1 BioCurrents Research Center and 2 Laboratory for Reproductive Medicine, 3 Marine Biological Laboratory, Woods Hole, Massachusetts 02543; and 4 Women and Infants Hospital, Providence, Rhode Island 02905
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ABSTRACT |
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This review introduces new developments in a technique for measuring the movement of ions across the plasma membrane. With the use of a self-referencing ion-selective (Seris) probe, transport mechanisms can be studied on a variety of preparations ranging from tissues to single cells. In this paper we illustrate this versatility with examples from the vas deferens and inner ear epithelium to large and small single cells represented by mouse single-cell embryos and rat microglia. Potassium and hydrogen ion fluxes are studied and pharmacological manipulation of the signals are reported. The strengths of the self-referencing technique are reviewed with regard to biological applications, and the expansion of self-referencing probes to include electrochemical and enzyme-based sensors is discussed.
ion-selective electrodes; self-referencing probes; vas deferens; embryos; microglia; inner ear
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INTRODUCTION |
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THIS REVIEW INTRODUCES the application of a new technique for studying tissues and cells by monitoring the movement of molecules in close proximity to the plasma membrane with the use of self-referencing probes. The principle underlying this approach has long been established, but the recent incorporation of different sensors provides novel approaches to cell physiology. Here we aim to draw attention to these new developments by presenting selected examples where detection of hydrogen and potassium ion flux is providing biological data from systems as diverse and complex as epithelia and single cells. We conclude by discussing opportunities for self-referencing electrochemical and biosensor probes making possible detection of molecules such as oxygen, nitric oxide, and glucose moving across the membrane of single cells. In this review the focus is on applications of this methodology. The technical details have recently been reviewed elsewhere (61).
There is a clear discontinuity between the ionic composition of the
inside and outside of cells. In the case of potassium, the internal
activity of this ion ([K]i), is more than 50 times that
of the bulk medium. However, the boundary between a living cell and the
surrounding medium is far from simple. It is easy to lose sight of the
fact that there can be a considerable disparity in the ion activities
in close proximity to the cell membrane and those in the bulk medium.
Boundary conditions and surface charges immediately at the interface
between the cell membrane and the medium can, in theory, drastically
alter the ionic composition of the cell-to-medium boundary (46,
47). Surface and boundary voltages, and their related effects on
ion accumulation, have been extensively modeled (12) and
are largely confined within the Debye length (~8 Å in saline). This
is a very difficult region in which to make direct measurements.
However, there are other disparities between the immediate environment
of a cell and those of the bulk medium that are amenable to direct
measurement. As ions, or molecules in general, move between a cell and
its immediate surroundings, there inevitably exists a diffusion
gradient within micrometers of the cell surface. If transport is steady
and maintained over the long term, a chemical gradient will be
established (Fig. 1). For example, in the
case of a net influx, there will be a depletion surrounding the site of
transport. This gradient can be measured noninvasively with a suitable
probe positioned within its boundaries, revealing new facets of
cellular transport mechanisms that complement the more familiar
approaches to cellular electrophysiology.
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One approach to assessing the movement of ions in proximity to the cell membrane requires the sampling of the local activity at more than one point with the use of multiple electrodes. This is difficult, not because there is a lack of suitably selective sensors but because all electrodes suffer from pronounced drift. This drift is of such a magnitude that it reduces the application of ion sensors to comparatively large changes in ion activity, such as between the experimental medium and within the cytosol of the cell. One solution that greatly enhances sensitivity for the extracellular detection of ion gradients is to borrow an older approach for extracellular detection of current densities and use a single self-referencing probe. As previously published for self-referencing calcium ion flux detection (61), a glass microprobe, front-filled with an ion-selective liquid membrane, is moved in a square-wave translation such that the sensor rests for 1 s at each of two positions ~10 µm apart. Via this method, ion activity can be locally sampled at known positions from each other and the cell membrane. One of these positions can be within a micrometer of the cell membrane, but both should lie within the diffusion gradient, thereby allowing the calculation of ion flux. This is the basis of a noninvasive, self-referencing, chemically selective probe and is the subject of this review.
Self-referencing probes have a relatively long history, particularly with regard to voltage detection. The first example of a noninvasive, self-referencing voltage probe, applied to a biological system, was published in 1950 (5), when an ingenious vibrating platinum probe was applied to the measurement of surface voltages from plants. This pioneering study preceded the subsequent development of other aerial probes achieving similar voltage resolutions (3, 20, 53). Most importantly, these first authors foresaw the use of this device in conductive media in which diffusion potentials could be measured. Despite an early report on the measurement of external voltage fields in a liquid medium (15), it was not until the seventies that a complete description of a self-referencing voltage probe appeared (24). In that design the probes had a sensitivity of low nanovolts over sampling distances of tens of micrometers. That device, commonly referred to as the "vibrating probe," achieved its voltage resolution by minimizing the impact of drift through the simple expedient of using a single electrode to compare voltages at two positions micrometers apart. The probe capacitively coupled to the external voltage field and used phase detection to isolate signals coherent with the frequency of vibration. The differential voltage acquired through this technique could be converted into a current through Ohm's law. The technique has been reviewed on several occasions, most recently in 1990 (48).
There were indications that the same principle of self-referencing could be applied to the selective detection of ion activity and, therefore, ion flux across the plasma membrane. For example, pH gradients can be observed along the length of fungal hyphae by using a pH electrode to locally sample the activity of hydrogen ions and comparing these values with those in the bulk medium (19). The electrode was positioned at the recording site by manually operated positioners. In 1987 a significant step was made in producing an automated self-referencing, ion-selective (Seris) probe when an artificial gradient was accurately followed beyond the levels possible with the use of static electrodes (23), a concept that found its first biological application in 1990 (33). Both of these early studies (23, 33) focused on calcium and established the Seris probe as a powerful new tool for monitoring the net trans-plasma membrane movement of this important ion. This experimental approach has subsequently found numerous and compelling applications in calcium detection, where results not only support and reinforce data acquired through other methodologies but offer new insights into cellular regulation of calcium transport and the role this ion plays in cell physiology (see Refs. 60-62 for review). The ability to detect the activity of an ion, inside or outside a cell, depends on the suitability of the sensor. In the application discussed here, ion-selective liquid membranes are used. These membranes have seen extensive application in cell biology but can be restricted by complicated additive voltages not dependent on the ion being targeted. The complications, for example, can come from junction potentials, direct voltage detection, and nonspecificity of the ionophores (1). The problems are encapsulated in the Nikolsky-Eisenman and Nernst equations, which define the selectivity and performance of a liquid membrane.
The application of these two pivotal equations and how a Seris probe can be built and applied to the local detection of ion activity was dealt with in depth in a previous paper (61), in which the subject was the detection of calcium ion movements. Several important features of Seris probes were dealt with in that paper, notably drift characteristics, response times, and the problem of contaminating chemicals. Most features involved in calcium detection apply generally to the measurement of all ions and are not repeated here. The purpose of the current review is to draw attention to the application of the Seris approach to the detection of hydrogen and potassium ions in the study of single cells and epithelial structures. It is worth noting that the application of this technique can be expanded to include any ion where a suitable ion-selective membrane and physiological subject coincide [see, for example, cadmium detection (51)].
The diversification of the self-referencing ion probes from calcium to other molecules was not led by demand from the animal sciences but from botany (31). The attraction lies in an inherent advantage of a noninvasive technique in plant studies because it avoids the need to penetrate the relatively rigid cell wall, where the contents are often under pressure. Plant sciences have made considerable use of self-referencing technology, and a list of probe-related publications illustrating the diversity of biological applications, including uses in mycology and botany, can be found at www.mbl.edu/BioCurrents. The first Seris plant study (31) demonstrated that the commercially available hydrogen and potassium ion liquid membranes [such as Fluka hydrogen ionophore I (tridodecylamine) in cocktail B and Fluka potassium ionophore I (valinomycin) in cocktail B] could be used in a manner similar to the liquid membrane for the detection of calcium. The results in this case showed pronounced hydrogen and potassium ion fluxes around roots. Although the probe was originally conceived for single cell studies, these results foreshadowed an unexpected strength of the Seris probes: their ability to aid in the characterization of ion-transporting mechanisms embedded within epithelial structures. Our first two examples, the mammalian vas deferens and the cation-transporting epithelia of the inner ear, illustrate epithelial applications. Our second two examples move from the tissue to smaller targets, individual cells, represented by the mouse single-cell embryo and primary cultures of single microglia. These examples illustrate the ability of a Seris probe to "microsample" the ion activities in close proximity to cellular structures, measuring the diffusion gradient and ion fluxes.
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PROTON FLUX FROM THE VAS DEFERENS |
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Mammalian spermatozoa are maintained in a quiescent state as they
pass through the caudal epididymis and vas deferens. An acidic pH,
~6.5 (11, 35), is essential to achieve this immobility (22, 25), but the mechanism for achieving this acidity has been unclear. Studies performed before those under review here used
perfused epididymis and cultured cells to implicate a role for an
apical Na+/H+ exchanger in establishing the
acidic environment (2). However, studies in which
antibodies were used against the vacuolar (V-type) hydrogen pump, a
nonphosphorylating H+-ATPase, beautifully demonstrated the
presence of this pump in the region of the apical membrane of cells
within the proximal to middle part of the vas deferens (Fig.
2, A and D)
(9). This finding raised the possibility that this pump
plays a role in luminal acidification.
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By taking a proximal section of the vas deferens and splitting it open
along its axis, it is possible to scan over the surface of the apical
epithelium with a hydrogen-selective Seris probe (H+
probe). A substantial flux of hydrogen ions is measured, showing regions of higher flux thought to occur over patches of pump-containing cells (9). Application of bafilomycin A1, a selective
blocker for the V-type pump, drastically reduces the hydrogen ion flux (Fig. 3), producing direct evidence that
the V-type pumps are responsible for most of the acidification in the
proximal region of the vas deferens.
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Hydrogen ion pumping requires a hydrogen ion source. In the mammalian testis and epididymis, studies have demonstrated the presence of carbonic anhydrase (CA) (13, 17, 18). At least two isoenzymes are found in the epididymis, the membrane-associated CAIV and the cytosolic CAII, with colocalization in the vas deferens between the V-type pumps and CAII (Fig. 2, B and C) (7, 9). Inhibition of CAII with acetazolamide eliminated the bafilomycin-sensitive component of the hydrogen ion flux (Fig. 3) (7), confirming a role for this enzyme in hydrogen ion generation in these cells. Of interest is the inability of compounds targeting the V-type proton pump or its performance to shut down the hydrogen flux entirely; only 60-80% of the signal is lost. A possible, but as yet unproven, explanation is that the residual flux is an indirect result of CO2 production by cellular respiration reacting with water outside the cell to produce hydrogen ions and bicarbonate. The probe will detect the generated hydrogen ions as a directional flux of protons coming from the cell surface.
By analogy with the kidney type A intercalated cells, we might expect
an electroneutral Cl/HCO3
exchanger
(AE1), coupled to basolateral Cl
channels and CAII, to
aid in the generation of hydrogen ions for apical pumping. However,
this exchanger could not be identified in the basolateral membranes
with an antibody that detects both the AE1 and AE2 isoforms
(9). This left the puzzling question as to the role of
Cl
and HCO3
transporters in the process
of vas deferens apical membrane proton transport. With the use of a
combination of chloride ion removal as well as the application of
4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and
diphenylamine-2-carboxylate (DPC), further studies with Seris
H+ probes have concluded that a
Cl
/HCO3
exchanger is not involved, but
the nature of the HCO3
transporter is not known
(7). The action of DPC in the absence of chloride
indicates the possible presence of a
Cl
/HCO3
channel (7).
Figure 3 presents a model of the proton pump (PP)-rich cell and a
summary of these results.
The difference in chloride dependence between the hydrogen ion-pumping cells of the proximal vas deferens and the intercalated cells of the kidney shows that there are differences in the mechanisms for generating an apical hydrogen ion flux (10). However, some processes and control mechanisms might still be common to the two tissues. An obvious comparison would be whether in the vas deferens, as in the kidney, the V-type pumps are trafficked to the apical membrane in a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent manner. This question has been examined in Seris studies in conjunction with immunohistochemistry and Western blotting before and after treatment with tetanus toxin (8). This toxin is known to cleave the vesicle-bound SNARE cellubrevin, preventing vesicle docking with the membrane. Cellubrevin colocalizes with the V-type hydrogen ion pumps (8), and tetanus toxin treatment inhibits the bafilomycin-sensitive proton secretion by 64% (Fig. 3). Western blotting demonstrated the expected cleavage of the cellubrevin in the intact preparation. Furthermore, treatment of the tissue with colchicine, a disrupter of the microtubule structure, causes a marked redistribution of the V-type H+- ATPase from the apical membrane to the cytosol (Fig. 2, E and F). These results strongly support the model of SNARE-dependent vesicle trafficking to the apical membrane.
One complication, unique to the measurement of hydrogen ion flux, is
the almost ubiquitous presence of a buffer in the bulk medium. This
will inevitably reduce the activity of hydrogen ions as they diffuse
from the site of transport, thus maintaining the bulk pH. Two attempts
have been made to accommodate the buffering effect, deriving a valid
and quantifiable flux measurement. The first attempt at this correction
(16) was used in the data discussed below for microglia
(59). This approach required an estimate for the diffusion
constant of the buffer. The second equation, derived by D. M. Porterfield (unpublished observations), reads as follows
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POTASSIUM SECRETION FROM THE VESTIBULAR DARK CELLS |
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Sensing static position and body motion involves the modulation of a transepithelial current through the inner ear neuroepithelium. This current is carried by potassium ions moving down the electrochemical gradients across the basolateral and apical membranes of the epithelium; the concentration of potassium in the inner ear lumen is 25-30 times that in the perilymph bathing the basolateral epithelial surface (56). Indirect evidence suggests that a specific cell type within the epithelium, the vestibular dark cells, establishes and maintains the transepithelial potassium gradient by transporting potassium across the basolateral surface into the cell cytoplasm and from there to the lumen of the inner ear (4, 30, 55). By employing vibrating voltage probe technology to derive near-field currents and Seris probes to measure the ionic composition in the proximity of epithelial cells, it was possible to conclusively identify vestibular dark cells as being responsible for transporting and concentrating potassium within the inner ear lumen (42). The molecules participating in the potassium transport across the vestibular epithelium were characterized (42, 43, 57, 65) by combining Seris technology with the use of a micro-Ussing chamber for measuring transepithelial voltage, resistance, and current and with the use of patch-clamp techniques for monitoring whole cell currents from individual epithelial cells, as well as with pharmacology. Seris technology has identified similar mechanisms underlying potassium transport across cochlear (acoustic) epithelium (65).
Initial electrophysiological investigation of the inner ear epithelium identified a transepithelial current by sealing the epithelium over the aperture of a micro-Ussing chamber. This current could be modulated by shifts in the ionic composition of solutions on either side of the epithelium (39, 40, 67). Although the micro-Ussing chamber (80-µm diameter) allowed determination of the electrogenic properties of the epithelium, the specific ionic species accounting for the electric current remained only an assumption until self-referencing technology was employed and a direct correlation to potassium ion secretion was demonstrated (42).
Relative current densities over the apical surface of epithelial cells
were measured with the use of the vibrating voltage probe, and a large
current (40-60 µA/cm2) was detected emanating from
vestibular dark cells (42, 65). Inhibition of the
Na+-K+-Cl cotransporter with
bumetanide or the Na+-K+-ATPase with ouabain
reduced the current, whereas increased basolateral potassium
concentrations enhanced the current. These data suggested that
potassium transport into the basolateral surface of vestibular dark
cells was critical in maintaining the apically directed current and
that perhaps potassium ions carried the apically directed current as
they become concentrated in the inner ear lumen (42). Employment of potassium-selective Seris probes (K+ probes)
directly demonstrated that vestibular dark cells were indeed secreting
potassium into the inner ear lumen (42). The activity of
potassium 20-240 µm above that of the epithelium was shown to be
elevated over that of the bulk medium, and positioning the Seris probe
in close proximity of vestibular dark cells revealed these cells to be
the source of the potassium efflux. Both bumetanide and ouabain caused
a decrease in the potassium activity near vestibular dark cells (Fig.
5) and established a model by which
potassium is transported into vestibular dark cells by the
Na+-K+-Cl
cotransporter and
Na+-K+-ATPase and subsequently secreted into
the inner ear lumen, where it becomes concentrated.
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Three classes of potassium channels have been described in the apical surface of vestibular dark cells, and each can contribute to the potassium efflux from vestibular dark cells into the inner ear lumen. Apical potassium channels include the slowly activating channel (IKs) (41), a nonselective cation channel (44), and a maxi-K+ channel (63). Modulation of the transepithelial voltage, resistance, current, and potassium efflux by disulfonic stilbenes (Fig. 5) suggested that the primary mechanism by which potassium moves across the apical surface of vestibular dark cells is through IKs channels (41, 57). Subsequent studies demonstrated that phospholipase C and protein kinase C are capable of modulating IKs channels and potassium efflux through the apical neuroepithelium (43). Interestingly, potassium efflux through the apical membrane also participates in cell volume regulation (66). Hyposmotic challenge of inner ear neuroepithelium resulted in an increase in the volume of the epithelial cells, followed by a compensatory volume decrease that returned cells to their original volume (67). The Seris K+ probe measured an elevation in potassium activity in the proximity of vestibular dark cells during compensatory volume decreases and established that the primary mechanism underlying this decrease is a change in osmotic pressure induced by potassium efflux through IKs channels in the apical surface (66).
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EMBRYONIC APOPTOSIS AND POTASSIUM FLUX |
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The studies on the inner ear neuroepithelium illustrate the
usefulness of the Seris technique for understanding the physiology of
cellular layers by measuring ionic activities in the proximity of cell
aggregates. The role of potassium in volume regulation of individual
cells undergoing cell death has also been investigated with the use of
Seris K+ probes (64). One characteristic of
apoptosis, a particular class of cell death, is pronounced cell
shrinkage (6, 28). Indeed, single-cell mouse embryos
treated with agents that induce apoptosis undergo rapid cell shrinkage
(2% decrease in volume per minute) (27, 64). With the use
of the Seris K+ probe, it was determined that, coincident
with shrinkage, the activity of potassium in the proximity of embryos
became elevated, and pharmacological dissection suggested that
potassium was fluxing through tetraethylammonium-sensitive potassium
channels (Fig. 6) (64).
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Movement of potassium across cell plasma membranes is critical to a wide variety of cellular processes, and the Seris technology is uniquely suited to noninvasively investigate the physiology of small tissues and individual cells by monitoring changes in the ionic composition of the media in their proximity. Next, we describe how single cells need not be relatively large, as are mouse embryos, but can be as small as 10 µm in diameter.
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MICROGLIA POTASSIUM REGULATION UTILIZES A MEMBER OF THE H+-K+-ATPASE FAMILY |
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The microglia are an interesting group of neural cells with features that set them apart from the other glia and neurons. Central to this is the developmental origin of microglia. Although still a matter of ongoing debate, the body of evidence now favors an ontogenetic relationship of microglia with mononuclear phagocytes (36, 21), a distinctly unique lineage for neural tissues. Microglia respond in specific manners to several pathological conditions and injury (32). Activated microglia can undergo oxidative bursts, facilitated oxygen radical production, and phagocytosis of pathogens and cellular debris. Microglia also interact with other cells of the brain, notably astrocytes, where inflammation initiates astrocytic release of cytokines such as granulocyte/macrophage colony-stimulating factor and macrophage colony stimulating factor, which in turn activate microglia. Potassium appears to be a key player in several microglial responses such that cytokine-induced proliferation and differentiation in microglia, for example, involve activation of an inwardly rectified potassium channel (Kir) (54, 58). Furthermore, injury to brain tissue can radically alter the microenvironment, raising external potassium concentrations and influencing transport mechanisms (14, 29). The potential significance to the microglia of potassium activity led to the study of the cellular mechanism behind potassium transport (59).
Initial application of a potassium-selective Seris probe to isolated
microglia from rat brain showed a clear K+ influx,
registered as a lower activity in proximity to the plasma membrane.
Close to the membrane, a drop of 9.43 ± 4.2 µM in the external potassium activity (
[K+]o) was
recorded, but the values within the sample showed an apparent bimodal
distribution with peaks at
6 and
15 µM. This
[K+]o is referred to by the authors as a
differential diffusion potential. Clearly, one possible source
of the change in the measured activity of potassium is influx through
the Kir channel. The presence of this channel was confirmed
in these isolated cells, but blocking the activity with 1 mM
Cs+ and 2 mM Ba2+ did not affect the
[K+]o gradient.
Kir presence was also examined by activating this channel
with voltage clamp and comparing the elicited currents with changes in
potassium ion gradients in the proximity of microglia recorded with the
use of the Seris technique. The clamp protocol included a two-step
hyperpolarization to 100 and
110 mV from a holding potential of
70 mV. Under these conditions the current density per driving force
was 76 × 10
4
pA · µm
2 · mV
1. The
voltage-sensitive inward potassium current generated a small but
measurable [K+]o depletion gradient. On the
basis of this Seris-derived [K+]o gradient, a
potassium flux can be calculated (61, 62) and converted
into the equivalent current. This can then be compared directly with
the concurrently measured clamp values. The Seris probe measured
0.61-0.77 of the current recorded through the whole cell patch.
This is in good agreement with the expected value, but there is still a
clear underestimation that may be attributable to the position of the
probe in relation to the membrane. A position-dependent response is
expected if a diffusion potential is being measured (61)
and has been demonstrated for the microglial data (59). It
should also be noted that the Seris probe is measuring a local flux
with a sensor of tip diameter between 2 and 4 µm. It is assumed that
the whole cell current is evenly distributed across the cell surface,
but this need not be the case. Clustering of channels may offer an
alternative explanation to the mismatch between the Seris probe
estimates of potassium ion influx and the monitoring with voltage-clamp
techniques. What is clear from these results is that noninvasive
measurement of the flux with a Seris probe closely follows the expected
value from voltage clamp. Coupling of these techniques in the future
can be expected to continue validation of the Seris measurements, not
only for potassium ions but for other ions where voltage-dependent
currents can be isolated.
The experiments described illustrate two important points. First, the
Seris probes can measure the expected whole cell current with an
acceptable level of agreement. Second, it is clear that Kir
does not contribute significantly to the standing potassium influx
measured in the cultured microglia. Because Kir does not contribute to the potassium flux measured in 64% of the cells examined, the question arises as to what does. The
Na+-K+-ATPase is an obvious candidate, but in
blocking experiments with both ouabain and strophanthidin, the latter
targeted ouabain-insensitive P-type ATPases (68, 69) and
the potassium influx gradient was undiminished. This result was
surprising given the neural environment of microglia, but in
considering their developmental origin, it seemed possible that these
cells were using a mechanism for potassium transport common to other
cell lineages. The H+-K+-ATPase was shown to be
responsible for the K+-influx gradient because it was
rapidly shut down by SCH-28080 and omprazole, compounds selective for
that pump (Fig. 7). A counter efflux of
hydrogen ions was also demonstrated with the use of a Seris
H+ probe and was inhibited by SCH-28080. Furthermore, an
antibody raised against the gastric form of the
H+-K+-ATPase bound to microglia in culture
(59).
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The physiological experiments performed with the Seris probe demonstrated that there is an ATPase, normally associated with the intestinal system, present in the microglia of the central nervous system. However, in some parameters the neural H+-K+- ATPase differs from the gastric variety despite sharing the common pharmacology described. Examining the regulation of the diffusion potential by this pump to changes in potassium concentration shows a maximal pumping activity at a [K+]o of 7 mM with a Michaelis-Menten constant of 3.67 mM. The transporter dependence on extracellular pH did not exhibit the expected Michaelis-Menten passive ion availability behavior. Saturation of the transporter by hydrogen ions was not achieved at pH values as low as 6.63.
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CONCLUSION |
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The study of the microglia revealed for the first time the
involvement of an H+-K+-ATPase in these
important reactive cells of the brain. The pump may be a new member of
the Na+-K+-ATPase superfamily. As with the
other studies described, the Seris technique was used to approach a
problem in cell physiology more effectively than could have been
achieved with other techniques. All studies produced unique data of
considerable biomedical importance. Each of these studies used sensors
available commercially in the form of liquid membranes incorporating
specific ionophores, and, therefore, the characteristics of these
ionophores limit the molecules that can be detected. However, the
principle of the self-referencing technique has the potential for
further applications by expanding the types of molecules detected. For
example, a self-referencing electrochemical probe, in this case
developed to measure local oxygen gradients, has been successfully used
with a single neuron and a plant cell (34). Subsequently,
the same methodology has been applied to single pancreatic -cells
(52) and diversified to include the detection of nitric
oxide and ascorbate from single cells (Pepperell J, Porterfield DM, and
Smith PJS, unpublished observations). Currently under
development is the incorporation of biosensors (26, 49)
onto the reactive surface of a self-referencing electrochemical probe.
Preliminary results have demonstrated the feasibility of this approach
for glucose detection (50), and we look forward to the
rapid diversification of self-referencing technologies to other enzyme
reactions with redox products.
In conclusion, the Seris technique has shown great versatility, noted here by the physiologically diverse types of cells studied and molecular components revealed. From a complex and heterogeneous cellular pavement to individual cells, nonelectrogenic events, as produced by the "gastric" H+-K+-ATPase, can be investigated. With the continued incorporation of new sensors, as well as the combination of the self-referencing approach with other methodologies, we can look forward to exciting observations in cellular transport physiology.
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ACKNOWLEDGEMENTS |
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The authors are supported by National Center for Research Resources (NCRR) Grant P41 RR-101395 (P. J. S. Smith), National Institutes of Health Grant KO-81099, and the Lalor Foundation (J. Trimarchi). The BioCurrents Research Center is a resource of the NCRR, which specializes in the design, development, and application of methods for the study of cell transport phenomena. As such, we encourage biomedical researchers who want to take advantage of the technology available to contact P. J. S. Smith. Information on the resource can be found at www.mbl.edu/BioCurrents.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. J. S. Smith, BioCurrents Research Center, 7 MBL St., Woods Hole, MA 02543 (E-mail: psmith{at}mbl.edu).
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REFERENCES |
---|
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---|
1.
Ammann, D.
Ion Selective Micro-Electrodes. New York: Springer-Verlag, 1986.
2.
Au, CL,
and
Wong PY.
Luminal acidification by the perfused rat cauda epididymidis.
J Physiol (Lond)
309:
419-427,
1980[ISI][Medline].
3.
Baikie, ID,
Smith PJS,
Porterfield DM,
and
Estrup PJ.
Multi-tip scanning Bio-Kelvin probe.
Rev Sci Instrum
70:
1842-1850,
1999[ISI].
4.
Bernard, C,
Ferrary E,
and
Sterkers O.
Production of endolymph in the semicircular canal of the frog Rana esculenta.
J Physiol (Lond)
371:
17-28,
1986[Abstract].
5.
Bluh, O,
and
Scott BIH
Vibrating probe electrometer for the measurement of bioelectric potentials.
Rev Sci Instrum
21:
867-868,
1950[ISI].
6.
Bortner, CD,
Hughes FM, Jr,
and
Cidlowski JA.
A primary role for K+ and Na+ efflux in the activation of apoptosis.
J Biol Chem
272:
32436-32442,
1997
7.
Breton, S,
Hammar K,
Smith PJS,
and
Brown D.
Proton secretion in the male reproductive tract: involvement of Cl-independent HCO3
transport.
Am J Physiol Cell Physiol
275:
C1134-C1142,
1998
8.
Breton, S,
Nsumu NN,
Galli T,
Sabolic I,
Smith PJS,
and
Brown D.
Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract.
Am J Physiol Renal Physiol
278:
F717-F725,
2000
9.
Breton, S,
Smith PJS,
Lui B,
and
Brown D.
Acidification of the male reproductive tract by a proton-pumping H+-ATPase.
Nat Med
2:
470-472,
1996[ISI][Medline].
10.
Brown, D,
and
Breton S.
H+ V-ATPase-dependent luminal acidification in the kidney collecting duct and epididymis/vas deferens: vesicle recycling and transcytotic pathways.
J Exp Biol
203:
137-145,
2000[Abstract].
11.
Caflisch, CR,
and
DuBose TD, Jr.
Direct evaluation of acidification by rat testis and epododymis: role of carbonic anhydrase.
Am J Physiol Endocrinol Metab
258:
E143-E150,
1990
12.
Cevc, G.
Membrane electrostatics.
Biochim Biophys Acta
1031:
311-382,
1990[ISI][Medline].
13.
Cohen, JP,
Hoffer AP,
and
Rosen S.
Carbonic anhydrase localization in the epididymis and the testis of the rat: histochemical and biochemical analysis.
Biol Reprod
14:
339-346,
1976[ISI][Medline].
14.
Colton, CA,
Jia M,
Li MX,
and
Gilbert DL.
K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels.
Am J Physiol Cell Physiol
266:
C1650-C1655,
1994
15.
Davies, PW.
Membrane potential and resistance of perfused skeletal muscle fibers with control of membrane current (Abstract).
Fed Proc
25:
332,
1966[ISI].
16.
Demarest, JR,
and
Morgan JLM
Effect of pH buffers on proton secretion from gastric oxyntic cells measured with vibrating ion-selective microelectrodes.
Biol Bull
189:
219-220,
1995
17.
Ekstedt, E,
and
Ridderstrale Y.
Histochemical localization of carbonic anhydrase in the testis and epididymus of the rabbit.
Acta Anat (Basel)
143:
258-264,
1992[Medline].
18.
Ekstedt, E,
Ridderstrale Y,
Ploen L,
and
Rodriguez-Martinez H.
Histochemical localization of carbonic anhydrase in the testis and epididymus of the boar.
Acta Anat (Basel)
141:
257-261,
1991[Medline].
19.
Gow, NAR,
Kropf DL,
and
Harold FM.
Growing hyphae of Achlya bisexualis generate a longitudinal pH gradient in the surrounding medium.
J Gen Microbiol
130:
2967-2974,
1984[ISI][Medline].
20.
Grahm, L,
and
Hertz CH.
Measurement of the geoelectric effect in coleoptiles by a new technique (Abstract).
Physiol Plant
15:
96,
1962.
21.
Hickey, WF,
Vass K,
and
Lassmann H.
Bone marrow-derived elements in the central nervous system: immunohistochemical and ultrastructural survey of rat chimeras.
J Neuropathol Exp Neurol
51:
246-256,
1992[ISI][Medline].
22.
Hinton, BT,
and
Palladino MA.
Epididymal epithelium: its contribution to the formation of a luminal fluid microenvironment.
Microsc Res Tech
30:
67-81,
1995[ISI][Medline].
23.
Jaffe, LF,
and
Levy S.
Calcium gradients measured with a vibrating calcium-selective electrode.
Proc IEEE/EMBS Conf
9:
779-781,
1987.
24.
Jaffe, LF,
and
Nuccitelli R.
An ultrasensitive vibrating probe for measuring steady electrical currents.
J Cell Biol
63:
614-628,
1974
25.
Jones, RC,
and
Murdoch RN.
Regulation of the mobility and metabolism of spermatozoa for storage in the epididymis of eutheran and marsupial mammals.
Reprod Fertil Dev
8:
553-568,
1996[ISI][Medline].
26.
Jung, SK,
Kauri LM,
Qian WJ,
and
Kennedy RT.
Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca(2+) in single islets of Langerhans.
J Biol Chem
275:
6642-6650,
2000
27.
Jurisicova, A,
Varmuza S,
and
Casper RF.
Programmed cell death and human embryo fragmentation.
Mol Hum Reprod
2:
93-98,
1996[Abstract].
28.
Kerr, JF,
Wyllie AH,
and
Currie AR.
Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.
Br J Cancer
26:
239-257,
1972[ISI][Medline].
29.
Kettermann, H,
Banati R,
and
Walz W.
Electrophysiological behavior of microglia.
Glia
7:
93-101,
1993[ISI][Medline].
30.
Kimura, RS.
Distribution, structure, and function of dark cells in the vestibular labyrinth.
Ann Otol Rhinol Laryngol
78:
542-561,
1969[ISI][Medline].
31.
Kochian, LV,
Shaff JE,
Kühtreiber WM,
Jaffe LF,
and
Lucas WJ.
Use of an extracellular, ion-selective, vibrating microelectrode system for the quantification of K+, H+, and Ca2+ fluxes in maize roots and maize suspension cells.
Planta
188:
601-610,
1992[ISI].
32.
Kreutzberg, GW.
Microglia: a sensor for pathological events in the CNS.
Trends Neurosci
19:
312-318,
1996[ISI][Medline].
33.
Kühtreiber, WM,
and
Jaffe LF.
Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.
J Cell Biol
110:
1565-1573,
1990[Abstract].
34.
Land, SC,
Porterfield DM,
Sanger RH,
and
Smith PJS
The self-referencing oxygen-selective microelectrode: detection of trans-membrane oxygen flux from single cells.
J Exp Biol
202:
211-218,
1999
35.
Levine, N,
and
Kelly H.
Measurement of pH in the rat epididymis in vivo.
J Reprod Fertil
52:
333-335,
1978[Abstract].
36.
Ling, EA,
and
Wong WC.
The origin and nature of ramified and amoeboid microglia: a historical review and current concepts.
Glia
7:
9-18,
1993[ISI][Medline].
37.
Malchow, RP,
Verzi MP,
and
Smith PJS
Extracellular pH gradients measured from isolated retinal cells.
Biol Bull
195:
203-204,
1998
38.
Marcus, DC.
Vibrating probes: new technology for investigation of endolymph homeostasis.
Keio J Med
45:
301-305,
1996[Medline].
39.
Marcus, DC,
Liu L,
and
Wangemann P.
Transepithelial voltage and resistance of vestibular dark cells epithelium from the gerbil ampulla.
Hear Res
73:
101-108,
1994[ISI][Medline].
40.
Marcus, DC,
Marcus NY,
and
Greger R.
Sidedness of action of loop diuretics and ouabain on nonsensory cells of utricle: a micro Ussing chamber for inner ear tissues.
Hear Res
30:
55-64,
1987[ISI][Medline].
41.
Marcus, DC,
and
Shen Z.
Slowly activating, voltage dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells.
Am J Physiol Cell Physiol
267:
C857-C864,
1994
42.
Marcus, DC,
and
Shipley AM.
Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe.
Biophys J
66:
1939-1942,
1994[Abstract].
43.
Marcus, DC,
Sunrose H,
Liu J,
Shen Z,
and
Scofield MA.
P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells.
Am J Physiol Cell Physiol
273:
C2022-C2029,
1997
44.
Marcus, DC,
Takeuchi S,
and
Wangemann P.
Ca2+-activated nonselective cation channel in apical membrane of vestibular dark cells.
Am J Physiol Cell Physiol
262:
C1423-C1429,
1992
45.
Marcus, NY,
and
Marcus DC.
Potassium secretion by non-sensory region of gerbil utricle in vitro.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F613-F621,
1987
46.
McLaughlin, S,
Mulrine N,
Gresalfi T,
Vaio G,
and
McLaughlin A.
Adsorption of divalent cations to bilayer membranes containing phosphatidylserine.
J Gen Physiol
77:
445-473,
1981[Abstract].
47.
McLaughlin, SGA,
Szabo S,
and
Eisenman G.
Divalent ions and surface potentials of charged phospholipid membranes.
J Gen Physiol
58:
667-687,
1971
48.
Nuccitelli, R.
Vibrating probe technique for studies of ion transport.
In: Noninvasive Techniques in Cell Biology, edited by Foskett JK,
and Grinstein S.. New York: Wiley-Liss, 1990, p. 273-310.
49.
Pantano, P,
and
Kuhr WG.
Dehydrogenase-modified carbon-fiber microelectrodes for the measurement of neurotransmitter dynamics. 2. Covalent modification utilizing avidin-biotin technology.
Anal Chem
65:
623-630,
1993[ISI][Medline].
50.
Pepperell J, Liu L, Keefe DL, and Smith PJS. A non-invasive
bioassay for real-time measurement of glucose uptake by single embryos.
Am Soc Reprod Med Ann Mtg Abstr. In press.
51.
Pineros, MA,
Shaff JE,
and
Kochian LV.
Development, characterization and application of a cadmium-selective microelectrode for the measurement of cadmium fluxes in roots of Thlaspi species and wheat.
Plant Physiol
116:
1393-1401,
1998
52.
Porterfield, DM,
Corkey RF,
Sanger RH,
Tornheim K,
Smith PJS,
and
Corkey BE.
Oxygen consumption oscillates in single clonal pancreatic -cells (HIT).
Diabetes
49:
1511-1516,
2000[Abstract].
53.
Sanger, R,
Karplus E,
and
Jaffe LF.
An aerial vibrating probe.
Biol Bull
179:
225,
1990.
54.
Schlichter, LC,
Sakellaropoulos G,
Ballyk B,
Pennefather PS,
and
Phipps DJ.
Properties of K+ and Cl channels and their involvement in proliferation of rat microglial cells.
Glia
17:
225-236,
1996[ISI][Medline].
55.
Schulte, BA,
and
Adams JC.
Distribution of immunoreactive Na+, K+ ATPase in gerbil cochlea.
J Histochem Cytochem
37:
127-134,
1989[Abstract].
56.
Sellick, PM,
and
Johnstone BM.
Production and role of inner ear fluid.
Prog Neurobiol
5:
337-362,
1975[Medline].
57.
Shen, Z,
Liu J,
Marcus DC,
Shiga N,
and
Wangemann P.
DIDS increases K+ secretion through an IsK channel in apical membrane of vestibular dark cell epithelium of gerbil.
J Membr Biol
146:
283-291,
1995[ISI][Medline].
58.
Shirihai, O,
Merchav S,
Attali B,
and
Dagan D.
K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors.
Pflügers Arch
431:
632-638,
1996[ISI][Medline].
59.
Shirihai, O,
Smith PJS,
Hammar K,
and
Dagan D.
H+ and K+ gradient generated by microglia H/K-ATPase.
Glia
23:
339-348,
1998[ISI][Medline].
60.
Smith, PJS
The non-invasive probes - tools for measuring trans-membrane ion flux.
Nature
378:
645-646,
1995[ISI][Medline].
61.
Smith, PJS,
Hammar K,
Porterfield DM,
Sanger RH,
and
Trimarchi JR.
A self-referencing, non-invasive, ion selective electrode for single cell detection of trans-plasma membrane calcium flux.
Microsc Res Tech
46:
398-417,
1999[ISI][Medline].
62.
Smith, PJS,
Sanger RH,
and
Jaffe LF.
The vibrating Ca2+ electrode: a new technique for detecting plasma membrane regions of Ca2+ influx and efflux.
In: Methods in Cell Biology. A Practical Guide to the Study of Ca2+ in Living Cells, edited by Nucitelli R.. San Diego, CA: Academic, 1994, vol. 40, p. 115-134.
63.
Takeuchi, S,
Marcus DC,
and
Wangemann P.
Maxi K+ channels in apical membrane of vestibular dark cells.
Am J Physiol Cell Physiol
262:
C1430-C1436,
1992
64.
Trimarchi, JR,
Liu L,
Smith PJS,
and
Keefe DL.
Non-invasive measurement of potassium efflux as an early indicator of cell death in mouse embryos.
Biol Reprod
63:
851-857,
2000
65.
Wangemann, P,
Liu J,
and
Marcus DC.
Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vacularis in vitro.
Hear Res
84:
19-29,
1995[ISI][Medline].
66.
Wangemann, P,
Liu J,
Shen Z,
Shipley A,
and
Marcus DC.
Hypo-osmotic challenge stimulates transepithelial K+ secretion and activates apical IsK channel in vestibular dark cells.
J Membr Biol
147:
263-273,
1995[ISI][Medline].
67.
Wangemann, P,
and
Shiga N.
Cell volume control in vestibular dark cells during and after a hyposmotic challenge.
Am J Physiol Cell Physiol
266:
C1046-C1060,
1994
68.
Xu, KY.
Inhibition of H+-transporting ATPase, Ca2+-transporting ATPase and H+/K+-transporting ATPase by strophanthidin.
Biochim Biophys Acta
1159:
109-112,
1992[ISI][Medline].
69.
Younes-Ibrahim, M,
Barlet-Bas C,
Buffin-Meyer B,
Cheval L,
Rajerison R,
and
Doucet A.
Ouabain-sensitive and -insensitive K-ATPase in rat nephron: effect of K depletion.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1141-F1147,
1995