ABSTRACT
The NIE-115 mouse neuroblastoma cell line was used to investigate the role of the
cholinergic receptor/ nitric oxide/cyclic GMP pathway in regulating neurite outgrowth.
Here evidence is presented that both carbachol and 8-Bromo-cGMP inhibit neurite
outgrowth. Nitric oxide donors, however, promote various forms of neurite outgrowth.
DEANO and Spermine NONOate were shown to promote neurite outgrowth from
lamellipodia, but had no effect on neurites that did not possess lamellipodia before
administration of the donors. SNAP was shown to allow growth of the main shafts of
neurites, but not that of lamellipodia or their extended neurites. This study suggests that
nitric oxide promotes growth through a cGMP-independent pathway. During activation
of cholinergic receptors, the stimulatory effects of nitric oxide appear to be masked by
other pathways in the cholinergic cascade.
INTRODUCTION
In the cholinergic receptor/nitric oxide/cGMP pathway, the muscarinic cholinergic
receptor, Ml, is coupled to the activation of nitric oxide synthase (NOS). NOS catalyzes
the conversion of L-arginine into L-citrulline and nitric oxide gas Nitric oxide, in turn,
has been shown to be coupled to the activation of guanylyl cyclase and the production of
cyclic GMP [3,5,1 1), as well as several other, less well-characterized actions [21.
Finally,
cyclic GMP acts as a signal molecule for opening and closing membrane channels
The present study investigates the role of nitric oxide in regulating neurite
outgrowth in the NIE-115 mouse neuroblastoma cell line. Along with other receptors,
NIE-115 cells express the Ml receptor. Previous studies conducted on the role of this
pathway in neurite outgrowth regulation have been inconsistent. While it has been
suggested that cGMP inhibits motility and causes neurite retraction in NIE-115 cells 111.
other research have found that cGMP promotes neurite extension and motility in rat dorsal
root ganglion (DRG) neurons [121. It has been shown that carbachol inhibits the growth-
promoting effects of forskolin in human neuroblastoma NB-OKI cells [71, yet
acetylcholinesterase promotes regeneration of neurites in cultured adult neurons of
Aplysia [10]. The effects of nitric oxide on neurite outgrowth have also been
controversial. While studies have shown that NO arrests growth of neuronal cells during
differentiation 6,9 and inhibits extension of regenerating axons of rat DRG neurons [131,
other research has indicated that nitric oxide promotes neurite extension and motility in rat
DRG neurons [121
The present study investigates the role of nitric oxide on neurite outgrowth by
examining three points along the MI receptor cascade: (1) at the level of the muscarinic
receptor, using the cholinergic agonist carbachol, (2) at the level of nitric oxide, using NO
donors DEANO, Spermine NONOate, and SNAP, and (3) at the level of cGMP using the
membrane permeable analog, 8-Bromo-cGMP
MATERIALS AND METHODS
Materials
S-nitroso-N-acetylpenicillamine (SNAP) and diethylamine nitric oxide, sodium salt
(DEANO) were purchased from Research Biochemicals International (Natick, MA)
Spermine NONOate was from Alexis (San Diego, CA, U.S.A.). Carbachol and 8-Bromo¬
cyclic GMP were from Calbiochem (San Diego, CA, U.S.A.).
Cell Culture
Mouse neuroblastoma clone NIE-115 cells (from UCSF Cell Culture Facility)
were grown in Dulbecco's modified Eagle’s medium (DMEM) (Sigma, Saint Louis, MO
U.S.A.) supplemented with 10% fetal bovine serum (HyClone, Logan, UT). Cells were
incubated at 37°C in a humidified atmosphere containing 10% carbon dioxide. Cells were
plated onto glass cover slips and placed in 35 mm plastic tissue culture dishes (Falcon,
Becton Dickinson Labware, Lincoln Park, NJ). Cells grown to-60% confluency were
then differentiated by treating with 2% dimethylsulfoxide (DMSO). All cells were
cultured for at least 3 days before use and fed one day before use.
Time-Lapse Video/OMDR Microscopy
Immediately before each experiment, a glass chip was selected from one of the
plastic culture dishes containing differentiated cells and placed in 10 mM HEPES buffer
(phosphate buffered saline, PBS) at pH 7.4. The chip was then transferred to a well that
fits into the stage of the microscope (Nikon Diaphot) and secured with a small amount of
petroleum jelly applied to the edges of the chip. This well was then placed onto the stage
and PBS was added immediately to prevent drying out of the cells. A field containing as
many distinct cells as possible, of varied morphologies and with visible processes, was
selected. Time-lapse video (Panasonic VHS) and optical disk (Panasonic OMDR)
recordings were made simultaneously, at a rate of one frame every two minutes, using
Nomarski D.I.C. optics and a Hammatsu C2400 SIT camera. A total of 69 frames was
recorded for each treatment, 9 frames before and 60 frames after stimulation The images
were acquired with a 20X bright field objective at low light intensities and at a controlled
temperature of 30°C. A pipeline image processor (Megavision, Santa Barbara, CA) was
used to digitize images, and its application programs were used for analysis of the entire
time series. Select images were converted into bitmap files and transferred to Adobe
Photoshop 3.0. Measurements and further analysis were done using Photoshop
applications. Digitally processed images were printed from an HP LaserJet AMV printer.
Controls
It was found that the length of time the cells were in the well for video time-
lapsing (-2.5 hours), coupled with the temperature of 30°C, increased the osmolarity of
the bathing solution by more than 25%. To control for this effect, all experiments were
done under constant perfusion (rate - 7.5 mL/hr) with the bathing solution.
Because different cells have varied basal motility, an 18-minute control period of
growth in PBS was recorded for each experiment prior to treatment, against which growth
during the 18 minutes after treatment was compared. The control period was used to
compare basal growth rates between experiments.
Growth in PBS was considered as the control for all the treatments. Triplicate
experiments using PBS were performed and recorded for the same total number of frames
as for the treatment experiments.
Neurite Outgrowth Assay
After the initial 18-minute PBS control period, the test compound was added.
Saline containing 100 uM DEANO was introduced into the chamber and allowed 20
minutes to react, after which constant perfusion with PBS was resumed. 1OOuM NO
donor Spermine NONOate was applied using constant perfusion. Nitric oxide donor S-
nitroso-N-acetylpenacillamine, at 10OuM in 1% DMSO, was also added using constant
perfusion. The cyclic GMP analog, 8-Br-cGMP, was added at a concentration of ImM
and allowed to react for 15 minutes before switching to constant perfusion at 20 uM.
Cells treated with carbachol were first incubated with Fura-2 (SuM) for 30 minutes on a
shaker table in the dark before experimentation. At addition of carbachol, the cells were
viewed with a 20X Fluor objective under UV light and video taped (Panasonic VHS) to
record calcium concentration increases in responding cells: the cells were recorded for
approximately 5 seconds at 340 nm before stimulation (to obtain background
fluorescence), and for one minute at 380 nm upon addition of carbachol. Carbachol was
added at an initial concentration of 1 mM and allowed to react for 15 minutes before
starting constant perfusion at 20 uM. Triplicate experiments were performed for each test
compound. Cells were time-lapse recorded for an additional two hours from time of
stimulation.
In addition to studying the entire time series as movies and using Megavision
program applications for image processing and individual frame analysis, images taken at
times -18, 0, 10, 18, 30, 60, 90, and 120 minutes relative to time of treatment were
analyzed and measured using Photoshop applications.
RESULTS
Cells grown in the PBS control exhibited a significant amount of growth, which
manifested itself in a variety of ways (see figure 1). Initiation and extension of neurites
from lamellipodia was observed. The number of neurites sprouting from each
lamellipodium varied, but they were oftentimes delicate and finely branched. Frequently,
instead of the neurites growing out from the ends of the lamellipodia, entire lamellipodia
would develop into these highly-branched neurites, suggesting that the process is one
involving the maturation of motile lamellipodia into neurites. Branches off the main shafts
of processes lengthened in PBS, as well as the main shafts themselves, regardless of the
original process length. Elongation and broadening of lamellipodia was also observed.
DEANO, Spermine NONOate, and SNAP Inhibit SpecificTypes of Growth
Cells grown in DEANO and Spermine NONOate exhibited the same types of
growth (see figures 2 & 3). In either treatment, neurites are seen to extend and elongate
from lamellipodia. As with cells grown in PBS, it is sometimes observed that entire
lamellipodia develop into thin, distinct processes. Also analogous to the control, these
neurites often appear delicate and finely branched. Both DEANO and Spermine
NONOate, however, appear to inhibit growth in the main shafts of processes.
Growth of neuronal cells treated with SNAP was different from that of cells
treated with DEANO and Spermine NONOate (see figure 4). Unlike DEANO and Sperno,
SNAP did not inhibit elongation of the main shafts of processes. As these processes
extended, they did not branch and remained thick in diameter. It was observed, however,
that SNAP did arrest the maturation and branching of lamellipodia.
Carbachol and cGMP arrest growth
The outgrowth exhibited by cells in PBS was almost entirely absent in carbachol-
and cGMP-treated cells. Almost all neurite outgrowth was inhibited upon stimulation
with carbachol (see figure 5). This result was observed both in cells that had a calcium
concentration increase upon stimulation (as detected with fura-2 imaging), as well as in
cells that did not. Likewise, almost all neurite outgrowth was arrested in cGMP-treated
cells (see figure 6).
Rapid Cell Death
A few examples of rapid cell death (RCD) was observed in the experiments. This
phenomenon is characterized by the speed with which the onset of dying becomes
apparent and RCD takes over the fate of the cell. The initiation of RCD can be detected
within two minutes - the time interval between frames.
The observed RCD can be divided into two distinct categories: flat cell death and
cluster cell death. Flat cell death (see figure 7) occurs to what the present study terms flat
cells -flat, thin, roughly-circular cells with broad, thin lamellipodia spread out along the
entire circumference. This type of RCD is characterized by the retraction of all processes
and an implosion into a round, compact form. Flat cells that die in this manner are in
contact with either the neurites or the soma of non-flat cells. The retraction that occurs
upon death is often observed to pull at attached neurites and cell bodies. Four instances of
flat cell death was observed: three in one SNAP experiment and one in a PBS control.
The second type of death, cluster cell death, occurs to undifferentiated cells growing in a
cloning group (see figure 8). Undifferentiated cells are defined here to be cells without
processes. This type of death is characterized by a sudden bursting and bubbling, followed
by continuous blebbing until the end of the time-lapse series In addition, it was found that
one cluster cell death was often followed by other cluster cell deaths within the same
group. Examples of cluster cell death were found in one SNAP, one Spermine NONOate,
and one DEANO experiment.
DISCUSSION
Rapid cell death
Due to the low frequency of rapid cell death, no conclusions can be drawn as to
the trends that may govern its occurrence. It is possible that the two morphologically
distinct types of RCD are governed by different mechanisms. Since it appears that only flat
cells and undifferentiated cluster cells are subject to RCD, it is conceivable that those
particular cell types possess or lack a certain quality, rendering them more susceptible.
This quality may be correlated with the developmental stage, environment, treatment,
and/or intercellular interactions
That one cluster cell death often triggers other similar deaths in the same group
suggests that there is intercellular communication between cells of the same cloning
cluster. This communication may occur via gap junctions. It is also possible that the cell
contents released by one cluster cell death triggers cluster cell deaths in susceptible cells in
the surrounding area.
Death of flat cells appears to be dependent on direct contact with the neurites
and/or cell bodies of non-flat cells. This suggests that non-flat cells may cause flat cell
death via a transmitted signal, and that this signal can travel through neurite and soma
membranes.
Concentration dependence of NO
The three NO donors release nitric oxide for different lengths of time, at varied
concentrations. DEANO, with a half life of 2.1 minutes, releases high concentrations of
NO for a short period of time. DEANO will have released most of its nitric oxide after 15
minutes. Spermine NONOate, with a half life of 39 minutes, releases lower concentrations
of nitric oxide for a longer period of time. The rate of release quickly plateaus, resulting
in a steady, low rate of release. SNAP is also a long term donor, releasing even lower
levels of nitric oxide for an even longer period of time
Since different results were obtained using Sperno and SNAP, although both are
slow-release molecules, this suggests that the effects of nitric oxide are concentration
dependent. SNAP, consistently releasing lower levels of NO, inhibits the extension of
thin, branching neurites from lamellipodia. In contrast, Sperno, releasing higher levels of
nitric oxide, inhibits the elongation of the main shaft of processes. That cells grown in
DEANO, the quick-release, high-level NO donor, and Sperno both exhibit the same
changes in growth behaviors suggests that the duration of stimulus is not as crucial as the
concentration of NO release.
Another cholinergic pathway masks effects of nitric oxide
Previous research has demonstrated that nitric oxide production is coupled with
the calcium concentration increase that occurs upon stimulation of MI muscarinic
receptors with carbachol [41. This study has found, however, that cells responding to
carbachol with a calcium increase, and hence producing nitric oxide, exhibit neither the
growth found in cells treated with nitric oxide donors nor that found in control conditions.
This observation, coupled with the fact that carbachol arrested growth even in cells that
did not show a calcium response, suggests that another cholinergic signal is involved in
regulating neurite outgrowth, and that this signal masks the effects of nitric oxide
Further evidence for this hypothesis lies in the effect of cGMP. It is widely
accepted that nitric oxide activates guanylyl cyclase and consequently stimulates the
production of cGMP (Moncada et al., 1991). However, the present study has shown that
cells stimulated with 8-bromo-cGMP, a membrane permeable form of cGMP, demonstrate
neither the growth found in cells treated with nitric oxide donors nor that seen in controls
This suggests that nitric oxide regulates neurite outgrowth through a pathway other than
CGMP.
Previous research has shown that the NIE-115 cell line express other receptors in
addition to the nitric oxide-cGMP coupled MI receptor. Yasuda et al have shown that
NIE-115 cells express 15% MI and 65% M4. The M4 muscarinic receptor is coupled to
the down regulation of cAMP, but not to a calcium response [14 Other research has
demonstrated that intracellular cAMP promotes neurite outgrowth, expansion of
lamellipodia, and motility [1. By this line of thought, stimulation of MA receptors by
carbachol might arrest growth by causing a decrease in intracellular cAMP levels. It has
also been shown that NIE-115 cells express nicotinic receptors [8]. Though not much is
known about these receptors, it is generally accepted that they possess distinct functional
properties. Their activation by carbachol may, in turn, play a role in neurite outgrowth
regulation.
CONCLUSIONS
In studying the role of the cholinergic receptor/nitric oxide/cGMP pathway in
regulating neurite outgrowth of the NIE-115 cell line, it was discovered that cells
stimulated with different nitric oxide donors exhibited varied forms of growth. It was
observed that cells treated with DEANO and Spermine NONOate expanded lamellipodia
and demonstrated maturation of lamellipodia through neurite formation and extension.
Growth of the main shafts of processes, however, was inhibited. Contrarily, it was
observed that in SNAP, the main shafts of processes elongated, generally remained
unbranched, and were thick in diameter. Growth of neurites from lamellipodia, however,
was arrested. It was also observed that both carbachol and cGMP arrested neurite
outgrowth. This study suggests that nitric oxide affects growth through a pathway other
than the cGMP pathway. Evidence suggests that cholinergic stimulation activates
additional pathways that mask the effects of nitric oxide, and that these other pathways
play a role in inhibiting neurite outgrowth.
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FIGURE LEGENDS
Figure 1. Nomarski D.I.C. ÖMDR images showing neurite outgrowth in PBS. A: An
image taken at start of PBS control. The cells possessed lamellipodia (a, b, c, d), as well
as neurites without lamellipodia. B: After two hours of PBS treatment, the cells showed
significant amount of growth. Neurites initiated and extended from lamellipodia (a, b in
A), lamellipodia expanded (d in A), and processes elongated (e in A). Frequently, entire
lamellipodia developed into fine, highly-branched neurites (c in A). Calibration bar in A,
20 um, and applies to A & B.
Figure 2. DEANO (100 um) inhibits growth of main shafts of processes. A: An image
taken at start of DEANO stimulation. B: Two hours after stimulation, lamellipodia had
initiated and extended thin, branched neurites (a in A). The main shafts of processes,
however, remained the same length (b in A). Calibration bar in A, 20 um, and applies to
A & B.
Figure 3. Spermine NONOate (100 um) inhibits growth of main shafts of processes. A:
An image taken at start of Spermine NONOate stimulation. B: Two hours after
stimulation, lamellipodia had initiated and extended neurites (a, b in A). The main shafts
of processes, however, did not elongate (c in A). Calibration bar in A, 20 um, and applies
to A & B.
Figure 4. SNAP (100 um) inhibits the growth of neurites from lamellipodia. A: An image
taken at start of SNAP stimulation. B: Two hours after stimulation, the main shafts of
processes had elongated while remaining unbranched and thick in diameter (arrows in A).
No neurite growth from lamellipodia is observed. Calibration bar in A, 20 um, and applies
to A & B.
Figure 5. Carbachol (1 mM during first 15 minutes of stimulation, 20 uM constant
perfusion afterwards) arrests growth. A: An image taken at start of carbachol stimulation.
B: Two hours after stimulation, no significant neurite outgrowth was observed.
Calibration bar in A, 20 um, and applies to A & B.
Figure 6. Cyclic GMP (1 mM during first 15 minutes of stimulation, 20 uM constant
perfusion afterwards) arrests growth. A: An image taken at start of cGMP stimulation. B:
Two hours after stimulation, no significant neurite outgrowth was observed. Calibration
bar in A, 20 um, and applies to A & B.
Figure 7. Flat cell death in PBS. A: An image taken at the start of PBS control: a and b
point to neurites of neighboring non-flat cells, c points to the cell body of a flat cell. B:
After 90 minutes of growth in PBS, neurites of neighboring non-flat cells (a, b in A) had
grown to make contact with the lamellipodia ruffle of the flat cell. C: After two hours of
growth in control conditions, the flat cell was dead. Note the characteristic compact,
round shape of a dead flat cell. Calibration bar in A, 20 um, and applies to A-C.
Figure 8. Cluster cell death in DEANO. A-B: Initiation of cluster cell death in cell 1 is
evident within 2 minutes (images A and B were taken two minutes apart, at the start of
DEANO stimulation). B-D: Progression of cluster cell death in cell 1 for the next 30
minutes, marked by bubbling and blebbing. E: Initiation of cluster cell death in cell 2, also
marked by explosive bubbling. E-F: Progression of cluster cell death in both cells until the
end of the two-hour time-lapse. Calibration bar in A, 20 um, and applies to A-F.
FIGURES


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