These cells can change length at acoustic frequencies when electrically stimulated as a consequence of the operation of voltage-sensitive membrane-bound molecular motors located along the length of the outer hair cell. Depolarization of the cell membrane results in cell shortening, and hyperpolarization results in elongation of the cell. The outer hair cell motor is neither Ca2+ nor ATP dependent, but its molecular identity is unknown. The simplest models in which the motor molecules bring about shape changes in the outer hair cell suggest that a specialized submembrane cortical lattice aids in force distribution during motility. The resulting cellular deformations alter the mechanics of the cochlear partition and result in sound amplification. Outer hair cells also undergo slow changes of shape, often occurring over several minutes. The underlying mechanism and physiological significance of this slow motility is less clear, but it may be Ca2+ and ATP dependent and under efferent control. There is no evidence to suggest that vestibular hair cells show a fast motility like that seen in outer hair cells; however, the somatic changes in vestibular cells take place on a time scale comparable with the slow motility of outer hair cells, suggesting a possible common mechanism.
The cytoskeleton of vestibular hair cells, which presumably defines cellular morphology, has been shown to consist of intermediate filaments and microtubules (32). A dense meshwork of actin filaments is present in the cuticular plate. Microtubules have also been found in the cuticular plate and in the cell neck, running parallel to the long axis of the cell. These filaments are thought to play a structural role, providing support to the cell and nucleus (32). Contractile proteins have been reported in the apex of mammalian vestibular hair cells, but their role, if any, in vestibular hair cell motility is not known (29).
In addition to the somatic shape changes described in vestibular hair cells, electrically induced and/or spontaneous deflections of hair bundles in the eel ampulla and in bullfrog saccular hair bundles also have been described (2, 28). Whether such deflections of the hair bundle in species having only type II hair cells are related to the somatic shape changes observed in isolated type I hair cells is not clear. However, because the type I cell is restrained by the calyx and neighboring cells in situ, it has been suggested that motile forces generated within the cell, perhaps under efferent influence, could result in a repositioning of the hair bundle and therefore alter the sensitivity of the cell (6, 12, 36).
We describe here experiments on type I hair cells isolated from guinea
pig vestibular organs that were designed to investigate possible
mechanisms underlying somatic shape changes. Perfusion with high
K+ has been used to elicit type I
hair cell shortening, as described previously (6, 16, 36). The effects
of ion substitutions and pharmacological agents on
K+-induced shape changes have been
tested. Our results provide the first evidence that changes in response
to high-K+ application indicate
ion and water uptake through a bumetanide-sensitive Na+-K+-Cl cotransporter in these
cells.
Members of the Na+-K+-Cl
cotransporter family have been described in numerous cell types and are
implicated in the maintenance of cell volume and in net transepithelial
salt movements (13, 14, 22), for which the stoichiometry for the
cotransporter is reported to be 1Na:1K:2Cl. Cotransporter activity is
known to be regulated under certain conditions by mechanisms involving phosphorylation (13, 22). In vestibular dark cells, stimulation of the
cotransporter with raised external
K+ results in an increase in
height (used as an index of volume) (34, 35). In many cells (e.g.,
epithelia, endothelial cells, nerve, and muscle), raised external
K+ results in cell swelling. This
is thought to be due to K+ or
Cl
entering through ion
channels or coupled transport, resulting in water influx and a
subsequent increase in volume. In type I cells, the
Na+-K+-Cl
cotransporter could
remove excess amounts of K+
hypothesized to accumulate in the intercellular space during vestibular
stimulation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of hair cells. Guinea pigs (female albino, weight 200-400 g) were killed by rapid cervical dislocation. Both bullae were removed, and the end organs (semicircular canals and utricles) were dissected out. Gerbil type I hair cells were also used in three experiments to test high K+ and hyposmotic stimuli when guinea pigs were not available. Gerbil cells were dissociated, using methods described previously (24). End organs were removed from Mongolian gerbils (55-70 g) anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg ip) and supplemented with ketamine (10 mg/kg im), followed by decapitation. Experiments were conducted in accordance with the "Guiding principles in the care and use of animals," as specified by the American Physiological Society. The otolithic membrane was mechanically removed from the utricle. End organs were placed in a low-Ca2+ phosphate-buffered solution, incubated with papain (0.17 mg/ml, 26-34 min), and washed in bovine albumin (0.5 mg/ml, normal phosphate-buffered solution), and the hair cells were mechanically dissociated as described previously (24). In some cases no enzyme was used, since papain is known to digest neurotransmitter cotransporters and certain ion channels (1, 23) and we wished to minimize alteration of membrane proteins. No obvious differences in responses were found between enzyme-treated and non-enzyme-treated cells. Cells were plated out onto glass chamber slides that had been coated with concanavalin A (0.5 mg/ml) so that cells adhered and did not wash away. After the isolation procedure, cells were left for ~20 min to allow settling to the base of the chamber. Thereafter, cells were perfused at ~0.7 ml/min with extracellular solution, using a peristaltic pump (Minipuls 2, Gilson International). Experiments were carried out at 22-24°C. As judged by morphological criteria (smooth opaque membranes, no nuclear membrane visible, and no granularity within the cytoplasm), hair cells remained healthy and were used in experiments for up to 7 h after extraction of the vestibular end organs from the labyrinth.
Pharmacological agents were pressure applied (Picopump PV800, World Precision Instruments) through glass pipettes with tip diameters of ~2 µm positioned at ~50 µm from the cell under investigation. Cell shortenings were evoked by pipette ejection of 125 mM K+ (high K+). Pharmacological agents were applied for 60 s. As shown in Figs. 2A and 4-7, control length was measured 1-10 s before test solution application, 60 s into application, and 45-60 s after termination of the test solution. Pressure ejection of normal extracellular solution produced no detectable length changes.Image acquisition and cell
measurements.
Cells were viewed with ×40 [0.75 numerical aperture
(NA)] or ×63 (0.9 NA) water immersion objectives on the
stage of an upright microscope (Axioskop, Zeiss) equipped with Nomarski
optics and a video camera (Wat-902, Watek). Images were recorded on an
S-VHS videotape recorder (Panasonic AG 4700). During each experiment, up to four images, recorded at regular intervals over time, were digitally stored on an image processor (Arlunya, TF600; Dindima) to aid
in motion detection. Before drug application, cells were identified
according to their neck-to-plate ratio (NPR) and neck-to-body ratio
(NBR) (27). This identification is based on previous measurements of
avian and mammalian type I and type II cells in fixed tissue (where
type I cells were identified by the presence of a nerve calyx) and
measurements on dissociated cells (27). Hair cell measurements were
made of the cell length, cuticular plate width, minimum neck width, and
maximum cell body width perpendicular to the long axis of the cell.
Cell length was measured as the distance from the center of the
cuticular plate to the base of the cell. The error associated with
length measurements was estimated to be 0.5%. Neck width was
measured at the narrowest point below the cuticular plate (27). All
type I cells studied here fell into the previously established group 1 category (NPR <0.70, NBR <0.64). Type II cells belonged to group 3 (NPR >0.70, NBR >0.58) (26, 27).
Solutions.
The bath solution contained (in mM) 145 or 148 NaCl, 2 or 5 KCl, 1.1 CaCl2, 2.0 MgCl2, 3 D-glucose, and either 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid or 2 NaH2PO4
and 8 Na2HPO4.
Unless otherwise stated, all solutions were adjusted to 305 mosM, and the pH was adjusted with NaOH or HCl to 7.4. High-K+ solution contained 125 mM
K+ substituted (equimolar) for
NaCl. Hyposmotic solution contained a lowered NaCl (120 mM). For the
hyperosmotic solution, sucrose was added to the normal extracellular
solution to increase the osmolarity to 350 mosM. Zero
Ca2+ solution contained no added
CaCl2 and 1 mM ethylene
glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA). In 0 Na+ solution,
Na+ was substituted (equimolar)
with
N-methyl-D-glucamine
(NMDG). In 0 Cl
solution,
Na+,
K+, and calcium gluconate and
MgSO4 were used to substitute
(equimolar) for chloride salts.
Statistical analysis. Mean values ± SD are shown. For statistical analysis, logarithms of the percent values were taken and compared, using a paired Student's t-test. In cases of a nonnormal distribution, a Wilcoxon signed rank test was performed.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hair cell shape changes in response to
high-K+
application.
An example of shortening and widening in response to high
K+ in an isolated type I cell is
shown in Fig. 1. Forty-five of
fifty (90%) type I cells shortened by >0.5% in response to high
K+. Two cells studied showed a
very pronounced shortening in response to
K+, to ~60%, but showed little
recovery and have therefore been excluded from the sample. The mean
length, as percent fraction of the control, was 96.7 ± 3.1%,
representing an average decrease in length of 0.8 µm, and the mean
width was 104.9 ± 5.7% of control in the presence of 125 mM
K+
(n = 48 cells; Fig.
2A), an
average increase of 0.4 µm. The cell length and cell
width in high K+, shown in the
distribution histograms of Fig. 2, B
and C, were statistically
significantly different from control (signed rank test and paired
t-test, respectively,
P < 0.001). Sixty seconds after the
termination of the K+ pulse, cell
length and width were 98.4 ± 2.8 and 101.4 ± 3.8% (n = 48), respectively, indicating a
partial recovery. Recovery values for length and width were
significantly different from values in high
K+ but were also significantly
different from control values (signed rank tests and paired
t-tests used for length and width
values, respectively; Fig. 2A).
Approximately one-third (34.9%) of cells that shortened in high
K+ recovered to lengths of 99.5%
or greater during the first minute after
K+ removal. During shortening, the
cell neck length typically decreased and width increased, while the
perinuclear region of the cell base increased in diameter. In some
cases, neck shortening was asymmetric, resulting in a change in the
angle of the cuticular plate as has been reported previously (6, 12,
36). These asymmetric shortenings were not analyzed further, because
the cells were stuck down and it was not possible to determine whether the tilting was inherent to the cell. The width of the cuticular plate
did not change during shortening. The mean control value for was
0.38 ± 0.1. In high K+,
= 0.48 ± 0.1, which was statistically significantly
different from the control (signed rank test,
P = 0.02, n = 9), indicating that type I cells
became significantly rounder during
high-K+ application. Cells
typically responded to repeated applications of high KCl (up to 3 times
tested) over periods of up to 20 min. The time course of length changes
for a cell during and after high-K+ application is shown in
Fig. 3.
|
|
|
Type I cell shortening is not dependent on Ca2+ influx. A previous report suggested that depolarization-induced shortening was dependent on external Ca2+, implicating a possible contractile mechanism in type I cells (12). Depolarization of the type I cell through elevation of external K+ could activate voltage-dependent Ca2+ channels, and the resulting Ca2+ influx might then trigger a Ca2+-dependent contraction of the cell. The effect of removal of external Ca2+ was therefore tested by using a solution containing high K+, nominally 0 Ca2+, and 1 mM EGTA. Removal of external Ca2+ has also been reported to reduce the concentration of intracellular Ca2+ in guinea pig type I hair cells (3). Ca2+ removal did not inhibit shortening in type I cells, as shown in Fig. 4A. Mean shortening in response to high K+ was 95.6 ± 4.0%, and cells subsequently recovered to 99.3 ± 3.0% (n = 4) of their original length. The same cells showed a mean shortening when external Ca2+ was absent to 95.9 ± 1.5%, which was not statistically significantly different from the high-K+ response, with recovery to 99.1 ± 2.4%.
|
Type I cell shortening is not blocked by externally applied
Ba2+.
Type I hair cells have a large K+
conductance that is active at potentials above approximately 90
mV and is blocked by externally applied
Ba2+ (24). To investigate whether
this conductance plays a part in type I cell shortening, external
Ca2+ was replaced with 5 mM
Ba2+.
Ba2+ did not inhibit but in fact
enhanced shortening in three cells tested (Fig.
4B). The mean cell length in high
K+-Ba2+
solution was 76.4 ± 14.7%, whereas the mean length in high
K+ was 97.4 ± 0.92%. Two of
the cells studied shortened to <75% of their control length during
application of high
K+-Ba2+
solutions and did not recover when returned to control solution (mean
length 78.7 ± 16.9%, n = 3).
Type I cell shortening to high
K+ is blocked by
removal of external
Na+ or
Cl.
To investigate a possible role for
Na+, external
Na+ was replaced with NMDG. The
mean response of nine cells tested with both high-K+ and
high-K+-NMDG solution is shown in
Fig. 5A.
In the absence of external Na+,
six of nine cells showed a small length increase when
K+ was applied, and in the
remaining three of nine cells, the response to high
K+ was reduced. The length in
K+-NMDG (100.1 ± 1.5%) was
statistically significantly different from the response to normal high
K+ (97.0 ± 1.2%,
n = 9, signed rank test,
P = 0.004). The mean width increase in
the presence of K+-NMDG was 103.3 ± 6.9% (n = 9) and was not
significantly different from the control mean width increase (108.8 ± 14.9%, n = 9; not shown).
|
Type I cell shortening is reduced by
bumetanide.
The dependence on Na+ and
Cl suggested that an
Na+-K+-Cl
cotransporter might be
involved in type I cell shortenings. To investigate this further,
bumetanide, a specific blocker of the cotransporter, was used. Control
responses to high-K+ application
and responses to high K+ with 10 µM (n = 4), 20 µM
(n = 3), or 100 µM
(n = 7) bumetanide are shown in Fig.
6. Results using the three different
concentrations were pooled, since there were no significant differences
in length or width values among the different bumetanide
concentrations. In response to high
K+ alone, cells shortened to 96.9 ± 2.0% of their original length and recovered to 98.4 ± 1.5%.
In contrast, the mean shortening in the presence of
K+ bumetanide was 99.1 ± 1.5%
and recovery was 99.9 ± 1.4%. Cells widened to 105.4 ± 4.3%
in the presence of K+ and
recovered to 100.9 ± 3.1% (n = 14). Cells widened to 103.6 ± 4.6% in response to potassium
bumetanide and showed no recovery (103.7 ± 6.7%,
n = 14). Cell length in response to
potassium bumetanide was significantly different from length in high
K+ (paired
t-test,
P = 0.001), and, although 9 of 14 cells gave smaller responses to K+
bumetanide than to high K+, cell
width was not statistically significantly different between the two
groups. Application of control solution (normal K+)
containing 100 µM bumetanide gave no statistically significant change
in length or width compared with control (paired
t-test, n = 4).
|
Type I cell shortening is not blocked by ouabain. Ouabain, a blocker of Na+-K+-ATPase, did not block K+-induced shortenings. The average shortening to high K+ applied in the presence of 0.5 mM ouabain was 96.0 ± 3.9% (recovery 97.3 ± 2.3%, n = 3) compared with a control response to KCl of 97.8 ± 0.21% (recovery, 99.1 ± 1.6%, n = 3).
Response to changes in tonicity.
External application of hyposmotic solution (255 mosM), shown in Fig.
7, resulted in type I cell shortening (mean
value after 60 s was 92.1 ± 8.9%,
n = 8; recovery 95.5 ± 5.5%,
n = 4) and widening (112.5 ± 3.9%, n = 7; recovery 103.4 ± 5.2%, n = 3). Both shortening and
widening in hyposmotic solution were statistically significantly
different from control values (signed rank test, P < 0.05 and paired
t-test,
P < 0.001, respectively), whereas recovery values were not (paired
t-test). In contrast, when the tonicity of the external solution was raised to 350 mosM, an increase in cell length accompanied by a decrease in diameter was observed (Fig.
7). The mean length after 60 s in hyperosmotic solution was 102.4 ± 2.2% and the mean width was 96.0 ± 2.5%. Both measurements were
statistically significantly different from control (signed rank test
and paired t-test, respectively,
P < 0.05). One minute after return
to normal osmotic strength, mean cell length and width were 98.6 ± 2.3 and 101.4 ± 3.4% (n = 8), respectively. Recovery values were not statistically
significantly different from control. The decreased from a control
mean value of 0.40 ± 0.1 to 0.38 ± 0.08 in hyperosmotic
solution, and this difference was statistically significant
(P < 0.05, paired
t-test,
n = 7). In hyposmotic solution cells
became more rounded. The
increased from a control value of 0.29 ± 0.04 to 0.33 ± 0.05, and this difference was also
statistically significant (P < 0.05, paired t-test,
n = 8).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results, in agreement with previously published observations (6,
16), show that vestibular type I cells respond to high
K+ by a shortening and widening of
the neck region and an increase in diameter of the base, resulting in
an overall decrease in cell length. Based on the sensitivity of the
K+-induced changes to bumetanide
and their dependence on externally applied
Na+ and
Cl, our results indicate
the presence of a bumetanide-sensitive Na+-K+-Cl
cotransporter in these
cells. In addition we show that type I cell shortening and widening
were produced by hyposmotic solution and were likely due to an increase
in cell volume through osmotic uptake of water (14). Although we did
not measure cell volume directly, it appears that the shortening and
widening in type I cells in response to hyposmotic solution and
K+, as described in outer hair
cells (7), involved an increase in cell volume. As type I cells
swelled, they tended to become more spherical. The simplest scheme for
the type I hair cell is one in which the neck region is modeled as a
cylinder closed at the top by the cuticular plate and inserting at the
base into a nearly spherical cell body (Fig.
8). In this configuration, an increase of
total cell volume will decrease the length of the neck region, provided
that the total membrane area is constant. There will be, to second
order, a small increase in the diameter of the cell body as volume
increases. Although the diameter of the neck and base of type I cells
changed, the cuticular plate width did not; this may be due to the
dense meshwork of actin and microtubules in this region (32).
|
Hyperosmotic solution resulted in an increase in length and decrease in width, expected if the type I cell were to shrink in volume. It therefore appears that, as described in outer hair cells, cell length is inversely related to volume (7). The cells were only exposed to solutions for 1-min periods; this was insufficient time to determine whether type I cells are capable of volume regulatory mechanisms in the face of osmotic changes (14). Preliminary results indicated that type II cells did not shorten significantly in response to high K+.
In vestibular dark cells of the semicircular canals and utricle and in
marginal cells of the stria vascularis in the cochlea, a basolateral
Na+-K+-Cl cotransporter is
thought to be involved in the uptake of
K+ from the perilymph (33, 34).
Secretion of K+ from the apical
surfaces of vestibular dark cells occurs through a slowly activating
K+ conductance (17). This
contributes toward maintaining a high concentration of
K+ in endolymph, the fluid that
bathes the apical surfaces and hair bundles of hair cells. Both
K+ uptake and secretion are
blocked by basolaterally applied bumetanide (18, 33). After
K+-induced swelling, mediated
through Na+-K+-Cl
cotransport,
vestibular dark cells showed a decrease in volume (35). This was found
to be based on K+ efflux through a
K+ channel sensitive to
Ba2+, lidocaine, quinine,
quinidine, and 4-aminopyridine (35). The mechanism for recovery of type
I cell length and width after termination of high
K+ is not known. However, a large
Ba2+-sensitive
K+ conductance has previously been
described in type I cells (24). After coapplication of
K+ and
Ba2+, type I cells showed large,
irreversible shortenings. Our results suggest, therefore, that this
K+ conductance is not required for
K+-induced cell shortening in
isolated cells but that it may be required for recovery.
Bumetanide reduced but did not completely inhibit
K+-induced shortening. In plasma
membranes isolated from dog kidney, radiolabeled bumetanide required
~20 min to reach maximal binding (9), and it is likely that the 60-s
exposures to bumetanide used here were insufficient to produce a
complete block in type I hair cells. Bumetanide is believed to inhibit
cotransport activity by binding extracellularly and preventing the
binding of Cl to an
intracellular anion site (19). Although the affinity for bumetanide
varies between cell types, the dissociation constant for mean saturable
binding is generally in the range 1-10 µM (13). At
concentrations one or two orders of magnitude greater than this,
bumetanide may block certain
Cl
conductances and other
transporters such as Na+-Cl
cotransport (8,
22). However, we saw no significant differences in the effects of 10, 20, and 100 µM bumetanide on type I cell dimensions, suggesting that
the effect was mediated through an Na+-K+-Cl
cotransporter.
Hair cells from the goldfish saccule and lagena have been reported to
lose Na+,
K+, and
Cl over a period of several
minutes when bathed in a solution in which NMDG was substituted for
Na+. However, no significant
volume decrease was observed, and it was suggested that NMDG could
permeate the cell membrane (21). A similar loss of osmolytes also may
have occurred in guinea pig type I cells during exposure to NMDG. In
addition, replacing external Na+
with NMDG would lead to an increase in intracellular
Ca2+ through inhibition of the
Na+/Ca2+ exchanger, thought to be present in
guinea pig type I cells and in goldfish saccular hair cells (3, 20).
The lack of type I cell shortening in the presence of NMDG also
suggests that a Ca2+ influx is not
involved. This is in agreement with results from a different study on
guinea pig type I cells, in which
K+-induced shortenings were
observed when no external Ca2+ (or
Mg2+ ) was present (16). In
contrast, no voltage-induced shortenings were seen in type I cells
bathed in 0 Ca2+ (12). A
comparison of the K+-induced
shortenings described here with voltage-induced shortenings (12)
indicates that the two processes have a similar time course, but
whether the underlying mechanism is the same remains to be tested.
Because the cotransporter is electroneutral, this would seem unlikely.
Our results do not support a
Ca2+-dependent microtubule-driven
contraction mechanism.
Ouabain had no effect on
K+-induced shortening, indicating
that
Na+-K+-ATPase
was not involved. In the guinea pig, immunocytochemistry using
antibodies to the 2-
and/or
3-subunit
isoforms showed no evidence for
Na+-K+-ATPase
in vestibular hair cells, and immunoreactivity was confined to the
calyx (15).
Na+-K+-ATPase
immunoreactivity was also reported in nerve terminals of the gerbil
vestibular system (30). In contrast, immunoreactivity to the
3- and
1-subunits of the
Na+-K+-ATPase
has been reported in the sensory cells of the rat crista and otolithic
organs (31).
Isolated type I cells have a large K+ current that is significantly activated at the zero-current potential of the cell (24). During positive deflection of the hair bundle, K+ will enter through transducer channels, leading to depolarization, and exit via basolateral K+ channels (Fig. 8). Estimates from mathematical modeling suggest that the K+ concentration in the restricted space between type I cell and the calyx nerve terminal, which envelops it in situ, could increase by up to 42 mM at the base of the hair cell (11). Although the concentration of K+ used in this study was somewhat higher than this, vestibular hair cell shortening has been reported in response to K+ concentrations as low as 25 mM (36). Osmolarity in the hair cell-calyx space could also increase significantly (10). In the intact epithelia, the consequences of type I cell changes in shape or volume are unclear. It has been suggested that depolarizations could drive active hair bundle deflections, altering mechanotransduction (6, 12, 36). An alternative view is that such changes in K+ concentration and osmolarity would be potentially damaging to both type I cells and their calyces. The concentration and regulation of K+ in the intercellular cleft remain to be determined experimentally. However, one possible function of the cotransporter, assuming it is located on the basolateral membrane of type I cells, could be reuptake of K+ into the cells to prevent continuous depolarization of the calyceal nerve terminals and deleterious effects on afferent coding.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Collingridge for the loan of a ×63 objective lens and Dr. A. Ricci for comments on the paper.
![]() |
FOOTNOTES |
---|
This work was supported by a National Institute on Deafness and Other Communication Disorders Post-Doctoral Fellowship Award (F32-DC-00169; to K. J. Rennie), a Wellcome Trust Programme Grant to J. F. Ashmore, and in part by a Claude Pepper Award (DC-01273; to M. J. Correia).
Present address of J. F. Ashmore: Dept. of Physiology, Univ. College London, Gower Street, London WC1E 6BT, UK.
Address for reprint requests: K. J. Rennie, Dept. of Otolaryngology, Rm. 7.102 Medical Research Bldg., Univ. of Texas Medical Branch at Galveston, Galveston, TX 77555.
Received 17 March 1997; accepted in final form 15 August 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amara, S. G.,
and
M. J. Kuhar.
Neurotransmitter transporters: recent progress.
Annu. Rev. Neurosci.
16:
73-93,
1993[Medline].
2.
Benser, M. E.,
R. E. Marquis,
and
A. J. Hudspeth.
Rapid, active hair bundle movements in hair cells from the bullfrog's sacculus.
J. Neurosci.
16:
5629-5643,
1996
3.
Chabbert, C.,
Y. Canitrot,
A. Sans,
and
J. Lehouelleur.
Calcium homeostasis in guinea pig type-I hair cell: possible involvement of an Na+-Ca2+ exchanger.
Hear. Res.
89:
101-108,
1995[Medline].
4.
Correia, M. J.,
A. J. Ricci,
and
K. J. Rennie.
Filtering properties of vestibular hair cells: an update.
Ann. NY Acad. Sci.
781:
138-149,
1996[Medline].
5.
Dallos, P.
Cochlear neurobiology.
In: The Cochlea, edited by P. Dallos,
A. N. Popper,
and R. R. Fay. New York: Springer, 1996, p. 1-43.
6.
Didier, A.,
L. Decory,
and
Y. Cazals.
Evidence for potassium-induced motility in type I vestibular hair cells in the guinea pig.
Hear. Res.
46:
171-176,
1990[Medline].
7.
Dulon, D.,
J. M. Aran,
and
J. Schacht.
Potassium depolarization induces motility in isolated outer hair cells by osmotic mechanism.
Hear. Res.
32:
123-130,
1988[Medline].
8.
Evans, M. G.,
A. Marty,
Y. P. Tan,
and
A. Trautmann.
Blockage of Ca-activated Cl conductance by furosemide in rat lacrimal glands.
Pflügers Arch.
406:
65-68,
1986[Medline].
9.
Forbush, B.,
and
H. C. Palfrey.
[3H]bumetanide binding to membranes isolated from dog kidney outer medulla. Relationship to the Na,K,Cl co-transport system.
J. Biol. Chem.
258:
11787-11792,
1983
10.
Goldberg, J. M.
Theoretical analysis of intercellular communication between the vestibular type I hair cell and its calyx ending.
J. Neurophysiol.
76:
1942-1957,
1996
11.
Goldberg, J. M.
Transmission between the type I hair cell and its calyx ending.
Ann. NY Acad. Sci.
781:
474-488,
1996[Abstract].
12.
Griguer, C.,
J. Lehouelleur,
J. Valat,
A. Sahuquet,
and
A. Sans.
Voltage-dependent reversible movements of the apex in isolated guinea pig vestibular hair cells.
Hear. Res.
67:
110-116,
1993[Medline].
13.
Haas, M.
The Na-K-Cl cotransporters.
Am. J. Physiol.
267 (Cell Physiol. 36):
C869-C885,
1994
14.
Hoffman, E. K.,
and
P. B. Dunham.
Membrane mechanisms and intracellular signalling in cell volume regulation.
Int. Rev. Cytol.
161:
173-262,
1995[Medline].
15.
Ichimiya, I.,
J. C. Adams,
and
R. S. Chimera.
Immunolocalization of Na+,K+-ATPase, Ca2+-ATPase, calcium-binding proteins, and carbonic anhydrase in the guinea pig inner ear.
Acta Otolaryngol. (Stockh.)
114:
167-176,
1994[Medline].
16.
Lapeyre, P. N. M.,
and
Y. Cazals.
Guinea pig vestibular type I hair cells can show reversible shortening.
J. Vestib. Res.
1:
241-250,
1991.
17.
Marcus, D. C.,
and
Z. Shen.
Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C857-C864,
1994
18.
Marcus, D. C.,
and
A. Shipley.
Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe.
Biophys. J.
66:
1939-1942,
1994[Abstract].
19.
Moore, M. L.,
J. N. George,
and
R. J. Turner.
Anion dependence of bumetanide binding and ion transport by the rabbit parotid Na+-K+-2Cl co-transporter: evidence for an intracellular anion modifier site.
Biochem. J.
309:
637-642,
1995[Medline].
20.
Mroz, E.,
and
C. Lechene.
Calcium and magnesium transport by isolated goldfish hair cells.
Hear. Res.
70:
139-145,
1993[Medline].
21.
Mroz, E.,
and
C. Lechene.
Extracellular N-methyl-D-glucamine leads to loss of hair-cell sodium, potassium, and chloride.
Hear. Res.
70:
146-150,
1993[Medline].
22.
O'Grady, S. M.,
H. C. Palfrey,
and
M. Field.
Characteristics and functions of Na-K-Cl cotransport in epithelial tissues.
Am. J. Physiol.
253 (Cell Physiol. 22):
C177-C192,
1987
23.
Proks, P.,
and
F. M. Ashcroft.
Modification of K-ATP channels in pancreatic beta-cells by trypsin.
Pflügers Arch.
424:
63-72,
1993[Medline].
24.
Rennie, K. J.,
and
M. J. Correia.
Potassium currents in mammalian and avian isolated type I semicircular canal hair cells.
J. Neurophysiol.
71:
317-329,
1994
25.
Rennie, K. J.,
A. J. Ricci,
and
M. J. Correia.
Potential-driven movements of type I vestibular hair cells (Abstract).
Biophys. J.
66:
A169,
1994.
26.
Ricci, A. J.,
S. L. Cochran,
K. J. Rennie,
and
M. J. Correia.
Vestibular type I and type II hair cells. 2. Morphometric comparisons of dissociated pigeon hair cells.
J. Vestib. Res.
7:
407-420,
1997[Medline].
27.
Ricci, A. J.,
K. J. Rennie,
S. L. Cochran,
G. A. Kevetter,
and
M. J. Correia.
Vestibular type I and type II hair cells. 1. Morphometric identification in the pigeon and gerbil.
J. Vestib. Res.
7:
393-406,
1997[Medline].
28.
Rüsch, A.,
and
U. Thurm.
Spontaneous and electrically induced movements of ampullary kinocilia and stereovilli.
Hear. Res.
48:
247-264,
1990[Medline].
29.
Sans, A.,
P. Atger,
C. Cavadore,
and
J.-C. Cavadore.
Immunocytochemical localization of myosin, tropomyosin and actin in vestibular hair cells of human fetuses and cats.
Hear. Res.
40:
117-126,
1989[Medline].
30.
Spicer, S. S.,
B. A. Schulte,
and
J. C. Adams.
Immunolocalization of Na+K(+)-ATPase and carbonic anhydrase in the gerbil's vestibular system.
Hear. Res.
43:
205-217,
1990[Medline].
31.
Ten Cate, W. J.,
L. M. Curtis,
and
K. E. Rarey.
Na,K-ATPase and
subunit isoform distribution in the rat cochlear and vestibular tissues.
Hear. Res.
75:
151-160,
1994[Medline].
32.
Takumida, M.,
H. Miyawaki,
and
Y. Harada.
Cytoskeletal organization of the vestibular sensory epithelia.
Acta Otolaryngol. (Stockh.)
519:
66-70,
1995.
33.
Wangemann, P.,
J. Liu,
and
D. C. Marcus.
Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro.
Hear. Res.
84:
19-29,
1995[Medline].
34.
Wangemann, P.,
and
D. M. Marcus.
K+-induced swelling of vestibular dark cells is dependent on Na+ and Cl and inhibited by piretanide.
Pflügers Arch.
416:
262-269,
1990[Medline].
35.
Wangemann, P.,
N. Shiga,
C. Welch,
and
D. M. Marcus.
Evidence for the involvement of a K+ channel in isosmotic cell shrinking in vestibular dark cells.
Am. J. Physiol.
263 (Cell Physiol. 32):
C616-C622,
1992
36.
Zenner, H. P.,
and
U. Zimmermann.
Motile responses of vestibular hair cells following caloric, electrical or chemical stimuli.
Acta Otolaryngol. (Stockh.)
111:
291-297,
1991[Medline].
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |