ABSTRACT
Stimulation of NIE-115 mouse neuroblastoma cells with the neurotransmitter bradykinin
(Bk) leads to inositol 1,4,5-trisphosphate (IPz) production and calcium release from IPz-regulated
Ca“ pools located in the endoplasmic reticulum. This involves a specific G-protein second
messenger cascade. The release of intracellular Ca“ is preceded by a latent period of up to 30
seconds. This study investigated the contribution of IPz diffusion and the dynamics of IP;
receptor activation to the latency between surface receptor binding and Ca- release. Intracellular
injection of caged IPz plus the fluorescent calcium indicator fluo-3 was used investigate the basis
of the observed latency. Following injection, cells were exposed to a brief (170us) flash of light
(230 joules, 360nm wavelength) to photolyse the caged compound. Fluorescence video imaging
was used to follow the change in intracellular [Ca. The study showed that [Ca begins to rise
immediately (within 200ms) following uncaging of IPz by flash photolysis, and reaches maximum
levels within 1.5 seconds. The result indicates that neither IPz diffusion to its receptors nor delay
between IP3R activation and Ca" release contribute significantly to overall latency. Analysis of
Ca“ wave kinetics shows that in contrast to cells stimulated with neurotransmitter, injected cells
do not exhibit propagation of Ca“ waves following photolysis of caged IPz. This observation
suggests that the IPz receptor is distributed throughout the cytoplasm of the soma and major
neurites. Ca“ waves apparently travel through this IPz receptor rich medium. The constant rate
in which Ca“ waves travel shows that propagation is an active process rather than a diffusional
process. The mechanism of propagation could involve either IPz production or Ca"-induced Ca-
release at the wave front.
INTRODUCTION
Metabotropic neurotransmitter receptors are linked via second messenger pathways to
their intracellular effector targets. The bradykinin (Bk) B2 receptor subtype is coupled by the G-
protein Gyii to a wide variety of second messenger cascades, with one prominent effect being the
activation of the membrane-bound enzyme phospholipase C (PLCB1). PLC hydrolyzes
phosphotidyl inositol 4,5-bisphosphate (PIP2) in the plasma membrane to form inositol 1,4,5-
trisphosphate (IPz) and diacylglycerol (DAG) [Rhee et al., 19891, both of which act as
intracellular messengers. IPz diffuses in the cytosol to receptors on the surface of the endoplasmic
reticulum. Activation of the IPz receptor leads to release of Ca into the cytosol from Ca
storage compartments of the endoplasmic reticulum.
Surface receptors coupled to second messenger pathways characteristically mediate
responses which are slow in both onset and duration [Hartzell, 1981]. Response latencies as long
as 30 seconds in duration have been observed for B2 receptor activation in neuroblastoma cells
[Coggan et al., 1995]. The existence of such an appreciable delay between surface receptor
binding and cellular response has important consequences for the synaptic activation of cells
Described here is an investigation of possible origins of the latency of responses mediated by the
phosphoinositide signaling pathway in NIE-115 mouse neuroblastoma cells, a sympathetic
neuronal cell line which express a number of G-protein coupled receptors including B2, Hj, Mi,
and Ma receptors.
It has been suggested that the limiting factors determining response latency at high agonist
concentrations arise from stages in the phosphoinositide signaling cascade located between
receptor binding and IPz liberation. It was further suggested that at low agonist concentrations,
an additional delay is introduced due to intracellular levels of IPz rising slowly to a threshold
before triggering calcium release [Miledi et al., 1988). To test the prior theory, NIE-115 cells
were intracellularly injected with a photo-releasable IPz (caged IPz) and an appropriate fluorescent
Ca indicator (fluo-3)
Photolabile caged compounds are inert precursors of biologically active molecules that can
be loaded into cells and subsequently released in their active form. Uncaging is accomplished by a
flash of light (360nm) that triggers the breaking of photolabile bonds to release the bioactive
molecule. The absorption of photons excites the inert parent compound to a higher energy state
where it undergoes a reaction leading to the formation of a stable, physiologically active
biomolecule. Measurements of response latencies evoked by intracellular injection of bioactive
molecules are complicated by uncertainties regarding micropipette tip positioning, and also by the
difficulty in controlling injection volume. The use of caged compounds results in more accurate
measurements by eliminating such uncertainties, and by presenting the opportunity of both
controlling release and guaranteeing even distribution throughout the cell by simple diffusion
[Miledi et al., 19881
In this study, injected caged IPz was released by flash photolysis and changes in latency
preceding Ca“ release from internal stores was measured through fluo-3 imaging. The results
were then compared to response latency measured following surface receptor stimulation with
bradykinin. The objective was to determine whether the time of IPs diffusion to its receptors on
the ER and/or delay between receptor activation and Ca“ release significantly contributed to the
overall response latency. IPz has been shown to have a measured diffusion coefficient (D) of
283um'/s and a effective range lifetime of 24um [Allbritton et al., 19921. Thus for cells smaller
than 20um, IPz acts as a global messenger.
IPz-dependent Ca“ release occurs in an organized manner, often taking the form of a
wave passing throughout the cell [Gilkey et al., 1978). Investigation into the Ca* wave
phenomena following agonist stimulation has led to a theory of wave propagation that relies on
the observation that Ca“ itself can act as a coagonist in triggering IPz-induced Ca- release Finch
et al., 1991). The model suggests a positive feedback loop that operates when intracellular IP,
levels are at threshold and if IPz-bound receptors are spaced closely enough that Ca- release
beginning at one receptor can bring neighboring IPz receptors to threshold for Ca release. The
local increase in Ca“ allows for further propagation of the Ca“ wave as the cycle continues down
the length of the cell [Wang et al., 1995). To test the model's assumption that IPz receptors are
distributed throughout the cell, analysis of wave propagation kinetics was conducted on cells
intracellularly injected with caged IPz.
MATERIALS AND METHODS
Cell Culture
NIE-115 mouse neuroblastoma cells were obtained from the UCSF Cell Culture Facility,
maintained without antibiotics in Dulbecco's Modified Eagle Medium (Sigma Chemical Co., St.
Louis, MO) supplemented with 10% bovine serum, and grown to passages 6-8. For
experimentation, cells were plated on glass coverslips. To initiate differentiation, the medium was
replaced with medium containing 2% dimethylsulfoxide. Cells were used 6-14 days following
differentiation [Wang et al., 19951.
Microinjection of cells
After rinsing in Hank's buffer, pH 7.4, the cells were transferred to a heated chamber
(30°C) for microinjection. FFP-18, K salt (TEFLABS, Austin, TX) and fluo-3, pentaammonium
salt (Calbiochem-Novabiochem Corp., La Jolla, CA) were injected at 1mM concentrations using
an Eppendorf micromanipulator 5171 (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany).
D-myo-Inositol 1,4,5-trisphosphate, P“-1-(2-nitrophenyl) ethyl ester, sodium (caged IPz,
trisodium salt - Calbiochem-Novabiochem Corp., La Jolla, CA) and fluo-3, pentaammonium salt
(Calbiochem) were each dissolved at concentrations of ImM in saline containing SmM HEPES,
pH 7 at 20°C and microinjected using the Eppendorf micromanipulator. Injected caged IPz was
allowed to evenly distribute throughout the cell for 10 minutes or more. Assuming that injection
volumes were no more than 10% of the total cell volume, the concentration of each compound in
cells was no more than 1OOUM.
AM loading of cells
Cells were loaded with the low-affinity Ca" indicator mag-fura-2 by incubation in saline
containing 5uM mag-fura-2/AM and 0.0025% Pluronic F-127 (Molecular Probes) for 45 minutes
at 22°C.
Calcium imaging
Following loading, cells were transferred to a heated chamber (30°C) on the stage of a
Nikon Diaphot epifluorescence microscope with 20x and 40x Fluor objectives (Nikon),
Hamamatsu C2400 silicon-intensified target camera, and a Panasonic VHS video tape recorder.
Excitation illumination by a Xenon arclamp was filtered through lOnm bandpass interference
filters at 340 or 380nm wavelengths for FFP-18 and 525nm wavelengths for fluo-3. Wave
analysis was done off-line on a Mega Vision 1024XM pipeline image processor (Mega Vision,
Santa Barbara, CA) frame by frame from data transferred from tape to a Panasonic TQ-2028F
optical disk recorder.
For FFP-18 injected cells, Ca“ kinetics were determined by monitoring cells continuously
for up to 60 seconds at 380nm excitation after initially obtaining Fzao/F3so. For fluo-3 injected
cells, Ca kinetics were determined by monitoring cells for up to 60 seconds at 525nm excitation
wavelengths. For caged IPz + fluo-3 injected cells, a brief (170us), maximum intensity flash (230
Joules, 360nm) from a Xenon Strobex lamp (Chadwick-Helmuth Co., El Monte, CA) was
focused directly onto the cell field to photolyse the caged IPz. Following flash illumination, Ca-
kinetics were resolved by monitoring cells for up to 30 seconds under 525nm light. A stroboflex
shutter was closed during the flash to protect the SIT camera. The reopening of the shutter is
associated with a time delay of 100ms. An additional delay of approximately 200ms is associated
with the delay in camera response time following shutter reopening.
Neurotransmitter application
Bradykinin (1OOnM) was applied by total replacement of the chamber volume using a
manually-controlled perfusion device.
Calcium and calcium wave analysis
Increases in intracellular Ca“ were observed following stimulation with 100nM
bradykinin. Responsive cells were identified by eye, reviewing the data played back at 1-4 times
normal speed. Changes in [Ca“ were measured in regions of interest corresponding to the
center of individual cell bodies. Baseline fluorescence, Fo, was defined as the average
fluorescence within the region of interest over 5-10 frames just prior to agonist application. The
fluorescence signal was converted to units of AF/F.
Ca“ waves were evoked in similar fashion as changes in intracellular Ca“. Waves were
identified by eye, viewing data played back at 1-4 times normal speed. For quantitative analysis of
wave motion, a line segment passing through the cell in the direction of wave propagation was
selected. Fluorescence was averaged over 1 to 3 pixels on either side of the line at a rate of 30
frames per second. Normalized lines of AF were stacked to generate images in which the y-axis
represented time and the x-axis represented location along the scanned line segment, yielding a
graph of wave position against time.
RESULTS
Properties of Ca indicators
Due to the wavelength needed to release caged IPz, my studies used fluo-3 as a
fluorescent Ca“ indicator. Fluo-3 is excited at longer wavelengths (525nm) than fura-2 and
related indicators (FFP-18, mag-fura-2), which are excited in the 340-380nm range, the same
spectral range in which caged compounds are photo-released. The lipophilic tail of FFP-18 allows
it to function as a near-membrane Ca“ indicator upon injection, allowing one to take advantage of
the fact that Ca“ levels near membranes rise faster and higher than in the cytosol. Mag-fura-2, a
low-affinity Ca“ indicator, presents the advantage of exhibiting spectral shifts at higher Ca-
concentrations (up to 100uM), allowing resolution of greater peaks in [Ca following
stimulation.
Latency following bradykinin stimulation
The activation of B» receptors by bradykinin in cells injected with FFP-18 (ImM
micropipette concentration) or mag-fura-2 (lmM) results in a latent period (mean latency = 9.06
seconds) preceding a rise in intracellular [Ca (Fig. 1-2). [Ca increases to approximately
2uM (an estimated 20 fold increase from resting Ca“ levels) within 2-5 seconds following initial
response. The increase in [Ca is followed by a characteristic falling phase where fluorescence
exhibits a smooth decay to resting levels. The decay of mag-fura-2 appears more rapid than the
decay of FFP-18. The high affinity of FFP-18 for Ca“ causes it to reach saturation well before
peak Ca is attained, accounting for the slower apparent decay of the signal.
Latency following flash photolysis of caged IP;
Flash photolysis of cells injected with caged IPz plus fluo-3 (ImM micropipette
concentrations) does not exhibit a latent period prior to the rise in intracellular [Ca (Fig. 3).
Photolysis occurs instantaneously following shutter closure (see Materials and Methods) and is
represented by a decline in fluorescence. Response to uncaging in the single cell, illustrated in Fig
3., is characterized by an immediate rise (within 200ms) in [Ca, with peak levels reached in less
than 1 second. Peak [Ca corresponds to approximately 600nM (an estimated 6 fold increase
from resting levels). An averaged response to flash photolysis of caged IPz (Fig. 4) shows similai
results, but exhibits a noticeably slower rise to maximum Ca“ levels (2-3 seconds following
photolysis). The attainment of peak [Ca is followed by a falling phase where fluorescence
exhibits exponential decay to resting levels (Fig. 5). The time to one-half decay was measured to
be 4.06 seconds.
Kinetics of rising phase following flash photolysis
Analysis of the response kinetics following flash photolysis reveals a 200ms period where
Ca“ imaging is not recorded (Fig. 6). The shutter reopening and delay associated with camera
response accounts for this lag period (see Materials and Methods). Recording begins within 1
second at which time [Ca are near peak levels. Thus, the initial rising phase following
photolysis is undocumented due to recording limitations. An average of response kinetics to
uncaging of IPz (Fig. 7) shows the same 200ms loss in imaging, but a slightly longer time interval
to peak [Ca of 2-3 seconds following uncaging.
Comparison of latency following agonist stimulation
A comparison of latencies to Ca“ release in the same neuronal cell following flash
photolysis of IPz and stimulation with bradykinin is shown in Fig. 8. Flash photolysis of IPz
results in a rise in intracellular [Ca within 200ms of uncaging. In contrast, stimulation with
bradykinin results in a latent period in which Ca" levels do not appreciably change, followed by a
rise in intracellular Ca“. Both responses are characterized by a falling phase where fluorescence
decreases either linearly (neurotransmitter) or decays exponentially (IPz) to resting levels.
Bradykinin stimulation elicits Ca wave propagation
The activation of the B2 receptor with agonist elicits a Ca- wave in a cell injected with
FFP-18. The Ca“ rise begins with a delay of 10-11 seconds after agonist application and spreads
across the cell as a defined wave-front. An example is shown in Fig. 9. The cell is stimulated
with bradykinin which invokes a Ca" wave beginning in the soma and spreading toward the
growth cone. A second wave originates in the growth cone and propagates toward the soma.
Wave speed is determined by measuring the time difference as a wave front passes two points in
the cell. The data suggests that each wave front travels uninterrupted for at least 30um at a speed
of 42um/s. Upon collision, the two waves annihilate each other (arrow). Annihilation is
consistent with the proposed theory of an active process underlying wave propagation through an
excitable medium [Wang et al., 19951.
Flash photolysis of caged IPz abolishes wave propagation
The activation of the IPz receptor through flash photolysis of caged IPz does not lead to
wave propagation (Fig. 10). Following photolysis (time-O), the change in fluorescence occurs
throughout the cell indicating that a Ca“ wave does not propagate. This result is consistent with
the working model of wave propagation that assumes the IPz receptor is distributed throughout
the cell, and not localized to a particular area.
DISCUSSION
Ca indicators exhibit unique decay phases
The use of FFP-18 as a Ca“ indicator is advantageous in allowing one to monitor changes
in Ca near intracellular membranes. However, its high affinity for Ca“ causes it to reach
saturation well before the Ca" signal peaks during stimulation with bradykinin. In contrast, mag¬
fura-2, a low-affinity Ca indicator, presents the advantage of displaying spectral shifts at high
Ca“ concentrations, allowing one to better resolve peak intracellular Ca. The difference in
binding affinity between these two indicators leads to fluorescence decay patterns that are unique
to the indicator following neurotransmitter stimulation. The slow decay to resting levels observed
in a cell injected with FFP-18 and stimulated with bradykinin (Fig. 1) suggests that FFP-18
reaches saturation well before maximum concentrations of intracellular Ca“ (2uM) are reached.
Even as Ca continues to decrease and return to resting levels, Ca remains well above FFP-
18 saturation concentrations for a significant period of time, leading to the slow apparent decay.
FFP-18 is therefore better employed as an indicator when changes in Ca- are small (nanomolar
range).
Mag-fura-2 exhibits a rapid exponential decay to resting levels following attainment of
peak [Ca (Fig. 2). The indicator's low affinity for Ca allows it to achieve better resolution of
the 2uM peak in Ca following stimulation with bradykinin, thus making it an indicator of
choice in imaging experiments where changes in intracellular Ca“ are large (micromolar range).
Flash photolysis of caged IPz eliminates latency
Stimulation of neuronal cells with the neurotransmitter bradykinin results in a latent period
as long as 30 seconds in duration prior to release of Ca- from IPz receptor-regulated Ca storage
compartments in the ER (Fig. 1-2). By direct release of IPz into the cytosol using flash
photolysis, this latency is shown to be eliminated (Fig. 3-4). Within 200ms of photolysis,
intracellular [Ca begins to increase, reaching peak Ca" levels within 1-2 seconds, following
which levels decay exponentially. The time needed to reach half decay was measured to be 4.06s.
This decay time most likely correlates to the degradation time of IP3. This compares favorably
with biochemical measure of IPz degradation of approximately 9 seconds WWang et al., 19951
Prior to this half decay point, it is likely that several IPs receptors are still being activated by IP;
molecules remaining in the cytosol. Past the half decay point, it is postulated that all IP3 released
by flash photolysis has degraded, IPz receptors are inactive, and Ca- levels slowly return to
resting levels.
The results suggest that the latency observed prior to Ca release is characteristic of the
pathway (Fig. 6). They further suggest that neither the time of IPz diffusion from its site of
production to its receptors on the ER nor the time to Ca“ release following IPz receptor
activation significantly contribute to the latency observed following stimulation with
neurotransmitter. It is postulated that the latency arises earlier in the phosphoinositide signaling
pathway, perhaps at the level of IPz formation through hydrolysis of phosphotidyl inositol 4,5
bisphosphate (PIP2) by phospholipase C (PLCB1).
Cooperativity at the IP; receptor
In this set of studies, the exact delay to Ca“ release from storage pools could not be
resolved due to limitations in the fluorescence video imaging equipment. In particular, the 200ms
delay following photolysis before imaging is accounted for by the 10Oms shutter speed as well as
delay associated with camera response following shutter reopening (see Materials and Methods).
To avoid such limitations, a rapid photodetector such as a photodiode or PMT needs to be
employed.
Research on the IPz receptor suggests that the receptor exhibits the property of
cooperative binding [Meyer et al., 1988). The theory states that the binding of one IPs molecule
to the IPz receptor facilitates the binding of other IP3 molecules. In order for the receptor to
activate and thus serve as a channel for Ca“ release into the cytosol, 3 to 6 molecules of IPz need
to be bound. The highly cooperative opening of Ca" channels by nanomolar concentrations of
IPz enables cells to detect and amplify extremely small changes in the concentration of this
intracellular messenger in response to various stimuli.
The results from the uncaging experiments performed in this study support the theory of
cooperativity at the IPz receptor. Release of relatively high intracellular concentrations of IP;
(100nM) by flash photolysis results in a rapid increase in intracellular [Ca. Taking cooperative
binding into consideration, it is concluded that the undocumented portion of the rising phase due
to the speed limitations of the imaging equipment (see Materials and Methods) represents the
lower foot portion of the sigmoidal curve characteristic of cooperative binding
Gradual rising phase following flash photolysis suggests regeneration
An averaged cellular response to flash photolysis of caged IPz exhibits a rapid increase in
[Ca following uncaging, but a noticeably slower rise (1-2 seconds) to peak Ca levels (Fig. 4).
This gradual rise to maximum intracellular [Ca suggests a regenerative process at the level of
IPz receptor activation. Rather than achieving peak cytosolic Ca levels immediately, the peak is
attained through repeated activation of IP3 receptors. Researchers have theorized that
submicromolar concentrations of Ca“ can cause IPz receptors to reach threshold by altering the
receptor's binding affinity for IPz, thus activating the receptors and releasing additional Ca- into
the cytosol. This theory of a Ca“-induced Ca“ release at the IPz receptor (Finch et al., 1994 can
explain the apparent regenerative process observed following uncaging of IP3.
Flash photolysis of caged IPz abolishes wave propagation
The working model of Ca“ wave propagation in neuronal cells suggests an active process
underlying wave propagation. This process may involve an interaction between cytosolic Ca-
and IPz receptors at the wave front [Wang et al., 1995]. The model is based on the assumption
that IPz receptors are distributed throughout the entire cell, and not localized to a particular area.
Stimulation with the neurotransmitter bradykinin elicits Ca" wave propagation in neuronal cells
(Fig. 9). Cells injected with caged IPz and exposed to flash photolysis, however, do not
propagate Ca“ waves (Fig. 10). The result indicates that the model's underlying assumption of a
wide cellular distribution of IPz receptors is correct. The result further suggests that Ca- waves
travel through this excitable IPz receptor rich medium.
Two mechanisms underlying the active wave propagation can be postulated. If IPz is
produced at the wave front, it can act to sustain propagation through activation of subsequent IPz
receptors down the length of the cell. Alternatively, if Ca" interacts with IPs receptors as the
model suggests, it can invoke a Ca“-induced Ca“ release at the wave front to sustain propagation
through the cell.
Future investigation
It has been suggested that IPz needs to reach sufficient threshold levels in order to activate
IPz receptors and subsequently cause Ca“ release into the cytosol (Parker, 1988). In order to
measure and quantify this hypothetical IPz threshold, continued experiments using flash photolysis
of lower concentrations of caged IPs need to be conducted. Furthermore, it would be equally
beneficial to stimulate cells with neurotransmitter, uncage IPz at varying intervals during the latent
period, and measure changes in latency to Ca“ release. Such studies could potentially determine
if it is [P itself which regulates and determines latency. In addition, other steps in the
phosphoinositide signaling pathway merit further investigation to determine their roles in the
latency to Ca“ release following neurotransmitter stimulation. Essential steps to investigate are
PIP2 hydrolysis, PLCBI activation, and Gyii turnover.
Finally, to determine the possible coagonist relationship between cytosolic Ca and IP-
receptors, one would measure changes in cytosolic [Ca via fluorescence video microscopy in
response to flash photolysis of caged Ca“ compounds (DM-nitrophen, NITR-5)
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FIGURE LEGENDS
FIGURE 1: Calcium signal in response to bradykinin with high-affinity indicator. Example of
the dynamics of Ca“ release from a cell injected with FFP-18, K salt (ImM micropipette
concentration) and stimulated with bradykinin (100nM). Agonist is delivered at time-O, followed
by a 10-11 second latent period in which [Ca do not appreciably change. The latency is
followed by a 20 fold increase in [Ca over a 2-3 second time period. Intracellular Ca then
exhibits a slow decay, presumably toward resting levels.
FIGURE 2: Calcium signal in response to bradykinin with low-affinity indicator. Example of
the dynamics of Ca“ release from a cell loaded with mag-fura-2/AM (SuM) and stimulated with
bradykinin (100nM). Following stimulation (time-O), [Ca*1 do not appreciably change for a
period of 6-7 seconds. The latent period is followed by a 25 fold increase in cytosolic Ca over
a time interval of 2-3 seconds in which peak Ca“ levels are resolved. Following attainment of
peak [Ca-, a rapid decay to resting levels is observed.
FIGURE 3: Calcium signal in response to flash photolysis of caged IPz. Cells injected with
caged IPz plus fluo-3 (ImM micropipette concentrations) exhibit an instantaneous increase in
[Ca following flash photolysis. The flash used to uncage IPz is represented by the sudden
decrease in fluorescence caused by shutter closing. Cytosolic [Ca“ begins to rise within 200ms
following exposure to the flash, and attain peak levels within 1 second following flash.
FIGURE 4: Average response to flash photolysis of caged IPz. An average response of cells
(n-3) injected with caged IPz plus fluo-3 exhibits an immediate increase in cytosolic Ca
following flash photolysis. Rise begins within 200ms following flash exposure, with peak levels
gradually attained 1-2 seconds later.
FIGURE 5: Falling phase kinetics following flash photolysis of caged IPz. Following attainment
of peak cytosolic [Ca, fluorescence decays exponentially to resting levels. The solid line
represents actual fluorescence decay while the dashed line represents an exponential function
curve-fitted to the data.
FIGURE 6: Rising phase kinetics of [Cd in response to flash photolysis of caged IPz. Ca'
imaging was not documented for the initial 200ms following flash photolysis (time interval
indicated by vertical, dashed lines). The time to shutter reopening as well as the delay response of
the camera accounts for this delay period. Recording begins (*) when cytosolic Ca is already
near maximum, which is attained within 1 second following recording.
FIGURE 7: Average rising phase kinetics of Ca1 in response to flash photolysis of caged IPz.
An average kinetic response of cells (n-3) exposed to flash photolysis shows the 200ms blanking
of the recording (indicated by vertical, dashed lines), but a slightly longer time to peak [Ca (2-3
seconds) compared to the single cell response.
FIGURE 8: Latency following flash photolysis of IPz versus bradykinin stimulation. The solid
line represents a cell injected with caged IPz plus fluo-3 (ImM micropipette concentrations) and
stimulated by flash photolysis (indicated by drop in fluorescence). The dashed line represents the
same cell stimulated with a saturating concentration of bradykinin (100nM) moments later. Flash
photolysis of IPz results in a rise in [Ca within 200ms of uncaging. Stimulation with bradykinin
(time-O) results in a 5-7 second latency before Ca“ release in which cytosolic Ca does not
appreciably change. The peak level attained following bradykinin stimulation is lower than
observed following neurotransmitter stimulation due to insufficient time to refilling of Ca
storage pools as well as IPz receptor desensitization. Following peak Ca, fluorescence values
decay either linearly (neurotransmitter) or exponentially (IPz) to resting levels.
FIGURE 9: Calcium wave propagation in bradykinin stimulated cells. Ca rise begins with a
delay of 10-11 seconds after agonist stimulation (time-O) and spreads across the cell in a defined
wave-front (varying colors represent varying levels of fluorescence). Two separate waves
originate at opposite ends of the cell (soma and growth cone) and propagate toward one another
at 42um/s. Upon collision, the waves annihilate each other (arrow).
FIGURE 10: Flash photolysis of IP3 does not cause wave propagation. Following flash
photolysis (time-O), fluorescence (black) increases evenly throughout the cell, indicating
simultaneous Ca“ release and a lack of wave propagation.
FIC
1
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Time (seconds)
FIGURE 2
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Time (seconds)
FIGURE 3
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Time (seconds)
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1.025
1.015
1.005
0.995
0.985
0.975 +
0.0
0.5
1.0
FIGURE 6

VV
—
k kava-


1.5 2.0
2.5 3.0 3.5
Time (seconds)
1.075 -
1.065
1.055
1.045
1.035
1.025
1.015
1.005

0.995
0.985
0.975
0.0
0.5
1.0
NA
FIGURE 7

1.5
2.0
Time (seconds)
V
2.5 3.0 3.5
FIGURE 8
1.150
N
1.125
W
1.100 -
V
.*
1.075
. .
1.050
Hene.
1.025
........
1.000
0.975
0.950 -
0.925
0.900
0.875
Flash

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Time (seconds)
FIGURE 9
k
11

FIGURE 10

Fa


14
38
Distance along axon (uni)