Biology Reference
In-Depth Information
When Ca
2
þ
electrodes cannot be practically used, one may still be able to use
optical indicators such as the fluorescent indicators fura-2, indo-1, Fluo-4, Fluo-
5N for selected [Ca
2
þ
] ranges, or the metallochromic dyes antipyralazo III, mur-
exide, or tetramethylmurexide for higher free [Ca
2
þ
](K
d
200
m
M, 3.6, and
2.8 mM, respectively,
Ohnishi, 1978, 1979; Scarpa et al., 1978
). Of course, these
indicators require calibration too.
A general potential complication with Ca
2
þ
bu
ers is that they may alter the very
processes one is interested in studying with Ca
2
þ
bu
V
ers. For example, EGTA and
other Ca
2
þ
-chelators have been documented to increase the Ca
2
þ
sensitivity of the
plasmalemmal and SR Ca
2
þ
-ATPase pumps and also of Na
þ
/Ca
2
þ
exchange
(
Berman, 1982; Sarkadi et al., 1979; Schatzmann, 1973; Trosper and Philipson,
1984
). For example, 48
m
MEGTAdecreased the apparent K
Ca
of Na
þ
/Ca
2
þ
exchange
in cardiac sarcolemmal vesicles from 20 to 5
m
MCa
2
þ
(
Trosper and Philipson, 1984
).
These points above are not meant to discourage one from using Ca
2
þ
bu
V
ers,
but simply to point out some of the potential problems that one might encounter.
Being aware of what might occur can help troubleshoot, when things do not make
sense. Clearly, the use of Ca
2
þ
bu
V
er solutions is essential for the understanding of
Ca
2
þ
-dependent phenomena. Our aim here is to provide helpful information.
V
C. Software Programs
While the above spreadsheet is useful for very simple Ca-EGTA or Ca-BAPTA
solutions used for calibrations, it is not su
ers that one
typically uses experimentally (which include Mg
2
þ
in addition to Ca
2
þ
and multiple
anionic species like ATP that bind Ca
2
þ
and Mg
2
þ
). Several computer programs
have been described (
Bers et al., 1994; Brooks and Storey, 1992; Fabiato, 1988;
McGuigan et al., 1991; Schoenmakers et al.., 1992; Taylor et al., 1992
), but we will
focus on, MaxChelator developed by one of the authors (CWP Bers et al., 1994). We
have seen above that care is needed in using Ca
2
þ
electrodes. This is equally true for
any software used to determine free metal concentrations in the presence of chela-
tors. In both cases, careful measurement of environmental conditions is needed:
temperature, pH, and ionic strength, as well as attention to the quality and accuracy
of measurement of all reagents. In addition, software is a
Y
cient for more complex bu
V
ected by the choice of
stability constants, quality of the code, and the particular algorithms used, and of
course, the understanding of those using the software (being dependent on personal
knowledge and the ease of use of the software).
Fabiato and Fabiato (1979)
broke ground for average users by publishing their
paper on using a hand held programmable calculator to determine free [Ca
2
þ
]or
[Mg
2
þ
] in the presence of EGTA. Before then complicated and user unfriendly
software running on main frames and mini computers was all that was available.
Use of Ca
2
þ
electrodes was also just starting and not easy for most labs to
implement. The Fabiato code opened this door, but was somewhat limited.
Richard Steinhardt's lab used the Fabiato paper to write a version for the Apple
2e, and one of us (CWP) further developed this to a program known as the
V
