Determination of pH by microfluorometry: intracellular and interstitial pH regulation in developing early-stage fish embryos (Danio rerio)
Department of Animal Physiology, Humboldt-Universität zu Berlin, D-10115, Germany
* Author for correspondence (e-mail: andreas.moelich{at}rz.hu-berlin.de)
Accepted 9 September 2005
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Summary |
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Intracellular pH (pHi) in Danio rerio embryos between 1k-cells stage and the end of epiboly was found to be well regulated to a mean value of 7.55±0.13 (± S.D.), a range distinctly more alkaline than typical values for adult fish but in accordance with embryonic pHi of a few non-fish species shortly after fertilization. Also, interstitial pH (pHint) was significantly higher (8.08±0.25) than values for extracellular pH in adult fish. Distributions of HCO3- across membranes and between interstitium and ambient fluid compared with respective potentials strongly suggest that pH in these early stages of ontogeny is already adjusted by active transfer processes. Non-respiratory changes in ambient pH between 7.7 and 8.5 did not significantly affect pHi, a result potentially attributable to low membrane leakage rate or to the potency of active transfer mechanisms. In order to assess the pH regulatory systems more quantitatively, embryos were exposed to ambient changes of carbon dioxide partial pressure (PCO2). The direct impact of PCO2 changes on cell pH was alleviated by cell non-bicarbonate buffering and subsequent rapid, almost complete, compensation by changes in cell [HCO3-] as an expression of transmembrane transfer of acid-base relevant ions. On the basis of these results, we conclude that the regulatory potency of embryonic cells is well developed, is active to resist extensive homoiostatic stress and is efficient to maintain critical metabolism in adverse conditions, even at early stages of ontogeny.
Key words: intracellular pH, interstitial pH, ontogeny, regulation, transmembrane transfer, microfluorometry, in vitro calibration, intracellular microelectrode, Danio rerio, zebrafish
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Introduction |
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In fish and amphibians, much more than in mammals, birds and reptiles,
embryos are exposed to challenges of pH regulation by direct contact with the
aqueous ambient medium. With all changes in environmental composition, pH in
the cells has to be maintained at values compatible with enzymatic function
throughout development from single cells to multicompartmental animals. Not
only the natural variability of the environment but also more recent and more
severe impacts of coal mining and industry emissions provide considerable
stress for developing organisms (e.g. Duis
and Oberemm, 2000; Ingersoll
et al., 1990
). Numerous studies have focused on survival of fish
eggs and fry in moderately and severely acidified waters (e.g. Johansson et
al., 1973
,
1977
;
Lacroix and Townsend, 1987
).
Mortality rises steeply at a water pH below 5, but loss of equilibrium and
subsequent death is generally attributed to a net loss of ions from the body
fluids, particularly at low water [Ca2+]
(Potts and McWilliams, 1989
;
Wood et al., 1990a
), rather
than to changes in body fluid pH.
The projected pattern of extensive ion loss as a cause of mortality may
ultimately be related to lack of energy to drive ion transfer mechanisms, due
to the inability to cope with the challenge for pHi regulation (see
above). In adult fish, extensive studies regarding acid-base regulation have
been performed on a number of species and aspects
(Claiborne, 1998; Heisler,
1986b
,
1993
), but, except for
confusing results from a few studies at fertilization
(Epel, 1997
), hardly anything
is known as to the regulatory facilities and capacities of cellular pH
regulation during early development (1k-cells stage and above).
The present study has been designed to shed some light on pH regulation in
intracellular and interstitial spaces during the early stages of ontogeny.
Early stages of fish embryos provide some unusual and complicating features as
compared with adult fish. The interstitial space is difficult to access for
sampling, and acid-base-relevant ion exchange between body compartments, a
common mechanism in adult fish (cf. Heisler,
1984,
1986b
), is hampered during
early ontogeny according to the delayed development of a defined extracellular
space and the absence of circulation. Due to the lack of specialized
structures, gas exchange is completely passive by diffusion through the
external surface, and ion transfer between organism and environment is locally
reduced to the external membranes of outer cell layers, rather than being
performed by epithelial organs. With continuing ontogeny, the general
regulatory capacity of the organism is expected to rise due to the development
of gills, kidneys and other specialized structures.
This study was performed in embryos of zebrafish (Danio rerio),
serving as an experimental model for pH regulation of developing fish. This
species was chosen because of its wide use in ontogenetic research, with a
well-founded background on care, breeding, preparation and development, as
well as for its speed of embryonic development (3.7 days at 25°C; cf.
Kimmel et al., 1995;
Westerfield, 1995
). Because of
the requirement for non-destructive and quasi-continuous measurement over a
relatively long time, pH was determined by application of
laser-scanning-microfluorometry with specific dextran-coupled fluorescent dyes
injected into studied fluid compartments before experimentation. After
adaptation of this method to the very particular requirements of multicellular
objects kept in an environment allowing normal ontogeny, the embryos were
challenged by various external pHs, induced by either metabolic or respiratory
means, in order to study characteristics of cellular and interstitial pH
(pHint) regulation.
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Materials and methods |
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Mating fish were separated in pairs in small tanks (4 litres) during
the light period. Fertilized eggs were collected the next morning after
light-cycle dawn and transferred into embryonic medium (in mmol
l-1: NaCl 13.7, KCl 0.54, CaCl2 1.3, MgSO4 1,
NaHCO3 5; modified from
Westerfield, 1995
). For
experimentation, embryos were mechanically dechorionated. Adequate progress of
embryonic development was checked by comparison with charts for standardized
stages of development vs time after fertilization (recalculated for
the experimental temperature of 25°C;
Kimmel et al., 1995
). (More
detailed information on developmental stages of zebrafish, including pictures,
is readily available at
http://zfin.org;
Sprague et al., 2001
.)
Procedure
Intracellular and interstitial pH were measured, and further development of
embryos was studied on the stage and in the light path of an inverse
microscope assembly (Axiovert 135 M with attached LSM 410; Carl Zeiss, Jena,
Germany). Water-soluble fluorescent dyes were pressure-injected through glass
microcapillaries (tip diameter 1 µm) into single cells of second or
third layer or into the interstitium of embryos 2-3 h after fertilization
(16-cell to 256-cell stages). Long-term compartment retention of dyes was
promoted by using dextran-bound indicators, a measure also suitable to reduce
compartmentalization, bleaching and cell damage by photolysis
(Bright et al., 1989
). These
dyes proved to be stable for 8 h, until embryos underwent or finished
epiboly.
Ambient conditions of the embryos were adjusted by continuous superfusion with embryonic medium adjusted in pH, ionic composition and respiratory gases. Embryos were kept in position over small funnel-like chamber exits by slight medium suction (Fig. 1), provided by a peristaltic pump. The resulting encircling flow for each individual proved suitable to provide well-defined media flow along the embryonic surface, preventing diffusion limitation on the basis of extensive unstirred layers. Unstirred layers and other elevations of resistance to diffusional gas exchange were found to cause unreproducible and largely drifting pH patterns, as observed in relevant preliminary experiments. In order not to affect the media composition by gas exchange with the ambient air, the air space of the chamber was flushed with the same gas as used for media equilibration.
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Adequate development of embryos under severe hypercapnia was studied in a separate series. Two groups of dechorionated embryos of similar stage were exposed in embryonic medium to PCO2s of 2.4 mmHg (0.32 kPa; N=20) and 24.6 mmHg (3.28 kPa; N=20). Ontogenetic progress was monitored and compared between the two groups as well as with non-dechorionated, completely unhandled embryos at normocapnia after 3 and 24 h of exposure.
Analytical approach
Microfluorometric determination of pH
Embryonic pHi and pHint were determined by
single-excitation/dual-emission or dual-excitation/single-emission fluorometry
using pH-sensitive dyes coupled to 10 kDa dextran molecules (Molecular Probes,
Eugene, OR, USA). Dyes were excited by laser light of an appropriate
wavelength, and the emission was monitored by high-sensitivity
photomultipliers. Laser bands of excitation were selected, and emission
channel ranges were discriminated by wavelength-selective beam splitters,
high-pass, low-pass and notch interference filters, where appropriate.
On the basis of published wavelength spectra, two fluorescent dyes were
originally selected as suitable for the project: SNAFL-2
{Seminaphthofluorescein; 9-chloro-3,10-dihydroxy-spiro
[7H-benzo(c)xanthene-7,1'(3'H)-isobenzofuran]-3'-one} and
SNARF-1 {Seminaphthorodafluor;
10-dimethylamino-3-hydroxy-spiro[7H-benzo(c)xanthene-7,1'(3'H)-isobenzofuran]-3'-one}.
Differentiation between ionized and non-ionized forms of the indicator was
achieved for SNAFL-2 by dual-wavelength channel excitation by subsequent
selective exposure to laser lines at 488 nm (`extended blue' argon) and 543 nm
(helium-neon), respectively, selected by an AOTF
(acousto-optical-transmission-filter) wavelength-selective shutter system for
each 4 s. The resulting fluorescence was picked up in one common emission
channel at wavelengths >570 nm. Single-wavelength excitation of SNARF-1 by
an extended blue argon laser at 488 nm results in fluorescent emission picked
up simultaneously for the ionized dye form at wavelengths of 590-610 nm and
that of the non-ionized form at >630 nm. The ratio of fluorescent
activities is accordingly a function of pH at the indicator site (for details,
cf. Whitaker et al., 1991).
Ratiometric determination carries the advantage of being independent of the
absolute concentration of the dye as well as of variable optical density of
apparatus light path and biological matrix.
Optical stability of instrumentation
Short- and long-term stabilities of the optical system (including lasers,
light path components and detectors) were checked in a series of preliminary
experiments. Solutions of 2-5 µmol l-1 SNARF-1 or SNAFL-2 in 100
mmol l-1 phosphate buffer at constant pH (7.2) were exposed to
laser excitation in a temperature-controlled sample chamber, and the emissions
were monitored as a function of time every 5 min for 4.5-5 h. Emission
intensities were corrected for autofluorescence (essentially, photomultiplier
offset and noise) and normalized to the initial value. In order to
differentiate between instabilities brought about by variability of laser
intensity and changes of fluorescent dye characteristics by bleaching or local
aggregation of attached dextran, measurements were referenced to a fluorescent
uranium glass standard (type F53; Carl Zeiss), with its fluorescent quantum
efficiency being only affected according to the decay of radioactivity
(half-life
4.5x109 years).
Choice of indicator type
With dual-excitation, single-emission dyes like SNAFL-2, the differential
variability between two excitation sources (488 nm and 543 nm) is directly
transmitted into the emission intensity ratio. Long-term instability can be
corrected for by carrying along a fixed fluorescent reference (e.g. uranium
glass; see above), but in this specific case the long-term instability was
superposed by additional short-term variability (flutter) of excitation
intensities. Due to direct transmission of excitation flutter into four
non-simultaneous determinations of fluorescent emission, the successive
two-standard and two-sample excitations required for fixed standard
referencing will, in the worst case, duplicate the maximal error rather than
correcting short-term fluctuations.
Consequently, the present project was conducted utilizing the dual-emission dye SNARF-1. Simultaneous dual emission cancels instabilities of excitation sources and light path, but optical instabilities of the required two emission paths cannot be eliminated by any means. The magnitude of fluctuations in the emission path, however, was relatively small as compared with that originating in laser excitation.
The SNARF-1 fluorescence ratio (R) relating to absolute pH
calibration (see below) calculates:
![]() | (1) |
where F1sam and F2sam are sample fluorescence intensities at wavelengths 1 or 2, respectively, and AF is autofluorescence.
Calibration of the optical system
In biological fluids, dyes are subject to modification of their
physicochemical and optical characteristics due to the presence of proteins,
nucleotides and other binding and interfering matter. Accordingly, in
situ calibration is advantageous, as long as the prevailing pH is
precisely known.
Ionophore calibration
In situ calibration procedures are based on equalizing
intracellular with extracellular conditions by application of ionophores such
as nigericin (e.g. Thomas et al.,
1979), effecting a transmembrane K+/H+
exchange with relative selectivity
(Pressman, 1976
). However, in
multicellular/multilayer preparations, the expected effect is usually not
achieved, as indicated by preliminary experiments. In intact tissues and
organisms, the procedure is probably hampered by long diffusion pathways and
tightly interlaced cell layers, resulting in variable and unpredictable
intracellular pH and ionic concentration values (N. Heisler and J. Wasser,
unpublished; N. Heisler and N. Gonzalez, unpublished). As a further major
obstacle, organisms are frequently destroyed by application of nigericin and
the required extremely high extracellular [K+] (A. Mölich and
N. Heisler, unpublished).
In vitro calibration
In order to avoid obstacles and limitations of in situ ionophore
calibration, the effect of biological fluid components on physicochemical and
optical characteristics of SNARF-1 was mimicked in vitro. The optical
system was calibrated with SNARF-1 standards, modified by addition of either
100 mmol l-1 inorganic phosphate, 2.5, 5 or 10% bovine serum
albumin (BSA; Fraction V; Sigma-Aldrich, Deisenhofen, Germany) and/or 150 mmol
l-1 KCl. pH-adjusted, modified standards were used for in
vitro calibration of the optical system before determination of pH in
embryonic cells and interstitium. The quality of simulation was simultaneously
determined in the same cell or the interstitium by microelectrodes as an
independent reference (see below).
Although the behaviour of SNARF-1 in biological fluid compartments could well be approximated by an aqueous in vitro mixture of cellular components, mock fluids could not be used for calibration because of a pronounced instability of the protein-containing solutions. Accordingly, the optical technique was calibrated in vitro with 100 mmol l-1 phosphate buffers. The in vivo-induced property shifts of SNARF-1 were corrected for by mathematical post-processing, based on the relationship established from in vivo comparison of the in vitro phosphate-buffer-calibrated optical system with simultaneous pH-sensitive intracellular or interstitial microelectrode measurements at the same site.
pH-sensitive microelectrodes
pH-sensitive liquid ion exchanger microelectrodes (LIX) were built with
slight modification of methods described by Voipio et al.
(1994). In brief,
double-barrelled monofilament borosilicate glass tubing (type 2GC150FS7.5;
Clark Electromedical Instruments, Pangbourne, UK) was pulled to tip diameters
of
1 µm. After silanizing one barrel by exposing the tip to vapor of
n,n-dimethyltrimethylsilylamin (Fluka, NeuUlm, Germany), the
electrode was dry-bevelled to a point (tip diameter 2-5 µm) at an angle of
about 45°. The silanized barrel was backfilled with buffer solution (NaCl
100 mmol l-1, Hepes 50, NaOH 25, pH 7.8), and the tip filled with
H+-ionophore (Hydrogen Ionophore I-Cocktail B; Fluka) by gentle
suction. The unsilanized barrel was filled with 150 mmol l-1 KCl,
serving as a reference for the pH electrode. The two barrels were each
connected to high-impedance voltage followers (1015
; AD515A
operational amplifiers; Analog Devices, Norwood, MA, USA) by chlorinated
silver wires. The potentials of both electrode channels, referenced to an
extra-embryonic double agar-bridge Ag/AgCl electrode, were amplified
independently using high-stability laboratory isolation amplifiers
(custom-made; N. Heisler and H. Slama, unpublished) and were recorded by
computer-aided data acquisition (A/D-converter, DAS 1602; Keithley, Taunton,
MA, USA; software, Test Point 3.3; Capital Equipment Corp., Billerica, MA,
USA). The pH signal as the difference of the two channels was calibrated with
100 mmol l-1 phosphate buffers.
Analysis of optical data
Fluorescence intensities were scanned in matrices of 128x128
locations, with each location digitized with a resolution of 8 bit. The
biological projection area represented by each scan field is approximately
450x450 nm with the generally used 20x/0.40 Zeiss LD-Achroplan
lens. The obtained matrices of fluorescence intensities were stored as raster
images in tag-based image file format (TIFF; 8 bit intensity). The overall
sensitivity of the analog detection system was carefully adjusted by
photomultiplier analog gain and anodes high voltage to allow dynamic pH
changes without clipping intensities by exceeding the range of digitization (8
bit). According to the general range adjusted during experimentation (1
pH unit/8 bit) the theoretical resolution of the apparatus is
0.004 pH
units.
Intensity ratios were calculated for each individual scan field (Eqn 1).
Since dividing two groups of normally distributed data by each other results
in a logarithmic distribution, all subsequent averages were calculated from
log-transformed data. Average ratios accordingly represent true average pH of
the area utilized for calculation. The obtained raw average pH data were
referenced to the calibration data obtained by fluorescent measurement of
SNARF-1 in 100 mmol l-1 phosphate buffer of specified pH and were
corrected according to mathematical post-processing (pp) for the correlation
between optical phosphate buffer calibration and microelectrode response (Eqns
2, 3; see also above) for the intracellular space
(r2=0.978, N=213):
![]() | (2) |
and for the interstitial space (r2=0.879,
N=111):
![]() | (2) |
These relationships are independent of the optical equipment used and may be applied to similar biological approaches.
Calculations
Calculations based on the Henderson-Hasselbalch equation were conducted
using constants derived from the relationships of Heisler
(1984,
1986a
,
1989
), applying the
experimental temperature of 25°C and parameters for typical fish cell
fluid (molarity, 0.281 mol l-1; ionic strength, 0.220;
[Na+]=0.012 mol l-1; [protein]=200 g l-1) and
interstitial composition (molarity, 0.270 mol l-1; ionic
strength=0.140; [Na+]=0.130 mol l-1; [protein]=0 g
l-1). Bicarbonate concentrations were calculated from measured pH
and applied PCO2. According to short diffusion distances
and high CO2 diffusivity, intraembryonic PCO2
was assumed to be equal to ambient values. Apparent nonbicarbonate buffer
values (ßNB,app) were calculated as ratios of deflections of
pH and [HCO3-]
(Heisler, 1986a
). Maximal
deflections of measured pH after about 10 min indicated that equilibration to
the new PCO2 was safely complete after this time
period.
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Results |
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Partial compensation of this most disturbing phenomenon was possible by carrying along a uranium-glass standard, which reduced long-term drift of the detection ratio. Short-term variability could not be compensated and resulted in a variability of the fixed standard-corrected ratio of ±6% (Fig. 2B). This amount of noise is still three times as large as for measurement of SNARF-1 (±2%; single excitation, 488 nm) with dual-emission detection at 570-590 nm and >630 nm, without uranium glass standard (Fig. 2A).
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Acid-base parameters of embryonic zebrafish
Intracellular and interstitial pH in the studied early stages of embryonic
development during control conditions were maintained relatively constant at
7.55±0.13 and 8.08±0.25, respectively
( ±
S.D.). Non-respiratory changes of ambient pH induced by
varying ambient bicarbonate concentration did not affect cell pH, at least not
in the tested, slightly alkaline range of 7.5 to 8.5. By contrast, changes in
ambient PCO2 were transmitted immediately to the embryonic
fluid compartments, effecting rapid changes in pHi and
pHint (Figs 4,
5: different levels of
hypercapnia; Figs 6,
7: post hypercapnia). Upon
exposure to a new PCO2, maximal deflection of
pHi and pHint was attained after
10 min. In spite
of the extensive (10-fold) change in PCO2, the maximal
average pH deflection was limited to
0.25-0.65 units in intracellular and
0.35-0.9 units in interstitial fluid compartments, equivalent to apparent
ßNB,app values of 10-25 meq. pH-1 l-1
and 0-85 meq. pH-1 l-1, respectively. In the following
two hours, pH was returned towards the original value, with the majority of
the theoretical shift compensated by changes in compartmental
[HCO3-] (75-96%; cf.
Heisler, 1986c
) (Figs
4,
5,
6,
7).
[HCO3-] changed between 5.6- and 9-fold in intracellular
compartments and between 2.5- and 8.9-fold in the interstitial space. The
compensation process started to level off but was not complete after 2 h of
exposure to the new PCO2. At this time,
ßNB,app values were attained between 37 and 170 meq.
pH-1 l-1 for intracellular space and between 44 and
infinity for the interstitium.
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Development of zebrafish embryos in hypercapnic environments
Comparison of two groups of zebrafish developing under moderately
(PCO2=2.4 mmHg, 0.32 kPa) and severely
(PCO2=24.6 mmHg, 3.3 kPa) hypercapnic conditions with
control, unmanipulated animals at normocapnia revealed no significant
differences in ontogenetic development during the observation period of 24 h,
in spite of largely different environmental pH (7.93 and 6.92 for
PCO2 of 2.4 and 24.6 mmHg, respectively). The observed
mortality of 50% during the 24 h monitoring period was higher than during
normal undisturbed growth but was the same in both observation groups. Reduced
survival in this experimental series has to be attributed to extensive and
repeated handling and agitation in the aeration chamber of sensitive and
fragile embryos deprived of their natural protective cover by dechorionation.
Although transition of the surviving embryos to specific stages was slightly
delayed (
15%) compared with standard charts (corrected for 25°C;
Kimmel et al., 1995
), the
pattern of developmental transitions was identical to control groups of
animals undisturbed by any experimental manipulation. Also, deviations from
the standard morphology of embryos were not found at any time.
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Discussion |
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Methodological resolution as a function of optical stability
One of the most critical factors for optical ion analysis is constancy of
used instrumentation. However, fluorescence intensities and, in particular,
laser excitation intensities measured in the course of the present study under
highly controlled conditions (advanced optical bench, decoupled from any
vibrational disturbance and floating on a gas-suspended large mass base) were
extremely unstable, an amazing fact in face of the wide use and the popularity
of confocal laser scanning microfluorometry for quantitative investigations.
Comparative tests on five confocal laser scanning microscopes (four different
models of two brands) resulted in similar instabilities of signal intensities
(A. Mölich and N. Heisler, unpublished), suggesting that the extensive
light processing and the stabilization of laser intensities in confocal
microscopes are still grave common problems. Most astonishingly, no report
concerning this evidently major methodological limitation was found in the
literature.
The obvious deficiency of instrumentation and the resulting error of up to 80% becomes particularly apparent during dual excitation but is partially corrected for by carrying along a fluorescent uranium glass standard with each determination, thus reducing the extreme long term instability to approximately ± 6%. The residual variability is related to equivalent short-term laser intensity variation, picked up due to the time delay between four required emission measurements (for each of two excitations at two different wavelengths for sample and standard). Thus, with present instrumentation, single-excitation/single-emission as well as dual-excitation/single-emission dyes are deemed unsuitable to provide the pH resolution required for scientific research.
Dual-emission measurement upon single excitation will cancel out any instabilities related to the excitation path, including laser and further optical equipment like AOTF, lenses, splitters, filters, etc. Accordingly, ratios obtained in the course of the present study were improved considerably with the dual-emission dye SNARF-1 as compared with dual-excitation measurements. To our surprise, however, even simultaneous recording with two emission detectors, eliminating the influence of any laser variability, did not reduce the obtained ratio noise to the low levels expected from passive optical components on an advanced, gas-floating optical bench. The residual noise is probably attributable to mechanical instabilities between optical components of the emission light paths. Unfortunately, these variabilities reduced resolution of the method far below the possible limit. The observed maximal variation of the emission ratio (±2%) is equivalent to ±0.020 pH units as compared with the theoretical resolution of 0.004 units, the limit by 8-bit digitization.
Although the available instrumentation provides relatively coarse
restrictions as compared with precision electrode analyses (<0.001;
Heisler, 1978), the present
study could not have been conducted with anything other than optical
techniques. Repeated sampling in a developing organism is clearly impossible
with indicator distribution techniques. Microelectrodes, used to calibrate the
optical techniques in absolute terms, could not be kept in dividing cells of a
growing embryo for more than 30-40 min, definitely not for the 8 h of a full
experimental run. Also, microelectrodes could not be produced that were stable
enough to be maintained for measurement at a site in a high-protein
environment for such a long time without recalibration.
Calibration of optical methods
One major problem in applying microfluorometric techniques is calibration
of the indicator emission signal. The interaction of dyes with numerous
biological modulators at the site of measurement renders in situ
calibration with ionophores like nigericin an advantageous procedure, which is
actually widely used in isolated culture cells. In excised tissues and
multicellular organisms, however, nigericin application is hampered by many
factors. In embryos, integrity and viability is impaired by nigericin, and
application often results in complete disintegration of the organism, related
to loss of adequate membrane potential, pHi or other cell
functions. Further problems are provided by the necessity to achieve a defined
nigericin concentration at the site of calibration, in order to equalize pH
and ionic concentrations at the studied interface. In intact organisms or
tissue chunks, diffusion of nigericin is inhibited by long diffusion paths or
interlaced epithelia. Applying the technique to embryos, circumventing the
precursors of epithelial cells by direct injection of nigericin is most
difficult, if not impossible. Also, the imperatively required measurement of
`extracellular' pH is hard to conduct before an adequately large defined
extracellular volume has developed during ontogeny. A major drawback of
nigericin is contamination of experimental equipment, hard to get rid of and
largely affecting consecutive experiments
(Richmond and Vaughan-Jones,
1993).
The accuracy of nigericin calibration, i.e. equalization of ionic
composition between two fluid compartments, is not comparable with other
techniques applied in acid-base physiology (cf.
Heisler, 1989). Even in single
culture cells, pHi differences of 0.15
(Nett and Deitmer, 1996
) or
0.06-0.11 (Chaillet and Boron,
1985
) have been reported in comparison with microelectrodes,
differences larger than the maximal error after in vitro calibration
with mock intracellular fluid (0.07; for calibration with 10% BSA + 150 mmol
l-1 KCl; see above and Fig.
3). Smaller differences observed in comparison with indicator
distribution techniques (0.03, Pärt
and Wood, 1996
; 0.05, Thomas
et al., 1979
) are biased by the inherent alkaline shift error of
such techniques (cf. Heisler,
1989
; Hinke and Menard,
1978
). For multicellular/multilayer organisms or preparations,
literature data of direct comparison of nigericin with microelectrodes are not
available.
As a consequence, for this study the optical system was calibrated in vitro with standardized 100 mmol l-1 phosphate buffers, and the data obtained from in vivo measurements were adjusted by mathematical post-processing based on in vivo simultaneous, at-site comparison with microelectrodes. After having established the correlation between the two techniques, this type of calibration is much easier, while providing much higher accuracy than any other approach. Since specific characteristics of the optical light paths of used instrumentation are accounted for by the procedure, the obtained relationships (cf. Eqns 2 and 3) can be extrapolated to other systems similar in their intracellular composition. This essentially applies to vertebrate cells and interstitial fluid, respectively, with similar [protein], [K+], ionic strength, etc., and SNARF-1 measurements with single excitation (488 nm) and dual emission (570-590 nm and >630 nm).
This approach of directly correlating microelectrode readout with optical
data carries the advantage of eliminating any differences between the two
techniques. Microelectrodes are considered as a most reliable basic reference,
in spite of the fact that electrodes may certainly be subject to spurious
bridge potentials, cross sensitivities and effects of media composition (e.g.
Ammann et al., 1981;
Siggaard-Andersen, 1961
). The
microelectrode assemblies utilized in the present study were tested thoroughly
before use but did not indicate any cross-sensitivities such as to variable
CO2 and [HCO3-], high [K+], high
BSA concentration, ionic strength and other factors in the physiological pH
range. The observed typical non-linear response and media sensitivity of
individual electrodes below pH 6 (Ammann et
al., 1981
) is irrelevant on the basis of the utilized pH range
between 7.0 and 8.2. Referencing the used microelectrodes with NBS (National
Bureau of Standards) phosphate buffer solutions with an accuracy of
±0.005, the directly correlated optical techniques are considered to
provide comparable accuracy. The repeatability of the approach was determined
from time series of standard measurements to be ±0.020 (maximal
deviation), with this limitation compared with the theoretical resolution by
8-bit digitization of 0.004 mainly attributable to instabilities of optical
apparatus.
Acid-base regulation in embryonic zebrafish
Intracellular pH is one of the key parameters for modulation of many
cellular functions, including general cellular metabolism via enzyme
activities, cell aggregation and cytoskeleton formation (for a review, see
Busa and Nuccitelli, 1984;
Putnam and Roos, 1997
). Thus,
development of pH regulation represents an important step of ontogeny. Under
control steady-state conditions, pHi is apparently adjusted to a
fairly constant setpoint value (7.55±0.13). The transmembrane pH
difference between intracellular space of not yet differentiated embryonic
cells and the interstitial space (8.08±0.25) of about 0.5 units at
25°C is comparable with the conditions in muscle tissues of adult fish
(
0.6; Heisler, 1986b
,
1993
). However, the absolute
level of pHi is higher by 0.3-0.4 than in adult fish (general range
in muscle, with few exceptions, 7.05-7.30 at 25°C;
Heisler, 1986b
), leading to
more than twice as high embryonic [HCO3-] for the same
PCO2. Thus, for a given effect in pH, twice as much
bicarbonate has to be transferred across membranes and `epithelia'. On the
other hand, a higher level of bicarbonate provides a higher bicarbonate buffer
value (Heisler, 1986a
,
1989
), which may become useful
under conditions of transient anoxia with production of organic acids
dissociating H+ ions.
Regulation of pH is generally based on passive distribution of
HCO3- on the basis of the membrane potential, eventually
supplemented by active transfer processes for final adjustment deviating from
passive distribution (cf. Heisler,
1986a). At the observed levels, adjustment of embryonic
pHi is impossible on the basis of passive mechanisms alone. The
equilibrium potential for HCO3- transmembrane
distribution in steady-state embryos, calculated from the Nernstian
relationship, varies between -20 and -40 mV (Figs
4,
5). This is significantly
different from typical membrane potentials of fish embryonic cells (-60 to -70
mV, Bregestovski et al., 1992
;
-71 mV, Buss and Drapeau,
2000
). These relationships strongly suggest an energy-demanding
process for active distribution of bicarbonate.
While active transport is definitely involved in pHi adjustment,
the regulatory capacity of the process is difficult to evaluate. During steady
state, the required activity depends largely on the rate of
HCO3- leakage (or of other acid-base relevant ions)
across the membrane. Thus, with tight compartmental interfaces, the active
process may suffice regulatory demands in spite of an extremely low capacity.
This also holds for the apparent independence of cellular pH during metabolic
(non-respiratory) challenges of ambient pH found in the present study, results
confirmed in a variety of fish embryos (e.g. Johansson et al.,
1973,
1977
).
Direct challenge of cellular and interstitial pH regulation by changes of
ambient PCO2 is readily transmitted to the intracellular
fluid compartments, independent of ionic permeation characteristics of
membranes. The maximal deflection of pH after changes in
PCO2 may be taken as an expression of available
non-bicarbonate buffering in the respective compartment, but only with caution
(ßNB,app; cf. Heisler,
1986a). Values obtained in cells at this stage (10-25 meq.
pH-1 l-1 cell H2O) are lower than chemical
nonbicarbonate buffer values of adult fish specimens (33-49 meq.
pH-1 l-1 cell H2O; cf.
Heisler, 1986a
). Deviations
between embryos and adults are probably attributable to exchange of
HCO3- equivalents with the environment as an expression
of not yet adjusted thresholds at membrane and `epithelial' levels, as was
found in Scyliorhinus (Heisler et
al., 1976
). Interstitial ßNB,app values at maximal
pH deflection are generally much higher than the expected rather low chemical
buffering ability of this fluid space (cf.
Heisler, 1986a
) and are
definitely an expression of bicarbonate originating from other fluid
compartments such as cells, yolk and/or surrounding embryonic fluid. During
the following
2 h, large changes in bicarbonate in both intracellular as
well as interstitial spaces indicate transfer of acid-base relevant ions for
compensation of these two fluid compartments from the ambient fluid as the
only available significant source
(Heisler, 1984
). The extremely
small interstitial space (estimated to be 5-8% of the organism at this
ontogenetic state) functions as a transit fluid volume rather than providing
bicarbonate for the compensation of the intracellular space. As a result of
bicarbonate accumulation, the ßNB,app after 2 h is largely
elevated in both studied compartments.
Net transfer of bicarbonate from interstitial to intracellular space is definitely an active process for the two series of PCO2 elevation (0.73 to 7.4 and 2.4 to 24.6 mmHg). The equilibrium potential of bicarbonate shifts during high PCO2 from the control range of -30 to -40 mV closer to zero, thus even enhancing the electrochemical force against compensatory cellular influx of HCO3-. Accordingly, the kinetics for bicarbonate accumulation during hypercapnia are slower than the release of bicarbonate equivalents from the cells after return to low-level PCO2s (cf. Figs 4 and 5 vs Figs 6 and 7). In these post-hypercapnia series, the equilibrium potential returns towards the -30 to -40 mV steady-state range, which still provides considerable electrochemical force for compensatory efflux of bicarbonate from the cells. It remains questionable, however, whether the high rate of bicarbonate-equivalent efflux can take place on the basis of passive diffusion alone. More likely, the efflux is supplemented by an active transfer enhancing the kinetics of pH normalization. The finally attained steady-state distribution is definitely maintained by active processes anyway (see above).
Also, comparison of bicarbonate distribution potentials between
interstitium and ambient fluid with actual data yields evidence as to the
nature of involved transfer mechanisms. Potentials in adult freshwater fish
are found in the range of slightly negative to slightly positive values (-5 to
+10 mV, inside to outside), depending upon a variety of factors, such as
[Na+], [Cl-] and, in particular, [Ca2+]
(Eddy, 1975;
Kerstetter et al., 1970
;
Kerstetter and Kirschner,
1972
). The HCO3- equilibrium potentials at
low-level PCO2 calculated for the present study are close
to this range. During changes in PCO2, however, the
equilibrium potential is largely shifted to attain values between -20 and +55
mV. Differential diffusion of Na+ and Cl-, responsible
for the interstitial/ambient potential, is largely affected by the presence of
Ca2+ but is relatively insensitive to changes in
PCO2 and [HCO3-]. The
transepithelial potential in goldfish is shifted by only 5 mV upon 100-fold
changes of the bicarbonate distribution ratio
(Eddy, 1975
). These
interrelationships strongly suggest that HCO3- is
distributed between embryonic interstitium and ambient fluid by active
processes located in the outer cell layer (structurally not yet `epithelium')
of the embryo.
Quantification of acid-base relevant ion fluxes between cells, interstitium
and ambient water is difficult on the basis of the present approach. The
observed kinetics of changes in intracellular [HCO3-]
and pH, however, suggest much higher transfer rates than typically found in
adult fish exposed to hypercapnia (Heisler,
1982,
1984
,
1986b
,
1993
). This may, at least
partially, be related to relatively high [Na+],
[HCO3-] and [Ca2+] in the ambient fluid
(embryonic medium; in mmol l-1: [Na+] 19,
[Ca2+] 1.3, [HCO3-] 5), supporting acid-base
relevant ion exchange (cf. Heisler,
1999
), but transfer of bicarbonate equivalents to the
intracellular compartment will also be advanced by the small capacity and
accordingly quick compensation of the interstitial space, supporting cellular
adjustments.
Evidently, the stress of hypercapnia is suitable to activate membrane ion
transfer processes in embryos, but, although the observed rate is high as
compared with adult fish, it may not represent the full capacity of
mechanisms. As is well known from similar experiments in adult fish, the rate
of ion transfer processes largely depends on the nature of stress (e.g.
temperature changes vs severe lactacidosis; Heisler,
1978,
2004
;
Holeton et al., 1983
),
suggesting graded reaction to different stimuli.
Abrupt changes of intracellular pH are initiated in oocytes by sperm
fusion. Data in zebrafish are unavailable, but a few literature reports
indicate pHi alkaline shifts of 0.2-0.3 in Xenopus embryos
(e.g. Webb and Nuccitelli,
1981), to attain a much higher range of 7.6-7.7 than found in
adult specimens (7.0-7.1; e.g. Boutilier et
al., 1987
). Even slightly larger shifts of 0.3-0.4 units are found
in sea urchin and clam oocytes (e.g.
Johnson and Epel, 1981
;
Dubé and Eckberg,
1997
). The alkalinization upon sperm fusion is considered to
follow activation of a Na+/H+ exchanger in these species
(Dubé and Eckberg,
1997
; Gusev,
2001
). During further embryonic development, the
Na+/H+ exchanger, as well as HCO3-
related transporters, is known to be modulated by a variety of growth factors
(Moolenaar, 1986
;
Boron and Boulpaep, 1989
).
Although pHi is a proven factor of large importance in energy
metabolism, in embryos the effect of shifts in pH on further development is
diverse, the range spanning from hardly any effect in Xenopus (e.g.
Stith and Maller, 1985), an
optimized protein synthesis at elevated pH in sea urchins
(Rees et al., 1995
) to a
pronounced correlation of development with pHi in echiuroids
(Gould and Stephano, 1993
). In
zebrafish, general ontogeny was not affected at all by a wide range of
hypercapnia and the associated changes in pH. Evidently, numerous factors are
involved in this complex pattern of regulation, and their interaction has to
remain the subject of further experimentation.
Conclusions
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Acknowledgments |
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References |
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