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The porphyrin handbook - Kadish K.M.

Kadish K.M. The porphyrin handbook - Academic press, 2000. - 368 p.
Download (direct link): kadishsmishgulilard2000.djvu
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into a constant volume of freshly boiled and deoxygenated buffer (pH 7.4
at 37 C).82 The current generated after each addition of NO is measured,
and the resulting plot of current versus concentration should be linear.
The detection limit of a correctly prepared sensor should be about 10 nM
for a single-fiber sensor, and 1 to 5 nM for multifiber catheter-
protected sensors for in vivo measurement. The detection limit of the
cell-culture macrosensor is 5 nM. The response time (time for signal
increase from 10-75%) of porphyrinic sensors is
0.1 ms for micromolar NO concentrations, and 10 ms for nanomolar
concentrations.
5. Single-Cell Measurements of Nitric Oxide
By use of a manual or motorized computer-controlled micromanipulator with
0.2 | x-y-z-resolution, the porphyrinic sensor can be implanted into a
single cell, or placed on the surface of the cell membrane, or kept at a
controlled distance (0.2-10 |) from a NO-generating cell. -(,-4<>--85
When the tip of the sensor touches the cell membrane, a transient small
electrical noise is observed. This is a good indicator of zero distance
from the cell, and from this point the sensor can be moved out from the
surface by 0.2 mm increments controlled by computer. Injection of NO
agonists can be done with micro-, nano-, or femto-injectors. Injection of
larger volumes of agonists with a microinjector will cause a "jet effect"
and the first release of NO will be due to shear stress. The shear stress
peak of NO will be followed by a NO peak resulting from the effect of the
NO agonist. The time resolution of these two peaks can be small,
therefore, they may be observed only with a high-
44/Porphyrin-Based Electrochemical Sensors
247
a)
b)
time, s
Figure 13. Amperogram of NO recorded with porphyrinic sensor (diameter 1
) placed 5 2 from the surface of an isolated single endothelial
cell (a) and endothelial cell in cell culture (b). NO release was
stimulated from rabbit aorta endothelial cells with calcium ionophore.
speed recording device (e.g., computer-controlled acquisition system);
with chart recorders these two peaks will be indistinguishable. Injection
of the agonist with a nano- or femto-injector will reduce the jet effect
and subsequent NO release due to shear stress.
Figure 13 shows the pattern of NO release (amperogram) from a single
endothelial cell isolated from the culture, and the pattern of NO release
from a single cell still in the culture. The sensor was placed in close
proximity to the cell membrane (2 0.5 ). This figure shows that the
peak concentration of NO on the membrane of a single cell isolated from a
given endothelial cell culture will be the same as the peak concentration
of NO measured on the membrane of a single cell in the same culture.
However, the duration of the plateau is significantly extended when
measurements are done in cell culture. This difference is due to the
horizontal gradient in NO concentrations, which is much smaller in cell
culture. The depletion of NO from the membrane is slower when the cell is
surrounded by other cells that release NO in culture. In a single
isolated cell, the gradient of NO concentration between the surface and
bulk solution is high all around the cell; therefore, the process of NO
depletion is relatively rapid.
The height of the peak of NO release depends on the distance from a
membrane surface (Figure 14). The highest NO concentration is observed on
the cell membrane (650 30 nM), with the concentration decreasing
exponentially with the distance from the cell membrane. At about 50
from the cell membrane, the NO concentration was 15 nM (single isolated
cell) and at a distance greater than 50 from the cell membrane, NO is
not detectable by the porphyrinic sensor. The decrease of NO
concentration from the cell surface is not so rapid when the cell is
surrounded by another cell from the cell culture. However, at a distance
greater than 150 from the cell membrane (in cell culture), NO is also
not detectable by the porphyrinic sensor.
From an analytical standpoint, the detection of NO at the site of
highest concentration, the surface of the endothelial cell membrane, is
the most convenient and accurate method for measurement of endogenous NO.
Due to the hydro-
distance,
Figure 14. Exponential decrease of NO concentration with increasing
distance of porphyrinic sensor from the membrane of single isolated
endothelial cell (dashed line) and single endothelial cell in cell
culture (solid line). Rabbit aorta endothelial cells were stimulated with
calcium ionophore.
a)
b)
NO
200 nmol/L

time, s
Figure 15. Typical amperograms showing changes of NO concentration near
the surface of the endothelium of aorta of normotensive (WKY rat) (a) and
spontaneously hypertensive rats (SHR rat) (b). NO was stimulated with
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