Second messenger mechanisms of neuronal activities:
cGMP-mediated neurite initiation and contact-related cell death
in NIE-115 neuroblastoma cells
Erin J. Gourley
Biology 175H
Professor: Dr. Stuart Thompson
June 4, 1993
In NIE-115 neuroblastoma cells, observations made with time-lapse photography
under bright field microscopy provide insights into the molecular mechanisms behind two
distinct phenomena: neurite initiation and cell death. A statistically significant increase in
the incidence of neurite initiation occurs upon elevation of intracellular concentrations of
CGMP by use of a membrane permeant derivative: dibutyryl cGMP. Data are consistent
with a model describing cGMP production by nitric oxide activation of guanylyl cyclase as
at least in part responsible for cGMP-mediated neurite initiation both in the absence and
presence of carbachol stimulation
The neuroblastoma cells exhibit a novel form of neuronal death when exposed to
carbachol. Deaths occur upon contact of a growing neurite from one cell onto the soma of
another; in some instances the cell that dies is the target of the "contacting neurite", while in
others the owner of the contacting neurite dies. A proposal is offered concerning the
involvement of nitric oxide in this event.
The molecular events underlying the establishment of neuronal contacts during the
development of the nervous system are a fundamental topic in neuroscience. One guiding
principal in this field is that the shaping of the nervous system often follows a general
pattern of excessive growth accompanied by a pruning process that is implemented on a
variety of levels. This work describes observations on the process of synaptogenesis
(neurite initiation) and the pruning (cell death) process that leads to the final pattern of
connections.
A main focus of research on neurite initiation has been the involvement of
intracellular Ca24 concentration changes. The collective data are inconsistent, some cells
exhibit no Ca2+ dependence2, while others require influx11. It appears that Ca2+ is
important, although optimum Ca2+ levels above and below which neurite initiation will not
occur may be specific to each cell type. In NIE-115 neuroblastoma cell, Audesirk, et al.1
present data indicating modulation of neurite initiation by Ca2+ influx through L-type
channels. One of the main actions of cytoplasmic Ca2+ is activation of nitric oxide
synthase, which produces nitric oxide to stimulate cGMP formation through activation of
guanylyl cyclase. This study examines the effects of membrane-permeant derivatives of
Gourley, p. 2
CGMP, and cholinergic stimulation of a molecular cascade that begins with the activation of
phospholipase C and leads to CGMP production.
Investigations of physiological cell deletion and neurodegenerative disorders
indicate the involvement of processes like competition for trophic factors and programmed
cell death. This work describes a new potential mechanism behind these phenomena
observed under cholinergic stimulation. With the assumption that the events that occur in
the neuroblastoma cells are representative of those in neuronal systems, contact-related cell
death might be a new method of neuron elimination.
MATERIALS AND METHODS
Solutions
Normal External saline: 146 mM NaCl, 5.4 mM KCl, 1.8mM CaCly, 0.8 mM
MgSO4, 0.4 mM KH2PO4, 0.3 mM NazHPO4, 5 mM glucose, and 20 mM HEPES, pH
7.4.
All chemical solutions used were made by dilutiing the chemical into the NE saline.
Cell Culture and Differentiation
NIE-115 mouse neuroblastoma cell line was obtained from Department of Tissue
Culture at UCSF. The cells were passaged in Dulbecco's minimum essential medium
(DMEM) with 10% fetal bovine serum from Hyclone. Chips made from glass microscope
slides were added to the Falcon polystyrene tissue culture plates as the cells only adhere to
glass and the chips could be easily removed for cell observation and manipulation under the
microscope. Cells were maintained under humidity incubation with 90% air, 10% CO2 at
37°C. When they reached approximately 60% confluency, they were differentiated by
culturing in 2% dimethylsulfoxide (DMSO) as described by Kimhi, et als. All cells were
either fourth or sixth passage, and had been under 2% DMSO for a minimum of 12 days.
Gourley, p. 3
Recordings and Analysis
Immediately preceding each experiment, a glass chip was selected from the plates in
the incubator, rinsed with NE saline and transferred into a well that fits into the stage of the
microscope. The chip was secured onto the floor of the well by using a syringe to dot the
edges with hot petroleum jelly and the chamber was filled with 0.5 ml NE Saline. Äfter
selecting a field that contained neurons with and without processes, the cells were imaged
with a 40X bright field objective, Nomarski prism, and Hammatsu SIT camera. Frames
were taken at 60 second intervals and the images stored on optical disc. . Ten minutes of
incubation in NE saline were recorded for each experiment before the chamber was rinsed
and perfused with the appropriate one of the following solutions: 1 mM carbachol, ImM 8-
bromo-cAMP, 1 mM 8-bromo-cGMP, ImM dibutyryl CGMP, 1 UM NÉMM-L-arginine,
HM NéMM-D-arginine, or 1 UM NéMM-L-arginine/1 mM carbachol. All experiments
involved 2 hours of recording after perfusion with the appropriate solution, with the
exception of those using either of the N6MM-arginine isomers, which required an
additional hour of recording to take into account slow uptake and incorporation of the
amino acid derivatives by the cells. In the case of the coincubation with carbachol and
NéMM-L-arginine, the cells were incubated in 1 uM NéMM-L-arginine for one hour prior
to recording, recorded for 10 minutes in this solution, then perfused with the
carbachol/Né MM-L-arginine solution and recorded for the next two hours. Controls were
recorded for 130 minutes under NE saline.
Data Analysis
The incidence of new neurites was measured in each experiment by determining
which of the neurites present in the frame taken 120 minutes after perfusion were not
visible in the first frame after perfusion. The binomial test was used to determine statistical
significance in the analysis of the proportion of cells with new growth. No such function
could be performed in the analysis comparing number of new neurites to number of cells in
the field as the data collection for many experiments involved only a single field.
Gourley, p. 4
RESULTS
The neurite growth expressed as the average number of projections initiated per cell
in the field during a two hour incubation in each of the four given chemical treatments is
shown in Figure 1. A new neurite was defined as any projection that 1) was present in the
final recorded frame while undetectable in the first frame after chemical introduction, 2)
originated directly from the cell body, and 3) had a length-to-base ratio greater than unity
(to distinguish from lamellipodia). Incubations in ImM carbachol, ImM 8-bromo-cAMP,
ImM 8-bromo-cGMP, and ImM dibutyryl-cGMP all exhibited elevated incidence of
neurite initiation relative to the NE saline control, dibutyryl cGMP causes the most dramatic
contrast with greater than ten-fold increase above control. Figure 2 presents the results of
coincubation with ImM carbachol and IuM Né-monomethyl-L-arginine, compared against
controls with each isomer of the Né-MM-arginine derivatives, NE saline, and ImM
carbachol. Both the N°-MM-L-Arg control and the coincubation have an incidence lower
than NE saline.
Comparisons of the percentage of cells exhibiting new growth are shown in Figure
3. A statistically significant elevation in this percentage is found with the cells incubated
with dibutyryl CGMP.
In four of the twelve assays, what I describe as “death behavior" occurred during
the course of the assay. The percent of cells dying in each of these assays is given in
Figure 4. The phenomenon is characterized by bubbling and expansion of the cell body
followed by contraction of processes, and finally terminal dormancy with a shriveled
appearance. In all assays except Ne-MM-D-arginine, at least one incidence of cell death
appeared to be contact related. The one cell death observed in Carbachol 1 was coincident
with an initial contact by a neurite from another cell. The same occurred for one of the
three deaths observed in Carbachol 2; while no such contact was visible in the other two
cases, these latter two initiated the death behavior simultaneously, as determined by frame-
Gourley, p. 5
by-frame analysis of when the bubbling begins. In the case of the first cell death in
Carb/N6-MM-L-Arg, the dying cell was the one growing the process whose contact with
the soma of a second neuron appeared to be coincident with initiation of its death. The
other two deaths in this assay occurred simultaneously when a new neurite growing from
what I will call neuron A contacted neuron B, who already shared contacts with neuron C;
neurons A and C died while B appeared to remain viable.
DISCUSSION
Neurite Initiation
The 10-fold increase in the incidence of neurite initiation and the statistically
significant 4.5 fold increase in the proportion of cells with new neurites in the dibutyryl-
cGMP incubation are particularly interesting in light of the observation that a wide variety
of neurotransmitters elicit substantial elevations in cytoplasmic levels of cGMP7. Coupled
with this observation, the results here are consistent with a model that describes cGMP as a
molecule that links incoming chemical information to the development of processes used to
collect such information. This model of cGMP as the transducer between neurotransmitter
stimulation and neurite initiation can be extended to speculate that each different
neurotransmitter may coactivate additional cascades and/or use different pathways to elicit
this elevation in cGMP, and other elements of these pathways could determine the nuances
of a specific neurotransmitter's effect. Acting in concert with the general action of cGMP's
induction of neurites, the elements of the specific pathway used to induce cGMP elevation
might fine tune the neuron's response, i.e. by determining the degree of branching in the
neurites initiated or the receptors to be expressed in the neurite membrane.
The results of the experiments presented in Figure 2 illustrate that the elevation in
the incidence of new neurites under incubation in 1 mM carbachol is prevented when 1 uM
No-MM-L-arginine is also present. 1 HM N6-MM-L-arginine alone lowers neurite initiation
below that of the control while the biologically inactive isomer, 1 uM N6-MM-D-arginine,
Gourley, p. 6
elicits no such change. Since the L-arginine derivative acts as an competitive inhibitor of
nitric oxide synthase, and thus blocks the production of CGMP by nitric oxide, the data are
consistent with a model that describes cGMP production by nitric oxide as at least in part
responsible for the cGMP-mediated initiation of neurites in unstimulated cells, as well as
cells under MI muscarinic stimulation.
Further experiments are necessary to assign statistical significance to the analysis of
the incidence of new neurites with respect to the number of cells in the field. Additionally,
assays employing different methods to block the carbachol pathway would be more
informative, perhaps using carbachol with irreversible muscarinic antagonists or blocking
agents more specific to nitric oxide synthase than the N6-MM-L-arginine used here. Since
this derivative should in some way affect all systems requiring the natural amino acid,
analysis of its effects are problematic and can not be conclusively interpreted in isolation
from all these other systems. However, from the statistically significant increase in the
proportion of cells growing new neurites under dibutyryl-cGMP it can be concluded that
elevations in the intracellular levels of cGMP stimulate growth of new neurites in NIE-115
neuroblastoma cells.
These results suggest the potential significance of cGMP as a second messenger in
the development and plasticity of neuronal networks. Investigating the correlation between
cGMP levels and neurite initiation in actual brain and nervous tissues is the next critical step
in determining just how universal this role of cGMP is in actual nervous systems. Such
studies would be particularly interesting in the hippocampus in light of observations of
dramatic elevations in intracellular cGMP levels correlated with the induction of long-term
potentiation (LTP) - a model of learning and memory at the cellular level - in hippocampal
CA-1 cells. Finding a correlation of cGMP elevations with neurite initiation in the CA¬
cells would establish cGMP as a key messenger behind the long-term structural changes
characteristic of LTP.
Gourley, p. 7
Cell death
Attempts to assign responsibility for the contact-related cell deaths observed here
find membrane permeant molecules among the most attractive suspects. In some cases,
contact-related death occurs in cells possessing the neurite that makes the contact coincident
with the death behavior, while in others, cells that receive the contact die. It seems unlikely
that the growing neurite would express the receptors at its tip that would be necessary for a
neurotransmitter-mediated communication causing the death of the neurite containing cell,
although such a mechanism cannot be altogether ruled out. Nitric oxide (NÖ) is one such
diffusable molecule which has potential for cytotoxicity through reaction with superoxide
(O2-) to form peroxynitrite. Radi et al.8 report that potentially toxic levels of peroxynitrite
can be achieved in tissues under conditions when -NO and O2- production are stimulated,
due to a 100-fold increase in peroxynitrite for every 10-fold increase in -NO and O2
concentration. Since all of the contact-related deaths observed occurred in experiments
involving carbachol, carbachol-stimulated increase in -NO production would be an
appealing model, were it not for the observation of contact deaths in the coincubation with
the N°-MM-L-arginine derivative that should inhibit-NO production. However this result
does not rule out nitric oxide involvement in the incidents as any disruption of normal-NO
production could be detrimental to the cells through disturbance of the regulation of
enzymes involved in -NO production or quenching. For example, depression of -NO
production could cause an upregulation of the production of NO synthase that
overcompensates the competitive inhibition of the enzyme due to N6-MM-L-arginine, and
results in a net increase in the amount of -NÖ produced. Alternatively, the carbachol
stimulation could in some way be affecting the expression or activity of superoxide
dismutase or other antioxidants that protect the neurons from-NO toxicity.
Further experiments are necessary to better characterize the contact-related deaths
that have been observed; investigations of particular interest include incubations in other
cholinergic agonists and agents that manipulate -NO levels. This behavior may be specific
Gourley, p. 8
to the neuroblastoma cell line, which could be overexpressing inducible NO synthase or
underexpressing endogenous antioxidants. On the other hand, these NIE cells could be
exhibiting a natural mechanism utilized in nervous tissues for the process of selective
neuronal elimination. Programmed cell death and competition for synapses and trophic
factors are among the most favored models explored in studies of physiological neuron
deletion seen in development and neurological disorders. With a better characterization of
the contact-related cell deaths in the NIE-115 neuroblastoma cell line, investigations in
actual brain tissue may find this phenomenon to be a revealing new mechanism of neuronal
death that participates in the shaping of nervous systems or the pathogenesis of
neurodegenerative diseases.
Gourley, p. 9
REFERENCES
Audesirk, G., Audesirk, T., Ferguson, C., Lomme, M., Shugarts, D., Rosack, J.,
Caracciolo, P., Gisi, T., and Nichols, P., L-Type calcium channels may regulate
neurite initiation in cultured chick embryo brain neurons and NIE-115
neuroblastoma cells, Developmental Brain Research, 55 (1990) 109-120.
2Bixby, J.L. and Spitzer, N.C., Early differentiation of vertebrate spinal neurons in the
absence of voltage-dependent Ca2+ and Nat influx, Developmental Biology, 106
(1984) 89-96.
Bredt, D.S., and Snyder, S.H., Nitric oxide, a novel neuronal messenger, Neuron, 8
(1992) 3-11.
4Heintz, N., Cell death and the cell cycle: a relationship between transformation and
neurodegeneration? TIBS,18 (1993) 157-159.
»Kimhi, Y., Palfrey, C., Spector, I., Barak, Y., and Littauer, U.Z., Maturation of
neuroblastoma cells in the presence of dimethylsulfoxide, Procedings of the
National Acadamy of Science USA, 73, 2 (1976) 462-466.
6Mathes, C., Wang, S. S.-H., Vargas, H.M., and Thompson, S. H., Intracellular calcium
release in NIE-115 neuroblastoma cells is mediated by the MI muscarinic receptoi
subtype and is antagonized by MCN-A-343, Brain Research, 585 (1992) 307-310.
"Matsuzawa, H., and Nirenberg, M., Receptor-mediated shifts in cGMP and cAMP levels
in neuroblastoma cells, Procedings of the National Acadamy of Science USA, 72,9
(1975) 3472-3476.
8Radi, R., Beckman, J.S., Bush, K.M., and Freeman, B.A., Peroxynitrite Oxidation of
Sulfhydryls: The cytotoxic potential of superoxide and nitric oxide, Journal of
Biological Chemistry, 266, 7 (1991) 4244-4250.
9Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Forstermann, U., and Murad, F..
Ca21/calmodulin-regulated nitric oxide synthases, Cell Calcium,13 (1992) 427-434.
10Schubert, D., Humphreys, S., Baroni, C., and Cohn, M., In vitro differentiation of a
mouse neuroblastoma, Biochemistry, 64 (1969) 316-323.
11Suarez-Isla, B.A., Pelto, D.J., Thomson, J.M. and Rapoport, S.I., Blockers of calcium
permeability inhibit neurite extension and formation of neuromuscular synapses in
cell culture, Developmental Brain Research, 14 (1984) 263-270.
Gourley, p. 10
LENGENDS
Figure 1. Number of new neurites per cell in field under various chemical treatments.
Range of field size: 11-22 cells. NE Saline: incubation in normal external saline,
number of experiments (n) -3; Carbachol: incubation in ImM carbachol, n-2;
cAMP: incubation in ImM 8-bromo-cAMP, n-2; br CGMP: incubation in ImM
8-bromo-cGMP, n=1; db CGMP: incubation in ImM dibutyryl-CGMP, n=1.
Figure 2. Number of new neurites per cell in field under treatments including carbachol
and arginine derivatives. Range of field size: 19-35 cells. NE Saline: incubation
in normal external saline, number of experiments (n) = 3; D-Arg: incubation in
IUM N6-monomethyl-D-arginine, n=1; L-Arg: incubation in IuM N6¬
monomethyl-L-arginine, n-1; Carb/L-Arg: incubation in ImM carbachol, IuM
No-monomethyl-L-arginine, n=1; Carbachol: incubation in ImM carbachol, n-2.
Figure 3. Percent of cells in field possesing new neurites under various chemical
treatments. Incubations as described in figure 1 legend, control = NE saline and
CBC = Carbachol. Error bars acheived through bionomial test.
Figure 4. Percent of cells in field that die during course of 2-hour observation under the
respective chemical treatments; data does not distinguish whether deaths were
contact related. Carbachol 1 and 2 are the two experiments grouped as
Carbachol in all other firgures. All others (n-8) refers to the following
previously described experiments: NE saline, n-2; cAMP, n=1; db CGMP, n=1;
br CGMP, n=1; and L-Arg, n=1.
1.7 -
1.6 -
1.5
1.4
1.3 -
1.1
0.9 -
0.8 -
0.7 -
O.6 -
0.5
0.4
0.3 -
0.2
0.1
0.4
0.35
0.3
0.25 -
0.2-
0.15
005
Effects of Chemicals
on Neurite
lnittot
Corbachol
NE Soline
br CGMF
Figure 1.
Effects of Carbachol & Arg Derivatives
initiot
NE Soline
DArg
LAn
Card/L-Arg
Figure 2.
dD CGMP
Corbachol
Percent
cells
with
new
neurites
Control CAMP CBC dBCGMP 8-BrCGMP
Figure 3.
Cell Deaths Observed
100
90 -
80 -
70
60 -
30 -
20 -
10
Q -
All others (n=8) Corbochol 1
Corbochol 2 Corb/1-Arg
Figure 4.
D-Arg