Biology Reference
In-Depth Information
cellular environments. Moreover, about the time that indo-1 and fura-2 were
synthesized (
Grynkiewicz et al., 1985
), a Ca
2
þ
-ligand of improved selectivity
(ETH 129) was introduced (
Schefer et al., 1986
), which made it more realistic to
measure low Ca
2
þ
levels typical of intracellular environments, since the electrode
response was Nernstian until pCa 8-9 (depending on the ionic background).
However, the availability and popularity of the fluorescent indicators did to a
large extent eclipse and limit further use and characterization of ETH 129-based
electrodes.
II. Rationale
We will not present an extensive review of the advantages and disadvantages of
Ca
2
þ
-selective electrodes compared to fluorescent indicators, but the purpose of
this chapter is rather to show how the advantages of both approaches can be
combined for specific purposes. For example, a classical problem with Ca
2
þ
indicators is to convert the fluorescent signal to actual [Ca
2
þ
]. In fact, it is
commonly acknowledged that in vitro calibration curves of fluorescent calcium
indicators are not applicable to in vivo or in situ conditions, because the indicators
bind to intracellular constituents, which modifies their excitation/emission spec-
trum and decreases the apparent a
nity of Ca
2
þ
for the probe (
Blatter and Wier,
1992; Harkins et al., 1993; Hove-Madsen and Bers, 1992; Konishi et al., 1988
).
In vivo calibration curves are even more di
Y
cult to obtain and are highly depen-
dent on the cell type under study. Ca
2
þ
-selective electrodes are still one of the most
straightforward ways to measure and quantify Ca
2
þ
because (1) their behavior is
not appreciably altered by the intracellular milieu (ions and proteins); (2) they can
be easily included in an electrophysiological setup and do not require extensive
and expensive apparatus; (3) their linear (Nernstian) response simplifies the con-
version of the voltage signal to [Ca
2
þ
]
i
; (4) their behavior allows determination
of wide ranges of pCa (see above); and (5) the Ca
2
þ
ligand itself does not change
(or bu
Y
er) [Ca
2
þ
]
i
.
Thus, Ca
2
þ
-selective electrodes continue to be a good choice for the preparation
of calibration solutions for Ca
2
þ
determinations and for measurements of dissoci-
ation constants for Ca
2
þ
-binding compounds such as ethylene glycol bis(
b
-amino
ethyl ether)-N,N,N
0
,N
0
-tetraacetic acid (EGTA), indo-1, 1,2-bis(o-aminophenoxy)
ethane-N,N,N
0
,N
0
-tetraacetic acid (BAPTA), and oxalate under di
V
erent experi-
mental conditions (
Bers, 1982; Harrison and Bers, 1987, 1989; Hove-Madsen and
Bers, 1992, 1993a; Hove-Madsen et al., 1998
) Indeed, commercially available Ca
2
þ
electrodes are largely directed towards determination of the calcium concentration
in solutions or biological fluids, but Ca
2
þ
electrodes can also be used to measure
cellular Ca
2
þ
bu
V
ering and changes in the free [Ca
2
þ
] in cell suspensions (
Hove-
Madsen and Bers, 1993a, 1993b; Hove-Madsen et al., 1998
) or combined with
other electrophysiological techniques (
Kang and Hilgemann, 2004; Kang et al.,
2003
). Moreover, Ca
2
þ
electrodes are economical, easy to prepare, and they can
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