Fogarty, K. E.

Thorn, P.

Tuft, R.

University of Queensland eSpace

 52nd Annual Meeting
 
×
 
Society of General Physiologists
 
33
 
a
 
not only reveal how global Ca
 
2
 
1
 
 signals are composed (Bootman
et al. 1997), but also provide information about the functioning of
I(1,4,5)P
 
3
 
 receptors at a microscopic level in vivo. In this regard,
the influence of feedback regulation by Ca
 
2
 
1
 
 and the kinetics of
I(1,4,5)P
 
3
 
 binding in determining the time course of Ca
 
2
 
1
 
 puffs
are unknown. If the time course of Ca
 
2
 
1
 
 puffs is determined by
the time for which agonist remains bound to the IP
 
3
 
 receptor, one
might expect agonist affinity, which reflects in part the ligand dis-
sociation rate from the receptor, to influence the duration of
these events. Therefore, we compared puffs evoked by I(1,4,5)P
 
3
 
with those elicited by I(2,4,5)P
 
3
 
 and adenophostin A [AdA].
I(2,4,5)P
 
3
 
 has an 
 
z
 
100-fold lower affinity than I(1,4,5)P
 
3
 
 for the
 
Xenopus
 
 IP
 
3
 
 receptor (Callamaras and Parker. 1994. 
 
Cell Calcium.
 
15:66–76), whereas AdA is the most potent agonist yet identified,
with an affinity 10–100-fold greater than I(1,4,5)P
 
3
 
 (Takahashi et
al. 1994. 
 
J. Biol. Chem.
 
 269:369–372). Taking the dissociation con-
stants (
 
K
 
d
 
) of AdA, I(1,4,5)P
 
3
 
, and I(2,4,5)P
 
3
 
 under physiological
conditions to be 
 
z
 
45 nM, 
 
z
 
450 nM, and 
 
z
 
50 
 
m
 
M, respectively,
and assuming a diffusion-limited association rate of 
 
z
 
10
 
8
 
 M
 
2
 
1
 
 s
 
2
 
1
 
,
the half-times for dissociation of these agonists from the I(1,4,5)P
 
3
 
receptor would be 150 ms, 15 ms, and 140 
 
m
 
s, respectively.
 
Xenopus
 
 oocytes were microinjected with caged I(1,4,5)P
 
3
 
 and
the fluorescent Ca
 
2
 
1
 
 indicator Oregon-green 488 BAPTA-1 (final
concentrations 
 
z
 
5 and 40 
 
m
 
M, respectively) 1 h before recording
confocal linescan images at 22°C. The duration (at half-maximal
amplitude) of Ca
 
2
 
1
 
 puffs evoked by intracellular ionophoresis of
analogs were compared with those evoked by photorelease of
I(1,4,5)P
 
3
 
 within the same oocyte. Both I(2,4,5)P
 
3
 
 and adeno-
phostin A evoked discrete puffs before global increases in the cy-
toplasmic Ca
 
2
 
1
 
 concentration ([Ca
 
2
 
1
 
]
 
cyt
 
). Despite the lower affin-
ity of I(2,4,5)P
 
3
 
, the duration of I(2,4,5)P
 
3
 
-evoked Ca
 
2
 
1
 
 puffs was
similar to those evoked by I(1,4,5)P
 
3 
 
(151 vs. 130 ms, respectively,
 
n 
 
5 
 
110 events). Maximally effective concentrations of AdA
caused a slower increase in global [Ca
 
2
 
1
 
]
 
cyt
 
 than saturating con-
centrations of I(1,4,5)P
 
3
 
, suggesting that AdA diffused more
slowly than I(1,4,5)P
 
3
 
. However, Ca
 
2
 
1
 
 puffs triggered by AdA
were briefer than I(1,4,5)P
 
3
 
-evoked Ca
 
2
 
1
 
 puffs (80 vs. 150 ms, re-
spectively,
 
 n 
 
5 
 
23 events): opposite to the prediction that the du-
ration of elementary events may lengthen with agonist affinity.
We conclude that the duration of Ca
 
2
 
1
 
 puffs cannot be deter-
mined by agonist dissociation alone, but rather other regulatory
mechanisms must delimit the period of Ca
 
2
 
1
 
 flux through the IP
 
3
 
receptor. (Supported by NIH grant GM 48071 and a Wellcome
Trust Prize Fellowship to J. Marchant.)
 
87. Clustering of “Hot Spots” of InsP
 
3
 
-evoked Calcium
Release Determines the Patterns of Calcium Signals in
Pancreatic Acinar Cells K.E. FOGARTY,
 
‡
 
 R. TUFT,
 
‡
 
 and
P. THORN,* 
 
*Department of Pharmacology, Cambridge Univer-
sity, Cambridge, United Kingdom; and 
 
‡
 
Department of Physiol-
ogy, University of Massachusetts Medical School, Worcester, Mas-
sachusetts
 
 (Sponsor: John V. Walsh)
 
Pancreatic acinar cells have two modes of InsP
 
3
 
-induced cal-
cium signaling: local, within the secretory pole, and global. We
used high temporal resolution calcium imaging methods, cou-
pled with spot (4-
 
m
 
m diameter) photolytic release of caged InsP
 
3
 
,
to map out in detail the regional response of the cell to InsP
 
3
 
.
Furthermore, we used three-dimensional imaging and EGTA
buffering to examine the cellular location of the discrete events
that underlie the “hot spots” of calcium release.
Our results show hot spots of InsP
 
3
 
 responsiveness, with similar
InsP
 
3
 
 thresholds and calcium release kinetics, in all regions of the
acinar cell. However, the probability of observing hot spots was
much less in the basal pole than in the secretory pole. Within the
secretory pole, the apparent density of hot spots and the speed of
calcium release was too great to allow us to resolve discrete
events. We therefore turned to techniques of high speed three-di-
mensional calcium imaging coupled with the use of EGTA to
buffer calcium diffusion. EGTA (200 
 
m
 
M) slowed the rising
phase and the spread of the global calcium response to global re-
lease of caged InsP
 
3
 
. In the three-dimensional images, the pres-
ence of EGTA revealed evidence for a matrix of clustered, dis-
crete, calcium release events.
We conclude that the clustering of hot spots of InsP
 
3
 
-evoked
calcium release is a fundamental parameter in defining the cellu-
lar calcium signal. At low InsP
 
3
 
 concentrations, the calcium signal
is restricted to the regions containing a high density of calcium
release sites. At higher InsP
 
3
 
 concentrations, the calcium signal
can spread across the cell, travelling in a saltatory manner from
release site to release site.
 
88. Calcium Diffusion Coordinates Discrete InsP
 
3
 
-evoked
“Hot Spots” of Calcium Release in Pancreatic Acinar
Cells J.F. KIDD,* K.E. FOGARTY,
 
‡
 
 R. TUFT,* and P.
THORN,* *
 
Department of Pharmacology, Cambridge Univer-
sity, Cambridge, United Kingdom; and 
 
‡
 
Department of Physiol-
ogy, University of Massachusetts Medical School, Worcester, Mas-
sachusetts
 
InsP
 
3
 
-evoked calcium responses are proposed to result from the
summation of discrete release events, but how these relate to
downstream cell functions is still unclear. Pancreatic acinar cells
show a local calcium signal that regulates fluid secretion and exo-
cytosis and is thought to represent the coordinated activity of a
number of discrete “hot spots” of calcium release (Thorn et al.
1996. 
 
EMBO
 
 (
 
Eur. Mol. Biol. Organ.
 
) 
 
J
 
. 15:999) Our hypothesis is
that calcium acts to coordinate this hot spot activity by a process of
calcium-induced calcium release at the InsP
 
3
 
 receptor. We there-
fore predict that EGTA, a slow calcium buffer, would act to limit
calcium diffusion and give rise to asynchronous and autonomous
hot spot activity. We report here on the use of the Cl
 
(Ca)
 
 whole cell
current as a high temporal resolution (2 kHz) monitor of the re-
petitive calcium spikes induced by the injection of (2,4,5)InsP
 
3
 
.
Under conditions of low intracellular calcium buffering (50
 
m
 
M EGTA), the majority (92%) of spikes had a monotonic rising
phase. However, with increasing EGTA concentrations, from 100
to 300 
 
m
 
M, we observed a dose-dependent increase in the occur-
rence of discrete steps in the rising phase of the spikes (from 25
to 61%) and also the appearance of smaller amplitude spikes.
Control experiments indicated that the EGTA effects were due to
a primary action on the mechanisms of calcium release rather
than the detection of the calcium signal by the Cl
 
(Ca)
 
.
We conclude that EGTA is acting to reduce the coordination
between adjacent hot spots of calcium release that then release
calcium in an autonomous manner and give rise to discrete steps
and smaller events in the Cl
 
(Ca)
 
 current records. High speed, 3-D
imaging experiments confirmed that EGTA acts to restrict the
spread of the hot spots that, under high buffer conditions, be-
come recognizable as discrete units. (Supported by the Medical
Research Council, The Wellcome Trust, NSF.)
 
89. Local Calcium Signaling by Inositol Trisphosphate in
Purkinje Cell Dendrites ELIZABETH A. FINCH and


Clustering of "hot spots" of InsP(3)-evoked calcium release determines the patterns of calcium signals in pancreatic acinar cells

http://espace.library.uq.edu.au/view/UQ:234412/UQ234412_OA.pdf

Clustering of "hot spots" of InsP(3)-evoked calcium release determines the patterns of calcium signals in pancreatic acinar cells

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