Biology Reference
In-Depth Information
ionophore is actually engaged in Ca
2
þ
binding and transport. This problem
becomes evident when either ionophore is used in calibrating Ca
2
þ
indicators in
cells (see
Section V.B
).
The most significant di
V
erence between the two ionophores lies in the pH
dependence of their ability to transport Ca
2
þ
(
Liu and Hermann, 1978
). Transport
of Ca
2
þ
by Br-A23187 approaches a maximum at pH 7.5, whereas Ca
2
þ
transport
by ionomycin does not reach a maximum until pH 9.5. The pH at which half-
maximal transport is achieved is
8.2 for ionomycin.
Therefore, if one desires to increase transport of extracellular Ca
2
þ
into cells in
acidic media (pH
6.4 for Br-A23187 and
<
7.0), Br-A23187 is a much better choice than ionomycin.
2. Using Br-A23187 and Ionomycin
Ionomycin can be obtained as either the free acid or the Ca
2
þ
salt. Br-A23187 is
available as the free acid. All forms are soluble in dry DMSO, which can be used to
prepare stock solutions. Because these ionophores are very hydrophobic, they are
bound avidly by serum proteins. Serum proteins such as BSA, when present in the
medium, greatly reduce the e
ectiveness of ionophores and, if possible, should be
left out of the experimental medium when ionophores are to be used. Otherwise,
much higher concentrations of ionophore must be used. Br-A23187 and ionomycin
have been used at concentrations ranging from 10
7
to 10
5
M. In addition to
increasing Ca
2
þ
flux across the plasma membrane, Br-A23187 and ionomycin also
transport Ca
2
þ
out of intracellular calcium stores into the cytosol. Therefore, in
the presence of these ionophores, intracellular calcium stores are rapidly depleted
(
Kao et al., 1990
).
V
ering Changes in Intracellular [Ca
2
þ
]
1. Increasing Intracellular Ca
2
þ
Bu
V
ering Capacity by BAPTA Loading
When a change in [Ca
2
þ
]
i
(a Ca
2
þ
signal) is correlated with a biological process,
one can ascertain whether the Ca
2
þ
signal is essential in the process by blocking the
change in [Ca
2
þ
]
i
with a calcium chelator. By far the easiest way to introduce extra
Ca
2
þ
bu
D. Bu
V
ering capacity into cells is by incubation with BAPTA AM in Pluronic
dispersion. Compared with the AM esters of common Ca
2
þ
indicators, BAPTA
AM has much higher aqueous solubility—15
m
Mat25
C(
Kao et al., 1990
).
Therefore, BAPTA can be loaded e
V
ciently into cells via the AM ester. BAPTA
AM is loaded into cells in precisely the same way that AM esters of indicators are
loaded. Cells can be loaded with AM esters of BAPTA and an indicator simulta-
neously.
Figure 9
illustrates the e
Y
ect of intracellular BAPTA loading on normal
changes in [Ca
2
þ
]
i
.
Figure 9
A shows the changes in [Ca
2
þ
]
i
in a REF52 cell loaded
with Fura-2 in response to sequential application of 1
m
M bradykinin and 1
m
M
Br-A23187.
Figure 9
B shows the responses in a similar cell loaded with Fura-2 and
BAPTA. These results clearly demonstrate that the presence of su
V
cient BAPTA
practically eliminates the rapid and transient rises in [Ca
2
þ
]
i
elicited by an agonist.
Y
