Osmotic and glutamate receptor regulation of
c-Jun NH2-terminal protein
kinase in neuroendocrine cells
Rick
Meeker and
Alda
Fernandes
Department of Neurology, University of North Carolina, Chapel Hill,
North Carolina 27599
 |
ABSTRACT |
Expression of a c-Jun NH2-terminal protein kinase
(JNK), also known as stress-activated protein kinase (SAPK) in rodents, has been implicated in the ability of cells to respond to a variety of
stressors. In nonmammalian cells, JNK participates in the regulation of
cell volume in response to hyperosmotic stress. To explore the
possibility that JNK may participate in the transduction of osmotic
information in mammals, we evaluated the expression of JNK
immunoreactivity in neuroendocrine cells of the supraoptic nucleus. Low
basal expression of JNK-2 (SAPK-
) and JNK-3 (SAPK-
) was seen in
vivo and in vitro. During water deprivation, JNK-2 increased in the
supraoptic nucleus but not in the cortex. Osmotic or glutamate receptor
stimulation in vitro also resulted in an increase in JNK-2 that was
tetrodotoxin (TTX) insensitive and paralleled by increased nuclear
phospho-c-Jun immunoreactivity. A TTX-sensitive increase in JNK-3 was
seen in smaller neurons. Thus different JNK pathways may mediate
individual cellular responses to osmotic stress, with JNK-2 linked to
osmotic and glutamate receptor stimulation in magnocellular
neuroendocrine cells.
stress-activated protein kinase; vasopressin; oxytocin; rat; brain
 |
INTRODUCTION |
EXPRESSION of a
c-Jun NH2-terminal protein kinase (JNK), also known as
stress-activated protein kinase (SAPK) in rodents, has been implicated
in the ability of cells to respond to a variety of stressors
(6, 8, 11, 17,
26). For example, the role of JNK in the signal
transduction cascade that leads to apoptosis has been extensively
investigated (12, 16). However, evidence also
suggests that JNK may have a number of physiological roles unrelated to
cell death (4, 8). One physiological role of JNK that appears to have been highly conserved in the evolution from
single cell to higher organisms is the regulation of cell volume in
response to a hyperosmotic stress. In single-cell organisms, expression
of the JNK analog HOG-1 has been shown to be crucial for survival of
cells in hypertonic medium (8). Yeast cell mutants lacking
this gene fail to grow in response to osmotic stress induced by raising
the NaCl concentration to 0.9 M (8). The human gene,
JNK-1, was shown to rescue these cells, indicating that the protein has
highly conserved physiological properties (8).
In multicellular organisms, physiological systems have evolved to
control the extracellular ionic environment, thereby minimizing the
impact of unfavorable external environments. However, the high level of
functional conservation of the JNK protein suggests that higher
organisms could utilize this stress response to signal homeostatic
mechanisms that regulate extracellular osmolality. Hypothalamic
magnocellular neuroendocrine neurons, which specialize in the detection
and response to changes in extracellular osmolality (2),
may take advantage of this conserved regulatory response for the
control of body fluid balance. These cells respond to increases in
extracellular osmolality by virtue of intrinsic osmoreceptors (23) and extrinsic excitatory synaptic inputs originating
from remote osmosensitive cells (3). The intrinsic sensors
are thought to be mechanoreceptors responding to changes in cell volume
(23), whereas the extrinsic inputs are thought to be
largely due to the activation of postsynaptic glutamate receptors
(9, 14). In particular, the characteristic
bursting patterns that facilitate vasopressin secretion appear to be
highly dependent on the activation of
N-methyl-D-aspartic acid (NMDA) glutamate
receptors (15). However, the signal transduction processes
that follow osmotic and NMDA receptor stimulation and regulate cell
activation and vasopressin synthesis are still poorly understood. The
transcription factors c-fos and c-jun are both
increased in response to osmotic stimulation (13,
27) and have been excellent candidates for linking
cellular activity to transcriptional regulation. However, experiments
designed to correlate c-Fos protein levels with transcriptional regulation are complicated by the divergent temporal patterns for each
of these responses and the complex role of phosphorylation (1), a key step in transcription factor activation.
Because JNK is a major enzyme for the phosphorylation of c-Jun
(6, 17) and is widely expressed in rat brain
(4), it could serve as an essential regulatory step for
the control of transcriptionally active c-Fos/c-Jun heterodimers. To
begin to explore the possibility that JNK could be an important step in
the intracellular transduction of osmotic information, we evaluated the
expression of JNK immunoreactivity in the neuroendocrine cells of the
supraoptic nucleus (SON) of the hypothalamus. The following studies
illustrate that magnocellular neuroendocrine cells express low but
significant basal expression of both
- and
-isoforms of the
enzyme (SAPK-
/JNK-2 and SAPK-
/JNK-3) in vivo and in vitro and
that this expression increases with osmotic or glutamate receptor stimulation.
 |
MATERIALS AND METHODS |
Animals.
For the analysis of the effects of water deprivation on JNK
immunoreactivity, Long-Evans rats in each experiment were split into
two equal groups. One-half of the rats were deprived of water for
44 h starting at 1400, and the other one-half were fed
water ad libitum. This deprivation interval produces a maximal increase in vasopressin mRNA (18) and therefore would be expected
to correspond to an active period of transcriptional activity. At the
end of the deprivation interval, the rats were rapidly and deeply
anesthetized in their home cage with an isotonic preparation of
pentobarbital sodium (60 mg/kg of a 40 mg/ml solution) designed to
minimize environmental and osmotic stress. Each rat brain was then
perfused with a balanced salt solution followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Sections were cut at a thickness of
30 µm on a vibrating microtome, and alternate sections were stained
for SAPK-
/JNK-2 and SAPK-
/JNK-3 (the isoforms predominantly expressed in adult rat brain).
JNK immunohistochemistry.
The tissue was rinsed three times in 0.01 M PBS, pH 7.4, and incubated
in 0.6% H2O2 for 15 min. After three rinses in
PBS (5 min each), the tissue was incubated for 1 h in PBS
containing 3% normal goat serum (NGS). The tissue was then incubated
with rabbit antirat SAPK-
/JNK-2 (1:1,000, Upstate Biotechnology,
Lake Placid, NY), SAPK-
/JNK-3 (1:2,000 Upstate Biotechnology), or mouse anti-phospho-c-Jun (1:2,000, Santa Cruz, Santa Cruz, CA) for
24 h at 4°C. These concentrations were chosen to give a moderate basal signal with low background on the basis of initial studies that
examined dilutions ranging from 1:500 to 1:5,000. This allowed analysis
of increases or decreases in enzyme immunoreactivity, particularly in
the in vitro studies. On the next day, the tissue was rinsed three
times in PBS and incubated for 1 h in biotinylated goat
anti-rabbit (anti-mouse for the phospho-c-Jun antibody) IgG (1:200) in
PBS with 3% NGS. The secondary antibody was washed from the tissue and
the antigen was visualized using ABC reagent, with diaminobenzidine
(0.5 mg/ml) as substrate, and 0.01% H2O2.
Primary cultures of magnocellular neuroendocrine cells.
Punch cultures were prepared from fetuses harvested at embryonic
day 16-17 from pregnant female
Long-Evans rats under deep isoflurane anesthesia. After removal from
the uterus, the fetuses were transferred to a 60-mm dish containing
fresh HEPES-buffered Hanks' balanced salt solution (HBSS, pH 7.4) at
room temperature. The brain was dissected from each fetus, placed into
fresh sterile HBSS, and washed three times. The brains were then
transferred to complete culture medium (MEM) with glutamine and sodium
bicarbonate (GIBCO/BRL, Grand Island, NY) + 10% fetal bovine
serum + 20 µg/ml gentamicin. Punches through the region of the
SON were taken from the whole brain under a dissecting microscope, with
the bifurcation of the middle cerebral artery from the circle of Willis
as a marker for the general location of the SON. A blunt 23-gauge
needle with sharpened edges connected to a 1-ml syringe was used to
extract the tissue. The brains were kept totally immersed in culture
medium throughout the punching procedure. The core of tissue
("punch") was placed onto 25-mm round glass coverslips previously
cleaned and coated with 0.1 mg/ml poly-D-lysine. The
punches often fragmented into smaller pieces, providing several small
foci of cell growth on the coverslip. Each dish was placed in a
humidified incubator maintained at 36°C and 5% CO2, with
just enough medium to wet the tissue. Viable punches attached loosely
to the coverslip within 1-2 h. Additional medium was added to
cover and feed the tissue after 2-4 h. Cells were fed every
2-3 days with 50% medium exchange. Neurons migrated from the
punch, resulting in a field of neurons from the region of the SON that
could easily be stained and individually analyzed. Coverslips with
well-developed magnocellular neurons were used after 12-22 days in culture.
Data analysis.
The density of regional staining in the SON and single cell staining in
cultured neurons was analyzed semiquantitatively with a Bioquant Image
Analysis System (R & M Biometrics). Controls and experimental
conditions were always run at the same time under identical conditions.
Mean gray level values were calculated for the entire SON at a final
magnification of 424× or for single cellular perikarya at a
magnification of 1,220×. The gray level value was converted to optical
density (OD), and the ODs were averaged across all of the supraoptic
nuclei or cells in each condition.
 |
RESULTS |
JNK expression in vivo.
Both JNK-2 (SAPK-
) and JNK-3 (SAPK-
) showed weak-to-moderate
basal levels of immunoreactivity throughout the brain. In the hypothalamus, JNK-2 showed weak or moderate levels of staining, localized principally to the SON (Fig.
1A). Cells scattered through the ventral anterior hypothalamus, suprachiasmatic nucleus, and periventricular regions were also lightly stained (not shown). In the
dorsal frontoparietal cortex (Fig. 1C), stained cells were localized to the pyramidal cells in layer 5 (arrow), with
lighter staining in layers 2 and 3. Moderate
staining was also seen in the piriform cortex (arrow) and in cells
scattered through the amygdala (Fig. 1E, arrowhead).

View larger version (195K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of c-Jun NH2-terminal protein
kinase (JNK)-2 [stress-activated protein kinase (SAPK)- ] and JNK-3
(SAPK- ) immunoreactivity in rat supraoptic nucleus (SON;
A, B), dorsal cortex (C,
D), and piriform cortex/amygdala (E,
F). The SON had the highest staining in the hypothalamus for
both JNK isoforms. JNK-2 staining in the SON was generally moderate in
cells scattered throughout the nucleus (arrowheads). JNK-3 staining in
the SON was very light, with a few cells occasionally showing staining
just greater than background (arrowheads). Inset: background
staining in the SON adjacent to the optic chiasm (OC) in the absence of
primary antibody. Contrast has been enhanced to improve visualization
of the tissue. Magnocellular neurons (arrowheads) show no staining
under these conditions. Arrows show the location of the base of the
brain and help to illustrate that the control tissue has approximately
the same density as the region below the brain with no tissue present.
In the pyramidal cells of the dorsal cortex, particularly layer
5 (C, arrow) or the piriform cortex (E,
arrow), JNK-2 staining was moderate. JNK-3 staining in the cortex
(D, F) had a similar distribution to JNK-2 but
was generally more robust, with staining readily visible in
layers 2, 3, and 5 (D, arrows) of the
dorsal cortex and in the piriform cortex (F, arrow).
Calibration bars: A, B (right) = 100 µm; C, D = 500 µm; E,
F = 500 µm.
|
|
Immunoreactivity for JNK-3 in the hypothalamus was lighter than for
JNK-2 but had a similar distribution. Very light staining was generally
seen in neuroendocrine cells in the SON (Fig. 1B, arrowheads), periventricular regions, and the suprachiasmatic nucleus
(not shown). In the cortex and amygdala, staining was seen for both
JNK-2 (Fig. 1, C and E) and JNK-3 (Fig. 1,
D and F), although staining was generally more
robust for JNK-3. Individual cells were stained throughout the
pyramidal cells in layers 2, 3, and 5 of the
dorsal frontoparietal cortex (Fig. 1D, arrows) and within
the piriform cortex (Fig. 1F, arrow) and amygdala (Fig. 1F, arrowhead). Other regions with light to moderate
staining (not shown) included the hippocampus and paraventricular nucleus.
To test the ability of water deprivation to induce the expression of
JNK in the SON, rats were deprived of water for 44 h, and the
immunoreactivity for JNK-2 and JNK-3 in the SON was assessed under
carefully matched conditions. Water deprivation resulted in a
significant 40% increase (t =
2.82, P = 0.030, n = 7 pairs) in the staining intensity of JNK-2
and a larger but more variable 80% increase (t =
2.08,
P = 0.076, n = 8 pairs) in the staining of JNK-3 in the SON (Fig. 2). The
increase in JNK-2 staining in response to water deprivation (Fig.
3B) was due to more extensive and intense cellular immunoreactivity relative to controls (Fig. 3A). Although increased nuclear staining for JNK-3 was
apparent in a few cases (Fig. 3D), it was not a consistent
finding. Controls stained in the absence of primary antibody were
devoid of immunoreactivity. In addition to the neuronal stain, an
increase in JNK-2 immunoreactivity could be seen associated with the
ventral glial layer at the base of the SON (small arrows).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Optical density readings from the SON of rats deprived of
water for 44 h (dehyd.), relative to matched controls with free
access to water. Water deprivation resulted in a significant increase
in the staining of JNK-2 (* P = 0.030) and a more
variable increase in JNK-3 that just failed to reach significance
(P = 0.076).
|
|

View larger version (123K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of JNK-2 (A, B) and
JNK-3 (C, D) immunoreactivity within the SON of
control rats (A, C) or rats deprived of water for
44 h (B, D). In controls, scattered
magnocellular neurons were moderately stained for JNK-2 (A).
A more widespread and intense stain for JNK-2 was seen after water
deprivation (B). Very weak immunoreactivity for JNK-3 was
seen in some neuroendocrine cells in the SON (C). In a few
cases, such as the case illustrated in D, an increase in
nuclear staining for JNK-3 was apparent in the ventral SON (region rich
in vasopressin cells), but this was not a consistent finding. In
addition, increased immunoreactivity can be seen in the regions
surrounding the SON, suggesting a more general and widespread increase
in JNK-3 expression.
|
|
Neurons in other regions of the brain, such as the cortex, also
exhibited small increases in immunoreactivity, indicating that
increased JNK expression is not exclusively associated with neuroendocrine cells. However, the increase was small and restricted to
JNK-3 expression. An example of JNK-2 and JNK-3 staining in the dorsal
frontoparietal cortex at the level of the SON is illustrated in Fig.
4 for both control and water-deprived
rats. Moderate staining of large pyramidal cells was seen for JNK-2
(Fig. 4A) but did not change significantly after water
deprivation (Fig. 4B). Quantification of the changes in
JNK-2 staining intensity within the pyramidal cells of layers
3-5 demonstrated a 4.6% decrease in mean OD [0.359 ± 0.024 in controls vs. 0.342 ± 0.022 in water deprived; t
= 2.00, n = 7 pairs, not significant (NS)].
Scattered staining of pyramidal cells was also evident for JNK-3 (Fig.
4C), including robust nuclear staining in layers 2, 3, and 5 of the cortex. After water deprivation, a
small but consistent 10.6% increase in JNK-3 staining was seen (0.237 ± 0.029 in controls vs. 0.262 ± 0.028 in water
deprived; t = 2.677, n = 8 pairs,
P < 0.05), which was largely due to an increase in
nuclear staining (Fig. 4D).

View larger version (145K):
[in this window]
[in a new window]
|
Fig. 4.
Example of JNK-2 (A) and JNK-3 (C) staining
in the pyramidal cells of the dorsal cortex in normal rats and in the
cortex of rats deprived of water for 44 h (B,
D). Cortical cells showed equivalent or less staining for
JNK-2 after water deprivation (B), whereas increased JNK-3
staining appeared in pyramidal cell nuclei and cell bodies
(D). Calibration bar (lower right) = 50 µm
for all.
|
|
JNK expression in vitro in response to osmotic and glutamate
receptor stimulation.
To better understand the stimuli that might drive the expression of JNK
in the neuroendocrine cells, we examined the ability of extracellular
hyperosmolality and NMDA or metabotropic glutamate receptor stimulation
to directly increase cellular JNK immunoreactivity in vitro. Cultures
were analyzed after 1 or 24 h of exposure to evaluate acute and
chronic JNK expression. During exposure to 30 mosmol/kg excess NaCl,
cell size decreased acutely within 2 min of exposure and then recovered
to normal over a period of 20 min (Fig.
5). Examples of the results of a 1-h
exposure to 30 mosmol/kg excess NaCl in the presence or absence of NMDA
(50 µM) or to the metabotropic receptor agonist 1S,3R ACPD (100 µM) are illustrated in Fig. 6. Stimulation
with 30 mosmol/kg excess NaCl resulted in an increase in staining
intensity within the perikarya (arrows) and processes (arrowheads) for
both JNK-2 (Fig. 6B) and JNK-3 (Fig. 6D) relative
to untreated controls (Fig. 6, A and C).
Stimulation of the cells with 100 µM 1S,3R ACPD had little effect on
JNK-2 expression (Fig. 6E) and produced only a slight
increase in JNK-3 (Fig. 6G) relative to unstimulated controls (Fig. 6, A and C). Combined stimulation
with 1S,3R ACPD and 30 mosmol/kg excess NaCl resulted in a level of
immunoreactivity that was greater than osmotic stimulation alone for
JNK-2 (Fig. 6F) but not for JNK-3 (Fig. 6H).
Stimulation with 50 µM NMDA alone increased the expression of JNK-2
(Fig. 6I) but had a negligible effect on JNK-3 (Fig.
6K). Combined osmotic and NMDA receptor stimulation resulted
in strong expression of JNK-2 (Fig. 6J) but only a slight
increase in JNK-3 (Fig. 6L). Staining in the absence of the
primary antibody was negligible in each case (Fig. 6, M and
N for JNK-2 and JNK-3, respectively).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Decrease and recovery of neuronal size relative to the
pretreatment cell size after exposure to 30 mosmol/kg excess NaCl. A
significant decrease in cell size was observed within 2 min of
stimulation (t = 4.05, P < 0.01, 13 cells), followed by recovery to baseline by 20 min.
|
|

View larger version (146K):
[in this window]
[in a new window]
|
Fig. 6.
Examples of immunoreactivity for JNK-2 (left 2 columns) and JNK-3 (right 2 columns) in cultured
neuroendocrine cells exposed to vehicle (A, C),
30 mosmol/kg excess NaCl (B, D), 100 µM 1S,3R
ACPD (E, G), 100 µM 1S,3R ACPD + 30 mosmol/kg excess NaCl (F, H), 50 µM
N-methyl-D-aspartic acid (NMDA; I,
K), or 50 µM NMDA + 30 mosmol/kg excess NaCl
(J, L). In all conditions, staining was seen in
neuronal perikarya (arrows) and in proximal dendritic processes
(arrowheads) of magnocellular and other smaller neurons. Untreated
controls (A, C) generally showed a
light-to-medium staining intensity. Moderate increases in mean staining
intensities were seen for cultures treated with hypertonic medium
(B, D). Nuclear staining for JNK-3 was seen in
occasional cells (open arrows), similar to in vivo observations.
Stimulation with 1S,3R ACPD (E, G) had a
negligible effect on JNK-2 expression and increased JNK-3 only
slightly. Combination of osmotic stimulation with 1S,3R ACPD resulted
in staining that was greater than osmotic stimulation alone for JNK-2
(F) but not for JNK-3 (H). NMDA stimulation
produced strong staining for JNK-2 (I) but did not
significantly increase JNK-3 (K). Combined NMDA and osmotic
stimulation resulted in robust JNK-2 immunoreactivity and a small
increase in JNK-3. Some cells showed nuclear staining for JNK-3 when
stimulated with NMDA plus hyperosmotic medium (L, open
arrow), but this was seen in controls and was not a consistent
observation across all cultures. No staining was detected in the
absence of primary antibodies for JNK-2 (M) or JNK-3
(N). Calibration bar (lower right) = 50 µm
for all.
|
|
A summary of the mean neuronal JNK-2 and JNK-3 expression across
all cultures and conditions is provided in Fig.
7. Osmotic stimulation produced a rapid
average increase in JNK-2 immunoreactivity of 33% (t =
8.61, P < 0.002) relative to isosmotic controls. This osmotically induced increase was enhanced when combined with 1S,3R
ACPD (+64%, t =
10.8, P < 0.002)
but not with NMDA. 1S,3R ACPD in the absence of osmotic stimulation
resulted in a small increase in JNK-2 expression (+12%, t =
3.96, P < 0.002), whereas NMDA induced a 36% increase
in JNK-2 (t = 9.96, P < 0.002).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 7.
Summary of changes in neuronal staining for JNK-2 and JNK-3 in
cultured neuroendocrine cells treated with control medium
(C) or hyperosmotic medium (Osm) in the presence and absence
of 100 µM 1S,3R ACPD (ACPD) or 50 µM NMDA. Cultures were fixed with
4% paraformaldehyde 1 h or 24 h after stimulation and were
stained with antibodies to JNK-2 (SAPK- ) or JNK-3 (SAPK- ).
Optical density readings were measured from 50 individual neurons
sampled at random from 3-4 cultures. a, P < 0.05 relative to untreated controls; b, P < 0.05 relative
to osmotic or drug stimulation alone.
|
|
A slightly different pattern was evident 24 h after stimulation.
The effects of osmotic and NMDA stimulation were reduced [+13%,
t =
1.84, P = 0.069 (P = 0.018 by Mann-Whitney) and +16.5%, NS, respectively], whereas the
effect of 1S,3R ACPD alone was increased (+40.6%, t = 7.50, P < 0.002). The combination of osmotic and NMDA or
ACPD stimulation was significantly greater than the untreated control
condition or osmotic stimulation alone (+28-29%, t
values = 5.28-6.22, P values < 0.002).
Increases in JNK-3 were also evident after osmotic and glutamate
receptor stimulation, but with a different pattern from that for JNK-2.
Acute osmotic stimulation resulted in a 13% increase in JNK-3
immunoreactivity (t =
3.69, P = 0.008).
1S,3R ACPD increased JNK-3 by 20.2% (t =
5.16,
P < 0.002), whereas NMDA was ineffective (+3.3%, NS).
Because the measurements included both the cell body and the nucleus,
the increases could have been due to an increase in the number of cells
with nuclear staining. However, a breakdown of the relative number of
neurons with an immunoreactive nucleus showed similar numbers for
osmotically stimulated (53 ± 7%) and control rats (44 ± 10%), indicating that, at best, this could only partially account for
the increases seen. Combined osmotic and glutamate receptor stimulation
failed to have a significant effect on JNK-3 expression and showed
slight decreases relative to drug alone.
After 24 h of treatment, the osmotic effect was increased slightly
(+24.8%, t =
2.38, P = 0.019). Combined
stimulation with 1S,3R ACPD and high osmolality gave results similar to
osmotic stimulation alone (+20.1%, t =
3.03,
P < 0.003) with no significant direct contribution
from 1S,3R ACPD. NMDA had a small but nonsignificant effect at 24 h (+18.7%) but in combination with osmotic stimulation induced a
47.3% increase in JNK-3 relative to controls (t =
3.43, P = 0.019).
Because cells were selected at random from the cultures and therefore
do not all represent magnocellular neurons, we segregated the data by
cell size to evaluate whether the large magnocellular neurons responded
differently from other neurons in the culture. The criterion for a
magnocellular neuron was set at an area of 300 µm2.
Although few differences in response profiles were observed in large
vs. small cells after 1 h, different patterns were apparent after
24 h (not shown). At this time, osmotically induced increases in
JNK-2 (mean cellular OD) were restricted almost exclusively to large
cells (15.0 ± 5.9%), with small cells showing only a 1.0 ± 2.6% increase (t =
2.18, large vs. small cells,
P = 0.034). On the other hand, osmotically induced
increases in JNK-3 tended to be exclusive to small cells, although
results were variable and not significant (large cells,
0.3 ± 11.8%; small cells, +30.8 ± 9.4%, t = 1.57 large vs.
small cells, P = 0.122). A similar trend was seen for
NMDA stimulation, but it did not reach statistical significance.
The similarity between the effects of NMDA and hyperosmotic stimulation
on JNK-2 immunoreactivity raised the question of whether the
osmotically induced increase might be due to indirect activation or
facilitation of endogenous NMDA receptors. To test this possibility, cultures were stimulated with 30 mosmol/kg excess NaCl, or with 30 mosmol/kg excess NaCl in the presence of the NMDA receptor antagonist
aminophosphonovaleric acid (APV, 100 µM). The results, illustrated in
Fig. 8, show that under these conditions,
APV failed to block the increase in JNK-2 immunoreactivity associated
with osmotic stimulation, suggesting the presence of two independent pathways for JNK upregulation. In contrast, the increase in response to
NMDA (0.246 ± 0.019) was blocked by APV in these cultures
(OD = 0.087 ± 0.015, t = 5.24, P = 0.0001, not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8.
Failure of the NMDA receptor antagonist
aminophosphonovaleric acid (APV; 100 µM) to block the osmotically
induced increase in JNK-2 staining in cultured neuroendocrine cells.
Optical density readings were taken from 63-102 individual neurons
sampled from 3-4 cultures. Osmotic stimulation resulted in a
68.2% increase in staining intensity in cells exposed to high
osmolality and a 91.3% increase in cells exposed to high osmolality in
the presence of 100 µM APV. The increase in the osmotic + APV group
relative to the osmotic alone group was small (13.7%) but indicated
that JNK-2 may be expressed in response to the loss of basal activity
at the NMDA receptor.
|
|
Because the NMDA antagonist did not block the osmotically induced
increase in JNK, we evaluated the general contribution of synaptic
stimulation by incubating the cells in 1 µM tetrodotoxin (TTX) during
stimulation. Osmotically stimulated cultures had many more heavily
stained neurons (Fig. 9B)
relative to controls (Fig. 9A). The increase was
particularly apparent for the largest neurons, which were robustly
stained after osmotic stimulation (Fig. 9B, inset,
lower right). A similar increase in immunoreactivity was seen in
neurons challenged osmotically in the presence of TTX (Fig.
9D). Neurons exposed to TTX alone showed some increase in
immunoreactivity that was evident in small neurons (Fig. 9C, small arrows) but not large neurons (Fig. 9C, large arrow).
A population analysis of all neurons, illustrated in Fig.
10, shows the shift in the frequency
distribution from low to high density with osmotic stimulation
(
2 = 40.8, P < 0.0001). The
greatest shift was seen for combined stimulation with 30 mosmol/kg
excess NaCl and TTX (
2 = 46.1, P < 0.0001), indicating that the osmotic stimulation effectively increased
JNK-2 expression in the absence of synaptic activity. However, TTX
alone also produced a significant increase (albeit smaller) in JNK-2
immunoreactivity (
2 = 26.2, P < 0.0001). A more detailed analysis of the responses based on separation
of the cells into large (>300 µm2) or small (<300
µm2) subpopulations, summarized in Table
1, revealed that only the large neurons
retained osmotic responsiveness in the presence of TTX on the basis of
increases in JNK-2 immunoreactivity (
2 = 12.5, P = 0.014), whereas small neurons failed to show a
significant increase due to osmotic stimulation in the presence of TTX
(
2 = 5.96, P = 0.202).

View larger version (162K):
[in this window]
[in a new window]
|
Fig. 9.
JNK-2 immunoreactivity in cultured magnocellular neurons
after exposure to hyperosmotic medium in the absence or presence of 1 µM tetrodotoxin (TTX). Fifteen minutes after transfer of one-half of
the cultures to TTX-containing medium, cells were osmotically
challenged. After 12 h, cultures were fixed and stained for JNK-2
or JNK-3. The density of JNK-2 immunoreactivity was measured in neurons
sampled from each of 4 conditions: control (n = 186),
+30 mosmol/kg NaCl (n = 116), TTX alone
(n = 229), and +30 mosmol/kg NaCl in the presence of 1 µM TTX (n = 225). Neurons from control cultures
(A) showed a wide range of JNK-2 immunoreactivity, although
most cells had light-to-medium staining. The lightest staining tended
to be in the largest neurons (A, arrows). After exposure to
hyperosmotic medium (+30 mosmol/kg), JNK-2 immunoreactivity increased
in most cells and processes (B) and was particularly high in
the largest neurons (inset, lower right). Treatment with TTX
alone (C) tended to increase immunoreactivity in small cells
(arrows). Some large cells also showed increased immunoreactivity,
although most were still lightly stained (arrowhead). Osmotic
stimulation in the presence of TTX (D) resulted in robust
immunoreactivity similar to that seen in the absence of TTX
(B). Calibration bar (lower right) = 50 µm
for all.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 10.
Population analysis of neurons stained for JNK-2 showing shift of
frequency distribution to right (higher densities) in
cultures treated with TTX alone or osmotic stimulation. All
P values reflect results of a 2 analysis
relative to controls.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Analysis of relative effects of osmotic and synaptic stimulation on
JNK-2 and JNK-3 expression in large vs. small neurons
|
|
Analysis of the density of JNK-3 immunoreactivity in neurons under the
same four conditions revealed a similar pattern of results. Staining of
neurons was generally lighter than JNK-2, with moderate
immunoreactivity seen in occasional cells (not shown). Population
analysis of all neurons (Fig. 11)
illustrated the shift in distribution to higher densities associated
with osmotic stimulation. Osmotic stimulation resulted in increased
neuronal JNK-3 immunoreactivity relative to controls
(
2 = 20.6, P < 0.0001). TTX alone
resulted in a distribution almost identical to controls
(
2 = 1.06, P = 0.7879). Osmotic
stimulation in the presence of TTX was similar to controls and TTX
alone (
2 = 1.14, P = 0.767),
indicating a full reversal of the osmotic effect. A separate analysis
of small and large subsets of neurons in Table 1 revealed that osmotic
stimulation increased JNK-3 staining in small neurons (P
values < 0.0001) but not large neurons. This effect of
osmotic stimulation in small neurons was completely reversed by the
presence of TTX.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 11.
Population analysis of neurons stained for JNK-3 showing shift of
frequency distribution to right (higher densities) in
cultures treated with hyperosmotic medium in the absence
(n = 109) or presence of TTX (n = 188).
TTX alone (n = 126) failed to affect JNK-3 staining
relative to controls (n = 174) and fully reversed the
effects of osmotic stimulation. All P values reflect results
of a 2 analysis relative to controls.
|
|
The increase in JNK induced by osmotic and glutamate receptor
stimulation would be predicted to give rise to phosphorylation of
c-Jun. Cultures treated with hyperosmotic medium and or NMDA for
24 h were evaluated for phospho-c-Jun immunoreactivity in the
nuclei of large cells. The results, summarized in Fig.
12, illustrate that under control
conditions 14% of the large cells had immunoreactive nuclei. This
value increased significantly to 30% (P = 0.0038, t-test) in the presence of 30 mosmol/kg excess NaCl and 22%
(P = 0.0531, t-test) after stimulation with
NMDA. The combination of NMDA and osmotic stimulation gave an increase of 36% (P = 0.0169, t-test).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 12.
Increased nuclear staining for phospho-c-Jun in cultured
magnocellular neurons stimulated with hypertonic medium (Osm, +30
mosmol/kg NaCl), NMDA, or NMDA + hypertonic medium. Nuclear
phospho-c-Jun staining was greatest for the combination of osmotic and
NMDA receptor stimulation, followed by osmotic stimulation and then
NMDA alone (all values * P < 0.05, t-test).
|
|
 |
DISCUSSION |
It is well established that the phosphorylation of the
transcription factor c-Jun by JNK is a key event in the cellular
response to stress (6, 10, 17).
In addition, the activation of JNK may be an early step in the
molecular cascade leading to apoptosis (7,
12, 16, 31). However, in
contrast to the apoptotic cascade, our results demonstrate that, in
mammalian cells, increased expression of JNK may have a protective
physiological role under conditions of osmotic stress. In addition, it
is clear that JNK is expressed in a variety of neurons under
nonpathological conditions. The role of this basal JNK is poorly
understood, although it seems likely that the cellular stress response
has been adapted to various functional outputs in different cells. In
lower organisms, the jnk gene homolog hog-1 is
part of the signal transduction pathway that protects cells from lethal
dehydration in hypertonic environments (8). This pathway
is linked to osmoreceptors, which are in turn coupled to the activity
of MAP kinases (24). Expression of the human JNK-1 protein
in yeast mutants lacking the hog-1 gene restored the ability
to grow in hyperosmotic media (8). Thus this protein
kinase is structurally and functionally conserved from yeast to
mammals, suggesting that the stress response has been adapted to the
more complex functions of specialized cells in higher organisms.
Our experiments showed that neuroendocrine cells of the SON and
paraventricular nucleus exhibit JNK immunoreactivity, which increased
in response to water deprivation. The most consistent increase in JNK
expression in the magnocellular neuroendocrine cells by osmotic or NMDA
receptor stimulation in vivo and in vitro was seen for the JNK-2
isoform. In contrast, JNK-3 was more likely to increase in the cortex
in response to water deprivation or in smaller hypothalamic neurons
osmotically challenged in vitro. These opposite response patterns
suggested that the two JNK isoforms may be differentially regulated in
different cells. These results also indicate that many types of neurons
are capable of responding to physiological increases in extracellular
osmolality. The widespread nature of the response is consistent with
previous reports of abundant JNK mRNA in rat cortex, hippocampus, and
other regions throughout the brain (5). Functional and
cellular specificity for neuroendocrine regulation may be dictated, at
least in part, by the preferential expression of the JNK-2 isoform in
response to osmotic and glutamate receptor stimulation, particularly
over sustained periods of time. Little information is available to indicate how this difference might translate to differences in the JNK
signal transduction pathway within the neuroendocrine cells. Increases
in nuclear phospho-c-Jun parallel the upregulation of JNK, but the
significance of this effect on vasopressin expression is presently
unclear, because the vasopressin gene does not have an activating
protein 1 (AP-1) binding site in the promoter (21), a
preferred target of the Fos-Jun complex. Other targets of phospho-c-Jun or the phosphorylation of other transcription factors such as ATF-2
(10), which bind to cAMP-regulating elements, may be
involved in the signal transduction cascade associated with
hyperosmotic stimulation.
Increases in JNK expression were induced in cultured neurons in vitro
in as little as 1 h, indicating that this response within the SON
as well as in other brain regions can emerge rapidly with changes in
extracellular osmolality. This timing is similar to the in vivo
increases in the transcription factors c-Fos and c-Jun (13, 25) associated with hyperosmolality and
hypovolemia. Message and protein levels for c-Fos increase rapidly
after stimulation, beginning at ~15 min and reaching a peak by 1 h (13). Protein levels may persist for several hours
(13) but must be phosphorylated and dimerized to be active
at the AP-1 site on the target gene (1). The increased
expression of JNK within 1 h of osmotic stimulation would be
expected to facilitate the phosphorylation of its preferred substrate,
c-Jun (6), and facilitate signal transduction. Thus JNK is
well poised to provide a key step in the functional activation of the
Fos-Jun complex in magnocellular and other neurons. This possibility is
supported by the increase in nuclear phospho-c-Jun, which we observed
in vitro after osmotic stimulation.
Role of glutamate receptors in JNK expression.
The osmotic activation of neuroendocrine cells is under the control of
both intrinsic and extrinsic stimuli. Increases in extracellular
osmolality can activate intrinsic osmoreceptors that induce
nonselective cationic currents (2, 23). In
addition, a wide variety of synaptic inputs are thought to provide the
extrinsic driving force for activation of the neuroendocrine cells
(3). Much of the excitatory activity from extrinsic
sources is provided by glutamate (9, 14,
20, 22, 29, 30).
NMDA receptors, in particular, are responsible for the induction of
bursting activity associated with hormone release (15).
Either osmotic or glutamate receptor stimulation could potentially
mediate the JNK response under physiological conditions. Indeed, in our
cultures, both NMDA and osmotic stimulation increased JNK-2. The
metabotropic glutamate receptor agonist 1S,3R ACPD also increased JNK-2
as well as JNK-3. These observations are consistent with other studies that have shown increases in JNK activity with osmotic (8, 11, 26) or glutamate receptor stimulation
(28). In rat striatum, glutamate receptor stimulation not
only activated JNK and AP-1-mediated transcription but also induced the
expression of transcription factors such as c-fos,
fosB, c-jun, and junB
(28). Peak activity of JNK induced by glutamate
occurred 1-2 h after stimulation (28). Glutamate,
dopamine, and forskolin were all shown to increase c-fos
mRNA, but only glutamate was capable of increasing c-jun mRNA and JNK activity. The glutamate-induced increases in JNK activity
were almost completely blocked by the NMDA glutamate receptor
antagonists MK-801 and APV (28). Because both NMDA and
osmotic stimulation increased JNK expression in our cultures, it was
possible that the osmotic effect might be mediated through activation
of NMDA receptors. However, blockade of NMDA receptor activity with 100 µm APV had no effect on osmotically induced JNK expression. Thus both
NMDA and osmotic stimuli appear to be capable of stimulating JNK
expression in neuroendocrine cells through independent pathways.
Role of synaptic stimulation in JNK-2 and JNK-3 expression.
Differences in the expression of JNK-2 and JNK-3 continued to emerge
when cultures were stimulated in the presence of 1 µM TTX to block
release of endogenous transmitters. A significant osmotically induced
increase in JNK-2 expression was maintained in large cells in the
presence of TTX, whereas the osmotic expression of JNK-3 was completely
suppressed by TTX. Thus osmotic stimulation of JNK-3 expression
appeared to be dependent on synaptic stimulation, whereas JNK-2
expression was at least partially independent of synaptic activity. The
relative contribution of synaptic vs. nonsynaptic stimulation of JNK-2
is difficult to extrapolate from these experiments. JNK-2
immunoreactivity in the presence of TTX was 80% of osmotic stimulation
alone. However, TTX alone increased JNK-2 staining, suggesting that JNK
expression may also be sensitive to the loss of synaptic activity. Thus
nonsynaptic stimulation may account for as much as 80% or as little as
42% of the osmotic response, depending on which baseline is the
appropriate comparison. Nevertheless, the presence of a significant
nonsynaptic increase in JNK-2 restricted to large cells supports the
possibility of a direct osmotic signal transduction pathway in
magnocellular neuroendocrine cells. This pathway may constitute a
relatively unique link between synaptic and osmotic activation of
neuroendocrine cells and the coordinate regulation of transcriptional activity.
 |
ACKNOWLEDGEMENTS |
We thank Elliott Brown, whose experiments contributed to portions
of this manuscript.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grant
NS-13411.
Address for reprint requests and other correspondence: R. Meeker, Dept. of Neurology, 781 Burnett-Womack, CB #7025, Univ. of
North Carolina, Chapel Hill, NC 27599 (E-mail:
rbmeeker{at}med.unc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 16 September 1999; accepted in final form 6 April 2000.
 |
REFERENCES |
1.
Abate, C,
Baker SJ,
Lees-Miller SP,
Anderson CW,
Marshak DR,
and
Curran T.
Dimerization and DNA binding alter phosphorylation of Fos and Jun.
Proc Natl Acad Sci USA
90:
6766-6770,
1993[Abstract].
2.
Bourque, CW,
Oliet SH,
and
Richard D.
Osmoreceptors, osmoreception, and osmoregulation.
Front Neuroendocrinol
15:
231-274,
1994[ISI][Medline].
3.
Bourque, CW,
and
Renaud LP.
Electrophysiology of mammalian magnocellular vasopressin and oxytocin neurosecretory neurons.
Front Neuroendocrinol
11:
183-212,
1996.
4.
Carboni, L,
Carletti R,
Tacconi S,
Corti C,
and
Ferraguti F.
Differential expression of SAPK isoforms in the rat brain. An in situ hybridisation study in the adult rat brain and during post-natal development.
Brain Res
60:
57-68,
1998.
5.
Carletti, R,
Tacconi S,
Bettini E,
and
Ferraguti F.
Stress activated protein kinases, a novel family of mitogen-activated protein kinases, are heterogeneously expressed in the adult rat brain and differentially distributed from extracellular-signal-regulated protein kinases.
Neuroscience
69:
1103-1110,
1995[ISI][Medline].
6.
Derijard, B,
Hibi M,
Wu IH,
Barrett T,
Su B,
Deng T,
Karin M,
and
Davis RJ.
JNK 1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.
Cell
76:
1025-1037,
1994[ISI][Medline].
7.
Estus, S,
Zaks WJ,
Freeman RS,
Gruda M,
Bravo R,
and
Johnson EM, Jr.
Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.
J Cell Biol
127:
1717-1727,
1994[Abstract].
8.
Galcheva-Gargova, Z,
Derijard B,
Wu IH,
and
Davis RJ.
An osmosensing signal transduction pathway in mammalian cells.
Science
265:
806-808,
1994[ISI][Medline].
9.
Gribkoff, VK,
and
Dudek FE.
The effects of the excitatory amino acid antagonist kynurenic acid on synaptic transmission to supraoptic neuroendocrine cells.
Brain Res
442:
152-156,
1988[ISI][Medline].
10.
Gupta, S,
Campbell D,
Derijard B,
and
Davis RJ.
Transcription factor ATF2 regulation by the jnk signal transduction pathway.
Science
267:
389-393,
1995[ISI][Medline].
11.
Han, J,
Lee JD,
Bibbs L,
and
Ulevitch RJ.
A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells.
Science
265:
808-811,
1994[ISI][Medline].
12.
Herdegen, T,
Skene P,
and
Bahr M.
The c-Jun transcription factor
bipotential mediator of neuronal death, survival and regeneration.
Trends Neurosci
20:
227-231,
1997[ISI][Medline].
13.
Hoffman, GE,
Smith MS,
and
Verbalis JG.
c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems.
Front Neuroendocrinol
14:
173-213,
1993[ISI][Medline].
14.
Hu, B,
and
Bourque CW.
Functional N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors are expressed by rat supraoptic neurosecretory cells in vivo.
J Neuroendocrinol
3:
509-514,
1991[ISI].
15.
Hu, B,
and
Bourque CW.
NMDA receptor-mediated rhythmic bursting activity in rat supraoptic nucleus neurones in vitro.
J Physiol (Lond)
458:
667-687,
1992[Abstract].
16.
Ichijo, H,
Nishida E,
Irie K,
ten Dijke P,
Saitoh M,
Moriguchi T,
Takagi M,
Matsumoto K,
Miyazono K,
and
Gotoh Y.
Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways.
Science
275:
90-94,
1997[Abstract/Free Full Text].
17.
Kyriakis, JM,
Banerjee P,
Nikolakaki E,
Dai T,
Rubie EA,
Ahmad MF,
Avruch J,
and
Woodgett JR.
The stress-activated protein kinase subfamily of c-Jun kinases.
Nature
369:
156-160,
1994[ISI][Medline].
18.
Meeker, RB,
Curras MC,
Stewart J,
Serje A,
and
Al-Ghoul W.
Functional activation of punch-cultured magnocellular neuroendocrine cells by glutamate receptor subtypes.
J Neurosci Methods
89:
57-67,
1999[ISI][Medline].
19.
Meeker, RB,
Greenwood RS,
and
Hayward JN.
Vasopressin mRNA expression in individual magnocellular neuroendocrine cells of the supraoptic and paraventricular nucleus in response to water deprivation.
Neuroendocrinology
54:
236-247,
1991[ISI][Medline].
20.
Meeker, RB,
Swanson DJ,
Greenwood RS,
and
Hayward JN.
Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalamus.
Brain Res
600:
112-122,
1993[ISI][Medline].
21.
Mohr, E,
and
Richter D.
Sequence analysis of the promoter region of the rat vasopressin gene.
FEBS Lett
260:
305-308,
1990[ISI][Medline].
22.
Nissen, R,
Hu B,
and
Renaud LP.
Regulation of spontaneous phasic firing of rat supraoptic vasopressin neurones in vivo by glutamate receptors.
J Physiol (Lond)
484:
415-424,
1995[Abstract].
23.
Oliet, SH,
and
Bourque CW.
Mechanosensitive channels transduce osmosensitivity in supraoptic neurons.
Nature
364:
341-343,
1993[ISI][Medline].
24.
Posas, F,
and
Saito H.
Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK.
Science
276:
1702-1705,
1997[Abstract/Free Full Text].
25.
Roberts, MM,
Robinson AG,
Fitzsimmons MD,
Grant F,
Lee W-S,
and
Hoffman GE.
c-fos Expression in vasopressin and oxytocin neurons reveals functional heterogeneity within magnocellular neurons.
Neuroendocrinology
57:
388-400,
1993[ISI][Medline].
26.
Rosette, C,
and
Karin M.
Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.
Science
274:
1194-1197,
1996[Abstract/Free Full Text].
27.
Sagar, SM,
Sharp FR,
and
Curran T.
Expression of c-fos protein in brain metabolic mapping at the cellular level.
Science
240:
1328-1328,
1988[ISI][Medline].
28.
Schwarzschild, MA,
Cole RL,
and
Hyman SE.
Glutamate, but not dopamine, stimulates stress-activated protein kinase and AP-1-mediated transcription in striatal neurons.
J Neurosci
17:
3455-3466,
1997[Abstract/Free Full Text].
29.
Steardo, L,
Steardo MD,
Mazzoccoli M,
Testa N,
and
Cuomo V.
Selective activation of glutamate receptor NMDA subtype induces plasma vasopressin increase in rats.
Acta Neurol
16:
235-239,
1994.
30.
Van den Pol, AN.
Glutamate and aspartate immunoreactivity in hypothalamic presynaptic axons.
J Neurosci
11:
2087-2101,
1991[Abstract].
31.
Xia, Z,
Dickens M,
Raingeaud J,
Davis RJ,
and
Greenberg ME.
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:
1326-1331,
1995[Abstract].
Am J Physiol Endocrinol Metab 279(3):E475-E486
0193-1849/00 $5.00
Copyright © 2000 the American Physiological Society