Biology Reference
In-Depth Information
Purities of 80-90% are typical for commercial samples of all the chelators, but
occasional batches of 60% purity or less have been seen; these also sometimes show
high degrees of toxicity. Whether such low purity is the result of poor synthesis or
storage is unclear. Nitr compounds decompose detectably after only 1 day at room
temperature, and exposure to ambient fluorescent lighting for 1 day causes detect-
able photolysis. Chelators should be shipped on dry ice and stored at
80
C in the
dark; even under these conditions they do not last forever. Repeated thawing and
freezing also degrades the compounds.
Some of the photolabile Ca
2
þ
chelators display a degree of biological toxicity in
some preparations. Commercial samples of nitr-5 have been seen to lyse sea urchin
eggs (R. S. Zucker and L. F. Ja
e, unpublished results) and leech blastomeres
(K. R. Delaney and B. Nelson, unpublished results) within minutes.
Zucker and
Haydon (1988)
found that nitr-5 blocked transmitter release within 10 min of
perfusion in snail neurons, whereas DM-nitrophen has no similar e
V
V
ect
(P. Haydon, unpublished results). These e
ects are not caused by the photoproducts,
since photolysis is not necessary for the problems to occur. DM-nitrophen has been
observed to reduce secretion in chroma
V
n cells; higher chelator concentrations,
photolyzed to give the same final [Ca
2
þ
]
i
level, caused less secretion (C. Heinemann
and E. Neher, unpublished results). The e
Y
ect was overcome partially by inclusion of
glutathione in the perfusion solution, as reported for the photoproducts of other
2-nitrobenzhydrol-based caged compounds (
Kaplan et al.,1978
). These signs
of toxicity have been observed sporadically; whether they are properties of the
chelators themselves or of impurities in the samples used is unclear. The chelators
have been applied successfully to a wide range of preparations without obvious
deleterious results, although subtle e
V
V
ects may have been missed.
IX. Biological Applications
A brief synopsis of the earliest biological applications of the caged Ca
2
þ
chela-
tors follows along with a much more selective sampling of the more recent and
extensive literature. This is included in this chapter because many of the original
papers include a wealth of detail about methodology and interpretation of Ca
2
þ
photorelease technology.
A. Ion Channel Modulation
1. Potassium and Nonspecific Cation Channels
The first and still one of the major applications of photosensitive Ca
2
þ
chelators
is analysis of Ca
2
þ
-dependent ion channels in excitable cells. In 1987, Gurney et al.
first used nitr-2,-5, and-7 to activate Ca
2
þ
-dependent K
þ
current in rat sympathetic
neurons. These researchers found that a single Ca
2
þ
ion binds to the channel with
rapid kinetics and 350 nM a
Y
nity.
