Biology Reference
In-Depth Information
diazo-2 revealed kinetics of muscle contraction and relaxation steps following the
binding and unbinding of Ca
2
þ
to troponin C (
Ashley et al., 1993
).
The first biological application of the caged chelator diazo-2 was in the study of
muscle relaxation.
Mulligan and Ashley (1989)
showed that rapid reduction in
[Ca
2
þ
]
i
in skinned frog semitendinosus muscle resulted in a relaxation similar to
that occurring normally in intact muscle, indicating that mechanochemical events
subsequent to the fall in [Ca
2
þ
] were rate limiting. However,
Lannergren and Arner
(1992)
reported some speeding of isometric relaxation after photolysis of diazo-2,
loaded in the AM form into frog lumbrical fibers. Lowered pH slowed relaxation
to a step reduction in [Ca
2
þ
]
i
(
Palmer et al., 1991
), perhaps accounting for a
contribution of low pH to the sluggish relaxation of fatigued muscle. In contrast
to frog muscle, photorelease of Ca
2
þ
chelator caused a much faster relaxation in
skinned scallop muscle than in intact fibers (
Palmer et al., 1990
), suggesting that, in
these cells, relaxation is rate limited primarily by [Ca
2
þ
]
i
homeostatic processes.
C. Synaptic Function
Action potentials evoke transmitter release in neurons by admitting Ca
2
þ
through Ca
2
þ
channels. Because of the usual coupling between depolarization
and Ca
2
þ
entry, assessing the possibility of an additional direct action of mem-
brane potential on the secretory apparatus has been di
cult. Photolytic release of
presynaptic Ca
2
þ
by nitr-5 perfused into presynaptic snail neurons cultured in
Ca
2
þ
-free media was combined with voltage clamp of the presynaptic membrane
potential to distinguish the roles of [Ca
2
þ
]
i
and potential in neurosecretion (
Zucker
and Haydon, 1988
), revealing no direct e
Y
V
ect of membrane potential on transmit-
ter release.
Hochner et al. (1989)
injected Ca
2
þ
-loaded nitr-5 into crayfish motor neuron
preterminal axons, and used a low-[Ca
2
þ
] medium to block normal synaptic
transmission. They found that action potentials transiently accelerated transmitter
release evoked by modest photolysis of nitr-5. However,
Mulkey and Zucker
(1991)
used fura-2 to show that the extracellular solutions used by
Hochner et al.
(1989)
failed to block Ca
2
þ
influx through voltage-dependent Ca
2
þ
channels.
When external Ca
2
þ
chelators or channel blockers eliminated influx completely,
spikes failed to have any influence on transmitter release, even when it was
activated strongly by photolysis of intracellularly injected Ca
2
þ
-loaded DM-
nitrophen.
Delaney and Zucker (1990)
confirmed at the squid giant synapse that in a Ca
2
þ
-
free medium, action potentials have no e
ect on transmitter release triggered by a
rise in [Ca
2
þ
]
i
upon photolysis of presynaptically injected DM-nitrophen. Flash
photolysis of DM-nitrophen produced a transient postsynaptic response resem-
bling normal excitatory postsynaptic potentials. The intense phase of transmitter
release was probably caused by the brief spike in [Ca
2
þ
]
i
following partial photol-
ysis of partially Ca
2
þ
-loaded DM-nitrophen. This response began a fraction of a
millisecond after the rise in [Ca
2
þ
]
i
, a delay similar to the usual synaptic delay
V
