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Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah 84112; and University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pHi) within single ventricular myocytes. The spatial distribution of pHi in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pHi were induced by local external application of NH4Cl, CO2, or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 µm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pHi gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.
confocal imaging; intracellular acidosis; ventricular myocytes
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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REGULATION OF INTRACELLULAR pH (pHi) in
cardiomyocytes is essential for the maintenance of normal cardiac
function. Many of the key processes involved have now been
characterized, including aspects of intracellular buffering and
sarcolemmal acid or base transport (17). Analyses of cardiac
pHi regulation have so far assumed that the spatial
distribution of H+ in the cytoplasm is uniform. There have
been suggestions, however, that under some circumstances significant
cytoplasmic pH nonuniformities may occur. For example, Vanheel et al.
(31) reported that large gradients of pHi measured with
pH-selective microelectrodes could be observed in multicellular cardiac
Purkinje fibers or in single skeletal muscle fibers that had been
exposed partially to solutions containing NH4Cl. Transient
cytoplasmic gradients of pHi have also been reported
recently in epithelial duodenal enterocytes after activation of
membrane acid transport, consistent with a low cytoplasmic mobility of
H+, at least in the absence of a catalyzed
CO2/HCO
3 buffer system
(28). The possible generation of cytoplasmic pHi gradients
in single cardiomyocytes has not so far been explored.
In the present work we characterize a dual-microperfusion system for selectively bathing a region of an isolated cardiomyocyte with weak acids (such as propionic acid or CO2) or bases (such as NH3). When used in conjunction with confocal imaging of pHi, this technique reveals that large spatial pHi gradients can be created rapidly and remain stable for extended periods of time (at least several minutes). We consider how these gradients are generated and discuss their physiological relevance. The microperfusion technique offers the possibility of observing the kinetics and functional effects of local acid movements within a cardiac cell, a subject of clinical relevance when the regional generation and spread of acid within the ischemic myocardium is considered. The technique may also provide a general, noninvasive method for inducing pHi nonuniformities in other cell types as well as in more complex multicellular preparations.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Solutions and Cell Bath
Cell bathing solutions were held at 37 ± 0.1°C in glass reservoir bottles that were completely sealed except for a small vent at the top and an exit port at the bottom. Solutions were delivered by gravity from the bottles to the cell bath through thermally jacketed gas-impermeable tubing. The temperature of the solutions in the bath, including the microperfusion system described below, was 36 ± 0.3°C. The 1-ml Plexiglas cell bath had a clear glass bottom and was mounted on the stage of an inverted microscope (Diaphot, Nikon). The HEPES-buffered normal solution continuously flowed through the bath at 4-6 ml/min, and solution depth was held at ~3 mm. The bottom of the bath was coated with laminin (Collaborative Research, Bedford, MA) to improve cell adhesion.The HEPES-buffered normal solution contained (in mM) 126 NaCl, 4.4 KCl, 1.0 MgCl2, 11.0 dextrose, 1.0 CaCl2, and 24.0 HEPES (titrated to pH 7.4 with 13.0 mM NaOH). The 80 mM sodium propionate (NaPr) solution [extracellular pH (pHo) 7.4] was prepared by equimolar replacement of NaPr for NaCl. The 15.0 mM NH4Cl solution (pHo 7.4) was prepared by adding the salt directly to the HEPES-buffered normal solution. The 20% CO2 solution contained (in mM) 126 NaCl, 4.4 KCl, 1.0 MgCl2, 11.0 dextrose, 1.0 CaCl2, and 18.5 NaHCO3, and it was continuously gassed with 20.0% CO2-80.0% O2 to give a pH of 6.80. Because the barometric pressure in Salt Lake City is typically 640 Torr, the resulting PCO2 of this solution was ~119 Torr.
Cell Isolation
As previously described (22), adult rabbit ventricular myocytes were obtained from New Zealand White rabbits (2-3 kg). In brief, animals were anesthetized with an intravenous injection of pentobarbital sodium (50 mg/kg). The heart was rapidly removed and attached to a Langendorff perfusion system. All perfusion solutions were held at 37°C and pH 7.4. Perfusion with a 0 mM Ca2+ solution for 5 min was followed by 20 min of perfusion with the same solution containing 1 mg/ml collagenase (class II, Worthington Biochemical, Freehold, NJ), 0.1 mg/ml protease (type XIV, Sigma Chemical, St. Louis, MO), and 0.1 mM CaCl2. The heart was then perfused for 5 min with the same solution containing no enzymes. The left ventricle was minced and shaken for 10 min and then filtered through a nylon mesh. The cells were stored at room temperature in the normal HEPES-buffered solution. All cells used in this study were rod shaped in appearance, had well-defined striations, and did not spontaneously contract. All experiments were performed within 2-5 h after isolation.Measurement of pHi
The pHi was measured in single myocytes using seminaphthorhodafluor (SNARF)-1 as the fluorescent indicator and a laser scanning confocal microscope (MRC 1024, Bio-Rad Microscience) to image the cells. Myocytes were equilibrated at 37°C for 10 min in the normal HEPES solution containing 13 µM SNARF-1-AM (Molecular Probes, Eugene, OR) as previously described (26). They were then placed in the cell bath, where they were bathed in the normal solution for at least 20 min before pHi measurements began. Excitation at 488 nm was provided by an argon/krypton mixed-gas laser (American Laser, Salt Lake City, UT). Emitted fluorescence was simultaneously collected by two photomultiplier tubes equipped with band-pass filters at 640 and 580 nm via a ×40 oil-immersion objective lens (NA 1.3). A transmitted light detector also provided a nonfluorescent image of the cell. Fluorescence ratios (640 nm/580 nm) and transmitted light images were acquired on-line at rates varying from ~1 to 2 s/frame. It is worth noting that pHi imaging along the z-axis of the myocyte with an oil-immersion objective (not done in this study) would cause skewing of the ratio and inaccurate determination of pHi. Use of a water-immersion lens avoids this difficulty.The emission ratio was calibrated as previously described (22, 26) using solutions of varying pH that also contained nigericin. The best-fit equation for the calibration curves from several myocytes was used to calculate pHi of the cells used in this study.
Confocal images were processed using NIH Image and Transform (Fortner Software, Sterling, VA) software packages. Images were stored on recordable compact discs.
Microperfusion System
To create regional differences in pHi in single myocytes, we used a microperfusion system (24, 25, 33). The device consists of a short length of custom-made, double-barreled square glass tubing (Wilmad Glass, Buena, NJ) attached to a modified miniature solenoid. The entire system is held in position in the cell bath with a precision micromanipulator. The internal width of each barrel is ~275 µm with an ~70-µm glass septum separating the barrels. Two microstreams simultaneously flowed at the same rate (~27 µl/s) from both barrels, creating a sharp boundary between the streams. The calculated linear velocity of the streams (flow rate/cross-sectional area) was ~36 µm/ms. One of the streams contained the normal HEPES-buffered solution, and the other one either 20% CO2, 80 mM NaPr, or 15 mM NH4Cl. A myocyte selected for study was positioned ~300 µm in front of the end of the barrels with its long axis oriented approximately perpendicular to the direction of microstream flow. This was accomplished by a combination of moving the microscope stage and rotating the bath. The entire cell was initially positioned in the normal HEPES stream. The stream boundary could be rapidly directed over the cell by lateral movement of the barrels, achieved by either electrically activating the solenoid or manually using the micromanipulator. Solenoid activation changed the bulk solution in ~4 ms, whereas manual switching required ~1 s. All experiments in this study were done with manual switching. Graduation marks on the micromanipulator identified the position at which the stream boundary traversed the cell. However, the boundary was not visible in most experiments, and its exact position on the cell was unknown. Substances can be included in one or both of the microstreams to help visualize the position of the boundary. pHi did not change when myocytes were first bathed at the normal bath flow rate of 4-6 ml/min and then exposed to a microstream of the same composition. Thus microstream exposure per se does not adversely affect membrane integrity. In previous studies, Spitzer and co-workers (25, 33) also found no adverse effects of the microstreams on cell properties.A schematic diagram of the relationship between the microstreams and
the cell is shown in Fig. 1A. Also
shown is an example of the stream boundary flowing over a pair of
physically attached myocytes (Fig. 1B). For visualization of
the boundary, the upper stream contained the normal solution, whereas
the lower stream and the bath contained a solution in which 80 mM of
NaCl was replaced with sodium gluconate. Figure 1C shows how we
used confocal imaging at 580 nm (excitation at 488 nm) to estimate the
width of the stream boundary. The upper stream contained normal HEPES
solution, and the lower stream contained 50 µM carboxy-SNARF-1
dissolved in the normal solution (Fig. 1C, right). All
conditions were the same as those for an actual cell experiment except
that no cells were present. The spatial profile of the fluorescence
image indicated that the stream boundary was ~10 µm wide (Fig.
1C, left).
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To determine the effect of cell orientation and geometry on the stream boundary, we exposed cells to a fluorescent boundary. The boundary consisted of one microstream with normal HEPES solution and another with 100 µM of 5- (and 6)-carboxy-4'-5'-dimethylfluorescein (Molecular Probes) dissolved in normal HEPES solution. To help visualize the cells, we labeled the sarcolemma with di-8-ANEPPS (Molecular Probes). Excitation was at 488 nm with emission signals collected at 530 nm. The stream boundary was positioned on the cell, and confocal scans were collected at several points along the z-axis.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Cell on Stream Boundary
It seemed possible that the geometry of the solution boundary might be influenced by the orientation and shape of the myocyte being superfused. If so, this could have affected our interpretation of how pHi gradients are formed. To test this possibility, we exposed myocytes of varied orientation and shape to a fluorescent boundary created by including fluorescein in one of the microstreams. Results from two myocytes are shown in Fig. 2. The images were acquired ~8 µm above the undersurface of the cell. The cell shown in Fig. 2A had a fairly flat surface, and even though it was oriented ~20° from the vertical, the solution boundary was well maintained across the width of the cell. Furthermore, scanning the field at different depths indicated a clean solution boundary at all levels (not shown). In contrast, the cell in Fig. 2B had an overall orientation of ~30° from the vertical and a complex geometry. The result was a disrupted solution boundary with turbulent flow occurring on the downstream side of the cell, probably reflecting complex eddy currents and a mixing of the two streams. Similar eddy currents were also evident when fields were scanned at different depths. Flat cells oriented closer to the vertical consistently had well-maintained boundaries (n = 5), so this orientation and shape was used in all subsequent experiments.
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pHi Gradient Induced by NH4Cl
The weak base ammonia (NH3) was used to induce selectively an intracellular alkalosis at one end of a myocyte (Fig. 3). The entire cell was initially exposed uniformly to the normal HEPES microstream, resulting in a uniform distribution of pHi with, in this case, a mean pHi in both measurement regions (R1, R2) of ~7.1 (Fig. 3, A and B). The stream boundary was then rapidly positioned across the width of the cell, thus exposing the upper pole (R2) to 15 mM NH4Cl while keeping the lower pole (R1) in the normal HEPES solution. The exact position of the boundary was not visible in these experiments. A rapid increase in pHi occurred in R2 as NH3 diffused into the cell and combined with H+ (3). Interestingly, this was accompanied by a simultaneous fall of pHi in R1 (the possible cause of this is considered in the DISCUSSION). Thus a longitudinal gradient of pHi was established that persisted for the period of partial exposure to NH3 (~100 s). Removal of the NH3 microstream, by reexposing the entire cell to the normal HEPES solution, rapidly restored pHi in both regions to a value close to the original control value. Thus removal of NH3 rapidly dissipated the pHi gradient.
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The marked spatial heterogeneity in pHi created by local application of NH4Cl is more readily appreciated by comparing the three-dimensional pH images shown in Fig. 3C. Exposure of the R2 pole of the cell to NH4Cl rapidly established a high local pHi that declined monotonically down the whole length of the cell with a pole-to-pole pHi difference of ~1.2 units. The same pattern of pHi heterogeneity was also observed in six other cells.
pHi Gradient Induced by CO2
We examined the effect on pHi of local application of the weak acid CO2 (Fig. 4). In this case the two microstreams consisted of a normal HEPES-buffered solution (pHo 7.40) and a HCO3-buffered solution equilibrated
with 20% CO2 (pHo 6.80). This solution
combination produced a boundary line that was faintly visible in the
transmission image (Fig. 4B, middle). When the entire
cell was in the HEPES microstream, pHi was fairly uniform
along its entire length (Fig. 4A, right). Selective
application of 20% CO2 to the upper part of the myocyte
elicited a local and rapid fall in pHi (R2) as CO2 diffused into the cell, generating intracellular
H+ and HCO3 (3). After 116 s of exposure to CO2 (frame 440), there was a monotonic and
persistent pole-to-pole pHi gradient of ~1.0 pH units
(Fig. 4A, right). Note also that there was a decrease
of pHi in a portion of the cell that was clearly bathed by
the HEPES-buffered solution. This portion extended from 26 µm (the
solution boundary) to 90 µm in the graph shown in Fig. 4A,
right. At distances beyond 90 µm, and thus well into the
region exposed to HEPES solution, pHi increased above
control levels. The same pattern of pHi changes was also
observed in four other cells.
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pHi Gradient Induced by NaPr
A regional intracellular acidosis could also be readily induced by partially exposing myocytes to NaPr (80 mM; pHo 7.4) (see Fig. 5). In this experiment the entire myocyte was first exposed to NaPr, which uniformly reduced pHi as uncharged propionic acid (pK 4.7) diffused into the cell and dissociated (8, 21). When NaPr was removed, pHi recovered to a higher value than in the control, presumably because of H+ extrusion during the NaPr pulse via Na+/H+ exchange. The subsequent recovery of pHi from alkalosis is most likely mediated by Cl/OH exchange (29). The upper
part of the myocyte was then selectively exposed to NaPr, resulting in
a decreased pHi at R2 but an increased pHi at
the opposite pole of the cell (R1). The stream boundary was not visible
in these experiments. The pHi completely recovered in both
regions when the entire myocyte was returned to the normal HEPES
microstream. The same pattern of pHi changes was also
observed in two other cells.
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Stability and Amplitude of pHi Gradient
The longitudinal pHi gradient set up across a weak acid or weak base boundary was maintained for as long as the boundary was in place. In the present work boundaries were imposed for no longer than 200 s, but within that time frame the intracellular gradient was stable (see e.g., Figs. 3-5). It seems likely, therefore, that a spatial pHi gradient would have persisted during more prolonged boundary exposures.The size of the longitudinal pHi gradient created by
propionate, NH+4, or
CO2/HCO3 boundaries is
summarized in Table 1. To compare the
effects of different acids or bases, we normalized the longitudinal
gradient, for convenience, to the initial change of pHi
produced during uniform exposure of the whole cell to
the same concentration of weak acid or base. Thus the presence of a
15 mM NH+4 boundary across a cell set
up a longitudinal rise in pHi of 0.9 pH units, a
value nearly twice as large as the rise of pHi
induced uniformly when the whole cell was exposed to 15 mM
NH+4. This comparison is illustrated in the
experiment shown in Fig. 6A. Similarly, the longitudinal pHi gradient set up by a
propionate boundary was 1.4 times larger than the whole cell fall of
pHi (see e.g., Fig. 5A), whereas the spatial
pHi gradient induced by a
CO2/HCO3 boundary
was about the same as the fall of pHi when the cell was
exposed uniformly to
CO2/HCO3 (not shown).
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An additional factor possibly influencing spatial pHi
gradients, and one not controlled for in the above experiments, is the precise location of the microstream border zone. We did not
systematically investigate this problem, but because the solution
border was clearly visible when
HEPES/HCO3 microstreams were used, we
tested the effect of shifting its position across the cell. This is
shown in Fig. 6B. In the first part of this trace, the majority
of the cell (the proximal end) was exposed to
CO2/HCO3, whereas in the
later part of the experiment the boundary position was readjusted so
that a smaller local area was exposed. In both cases a longitudinal
pHi gradient was rapidly established. It is notable,
however, that the magnitude of the proximal-to-distal gradient
increased by ~60% as the size of the local area exposed to
CO2/HCO3 was reduced. Thus
not only is the magnitude of the pHi gradient influenced by
the species of weak acid or base forming the border zone, but it also
appears to be influenced by the position of the border along the length
of the cell. Experiments with other weak acids or bases are required to
further assess the role of border position in pHi gradient formation.
A final observation is that the secondary slow recovery phase of
pHi during the NH4Cl or NaPr prepulse and the
typical pHi overshoot on
NH+4/propionate removal was always much more
pronounced with whole cell pulsing than with partial cell pulsing (see
e.g., Figs. 5A and 6A; n = 3 for propionate experiments, n = 6 for NH+4
experiments). A similar observation was also made in three of five
experiments in which CO2/HCO3 was added and removed.
In summary, local exposure of a myocyte to a weak acid or base boundary generates a spatially stable pHi gradient whose magnitude depends on the species of weak acid or base used, the dosage, and the relative position of the border zone across the cell. Furthermore, the well-known ability of the weak acid or base prepulse technique to base load or acid load a cell is severely compromised when the prepulsing occurs only locally.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Localized Weak Acid or Base Generates pHi Gradients
Little information exists concerning the spatial modulation of cardiac pHi. Confocal images of SNARF-AM-loaded cardiac cells previously indicated a uniform cytoplasmic pH punctuated by small alkaline pockets attributed to intramitochondrial spaces (7). In the present work, possible subcellular structures were sometimes evident in the ratiometric images (see Figs. 4B and 5B), but, with the exception of the region corresponding to the nucleus, their morphology was never clearly defined. More commonly, the resting pH was spatially uniform [see e.g., Fig. 3, A (middle) and C (top)], provided, of course, that the cells were being perfused uniformly with the same solution. It is possible that with longer SNARF-AM loading conditions (i.e., longer than the 10 min used in the present work), dye uptake into subcellular structures may become more pronounced, leading to a clearer resolution of these regions. This possibility was not explored.Using pH microelectrodes in multicellular Purkinje fibers that had been mounted in a two-compartment chamber, Vanheel et al. (31) reported that spatially confined changes of pHi could be induced by perfusing one compartment with NH4Cl. Although a similar result was achieved in skeletal muscle fibers, the spatial resolution of the measurements was >100 µm, roughly equivalent to one cardiac cell length, so that successive pHi measurements along the Purkinje fiber would have been made in different cells, raising the possibility that a lack of H+ mobility through gap junctions may have contributed to the spatial pHi confinement.
Our dual-microperfusion technique, in combination with confocal pH imaging, has enormously increased the spatial resolution of the experiment. We are now able, for the first time, to observe large longitudinal pHi gradients within the same cardiac cell exposed locally to NH4Cl. Large, persistent pHi nonuniformities are also readily achieved with local applications of weak acids such as CO2 and propionic acid. Unlike Vanheel et al., however, we did not find any significant and lasting pHi gradients after the removal of local weak acid or base. In our work, this resulted consistently in the rapid dissipation of any intracellular nonuniformity. We present below a qualitative model that can account for the spatial pHi changes on addition and removal of local weak acid or base. At present we cannot explain the particular discrepancies with earlier work.
Solution Separation in the Dual Microstream
The glass septum within the dual-perfusion apparatus separates the microstreams and will prevent diffusion of NH3, CO2, and propionate from one stream into the other. However, once the streams exit the barrels these molecules will start to diffuse across the solution boundary into the other microstream. This could conceivably produce a graded concentration profile of weak acid or base at the boundary that might then produce the observed gradients of pHi. This seems unlikely for two reasons. 1) The concentration profile of SNARF when incorporated into one stream drops from 100% to <2% over a 10-µm distance across the stream boundary, whereas the distances over which monotonic pHi gradients can be detected extend >90 µm beyond the boundary (see Fig. 4A, right). 2) At the flow velocity used in this study (36 µm/ms), the two microstreams would only have been in contact for 8 ms before reaching the cell 300 µm away, the distance used in this study. We estimate that at 8 ms the mean lateral displacement (
) of NH3, CO2,
and propionate into the uncontaminated microstream would be <7 µm. The diffusion distance () was calculated from the Einstein equation as = 
, where
D is the diffusion coefficient in water for NH3
(1.8 × 105 cm2/s; Ref.
20), CO2 (2.4 × 105
cm2/s; Ref. 30), or propionate (0.9 × 105 cm2/s, calculated from
limiting conductance; Ref. 19) and t is 8 ms. This simplified
calculation suggests that the concentration profiles for
NH3, CO2, and propionate are likely to have
been well maintained over the cell.
Provided that myocytes were flat and vertically oriented, the separation of the two solutions across the cell itself also appears to have been well maintained (e.g., Fig. 2). Unfortunately, it was not feasible to image solution separation and pHi simultaneously (indeed, it would have been most helpful to do so). We therefore cannot exclude entirely the possibility of extracellular solution mixing in some experiments. We also cannot exclude the possibility that unstirred extracellular microenvironments close to the cell surface, and beyond resolution of the confocal technique, have a composition different from that in the bulk microstreams. We must therefore exercise some caution over the precision of our solution-separation technique, but our results so far suggest that it is fairly clean, with separation probably being achieved at distances >10 µm or so from the solution boundary.
In summary, the large monotonic change of pHi induced along the length of a myocyte when bisected by two extracellular microstreams is unlikely to be caused simply by a graded extracellular mixing of the two solutions.
Mechanism Generating pHi Gradients
The general mechanisms likely to be responsible for the pattern of pHi changes observed with regional application of NH4Cl (Fig. 3), CO2 (Fig. 4), and NaPr (Fig. 5) are represented schematically in Fig. 7. A more formal, quantitative treatment of the model will require additional information regarding the intracellular diffusion coefficients of the relevant molecules. The passive pHi changes elicited by uniform application of a weak acid or base, to both cardiac and non-cardiac cells, have been well characterized (3, 8, 21). However, with local application, additional geometric factors must be considered. Specifically, the region of the cell bathed in solution free of weak acid or base represents an infinite sink for the continuous removal of NH3, CO2, and HPr that has been applied locally to the other end of the cell.
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NH4Cl. The rise of pHi at the end exposed to NH4Cl (proximal end) is accompanied by a fall of pHi at the other (distal) end, as shown in the sketch in Fig. 7A. The ammonium stream contains 15.0 mM NH+4 (pK 9.5; pHo 7.4) and a calculated NH3 concentration of 119 µM, which will drive the rapid diffusion of NH3 into the proximal part of the cell. Intracellular NH3 will associate with intracellular H+ to form NH+4, thus increasing proximal pHi (3). However, unlike the case for uniform exposure to NH4Cl, the local intracellular NH+4 and NH3 can now diffuse to the distal end of the cell. Longitudinal diffusion of intracellular NH3 will tend to increase pHi at sites just beyond the solution boundary (as the NH3 becomes protonated). However, most NH3 will simply diffuse out of the cell because there is no extracellular NH3 in this region. In contrast, longitudinal diffusion of intracellular NH+4 will tend to decrease pHi at more distal sites beyond the solution boundary. This is because NH+4, which cannot rapidly diffuse out of the cell, will dissociate into NH3 that readily escapes and H+ that is retained, thus acidifying the distal regions. Finally, the distal increase of intracellular H+ concentration ([H+]i) will be limited by longitudinal diffusion of H+ back down its concentration gradient toward the more alkaline, proximal end of the cell. These three diffusive movements (i.e., for NH3, NH+4, and H+) are indicated by the three arrows across the boundary region shown in Fig. 7A. If, for simplicity, one ignores longitudinal spread of NH3 (on the grounds that most of this will escape from the cell just beyond the junction), then the diffusion of NH+4 from left to right in Fig. 7A builds up a pole-to-pole pHi gradient that drives a backward movement of H+. The system will come into a steady state when the longitudinal NH+4 and H+ fluxes are equal.
High CO2.
The intracellular acidosis produced at the high-CO2,
proximal end of the cell is accompanied by a modest alkalosis at the distal end bathed in normal HEPES solution (Fig. 7B). The fall of proximal pHi results from intracellular hydration of
CO2 and subsequent dissociation to produce H+
and HCO3 (3). However, unlike the case
for uniform application of high CO2, in this experiment
intracellular CO2, H+, and
HCO3 can also move passively down
concentration gradients toward the HEPES-bathed, distal end of the
cell. The fall of pHi just beyond the solution boundary
(from 26 to 90 µm in Fig. 4A, right) is presumably
caused by the intracellular H+ movement and/or the
diffusion and subsequent hydration of intracellular CO2.
The rise of pHi at more distal sites will reflect the
dominance of CO2 efflux from the cell (into the
CO2-free perfusate) and the reversal of the hydration reaction.
NaPr.
The NaPr protocol elicits spatial pHi changes similar to
those with high CO2 (Fig. 6C). The fall of
pHi in 80 mM NaPr results from influx of
uncharged HPr and subsequent intracellular dissociation (8, 21).
Longitudinal intracellular diffusion of propionate (Pr) and its subsequent association with distal
H+ to form HPr, which exits from the cell down a
concentration gradient, will increase pHi in the distal
region. However, the extent and spatial distribution of the distal
alkalosis will be blunted by any longitudinal movement of intracellular
H+ and HPr from the proximal end of the cell.
Effects of sarcolemmal acid and/or base transporters.
For simplicity, the models presented in Fig. 7 have ignored possible
effects caused by sarcolemmal membrane transport of acid equivalents.
Although these cannot account for the overall pattern of
pHi changes that we observed, the acid loaders
(Cl/OH exchange,
Cl/HCO3 exchange)
and acid extruders (Na+/H+ exchange,
Na+-HCO3 cotransport) will
tend to attenuate the magnitude and time course of pHi
spatial gradients. We did not specifically block these transporters in
our experiments, but Na+-HCO3 cotransport and
Cl/HCO3 exchange
would have been largely inactive when both microstreams contained no
added CO2 or HCO3 (16,
29). It should be noted that, on local exposure to weak acid or base,
the resulting pHi gradient could be as large as 1.0 pH
unit, was established within tens of seconds of exposure, and remained
for the whole period of local exposure. Gradient formation is therefore
a much faster process than the time course of adjustment of
pHi via membrane transporters, which typically occurs over
periods of several minutes. The basic mechanism that sets up spatial
pHi gradients is thus too fast to be generated directly
through ion transporter activity, although subsequent spatial sculpting
of the gradient by transporters is not excluded.
Intracellular H+ Mobility
We did not attempt to estimate intracellular H+ mobility in the present work, but a recent report indicated a markedly low value in duodenal enterocytes (~1,000-fold less than in free solution) that is limited by the mobility of the intracellular intrinsic H+ buffers and that is accelerated in the presence of carbonic anhydrase activity and a CO2/HCO3
buffer system (28). Low values were measured in extruded molluscan
axoplasm (1) and predicted on theoretical grounds (13). It should be
noted in the models presented in Fig. 7 that the size of the
longitudinal pHi gradient in the steady state will be
dependent, in part, on H+ immobility; the slower the
mobility, the larger the predicted pHi gradient.
Amplitude and Long-Term Stability of pHi Gradients
Two striking features are the stability over periods of minutes of a locally induced pHi nonuniformity and the fact that such large spatial pHi changes (up to 1.0 pH unit in the present work) can be established. The schemes shown in Fig. 7 indicate that local stability occurs when the passive longitudinal fluxes of intracellular H+ (dashed arrow across border zone) are matched by equivalent longitudinal counterfluxes of NH+4 or cofluxes of HCO3 or Pr (solid
arrows across border zone). Because intracellular
NH+4, HCO3,
or Pr are ultimately derived from the local
extracellular weak acid or base microstream, they are constantly being
replenished as they flux down the cytoplasmic compartment, and it is
this constant replenishment that maintains the pHi
gradient. The constant replenishment represents a continuous injection
of acid or base equivalents into the proximal pole of the cell, and
this explains why very large pHi gradients can be
established. The magnitude of the proximal-to-distal gradient will
presumably depend on the longitudinal mobility of intracellular
H+ relative to that of the other intracellular solutes
crossing the boundary zone (Fig. 7) and on the ease of escape of the
uncharged solutes (NH3, CO2, or HPr) from the
distal pole. For example, the slower the intracellular H+
mobility or the faster the intracellular
NH+4, HCO3,
or Pr mobility, the larger the anticipated
pHi gradient established across the border zone.
Finally, during local microstream application, the continuous
longitudinal diffusion of intracellular NH+4, HCO3, or Pr from
the proximal to the distal end of the cell (Fig. 7) will prevent
significant proximal accumulation of these solutes. This in turn means
that removal of extracellular weak base or acid from the local
microstream will produce little or no net intracellular acid or base
loading, and local pHi will merely return monotonically to
its preexposure level (see e.g., Figs. 3-5). This is in clear contrast to the situation seen with whole cell weak acid or base prepulsing, in which intracellular accumulation of
NH+4, HCO3,
or Pr readily occurs during prolonged prepulsing,
resulting in large rebound intracellular acidification or
alkalinization on removal of extracellular weak acid or base. It is
unlikely, therefore, that the local addition and subsequent removal of
extracellular weak acids or bases can be used as a technique for
inducing spatial pHi nonuniformity in a cardiac myocyte.
Nonuniformity would seem to be confined to the prepulse situation and
not to the postpulse rebound.
Physiological Relevance of pHi Gradients
Changes in pHi have marked effects on cardiac electrical activity (15, 27), contraction (4, 18, 23, 26), and excitation-contraction coupling (2, 14). In these earlier studies single myocytes or multicellular cardiac preparations were uniformly exposed to conditions that altered pHi. Our microperfusion system provides a unique opportunity for studying the effects of local pHi changes on overall myocyte function. In this regard our results are relevant to myocardial ischemia. The large increases in PCO2 and [H+]i that accompany ischemia (6, 9, 12) are spatially confined if the ischemic episode is itself localized. This creates sharp gradients in both PCO2 and pHo at border zones (5, 32). Our present finding that a border zone for [CO2] is capable of sustaining a large and stable gradient of pHi, even within a single cell, raises the interesting possibility that ischemic border zones in vivo may also be associated with major standing gradients in myocardial pHi. Such gradients need not necessarily be established within single myocytes, although this could certainly occur. They may be established across macroscopic areas of myocardium at the border zone. Marked spatial heterogeneity in myocardial pHi may contribute to the tendency of border zones to promote arrhythmias (10).Gradients of pHi were observed recently in monolayers of colonocytes regionally (apical or basolateral surface) exposed to propionate (11). However, unlike cardiac myocyte preparations, this preparation has polarized Na+/H+ exchangers, and the monolayer itself acts to physically separate the two bathing solutions.
In summary, our dual-microstream system provides a simple, noninvasive technique for rapidly inducing steep pHi gradients in single cardiac myocytes. This microperfusion technique will be useful for studying the effects of regional acidosis on myocyte function and estimating intracellular ion diffusion rates. It may also be useful for inducing regional intracellular changes in other important ions and, perhaps, even for local extracellular agonist activation of sarcolemmal receptors.
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ACKNOWLEDGEMENTS |
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We thank Leona Montoya for secretarial assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-42873, HL-30478, HL-17682, and HL-42357 and awards from the Nora Eccles Treadwell Foundation (to K. W. Spitzer) and by a grant from the British Heart Foundation and the Wellcome Trust (to R. D. Vaughan-Jones).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. W. Spitzer, Univ. of Utah, CVRTI, 95 S. 2000 East, Salt Lake City, UT 84112 (E-mail: spitzer{at}cvrti.utah.edu).
Received 2 August 1999; accepted in final form 28 October 1999.
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REFERENCES |
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TOP
ABSTRACT
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METHODS
RESULTS
DISCUSSION
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