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1Burdon Sanderson Cardiac Science Centre, University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom; 2Dipartimento di Biologia Evolutiva e Funzionale, University of Parma, Parma 43100, Italy; and 3Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah 84112-5000
Submitted 27 March 2003 ; accepted in final form 7 May 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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) mobility. The apparent
diffusion coefficient for
,
was estimated in single ventricular myocytes isolated from the rat, guinea
pig, and rabbit. 
was
derived by best-fitting predictions of a two-dimensional model of
H+ diffusion to the local rise of intracellular [H+],
recorded confocally (ratiometric seminaphthorhodafluor fluorescence)
downstream from an acid-filled, whole cell patch pipette. Under
conditions, was similar
in all three species (mean values: 8–12.5 x
10–7 cm2/s) and was over 200-fold lower
than that for H+ in water. In guinea pig myocytes,
was increased 2.5-fold in
the presence of
buffer, in agreement with previous observations in rabbit myocytes.
mobility is therefore low
in cardiac cells, a feature that may predispose them to the generation of
pHi gradients in response to sarcolemmal acid/base transport or
local cytoplasmic acid production. Low
mobility most likely
results from H+ shuttling among cytoplasmic mobile and fixed
buffers. This hypothesis was explored by comparing the pHi
dependence of intrinsic, intracellular buffering capacity, measured for all
three species, and subdividing buffering into mobile and fixed fractions. The
proportion of buffer that is mobile will be the main determinant of
. At a given
pHi, this proportion appeared to be similar in all three species,
consistent with a common value for
. Over the pHi
range of 6.0–8.0, the proportion is expected to change, predicting that
may display some
pHi sensitivity.
intracellular pH; H+ diffusion
, working in concert with
intracellular, cytoplasmic buffers
(18,
29). As a result,
pHi is stabilized in the range of 7.1–7.3. Buffers represent
the first line of defence against intracellular acid-base disturbance
(3). They comprise
H+/OH–-titratable groups, most commonly imidazole
groups on intracellular proteins and dipeptides, as well as titratable groups
on other molecules such as taurine, various amino acids, inorganic phosphate,
and lactate (3,
10,
34,
38). All these groups
constitute the intrinsic buffer system of the cell. An extrinsic
CO2/bicarbonate buffer system (defined here as carbonic buffer)
also plays an important role (e.g., Refs.
3 and
18).
In addition to their function in minimizing changes of pHi,
intracellular buffers are intimately involved in the spatial regulation of
pHi. Because their collective buffering capacity is high
(20–60 mM), they bind almost all acids or bases introduced into the
cell. Consequently, intracellular H+
() cannot diffuse freely.
Instead, it is proposed to move via a process of shuttling on mobile buffers
(1,
14,
15,
34). This implies that the
properties of H+ movement will reflect buffer mobility and
reactivity. Intrinsic, intracellular buffers of lower molecular weight are
likely to be relatively mobile. Their combined buffering capacity in the
cardiac cell has been estimated as 11 mM/pHi unit, at a
pHi of 7.1 (34).
This is 
40% of the total intrinsic buffering capacity
(
int). Apparent
mobility will, however, be
low because competition for H+ binding from fixed buffer will
hamper net movement of acid via mobile buffer. In addition, mobile buffer
molecules are larger than H+ and will therefore diffuse more
slowly. A low apparent
mobility has potentially important consequences, as it means that sarcolemmal
fluxes of H+ will generate local pHi gradients that
dissipate more slowly than in unbuffered solution. Significant heterogeneity
of pHi will cause differential pH modulation of spatially
distributed proteins, which may affect the coordinated function of the
cell.
To evaluate the spatial characteristics of pHi in cardiac cells,
it is necessary to estimate the apparent
diffusion coefficient,
. Although this has been
done recently for the rabbit ventricular myocyte
(34), it has not been
estimated for any other cardiac cell. Confocal seminaphthorhodafluor (SNARF)
imaging of the spread of intracellular acid from a cell-attached micropipette
indicated that, for rabbit myocytes, H+ mobility was 250-fold
lower than in simple unbuffered solution. Intracellular mobility was
severalfold faster in the presence of carbonic buffer (2 x
10–6 cm2/s), but estimates of
were still nearly two
orders of magnitude lower than for unbuffered H+
(28).
In the present work, we compared
mobility in ventricular
cells isolated from the guinea pig, rat, and rabbit. One aim was to establish
whether low intrinsic
mobility, and its enhancement by carbonic buffer, is a general phenomenon. We
also compared int and its dependence on pHi in the
three species. Although there have been many reports of intracellular
buffering power in cardiac cells, there have been few quantitative
comparisons. Using our estimate of the H+-binding capacity of
mobile buffers in cardiac tissue, we mapped the likely intracellular
concentration and average pKa of fixed intrinsic buffer in cardiac
cells. We then considered how the relative concentrations of fixed and mobile
buffer contribute to the regulation of
mobility.
A preliminary report of this work has appeared in abstract form (41).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Rat and guinea pig ventricular myocytes were enzymatically isolated
according to a previously described procedure (see Ref.
17). Briefly, single
ventricular myocytes were isolated from albino guinea pigs and from Wistar
rats (killed by cervical dislocation) weighing 
400 and 300 g,
respectively, using a combination of enzymatic and mechanical dispersion (0.7
mg/ml collagenase, Boehringer Mannheim, and 0.04 mg/ml protease, Sigma; St.
Louis, MO). Rats and guinea pigs were killed humanely, according to UK Home
Office recommendations, by concussion and cervical dislocatoin. The cells were
finally suspended in HEPES-buffered Dulbecco's modified Eagle's medium
(culture medium) and kept at room temperature until use.
As previously described (26), adult rabbit ventricular myocytes were obtained from New Zealand White rabbits (2–3 kg). Animals were anesthetized with an intravenous injection of pentobarbitone sodium (50 mg/kg) and 0.5 ml heparin to prevent clotting, in accordance with national guidelines. The heart was rapidly removed and attached to a Langendorff perfusion system. The heart was digested with a solution containing 1 mg/ml collagenase (class II, Worthington Biochemical; Freehold, NJ), 0.1 mg/ml protease (type XIV, Sigma), and 0.1 mM CaCl2. The cells were stored until use at room temperature in the normal HEPES-buffered solution.
All myocytes used in this study had a rod shape, well-defined striations, and did not spontaneously contract. Experiments were performed within 24 h after isolation.
Solutions
Superfusion solutions were held at 37°C and delivered by means of a
peristaltic pump to the cell chamber. The 1-ml Plexiglas chamber had a clear
glass bottom and was mounted on the stage of an inverted microscope (Leica DM
IRBE). The temperature of the solutions in the bath was kept at 37°C by an
electrical temperature control circuit. Bathing solutions continuously flowed
through the bath at 2 ml/min, and the solution depth in the chamber was
held at 200 µm. The bottom of the bath was coated with
poly-L-lysine (Sigma) to improve cell adhesion.
HEPES-buffered Tyrode solution contained (in mM) 135 NaCl, 4.5 KCl, 1 MgCl2, 2 CaCl2, 11 glucose, and 20 HEPES adjusted to pH 7.4 with 1 M NaOH at 37°C. Bicarbonate-buffered Tyrode solution was identical except for the NaCl concentration, which was reduced to 120 mM, and for 22 mM NaHCO3, which was added instead of HEPES. Bicarbonate solution was equilibrated with 5% CO2-95% air.
For ammonium- or acetate-containing solutions, NaCl was replaced osmotically by ammonium chloride and sodium acetate. For chloride-free solutions, all chloride-containing salts (except ammonium) were replaced with salts of gluconic acid and pH was adjusted to 7.4 using 1 M NaOH (NH4Cl was replaced with NH4SCN). For sodium-free solutions, sodium salts were osmotically replaced by N-methyl-D-glucamine and pH was adjusted to 7.4 with 5 M HCl.
In some experiments, 30 µM cariporide (HOE-642, Aventis), a selective Na+/H+ exchanger (NHE)-1 inhibitor (24), structurally related to a previous inhibitor compound, HOE-694 (Aventis; 20), was added to the Tyrode solution.
The fluorophore stock solution was prepared by dissolving 1 mg SNARF-1-AM (Molecular Probes) in 1 ml DMSO to reach a concentration of 1.7 mM.
To acid load myocytes, suction pipettes were filled with an unbuffered isotonic solution containing (in mM) 140 KCl, 0.5 MgCl2, 5.5 dextrose, and 1.0 HCl (pH 3.0). To pipette load myocytes with unesterified carboxy-SNARF-1 (Molecular Probes), it was added to a final concentration of 400 µM in a filling solution containing (in mM) 140 KCl, 1.0 MgCl2, and 10 HEPES adjusted to pH 7.1 with 1 M NaOH. No attempt was made to compensate for the small reduction in pH of the filling solution caused by the acidic nature of the fluorophore.
Whole Cell Epifluorescence Measurement of pHi
Whole cell pHi was measured at 37°C in single myocytes previously equilibrated at room temperature for 10 min in culture medium containing 10 µM of the AM form of carboxy-SNARF-1 as the fluorescent pH indicator (see, e.g., Ref. 18). Cells were superfused in a chamber mounted on an inverted epifluorescence microscope. The dye was excited by light from a 100-W xenon lamp at 540 nm. Emission at 590 and 640 nm was measured by two photomultiplier tubes, filtered at 10 Hz, and digitized by an analog-digital converter (CED 1401, Cambridge Electronic Design). The ratio was averaged at 2 Hz (in-house software) and converted off-line to pH using the calibration curve obtained by the nigericin calibration technique (see Ref. 31).
Confocal Measurement of pHi
The pHi was measured at 37°C in single myocytes using the carboxy-SNARF-1 dye (5). A laser scanning confocal microscope, Leica DM IRBE, with Leica TCS NT software and a Leica x40, 1.25 numerical aperture, oil immersion, plano-apochromat objective lens were used to confocally image the cells. SNARF excitation was achieved with the 514-nm laser line of an air-cooled argon laser. Emitted fluorescence was simultaneously collected by two photomultiplier tubes equipped with band-pass filters at 640 and 580 nm. A transmitted light detector also provided a nonfluorescent image of the cell. The two fluorescence x-y images plus the transmission image were acquired on-line at a rate of one frame every 2 s. No z scan was performed, and pinhole size was kept constant at a fixed value of 1.29.
Confocal images were processed, and the fluorescence ratio (580/640 nm) was calculated off-line using NIH Image and Transform (Fortner Software; Sterling, VA) software packages. The ratiometric signal was converted to pH, as previously described, using the nigericin calibration technique (31). This requires bathing solutions of varying pH that also contain 10 µM of the ionophore nigericin. The best-fit equation for the calibration curves obtained from several myocytes was used to calculate pHi of the cells. Local pHi was measured in predefined regions of interest (ROIs) that were drawn as circles of 10 µm diameter, avoiding nuclei and the pipette tip. To derive space-profiles for pHi, only one ROI was drawn, with a long rectangular shape that fitted most of the cell area, avoiding the pipette tip.
Acid and SNARF pipette-loading procedure. Myocytes were exposed to
acid via whole cell patch pipette attachments, as previously described
(34). The pipettes were made
from borosilicate capillary tubing (Harvard Apparatus; Edenbridge, UK) and had
a resistance, when filled, of 1–2 M
. Transmembrane potential was
monitored by means of the bridge circuit of an Axoclamp 2B amplifier (Axon
Instruments; Union City, CA). Before a cell was contacted with the pipette
tip, the pipette potential was set to zero and the voltage drop across the
pipette was compensated with the bridge balance. Cells, previously loaded with
SNARF-AM as described above, were introduced into the perfusion chamber and
continuously bathed with Tyrode solution. Fluorescence (580 and 640 nm) and
transmission confocal images were recorded before (1 min) and during
pipette attachment and break in. Pipette attachments were made as close to the
end of a cell as possible and lasted from 1 to 7 min. A similar technique was
used to pipette load carboxy-SNARF-1 into myocytes.
Finite Element Method Model
The spatiotemporal characteristics of the intracellular proton concentration during pipette loading were analyzed by solving diffusion equations. Two-dimensional rather than three-dimensional, time-dependent equations are solved because the depth of the cell on a coverslip is small; hence variation of H+ concentration along the z-axis is likely to be much smaller than that along the other two axes.
The geometry of a myocyte was used to define a two-dimensional array of
points (x,y) called domain , with boundary points belonging to
. This array of points was generated by Delaunay triangulation
(MATLAB PDE Toolbox), whereby the density of the mesh depended on the
regularity of the geometry
(32), such that density
decreased where the structure had regular, rectangular geometry and increased
in regions where the boundary (i.e., cell membrane) became irregular. The
diffusion problem was solved using the finite element method (FEM) approach.
In FEM, piecewise approximations with simple functions at each point are
combined to provide a solution for the whole domain. Unlike previous
approaches (finite difference method; see Ref.
34), FEM permits considerable
flexibility in mesh density and distribution and optimizes computational time
in regions where it is not necessary to maintain a high mesh density. The
method is particularly well suited to our type of boundary condition.
The variable u is the concentration of protons above baseline
[change in concentration
(
[H+]i)]. The diffusion partial differential
equation (PDE) is
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The geometry and pipette location are defined according to the transmission image. The FEM algorithm implemented in the MATLAB PDE Toolbox (for an overview, see Ref. 32) is used to solve the diffusion PDE for a particular value of diffusion coefficient, D. Solutions can be obtained as a function of time or space, depending on the nature of the data. Because this diffusion PDE is linear, the solution is scaled by a constant to fit the magnitude of the results (equivalent to scaling the injection rate P). This is repeated for different values of D until the best fit is obtained according to the least-squares method. This best-fitting value of D is selected as the apparent proton diffusion coefficient. Fitting procedures are limited to the first 60 s of acid injection due to possible sarcolemmal acid extrusion and changes in the mobile-to-total buffer ratio (Eq. 2), which will alter the value of D (32).
Measuring Intrinsic Buffering Capacity
int was measured while cells were superfused in
HEPES-buffered solutions (nominally free of CO2/bicarbonate)
containing various concentrations of a salt of a weak acid (acetate) or a weak
base (ammonium). For experiments with guinea pig and rat myocytes,
ammonium-containing solutions were chloride free (plus 30 µM cariporide;
Aventis); they contained 40, 30, 20, 15, 10, 5, and 2.5 mM NH4SCN.
Acetate solutions contained 80, 60, 40, 20, and 10 (plus 30 µM cariporide).
For experiments on rabbit myocytes, solutions were sodium free (no cariporide
added) and contained 15, 12, 9, 5, 3, 2.5, or 1 mM NH4Cl. No
acetate experiments were performed with rabbit cells.
Cells were superfused in normal Tyrode followed by a switch to one of the
ammonium or acetate solutions. A series of solution changes was then made, in
which the extracellular weak acid or base concentration was stepped to
progressively lower levels, eventually returning to normal Tyrode solution.
This produced step changes of pHi in the alkaline and acid
directions, respectively (see Fig.
5A). The changes approached a steady state after
2–4 min. The pHi before and after the weak acid/base removal
step (
and
, respectively)
was noted. During ammonium removal, 
leaves the cell in the form of uncharged NH3, leaving behind
H+. The change in [H+]i is assumed to be
equal to the change in intracellular
concentration
([]i), which can be
estimated using the following rearranged Henderson-Hasselbalch equation
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] in the extracellular
solution ([]o) is
calculated from the total ammonium concentration
divided by [1 + 10(pHo –
pK), where pHo is extracellular pH].
int may then be computed
(23)
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int so determined
to the midpoint in the change of pHi induced by the step decrease
in extracellular weak acid/base.
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Statistical Analysis
Summarized results are presented as means ± SE. Statistical analysis was performed using Student's t-test for unpaired data and univariate ANOVA for three-species comparison by means of the software package SPSS 10.0.6 (SPSS; Chicago, IL). P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Figure 1 illustrates the
experimental protocol applied to a rat ventricular myocyte. The patch pipette
was filled with buffer-free, isotonic KCl adjusted to pH 3.0 and positioned
close to one end of the cell (Fig.
1A). The superfusate contained no added
and
was buffered with 20 mM HEPES (pH 7.4). It also contained 30 µM cariporide,
a high-affinity, selective inhibitor of sarcolemmal NHE-1 to prevent acid
extrusion during the course of the acid-loading protocol. A ratiometric image
of the cell loaded with SNARF is shown in
Fig. 1B. Superimposed
on this image are three ROIs positioned longitudinally downstream of the
pipette.
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After break in by the pipette, the myocyte acidified, as shown in
Fig. 2.
Figure 2 compares the time
course of pHi averaged in the three ROIs. The greater the distance
of the ROI from the pipette, the slower was the rate of acidification. After
break in, acidification was detected almost immediately above the noise level
in the proximal ROI, but it was not detected in the most distal ROI, 52 µm
downstream, until 30 s later. This indicates a slow spreading of acid
within the cytoplasmic compartment.
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In Fig. 2B, changes
of pHi in the three regions over the first 60 s have been converted
into increments in H+ concentration and best fitted by the FEM
model of two-dimensional diffusion described in METHODS. The
triangulated image of the cell used for the fitting procedure is illustrated
in Fig. 1C. The value
for obtained by the model
was 2.5 x 10–7 cm2/s.
Figure 2C
illustrates results from a similar experiment, but this time on a guinea pig
ventricular myocyte. The longitudinal profile for [H+]i
was extracted from the data at different times after break in. A gradient of
up to 0.3 pH units was apparent during the course of acid loading, with the
greatest [H+]i occurring at the site of the pipette.
These data were best fitted by the diffusion model assuming a value for
of 5.0 x
10–7 cm2/s. The good spatiotemporal
agreement between the model and the data shown in
Fig. 2, B and
C, indicates that acid movement in guinea pig and rat
cells conforms to a passive diffusive process, as reported previously for the
rabbit ventricular myocyte
(34).
Figure 3 shows average
values of obtained by
best fitting data from the rat, guinea pig, and rabbit using the FEM model.
Mean values ranged from 8.2 x 10–7
cm2/s in the rabbit to 12.1 x
10–7 cm2/s in the guinea pig, although
these differences were not statistically significant. For guinea pig and rat
cells, experiments were performed in normal Tyrode solution, with (n
= 10 and 5) and without 30 µM HOE-642 (n = 10 and 6). The paired
means with and without drug were not significantly different; therefore, they
were pooled in Fig. 3. Rabbit
experiments were performed in the presence of 1 mM amiloride (n = 6).
The data indicate that H+ mobility in ventricular myocytes is about
two orders of magnitude lower than for H+ in water [1.2 x
10–4 cm2/s
(33)].
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Imaging Intracellular SNARF Diffusion
Figure 4 illustrates the
results of an experiment designed to estimate intracellular SNARF mobility in
a guinea pig myocyte. As the fluorophore is a weak acid (pKa
7.6), we assessed whether it would significantly influence the
measurements of mobility.
SNARF was diffused into one end of a guinea pig myocyte from a cell-attached
micropipette containing 400 µM of the carboxylic acid form of the
fluorophore. Figure 4
illustrates longitudinal profiles of fluorescence obtained at different time
intervals after break in by the pipette. The profiles have been best fitted by
the FEM model. In three experiments, a mean value for the diffusion
coefficient of SNARF of 3.22 ± 0.86 x
10–7 cm2/s was obtained, comparable to
the value of 1 x 10–7 cm2/s
measured recently for SNARF mobility in the rabbit ventricular myocyte.
Assuming the AM-loading procedure for SNARF results in 400 µM of the
acid form of the dye inside the cell, as it does in the rabbit myocyte
(34), the fluorophore's low
buffering capacity (170 µM/pH unit; estimated assuming one
H+ binding site per SNARF molecule) and low intracellular mobility
means that it will spatially shuttle only small quantities of H+
during an acid injection procedure. With the use of the formalism derived by
Junge and McLaughlin (15) and
by Irving et al. (14), the
presence of the intracellular fluorophore would increase
in accordance with the
function
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is the apparent
diffusion coefficient of SNARF, SNARF is the buffering
capacity of the dye, and int is 24 mM at pHi 7.1
(see Fig. 5). The increase
would be 0.02 x 10–7 cm2/s, i.e.,
an increase in of <1%.
This increase will therefore have a negligible effect on the experimental
measurements of mobility.
Furthermore, the similar SNARF mobility measured in guinea pig and rabbit
myocytes strongly suggests that mobility in rat myocytes will also be
comparable. If so, the fluorophore will have a negligible impact on
mobility in ventricular
myocytes from all three species.
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Many previous studies have used AM-loaded
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) instead
of SNARF to evaluate pHi regulation in cardiac cells. Without
knowledge of the intracellular concentration and mobility of BCECF, it is not
possible to estimate whether it significantly affects
mobility. Given the
similarity in the molecular weight of SNARF and BCECF, however, and given the
similar loading protocols, it would not seem unreasonable to assume that both
fluorophores will influence
mobility by similar amounts.
Estimating Total, Mobile, and Fixed Intrinsic Buffering Capacity
Figure 5, A and
B, illustrates the experimental protocol used for
investigating the pHi dependence of total int. A
wide range of pHi values was achieved by superfusing various
concentrations of salts of a membrane-permeant weak base or weak acid
(ammonium and acetate salts, respectively). Successive reduction in the
concentration of these compounds in the superfusate results in a step-wise
titration of the cytoplasm by acid and base, respectively. The procedure for
calculating int is detailed in METHODS.
The graph shown in Fig.
5B plots data on int gathered from
guinea pig myocytes. Also plotted (blue curve) is the pHi
dependence predicted for intrinsic mobile buffer capacity
(mob). This has been assembled by summing the capacity of the
mobile buffers identified in cardiac tissue, listed recently by Vaughan-Jones
et al. (34; see also present
study, METHODS). The capacity of any individual mobile buffer was
estimated from its concentration (C) and pKa
 | (1) |
mob from the data points plotted for int
produces points predicted for fixed intrinsic buffer capacity
(fix). Interestingly, the characteristics of these latter
data conform to a buffer with a concentration of 68.3 mM, with a single
pKa of 6.1, as illustrated by the red curve in
Fig. 5B, which is a
least squares, best fitto the data. Summing this curve with that for the
mobile buffer gives the pHi dependence of int,
plotted as the continuous black curve in
Fig. 5B.
Figure 5C
illustrates the experimental results obtained for int in all
three species. Superimposed on the data are the individual curves for
int. These were generated, as above, by assuming that the
pHi dependence predicted for mob applies equally
in all three species. This allowed fix to be calculated. The
characteristics derived for the fixed buffer in the three species are listed
in Table 1. The pKa
appears to be very similar in all three cases (6.1–6.2), as is the
effective concentration (varying from 53 to 68 mM).
Effect of Carbonic Buffer on
Mobility
Figure 6 summarizes the
results for the estimation of
in guinea pig ventricular
myocytes superfused with Tyrode solution equilibrated with carbonic buffer (5%
CO2/22 mM ; pH 7.4). The
experimental protocol and analysis in these experiments were identical to that
employed previously for estimating intrinsic
mobility, with the
exception that the pipette filling solution was saturated at 37°C with 5%
CO2, to match the PCO2 inside and outside the
cell. The time course of the rise of [H+]i in proximal
and distal downstream regions, when best fitted by the FEM model, produced
values for that were up
to threefold larger than in the absence of carbonic buffer (i.e., with
HEPES-buffered superfusate). This indicates that, in the guinea pig
ventricular myocyte, the presence of a CO2/bicarbonate buffer
system facilitates intracellular proton mobility, as reported previously for
rabbit myocytes (34).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Mobility
in the Ventricular Myocyte
Intrinsic, mobility,
defined as that measured in the absence of carbonic buffer, appears similar in
rat, guinea pig, and rabbit ventricular myocytes, average values for
being 8–12 x
10–7cm2/s. A previous estimate in
rabbit ventricular myocytes was somewhat lower [4 x
10–7 cm2/s
(34)] but still falls within
the range of individual values recorded in the present study (2.9–22
x 10–7 cm2/s). The value for
is more than two orders
of magnitude lower than for H+ in unbuffered aqueous solution (1.2
x 10–4 cm2/s). A low
mobility is therefore
likely to be a universal feature of mammalian ventricular cells.
Low values for are
proposed to be caused by the presence of intracellular buffers that bind
almost all acid or base introduced into or generated within the cell.
Depending on net electrical charge and molecular size, intrinsic buffer
molecules are likely to exhibit a range of mobilities lower than for
H+. Large macromolecules, like proteins, display extremely low
intracellular mobility with apparent diffusion coefficients of
10–8 cm2/s
(37). They are, therefore,
effectively immobile (i.e., fixed) on the time scale of the present
experiments. Lower molecular weight intrinsic buffers like homocarnosine (a
cytoplasmic dipeptide containing histidine) and inorganic phosphate are likely
to have higher mobility coefficients. Because the overall capacity of
intrinsic buffer is high (20–60 mM depending on pHi), the
free diffusive flux of will
be negligible compared with that of H+ conjugated to mobile
buffers. H+ will therefore be moved from regions of high to low
concentration by means of a buffer shuttle, protonated buffer diffusing to
more alkaline regions where deprotonation occurs.
Geometric factors are also likely to play a role in determining a low
intrinsic mobility. In
muscle cells, macromolecular crowding within the cytoplasmic compartment is
thought to reduce the effective volume for diffusion by up to 50%, thus
reducing solute mobility (16).
This may be accentuated further by extensive sarcolemmal invagination, as
occurs with the t-system. It will be of interest, therefore, to explore
whether mobility is faster
in cardiac cells that lack a t-system, such as atrial
(13) and Purkinje myocytes
(8).
Estimates of
are unaffected by sarcolemmal NHE. Sarcolemmal acid extrusion, by
removing H+ from the cytoplasm, will limit their spatial spread.
This could potentially lead to an underestimate of
. While H+
transport undoubtedly occurs, transport proteins such as NHE did not appear to
distort the measurement of
as the estimate was
unaffected by adding NHE inhibitors to the superfusate (e.g., amiloride or
cariporide), a result similar to that obtained previously in rabbit
ventricular myocytes (34).
These observations are consistent with a recent mathematical analysis
indicating that NHE activity should not significantly distort estimates of
, provided fitting
algorithms are restricted to the first 60 s of acid loading, the time period
used in the present work (32).
Effects on acid loading caused by subcellular sequestration of H+
into organelles such as mitochondria also cannot be excluded but, over the
time scale of our experiments, such a mechanism, if sufficiently fast and
large, would appear as a cytoplasmic H+ sink that contributes to
our overall estimate of buffering capacity and hence would be accommodated by
the present analysis.
Facilitation of
Mobility by Carbonic Buffer
Carbonic buffer contributes to spatial pHi regulation. We found
that the mobility
coefficient in guinea pig myocytes is enhanced two- to threefold in the
presence of carbonic buffer, an effect that will help to reduce the magnitude
and duration of pHi gradients associated with local acid/base
disturbances within the cell. This result is similar to that reported recently
in rabbit ventricular myocytes where carbonic facilitation of
mobility was up to sixfold,
suggesting this is a general phenomenon in cardiac cells
(28). Similar observations
have also been made in intestinal enterocytes
(30). Carbonic facilitation of
mobility has been
questioned recently in neuronal cells
(25), although interpretation
of the evidence for this has been disputed
(32,
35).
The mechanism whereby CO2/bicarbonate buffer facilitates
mobility is proposed to
depend on a buffer shuttle, much as occurs with intrinsic buffer. In this
case, however, there is diffusion and hydration of intracellular
CO2 (or diffusion and dissociation of H2CO3),
leading to the appearance of H+ and
in regions distal to the local
acid disturbance, with back diffusion of
to neutralize some of the original
acid load (28,
35). The shuttle thus
effectively mediates a passive spread of
from proximal to distal
regions. In rabbit ventricular myocytes and murine duodenal enterocytes, the
enzyme carbonic anhydrase has been proposed to play a key role in maximizing
the turnover rate of this shuttle
(28,
30). The participation of
carbonic anhydrase in rat and guinea pig myocytes was not investigated in the
present work, although the expectation is that the enzyme will play a similar
role in these cells.
Under physiological conditions, the intrinsic and carbonic buffer shuttles
will independently mediate spatial movements of intracellular acid. Their
effects should be combined when computing contributions to
(28).
Intrinsic Buffers and the Regulation of Intrinsic
Mobility
The values for intrinsic buffering power reported here are within the range
of most previous estimates in cardiac tissue
(4,
36), including ventricular
myocytes isolated from the guinea pig
(17,
18,
40), rat
(11,
39), and rabbit
(21). More importantly, our
quantitative estimates of the pHi dependence of
int in the three species are also similar. This would be
consistent with the intracellular concentration and pKa of
individual constituent buffers being comparable among the species, thus also
accounting for the comparable values of
.
Fractional mobile buffer capacity. The influence of mobile and
fixed intrinsic buffer on
mobility is described by the function
(14,
15,
32)
 |
mob,i, and
fix,j is the capacity of the jth
fixed intrinsic buffer. At physiological pH, and pooling all mobile buffers
under one diffusion coefficient and all fixed buffers under zero mobility,
this equation can be approximated as
 | (2) |
int equals the summed capacity of all mobile and fixed
intrinsic buffers (equal to mob + fix). This
has been dubbed the "proton mobility" equation
(32).
Equation 2 indicates that a major determinant of intrinsic
mobility will be the
fraction of int that is mobile
(mob/int). As illustrated in the present
work, int may be estimated experimentally from measurements
of pHi, whereas mob may be computed theoretically
from the list of mobile buffers identified in cardiac tissue (Ref.
34 and the present study,
METHODS). While this list may not be entirely comprehensive, it
will nevertheless provide a reasonable estimate of the pHi
dependence of mobile buffering power (see blue curve in
Fig. 5B). With the use
of data shown in Figure 5, B and
C, one may compute the mobile buffer fraction
(mob/int) for a range of pHi
values. This has been compared in Fig.
7 for rat, rabbit, and guinea pig myocytes. Over the range of
pHi from 8.0 to 6.0, the mobile fraction of intrinsic buffering in
all three species is predicted to decline by 80%. If buffer mobility
(Dmob) were independent of pHi (see below), the
proton mobility equation would predict that the same acidosis should produce
an 80% decline of
mobility.
|
At present, there is no experimental evidence for the effects of
pHi on mobility
in cardiac cells, but measurements of
in extruded samples of
molluscan axoplasm suggest that it is decreased by acidosis
(1). Mobile buffers in axoplasm
differ significantly from those in cardiac cytoplasm [the former contains high
concentrations of amino acids such as glycine (180 mM) and aspartic acid (75
mM), whereas the latter contains more modest concentrations of dipeptides
(2–9 mM) such as homocarnosine and anserine
(34)]. Nevertheless, as
discussed above, there is a strong theoretical argument for a qualitatively
similar pHi dependence of
in the heart. While it
may not be feasible to extrude cytoplasm from cardiac myocytes without
affecting constituent buffers (particularly the fixed buffers), it may be
possible to estimate in
an intact cell after pipette loading of an intracellular base rather than
acid, thereby exploring more alkaline regions of pHi.
Fixed intrinsic buffer. A striking observation in the present
study is that the fraction of int attributed to fixed buffer
can be represented by a single component of pKa 6.1–6.2 and a
concentration of 50–70 mM (e.g., red curve in
Fig. 5B). This is in
contrast to the predicted mobile buffer curve that comprises contributions
from several individual buffers of much lower concentration, with overlapping
pKa values such that summed mobile capacity is relatively constant
over the physiological pH range. We conclude that fixed buffers in the cardiac
myocyte constitute a relatively homogeneous population. The pKa of
6.1–6.2 would, for example, be consistent with imidazole groups on
histidine residues. Given their effective concentration, these would most
likely be components of proteins that are of low cytoplasmic mobility or
anchored within the cell.
The fixed buffer curve (i.e., fixed buffer capacity vs. pHi) predicted in the present study is comparable to the intrinsic buffer component of low pKa first identified by Leem et al. (18). These authors approximated the pHi dependence of intrinsic buffering in the guinea pig ventricular myocyte by assuming two principal buffer populations, one of low average pK (6.03) and high concentration (84 mM) and one of higher average pKa (7.57) and more modest concentration (29 mM). These subdivisions are therefore reminiscent of the present proposals for fixed and mobile buffers (e.g., Fig. 5B). Indeed, Swietach et al. (32) have recently adopted the Leem description of intrinsic buffering in a computer model of two-dimensional diffusion of acid within a cardiomycyte. This was done by defining the low pKa component of buffering as fixed and the higher pKa component as mobile. The success of the simulation suggests that the broad division of intrinsic buffering into mobile and fixed components with differing pH-dependent characteristics may be correct.
Intracellular buffer mobility. Apart from the mobile buffer
fraction, Dmob is the other principal factor influencing
. The proton mobility
equation predicts that, at a pHi of 7.1, Dmob
in guinea pig myocytes will be 2.6 x 10–6
cm2/s, a value pooled for all mobile buffers. Possible effects of
pHi on Dmob have not been investigated, but
mobility will be independent of pHi only if the protonated and
nonprotonated forms of any buffer display identical diffusion coefficient
values. Given that protonation alters the net electrical charge on a molecule,
this may influence the mobility of a buffer within the intracellular
compartment, so that a pH sensitivity of Dmob cannot be
excluded.
Relevance to Physiological Acidosis and Ischemia
A of sufficient
magnitude is necessary to promote uniformity of pHi in the face of
a local acid/base disturbance. A reduced
during intracellular
acidosis will therefore increase the possibility of pHi
nonuniformity, at a time when acid efflux transporters operating locally at
the sarcolemma are strongly stimulated. At present, there are no experimental
data regarding possible pHi heterogeneity induced by acid transport
in cardiac myocytes, although nonuniformity is readily produced by other
maneuvers (27,
34). The physiological
consequences of pHi nonuniformity are also not known, but the
spatial coordination of local, intracellular processes such as sarcomere
contraction is likely to be compromised.
It is tempting to speculate that a decline of
may occur during the
severe metabolic acidosis associated with myocardial ischemia. Such
speculation, however, would be premature. Complex metabolic disturbances, such
as occur during ischemia, are associated with changes in the concentration of
certain intracellular mobile buffers, an effect independent of changes in
pHi. For example, during myocardial ischemia, there is a
considerable rise (from 2 to 30 mM) in the levels of inorganic phosphate
(see, e.g., Ref. 2), a molecule
with a principal pKa of 6.9. This might raise rather than reduce
mobility. In contrast, as
noted recently (28), because
acidosis during ischemia is also associated with a fall of
, this would
tend to reduce overall
mobility. Direct measurements of
under conditions that
simulate ischemic acidosis will therefore be required to explore these
possibilities further.
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DISCLOSURES |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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in
rabbit ventricular myocytes is regulated by carbonic anhydrase. J
Physiol 541:
159–167, 2002.
conditions.
Circ Res 75:
123–132, 1994.
of 2.5 x 10–7 cm2/s. C:
guinea pig ventricular myocyte. Longitudinal intracellular [H+]
([H+]i) profiles were recorded confocally at different
times (1, 10, and 26 s) after cell break in by the acid-containing pipette
(cell length 150 µm, width 23 µm). The spatial profiles are shown for a
ROI (137 µm long and 10 µm wide) positioned centrally along the
longitudinal axis of the cell. The pipette is positioned at 20 µm from the
left end of the cell. At any given longitudinal distance, the change in
[H+] has been averaged across the width of the ROI. Continuous
curves show results of the FEM model with a best-fitting
