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Published in final edited form as:
Neuroscience. 2010 March 17; 166(2): 455–463. doi:10.1016/j.neuroscience.2009.12.059.
Neuronostatin is co-expressed with somatostatin and mobilizes
calcium in cultured rat hypothalamic neurons
Siok L. Dun1, G. Cristina Brailoiu1, Andrei A. Tica1,2, Jun Yang3, Jaw K. Chang3, Eugen
Brailoiu1, and Nae J. Dun1
1Department of Pharmacology, Temple University School of Medicine Philadelphia PA 19140 USA
3Research
Division, Phoenix Pharmaceuticals, Inc., Burlingame, CA 94010 USA
Abstract
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Neuronostatin (NST) is a newly identified peptide of 13-amino acids encoded by the somatostatin
(SST) gene. Using a rabbit polyclonal antiserum against the human NST, neuronostatinimmunoreactive (irNST) cells comparable in number and intensity to somatostatin immunoreactive
(irSST) cells were detected in the hypothalamic periventricular nucleus. Fewer and/or less intensely
labeled irNST cells were noted in other regions such as the hippocampus, cortex, amygdala, and
cerebellum. Double-labeling hypothalamic sections with NST- and SST-antiserum revealed an
extensive overlapping of irNST and irSST cells in the periventricular nucleus. Pre-absorption of the
NST-antiserum with NST (1 µg/ml) but not with SST (1 µg/ml) abrogated irNST and vice versa.
The activity of NST on dissociated and cultured hypothalamic neurons was assessed by the Ca2+
imaging method. NST (10, 100, 1000 nM) concentration-dependently elevated intracellular Ca2+
concentrations [Ca2+]i in a population of hypothalamic neurons with two distinct profiles: 1) a fast
and transitory increase in [Ca2+]i, and 2) an oscillatory response. Whereas, SST (100 nM) reduced
the basal [Ca2+]i in 21 of 61 hypothalamic neurons examined; an increase was not observed in any
of the cells. Optical imaging with a slow-responding voltage sensitive dye DiBAC4(3) showed that
NST (100 nM) depolarized or hyperpolarized; whereas, SST (100 nM) hyperpolarized a population
of hypothalamic neurons. The result shows that NST and SST, though derived from the same
precursor protein, exert different calcium mobilizing effects on cultured rat hypothalamic neurons,
resulting in diverse cellular activities.
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Keywords
periventricular nucleus; arcuate nucleus; cortex; hippocampus
© 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
Corresponding author: Nae J. Dun, Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad Street,
Philadelphia PA 19140 USA, Tel: 215-707-3498, Fax: 215-707-7068, ndun@temple.edu.
2Current address: Andrei A. Tica, M.D., Department of Pharmacology, University of Medicine and Pharmacy, Craiova, Romania
Section Editor
Cellular Neuroscience: Dr. Menahem Segal, Weizmann Institute of Science, Department of Neurobiology, Hertzl Street, Rehovot 76100,
Israel
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Disclosure Statement: J.Y. and J.K.C. are employed by Phoenix Pharmaceuticals, Inc., which produces and markets neuronostatin and
neuronostatin antiserum used in this study. No other author has any financial relationship with the company.
Dun et al.
Page 2
Introduction
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Somatostatin (SST) is a 14-amino acid cyclic polypeptide originally isolated from extracts of
bovine hypothalamus based on its ability to inhibit growth hormone release from cultured rat
pituitary cells (Brazeau et al., 1973). Subsequent studies show that SST is, in addition to the
hypothalamus, widely distributed to neural and non neural tissues including the spinal cord,
autonomic and sensory ganglia, pancreas, stomach, intestine, and kidney. The original concept
that SST is primarily involved in regulation of growth hormone has since been considerably
expanded; for example, the peptide regulates the secretion of thyrotropin-releasing hormone,
glucagon and insulin, and modulates gastrointestinal activity. In addition to SST, the prosomatostatin yields two mature peptides, a 28-amino acid peptide (SS28) and a 12-amino acid
peptide (SS1-12) (Brazeau et al., 1973; Benoit et al., 1982, 1984; Goodman et al., 1983; Shen
et al., 1984). Immunohistochemical studies with antibodies selective against SS28 and SS1-12
show that these two peptides may be differentially processed within cortical neurons such that
SS28 is found predominately in the somata and SS1-12 in the nerve terminals (Morrison et
al., 1982, 1983).
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Recently, a 13-amino acid non cyclic amidated peptide, named neuronostatin (NST), was
isolated from porcine tissues, with a predicted sequence based on bioinformatic analysis of
evolutionary conserved sequences of pro-somatostatin (Samson et al., 2008). NST is found to
be present in the rodent brain, pancreas, stomach and small intestine (Samson et al., 2008).
While the tissue distribution of NST appears to be similar to that of SST, the ratio of NST and
SST present in the same tissue samples varies, suggesting that these two peptides may be
differentially processed in a given cell. Further, NST is found not to interact with the five
putative somatostatin receptors expressed in human embryonic kidney 293T cells nor does it
affect the basal or growth hormone releasing hormone-stimulated secretion of growth hormone,
implying NST and SST may interact with different receptors or binding sites.
The present study was undertaken to establish the expression pattern of NST and compare to
that of SST in the rat brain by immunohistochemical techniques, and to compare the membrane
potential and calcium mobilizing effects of these two peptides on cultured rat hypothalamic
neurons.
Experimental Procedures
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Male Sprague-Dawley rats, 7–8 weeks old, weighing 250–275 gm (Ace Animals Inc.,
Boyertown, PA) were used in immunohistochemical studies. Ca2+ or voltage measurements
were conducted on dissociated hypothalamic neurons harvested from 1 to 3 days old rats, which
were cultured for 5 days. Animal protocols were reviewed and approved by the Temple
University Institutional Animal Care and Use Committee.
Immunohistochemistry
Rats anesthetized with urethane (1.2 g/kg, IP) were intracardially perfused with 0.1 M
phosphate buffered saline (PBS) followed by 4% paraformaldehyde/0.2% picric acid in PBS.
Brains were removed, postfixed for 2 hr, and stored in 30% sucrose/PBS solution overnight.
Tissues were processed for neuronostatin immunoreactivity (irNST) or somatostatin
immunoreactivity (irSST) by the avidin-biotin complex procedure (Brailoiu et al., 2007). The
brain was embedded in agar and coronal sections of 40 µm were prepared with the use of a
Vibratome. Tissues were first treated with 3% H2O2 to quench endogenous peroxidase, washed
several times, blocked with 10% normal goat serum, and incubated in NST antiserum (1:1,000
dilution), a rabbit polyclonal raised against a conserved region of the human preprosomatostatin 31–43 (31LeuArgGlnPheLeuGlnLysSerLeuAlaAlaAlaAla-amide43) (Phoenix
Pharmaceuticals, Inc., Burlingame, CA). The NST antiserum, which exhibits 100% crossNeuroscience. Author manuscript; available in PMC 2011 March 17.
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reactivity with human NST, does not cross-react with somatostatin in radioimmunoassay
(Phoenix Pharmaceuticals, Inc). After thorough rinsing, sections were incubated in biotinylated
anti-rabbit IgG (1:200 dilution, Vector Laboratories, Burlingame, CA) for 2 hr, and rinsed with
PBS and incubated in avidin-biotin complex solution for 1.5 hr (1:100 dilution, Vector
Laboratories). Following several washes in Tris-buffered saline, sections were developed in
0.05% diaminobenzidine/ 0.001% H2O2 solution and washed for at least 2 hr with Tris-buffered
saline. Sections were mounted on slides with 0.25% gel alcohol, air-dried, dehydrated with
absolute alcohol followed by xylene, and coverslipped with Permount.
In the case of double-labeling experiments, the sequential labeling method with the primary
antiserum from different hosts was used (Dun et al., 1994; Brailoiu et al., 2007). Hypothalamic
sections were first incubated with NST antiserum (1: 350 dilution) and then with SST antiserum
(1: 500 dilution, a rat polyclonal from Chemicon International, Inc., Temecula, CA). Sections
were incubated with appropriate secondary antiserum conjugated to either fluorescein
isothiocyanate (FITC) or Texas Red, and examined under a confocal scanning laser microscope
(Leica TCS SP5) with excitation wavelengths set to 488 nm for FITC and 561 nm for Texas
Red in the sequential mode.
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For control experiments, hypothalamic sections were processed with NST antiserum preabsorbed with human NST (1 µg/ml) or SST (1 µg/ml) overnight. In addition, sections were
processed with SST antiserum pre-absorbed with SST (1 µg/ml) or NST (1 µg/ml).
Neuronal cell culture
Hypothalamic neurons were isolated from postnatal 1–3 days old rats by enzymatic digestion
with papain (Brailoiu et al., 2007). Cells were plated at a density of 103/mm2 in a NeurobasalA™ medium, supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml
penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA), and maintained at 37°C in
a humidified atmosphere with 5% CO2. Glial cell growth was inhibited by the mitotic inhibitor
cytosine β-arabino furanoside (1 µM) (Sigma-Aldrich, St. Louis, MO). Neurons cultured for
5 days were transferred to a medium without fetal serum 12 h prior to Ca2+ measurements.
Cytosolic Ca2+ concentrations
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Cytosolic Ca2+ measurements were performed as described previously (Brailoiu et al., 2006,
2007). Cells were incubated with 5 µM fura-2 AM (Molecular Probes, Eugene, OR) in Hanks'
balanced salt solution (HBSS) at room temperature for 45 min in the dark, washed three times
with dye-free buffer, and incubated for another 45 min to allow for complete de-esterification
of the dye. Coverslips were subsequently mounted in a custom-designed bath on the stage of
an Eclipse TE 2000-U Nikon inverted microscope equipped with a Roper Scientific CCD
camera. Cells were routinely superfused with HBSS at a flow rate of 1 ml/min. Fura-2
fluorescence (emission = 510 nm), following alternate excitation at 340 nm and 380 nm, was
acquired at a frequency of 0.33 Hz. Acquired images were analyzed offline using Metafluor
software. For Ca2+- free experiments, HBSS without Ca2+ and supplemented with 2.5 mM
EGTA was used.
Voltage measurements using DiBAC4(3)
Optical imaging using voltage sensor probes is a reliable approach in monitoring membrane
potential changes in neurons (Ebner and Chen, 1995; Kunkler et al., 2005; Brailoiu et al.,
2008). Briefly, hypothalamic cells were incubated for 30 min in HBSS containing 0.5 mM
DiBAC4(3). The fluorescence (excitation wavelength = 480 nm, emission wavelength = 540
nm) was continuously recorded at a rate of 10 points min−1. Background values (windows of
identical area placed beside the cells) were always subtracted. The dye partition between the
cell membrane and the cytosol is a function of membrane potential. Depolarization of the
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membrane leads to a sequestration of the dye into cytosol and is associated with an increase in
the fluorescence intensity; whereas, during membrane hyperpolarization the dye concentrates
in the cell membrane, leading to a decrease of cytoplasmic fluorescence intensity (Brauner et
al., 1984).
Calibration of DiBAC4(3)
Calibration of DiBAC4(3) fluorescence was performed using the Na+-K+ ionophore gramicidin
in Na+-free physiological solution (Brauner et al., 1984; Brailoiu et al., 2008). The osmolarity
was maintained constant by addition of N-methylglucamine. In the presence of gramicidin (1
µM), the Na+ concentration gradient is zero, and the membrane potential is approximately
equal to K+ equilibrium potential which is determined by the Nernst equation. The intracellular
K+ and Na+ concentrations were assumed to be 130 mM and 10 mM, respectively. The addition
of gramicidin with various concentrations of K+ to the cultured neurons alters the cell
membrane potential, thereby, altering fluorescence. According to the calibration
measurements, changes in DiBAC4(3) fluorescence intensity by 1.092 are equivalent to a
change in membrane potential of 1 mV.
Statistical analysis
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In calcium and voltage measurement experiments, statistical significance between groups was
tested with one-way ANOVA followed by Bonferroni test, p< 0.05 being considered
significant.
Results
Somatostatin immunoreactivity
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As reported earlier (Johansson et al., 1984; Vincent et al., 1985), somatostatin immunoreactive
(irSST) neurons were noted in the lateral septum, nucleus accumbens, amygdaloid complex,
hypothalamic periventricular nucleus, hippocampus, cortex, cerebellum and several brainstem
nuclei. In all four rat brains examined, strongly labeled, small diameter irSST cells of different
morphologies were detected throughout the cortex (Fig. 1A). Within the hippocampus,
moderately to intensely labeled irSST cells were noted in the CA1, CA2 and CA3 areas, as
well as, in the polymorphic layer of dentate gyrus (PoDG; Fig. 1B). Purkinje cells were strongly
labeled (Fig. 1C), so were Golgi cells in the cerebellum. In the hypothalamus, neurons in the
anterior periventricular nucleus (Pe) were strongly labeled (Fig. 1D and E); small diameter
irSST cells were also noted in the suprachiasmatic nucleus (Sch) (Fig. 1D). In addition to irSST
cells, numerous cell processes were noted in the median eminence, particularly the external
layer (MEE). Labeled cells and fibers were scattered in the medial preoptic nucleus (MPO),
the lateral septum, nucleus accumbens, amygdaloid complex, ventromedial hypothalamic
areas, as well as, the arcuate nucleus (Arc).
Neuronostatin immunoreactivity
Examination of the rat brains (n=6) revealed that irNST was conspicuously present mainly in
two areas: the hypothalamic periventricular nucleus (Pe) (Fig. 2A and B) and median eminence
(ME) (Fig. 2C). The number and intensity of irNST cells in the Pe was comparable to that of
irSST cells in the same area (Fig. 2A and B; compare with Fig. 1D and E), so were cell processes
in the ME (Fig. 2C; compare with Fig. 1F). In other regions of the rat brain including the
suprachiasmatic nucleus, cortex and hippocampus; the number of irNST cells appeared to be
fewer and less intensely labeled than that of irSST cells (Fig. 2C, D and E). The result shows,
with the exception of periventricular nucleus, a less abundant expression of irNST as compared
to that of irSST in the rat brain.
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Control experiments
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The specificity of NST antiserum and SST antiserum was evaluated by processing
hypothalamic sections with an antiserum pre-absorbed with either NST (1 µg/ml) or SST (1
µg/ml). In all three brains tested, hypothalamic sections processed with NST antiserum preabsorbed with NST amide peptide showed no positive labeling in the hypothalamic
periventricular nucleus and suprachiasmatic nucleus (Fig. 2F); whereas, immunoreactivity
could be detected in the hypothalamic periventricular nucleus processed with NST antiserum
pre-absorbed with NST free acid peptide or SST. Conversely, positively-labeled cells or cell
processes were not detected in hypothalamic sections processed with SST antiserum preabsorbed with SST peptide (1 µg/ml), but not with NST peptide. These results demonstrate
that NST antiserum was specific against NST amide peptide and that SST antiserum specific
against SST.
Double-labeling experiments
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Hypothalamic sections double-labeled with NST antiserum and SST antiserum revealed that
irNST cells in the hypothalamic periventricular nucleus express irSST and vice versa (Fig. 3A–
F). In the median eminence, there was extensive overlap of irNST and irSST fibers (Fig. 3G–
I). In most of the cortical areas sampled, there was extensive overlapping of irNST and isSST
neurons (Fig. 3 J–L). The intensity of irNST appeared to be less than that of irSST in some of
the cortical neurons (Fig. 3J–L).
Intracellular calcium concentrations in hypothalamic neurons
The neuronal activity of NST and SST was assessed by monitoring changes in intracellular
calcium concentrations [Ca2+]i or membrane potentials in cultured hypothalamic neurons.
Initial studies showed that NST amide was active; whereas, NST free acid was devoid of
calcium mobilizing effects on hypothalamic neurons. Consequently, NST amide, referred to
throughout the text as NST, was used in this study.
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The basal [Ca2+]i of dissociated rat hypothalamic neurons was 57 ± 5.8 nM (n = 539 cells),
which is close to the value reported in earlier studies (Connor, 1986; Brailoiu et al., 2007).
When superfused to cultured hypothalamic neurons, NST (100 nM) produced two patterns of
Ca2+ responses. In Ca2+-containing saline, NST (100 nM) induced a fast and transitory increase
in [Ca2+]i, with an average of 512 ± 4.3 nM in 14 of 194 hypothalamic neurons tested (solid
line, Fig. 4A1). In other 37 cells, NST induced an oscillatory response. The first peak
corresponded to an increase in [Ca2+]i of 396 ± 3.4 nM; a representative response (dotted line)
is shown in Fig. 4A. 1NST (10 and 1000 nM) also produced two types of Ca2+ responses: i)
single spike with a mean amplitude of 93 ± 2.7 nM (n=11) and 739 ± 5.6 nM (n=21), and ii)
oscillations with a mean amplitude of the first peak of 71 ± 2.9 nM (n=19) and 488 ± 4.7 nM
(n=35) (Fig. 4C). The increase was statistically significant (P < 0.01) compared to the basal
[Ca2+]i. In contrast, SST (100 nM) slightly reduced the basal Ca2+ by 23 ± 1.7 nM in 21 of 61
cells tested (Fig. 4B1).
In a Ca2+-free saline supplemented with 2.5 mM EGTA, NST (100 nM) also elicited two types
of responses. In 13 cells from 237 tested, NST produced a single, transitory increase in
[Ca2+]i by 256 ± 2.7 nM (solid line, Fig. 4A2). In the other 32 cells, NST (100 nM) initiated
Ca2+ oscillations. The peak of the first Ca2+ wave was 78 ± 2.3 nM above the basal; a
representative trace (dotted line) is shown in Fig. 4A2. On the other hand, administration of
SST in Ca2+-free saline caused no appreciable changes in [Ca2+]i in any of the 53 cells tested
(Fig. 4B2).
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Membrane potential changes
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The mean resting membrane potential of cultured hypothalamic neurons as monitored by
changes in DiBAC4(3) fluorescence was −48 ± 3.7 mV (n = 371), which was comparable to
that recorded by electrophysiological techniques (Stern, 2001). In 23 out of 257 cells tested,
NST (100 nM) produced a depolarization with a mean amplitude of 5.2 ± 1.6 mV (solid line;
Fig. 5A). In the other 7 cells, NST (100 nM) induced a hyperpolarization of 6.1 ± 2.2 mV
(dotted line; Fig. 5A).
In 114 cells tested, administration of SST (100 nM) induced a hyperpolarization of 9.8 ± 2.2
mV in 43 cells; a representative trace (dotted line) is shown in Fig. 5B.
Discussion
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Post-translational processing of prepro-somatostatin results in the formation of
somatostatin-28 (SS28), which is cleaved to yield two mature peptides somatostatin, also
known as somatostatin-14, and somatostatin1-12 (SS1-12) (Benoit et al., 1982, 1984;
Goodman et al., 1983; Zingg and Patel, 1983; White et al., 1985). The neural activity of SS1-12
appears to differ from that of the other two peptides. For example, SS28 and SST hyperpolarize
CA1 pyramidal neurons in vitro; whereas, a membrane response is not elicited by SS1-12
(Watson and Pittman, 1988). Recently, Samson et al. (2008) reported the isolation and
identification from porcine tissues a non cyclic, 13-amino acid amidated peptide, named
neuronostatin (NST), which is derived from the N-terminus of pro-somatostatin. The NST
antiserum and SST antiserum used in our study appear to be specifically directed against
neuronostatin and somatostatin, as hypothalamic sections processed with NST antiserum preabsorbed with NST, but not with SST, show no labeling and vice versa. NST is found to be
bioactive in a variety of tissues tested including gastrointestinal tract, hippocampus, cerebellum
and hypothalamus (Samson et al., 2008).
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Our immunohistochemical studies show for the first time that the expression profile of irNST
and irSST is similar in the hypothalamus; i.e., strongly labeled irNST and irSST neurons are
noted in the hypothalamic periventricular nucleus, as well as numerous cell processes in the
median eminence. Ishikawa et al. (1987) showed by a combination of retrograde labeling with
horseradish peroxidase and immunostaining of somatostatin cells that irSST neurons in the
rostral parts of the periventricular nucleus project their axons to the median eminence. Our
observation that numerous irNST cell processes traverse the median eminence is consistent
with the idea that they arise from cell bodies in the periventricular nucleus. In other areas of
the rat brain such as the suprachiasmatic nucleus, cortex and hippocampus, irNST appears to
be less abundantly expressed as compared to that of irSST. In spite of considerable efforts, for
example, irNST sections were processed side by side with irSST sections, rendering variability
of immunostaining a less likely contributing factor, it is recognized immunofluoresence
labeling is less than quantitative.
Using radioimmunoassay (RIA), a lower expressional level of NST as compared to that of SST
has been noted in the initial study by Samson et al. (2008). The ratio of NST over SST in
different brain regions, with the exception of cerebrum, was found to be less than 0.05. For
example, the hypothalamus, hippocampus, cerebellum and pons show a ratio of 0.05, 0.02,
0.05 and 0.01, respectively; the ratio is 0.91 for the cerebrum (Samson et al., 2008). Hence,
our immunohistochemical result seems to be in general agreement with their quantitative assay
in that fewer or less intensely labeled irNST cells are observed in the hippocampus, cerebrum,
cerebellum and cortex. There is an apparent discrepancy between immunohistochemical
detection and RIA relative to the expression of NST and SST in the hypothalamus. Our
immunostaining, while non-quantitative, shows that the number and/or intensity of irNST cells
appear to be comparable to that of irSST cells in the periventricular nucleus. A possible
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explanation is that in our study comparison was made only with respect to the hypothalamic
periventricular nucleus; whereas, the radioimmunoassay measured the entire hypothalamic
area, which may express few irNST cells in hypothalamic areas outside of the periventricular
nucleus. Neuronostatin is a non-cyclic, amidated peptide, and somatostatin is not, the presence
or absence of amidation enzymes or degrading enzymes may dictate the level of neuronostatin
and somatostatin expression in different populations of neuron (Samson et al., 2008).
In the rat brain, other neuroactive substances have been identified in SST-containing neurons;
for example, irSST is expressed in some of the cholecystokinin- (Somogyi et al., 1984) or avian
pancreatic polypeptide- (Vincent et al., 1982) containing neurons. Further, irSST is frequently
co-expressed with δ-aminobutyric acid and/or nitric oxide synthase in central neurons (Sloviter
and Nilaver, 1987; Dun et al., 1994). Whether or not NST-containing neurons in the
hypothalamic periventricular nucleus may represent a heterogeneous population of neurons as
in the case of SST-expressing neurons remains to be studied.
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Several earlier studies show that SST, SS28 and SS1-12 are released from neural and non neural
tissues in response to appropriate stimuli in a calcium-dependent manner (Iversen et al.,
1978; Bennett et al., 1979; Kewley et al., 1981; Bakhit et al., 1983; Sheward et al., 1984).
Whether or not NST is secreted from neurons and non neural cells in a calcium-dependent
manner remains to be investigated. Using the early oncogene c-fos expression as a biomarker
for cell activation, NST is found to be active in multiple sites, including cells of the
gastrointestinal tract, anterior pituitary, cerebellum, and hippocampus in vivo. NST by
intracerebroventricular injections increases blood pressure but decreases food and water intake
(Samson et al., 2008). NST appears to exert its biological effects by interacting with receptors/
binding sites that are different from those interacting with SST. For example, SST, but not
NST, was effective in stimulating the inhibitory G protein pathway in HEK 293T cells cotransfected with plasmids encoding a chimeric Gqi protein, the serum responsive elementluciferase reporter construct, and individual somatostatin receptors, indicating that NST does
not interact with the five known somatostatin receptors (Samson et al., 2008).
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Our functional studies of NST on cultured hypothalamic neurons revealed that the amidated
NST, but not the free acid, is biologically active, suggesting that the naturally occurring form
is an amidated peptide. The biological activity of NST or SST were evaluated in cultured
hypothalamic neurons by means of the calcium and voltage imaging techniques, which offer
the advantage of being able to assay the activity of many cells in one experiment as compared
to several cells by the whole-cell recording technique. A potential drawback is that the identity
of neurons being studied is not known. Results obtained here are consistent with the contention
that NST and SST interact with pharmacologically distinct sites on hypothalamic neurons
(Samson et al., 2008). Five types of receptor for somatostatin, SSTR1-SSTR5, have previously
been reported in several brain regions, including the hypothalamus (Beaudet et al., 1995;
Kumar, 2007; Patel and Srikant, 1997). Somatostatin has been reported to decrease [Ca2+]i by
inhibiting Ca2+ influx (Lussier et al., 1991) through T- and L-type channels (Chen et al.,
1990) in somatotrophs cells. In agreement with the previous reports, our results show that SST
decreased basal [Ca2+]i in cultured rat hypothalamic neurons. In contrast, NST in equi-molar
concentrations increased [Ca2+]i in hypothalamic neurons with two distinct profiles: a
transitory rise and an oscillatory increase of [Ca2+]i. Similar to other cells, neurons utilize both
Ca2+ influx through plasmalemmal Ca2+ channels and Ca2+ release from internal stores
(Berridge, 1998). NST-induced [Ca2+]i were reduced in Ca2+-free saline, suggesting NST
activates Ca2+ entry through plasmalemmal Ca2+ channels as well as Ca2+ release from internal
stores. Previous reports indicate that in the pituitary gland Ca2+ oscillations are associated with
growth hormone secretion (Holl et al., 1988; Lussier et al., 1991). The observation that NST
was unable to alter the basal or hormone-stimulated growth hormone secretion by cultured
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Page 8
anterior pituitary cells (Samson et al., 2008), suggests that the oscillatory Ca2+ response evoked
by NST may regulate activity other than growth hormone homeostasis in pituitary cells.
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Using the voltage sensitive dye as an indicator, hypothalamic neurons responded to NST with
two opposing membrane potential changes: a depolarization and a hyperpolarization in 9% and
3% of hypothalamic neurons tested. This is in general agreement with the report in which NST
elicits a hyperpolarization or depolarization in 31% and 42% of electrophysiologically
identified hypothalamic paraventricular neurons (Samson et al., 2008). The number of cells
responsive to NST, irrespective of hyperpolarization or depolarization, is significantly fewer
in our study as compared to that reported in an earlier study (Samson et al., 2008). A plausible
explanation is that NST binding sites/receptors may be highly localized to hypothalamic
paraventricular neurons, which can be electrophysiologically identified in a slice preparation
(Samson et al., 2008). In our case, recordings were randomly made from unidentified
hypothalamic neurons in culture, most of which may not be endowed with NST receptors/
binding sites.
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Transient elevation of cytosolic calcium concentrations [Ca2+]i serves as second messenger
signals controlling several neuronal functions from development to apoptosis (Augustine et
al., 2003). The physiological role of NST in the neural and non neural cells is largely not known.
The finding that NST and SST elicit different Ca2+ mobilizing responses in hypothalamic
neurons suggests that these two peptides, though derived from the same precursor, may produce
different biological activity in their target cells.
Abbreviations
Arc
[Ca2+]
arcuate nucleus
intracellular calcium concentration
i
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DiBAC4(3)
bis-(1,3-dibutylbarbituric acid) trimethine oxonol
HBBS
Hanks' balanced salt solution
MPO
medial preoptic nucleus
ME
median eminence
NST
neuronostatin
irNST
neuronostatin immunoreactivity
Pe
periventricular nucleus
PBS
phosphate buffered saline
PoDG
polymorphic layer of dentate gyrus
Sch
suprachiasmatic nucleus
SST
somatostatin
irSST
somatostatin immunoreactivity
Acknowledgments
This study was supported in part by NIH Grants NS18710 and HL51314 from the Department of Health and Human
Services. Neuronostatin antiserum and peptides were a gift from Phoenix Pharmaceuticals, Inc.
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Fig. 1.
Somatostatin (SST) immunoreactive cells and fibers in the rat brain. A, irSST cells and cell
processes in the motor cortex. B, irSST cells in CA1, CA2 and CA3 regions and polymorphic
layer of the dentate gyrus (PoDG). C, Purkinje cells of the cerebellum are irSST. D, irSST cells
are concentrated in the periventricular hypothalamic nucleus (Pe) adjacent to the 3rd ventricle
(3V); irSST-cells and processes are also present in the suprachiasmatic nucleus (Sch). E,
enlargement of the area outlined in D where strongly labeled irSST cells are located in Pe, and
fibers in medial preoptic nucleus (MPO). F, strongly labeled irSST fibers are present in median
eminence, particularly the external median eminence (MEE), and in arcuate nucleus (Arc).
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Abbreviations: ox, optic chiasm. Calibration bar: A, C, E and F: 50 µm; B: 250 µm; D: 100
µm.
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Fig. 2.
Neuronostatin (NST) immunoreactive cells and processes in the rat brain. A, irNST cells are
concentrated in the hypothalamic periventricular nucleus (Pe). B, strongly labeled irNST cells
are distributed to the periventricular nucleus, and a few lightly to moderately labeled cells and
processes in the suprachiasmatic nucleus (Sch). C, numerous irNST fibers are present in the
median eminence (ME) and arcuate nucleus (Arc). D, fewer or less intensely labeled irNST
cells are detectable in the motor cortex. E, moderately labeled cells are seen in the polymorphic
layer of the dentate gyrus (PoDG). F, irNST is not detected in a hypothalamic section processed
with an NST antiserum pre-absorbed with the peptide NST (1 µg/ml) overnight. Abbreviations:
3V, 3rd ventricle; f, fornix. Calibration bar: A to C, E, 50 µm; D: 25 µm; F: 100 µm.
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Fig. 3.
Confocal images of rat brain sections double-labeled with neuronostatin (NST, green
fluorescence) and somatostatin (SST, red fluorescence) antiserum. A and B, hypothalamic
periventricular cells located next to the 3rd ventricle (3V) are immunoreactive to NST and SST.
C, a merged image of A and B, where cells immunoreactive to both NST and SST appear
orange. D–E, higher magnification of the area outlined in A–C, where nearly all of the cells
are irNST and irSST. F, a merged image of D and E, where overlapping cells appear orange
color. G–I, dense fibers immunoreactive to NST and SST are seen in the median eminence,
mostly in the external layer. J and K, all 4 cortical neurons in the cerebral cortex are double-
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labeled, but the intensity of irNST appears to be lower than that of irSS. L, a merged image of
J and K. Calibration bar: A–C, and G–I, 50 µm; D–F and J–L, 25 µm.
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Fig. 4.
Calcium responses induced by neuronostatin (NST) and somatostatin (SST) in cultured rat
hypothalamic neurons. A1, NST increases cytosolic calcium [Ca2+]i with two profiles: fast and
transitory (solid line) and calcium oscillations (dotted line). B1, SST slightly reduced the basal
Ca2+. A2, in Ca2+-free saline, NST produced a fast and transitory (solid line) or an oscillatory
response (dotted line), with amplitude lower than those produced in Ca2+-containing saline.
B2, no change in [Ca2+]i was noted in response to SST in Ca2+-free saline. C, NST (10, 100
and 1000 nM) produced a concentration-dependent increase in [Ca2+]i with two profiles: single
spike and oscillations. Arrows denote the administration of neuronostatin or somatostatin.
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Fig. 5.
Changes in resting membrane potential induced by neuronostatin (NST) and somatostatin
(SST) in rat hypothalamic neurons. A, examples of NST-induced depolarization (solid line)
and hyperpolarization (dotted line); NST depolarized 23/257 neurons with a mean amplitude
of 5.2 ± 1.6 mV and hyperpolarized 7/257 neurons with a mean amplitude of 6.1 ± 2.2 mV. B,
example of SST-induced hyperpolarization; SST hyperpolarized 43/114 neurons, with a mean
amplitude of 9.8 ± 2.2 mV. Arrows denote the administration of neuronostatin or somatostatin.
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