Biology Reference
In-Depth Information
producing a modest but quantifiable step-like rise in response to a partially
photolyzing light flash, or a gradually increasing [Ca
2
þ
]
i
during exposure to steady
light. Subsequent flashes cause further increments in [Ca
2
þ
]
i
. These increments
actually increase because, with each successive flash, the remaining unphotolyzed
chelator is loaded more heavily with Ca
2
þ
. Eventually, unphotolyzed nitr-5 or
azid-1 is fully Ca
2
þ
-bound, and subsequent flashes elevate Ca
2
þ
by smaller incre-
ments as the amount of unphotolyzed chelator drops.
If a calibrated light source is used that photolyzes a known fraction of nitr in the
light path, or in cells filled with chelator and exposed either fully or partially to
light, then the mixture of unphotolyzed nitr and photoproducts may be calculated
with each flash (
Land ` and Zucker, 1994; Lea and Ashley, 1990
). The di
V
erent
ciencies of free and Ca
2
þ
-bound chelators must be taken into account.
Simultaneous solution of the bu
quantum e
Y
V
er equations for photolyzed and unphotolyzed
chelators and native Ca
2
þ
bu
ers predicts the [Ca
2
þ
]
i
. For su
V
Y
ciently high nitr-5
concentration (above 5 mM), the native bu
ect and usually may
be ignored in the calculation. Further, since [Ca
2
þ
]
i
depends on the proportion of
chelator loaded with Ca
2
þ
, the exact chelator concentration in the cell makes little
di
V
ers have little e
V
erence, at least in small cells or cell processes.
If the cell is large, the light intensity will drop as it passes from the front to the
rear of the cell. Knowing the absorbance of cytoplasm and chelator species at
360 nm, and the chelator concentration before a flash, the light intensity and
photolysis rate at any point in the cytoplasm may be calculated. A complication
in this calculation is that nitr-5 photoproducts have very high absorbance (Ca
2
þ
-
free photoproduct, 24,000 M
1
cm
1
and Ca
2
þ
-bound photoproduct,
10,000 M
1
cm
l
)(
Adams et al., 1988
). As photolysis proceeds, the cell darkens
and photolysis e
V
Y
ciency is reduced by self-screening. For azid-1 the situation is
reversed: its photoproducts have much lower absorbance (11,500 and
5000 M
1
cm
1
) for Ca
2
þ
-bound and free species or 1/3 and 1/4 of the respective
unphotolyzed forms. Thus light penetrates more deeply as photolysis proceeds.
Regardless, with estimation of the spatial distribution of light intensity from Beer's
Law, the spatial concentrations of photolyzed and unphotolyzed chelator can be
computed; from this calculation follows the distribution of the rise in [Ca
2
þ
]
i
. The
subsequent spatial equilibration of [Ca
2
þ
]
i
can be calculated by solving di
usion
equations, often in only one dimension, using the initial [Ca
2
þ
]
i
and chelator
distributions as the boundary conditions. E
V
ers, uptake,
and extrusion mechanisms on the rise in [Ca
2
þ
]
i
can be incorporated into the
calculations. Simulations of the temporal and spatial distribution of [Ca
2
þ
]
i
have
been devised (
Zucker, 1989
) and applied to experimental data on physiological
e
V
ects of endogenous bu
V
ects of [Ca
2
þ
]
i
; the predicted changes in [Ca
2
þ
]
i
have been confirmed with Ca
2
þ
-
sensitive dyes (
Land ` and Zucker, 1989
). Simplified and approximate models using
the volume-average light intensity to calculate volume-average photolysis rate and
average [Ca
2
þ
]
i
changes often su
V
ce when the spatial distribution of [Ca
2
þ
]
i
is not
important, for example, in small cells or processes or when estimating the change in
[Ca
2
þ
]
i
in a cell after di
Y
V
usional equilibration has occurred.
