16 T
the small intestine of aged rats, compared to young rats months old, n524 BN3F344 F1 rats were used in this
using the neuronal marker, PGP 9.5 [18]. In these latter study.
studies, neuronal counts that were corrected for age-associ- ated changes in intestinal length and circumference [18].
2.3. Catalytic activity of NOS A modest decrease in the contribution of nitrergic
innervation to NANC-mediated relaxation was observed
L
-Citrulline and NO are produced in a 1:1 ratio from with advanced age in the rat intestine [30,33]. Few studies
L
-arginine by the action of NOS. Production of NO was have examined the expression of NO synthase NOS with
3
measured in colonic tissue preloaded with
L
-[ H]arginine advancing age. The number of NADPH diaphorase a
3
and expressed as amount of
L
-[ H]citrulline formed in the histochemical marker for NOS containing neurons-posi-
tissue as described by Bredt and Snyder [5]. As previously tive neurons in the small intestine myenteric plexus of rats
described [16], the longitudinal muscles with adherent decreased modestly with age [28]. In contrast, Belai et al.
myenteric plexus LMMP preparations were obtained observed a significant increase in NADPH diaphorase-
from the mid-colon in young and aged rats and incubated positive neurons with age in the myenteric plexus of the
in a 1.5 ml organ bath for 30 min at 378C in the presence proximal colon, but not the ileum of the rat intestine [2].
of cofactors 1 mM NADPH, 10 mM FAD, 10 mM FMN, The discrepancies in these observations might be explained
and 10 mM tetrahydrobiopterin. LMMP preparations were by the possibility that NADPH diaphorase is a non-specific
3
further incubated with [ H]arginine 3 mCi ml for 4 min marker for NOS containing neurons [35].
at 378C. Immediately following the stimulation of ver- Therefore, based on available literature it is not clear
atridine 5 mM for 5 min, the reaction was stopped by whether advanced age is associated with a decrease in the
flash freezing in liquid nitrogen. The samples were stored total number of myenteric neurons or preferentially in-
3
at 2808C for subsequent measurement of
L
-[ H]citrulline. volves a reduction in a subpopulation of myenteric neu-
Muscle tissues were homogenized with a 1-ml straight wall rons. The goals of this study were 2-fold; 1 to examine
grinder. After centrifugation at 3000 rpm for 10 min at whether advanced age is associated with a loss of myen-
48C, the supernatant in 10 TCA was sonicated for 5 min. teric neurons in the rat colon using the neuronal marker,
After washing with water-saturated ethyl ether, the super- PGP 9.5, and 2 to examine whether advanced age is
natant was applied to a Dowex AG50WX-8 resin column associated with decreased expression of NOS in the rat
1 3
Na form and
L
-[ H]citrulline was eluted with Hepes colon.
buffer pH 5.5 and water, as previously reported [16].
3
L
-[ H]Citrulline in the effluent was measured by liquid
3
scintillation spectroscopy, and the production of
L
-[ H]cit-
2. Materials and methods
rulline in the colonic tissue was expressed as cpm mg tissue. The protein in the pellet was measured by the
2.1. Materials Bio-Rad method using BSA.
Aprotinin, diaminobenzidine, NADPH, flavin adenine dinucleotide
FAD, flavin
mononucleotide FMN,
2.4. NOS immunohistochemistry leupeptin, Triton X-100, and PMSF were obtained from
Sigma St. Louis, MO. Tetrahydrobiopterin was obtained LMMP preparations were obtained from the mid-colon.
3
from ICN Costa Mesa, CA.
L
-[ H]Arginine was obtained LMMP preparations were pinned and stretched on a
from New England Nuclear Boston, MA. Dowex silicone-coated dish and fixed overnight in 4 paraformal-
AG50W-X8 was obtained from Bio-Rad Laboratories dehyde. LMMP preparations were washed and rinsed in
Hercules, CA. Antibody to constitutive nNOS was 0.15 M PBS, and blocked in 0.15 M PBS that contained
obtained from Santa Cruz Santa Cruz, CA. Antibody to 5 normal goat serum, 0.1 BSA, and 0.1 Tween 20
PGP 9.5 was obtained from UltraClone Isle of Wight, for 30 min at room temperature. LMMP preparations were
UK. Anti-rabbit IgG and the avidin–biotin labeled kit then incubated with a polyclonal antibody raised in rabbits
Vectastain ABC kit were obtained from Vector Lab- against the purified soluble NOS extract from rat cere-
32
oratories Burlingame, CA. [ P]dCTP and random primer bellum at a dilution of 1:1000 in 0.15 M PBS for 18 h at
labeling system Rediprime were obtained from Amer- 48C. After washing in 0.15 M PBS, LMMP preparations
sham Arlington Heights, IL. TRIzol was obtained from were incubated with biotinylated anti-rabbit IgG for 1 h at
Life Technologies Gaithersburg, MD. AatII and dithio- room temperature. Horseradish peroxidase staining was
threitol were obtained from Boehringer Mannheim In- done using the Vectastain ABC kit. Diaminobenzidine and
dianapolis, IN. nickel chloride were used as chromogens.
The number of nNOS–positive cells in 50 ganglia were 2.2. Animals
counted in each preparation and the average number of nNOS–positive cells in one ganglion were determined in
Young 4–8 months old, n524 and aged 22–28 each preparation, as previously described [31,32].
T . Takahashi et al. Brain Research 883 2000 15 –21
17
2.5. Western blot analysis of NOS synthesis labeled probes for 16 hrs at 658C. The samples were
washed four times: twice in 23 SSC, 0.1 SDS for 5 min LMMP preparations were obtained from the mid-colon
at 658C, and twice in 0.23 SSC, 0.1 SDS for 5 min at in young and aged rats. Soluble homogenates of these
658C. The samples were autoradiographed with intensify- samples were prepared in lysis buffer containing 25 mM
ing screens at 2808C. Radioactivity of the tissues was Tris–HCl pH 7.4 with EGTA 1 mM, dithiothreitol 1
measured by an imaging analyzer. To confirm equivalent mM, leupeptin 10 mg ml, aprotinin 10 mg ml, PMSF
loading of RNA in the various lanes, the membrane was 1 mM, and Triton X 0.1. Western blot analysis of
washed and
rehybridized with
a probe
against LMMP preparations was performed, as previously de-
glyceraldehyde-3-phosphate dehydrogenase GAPDH. scribed [31,32]. Equal amounts of protein 20 mg in each
sample were separated by SDS–PAGE 7.5, w w, gel and transferred to a nitrocellulose membrane. All pro-
2.8. Statistical analysis cedures were done in Tris buffer 40 mM, pH 7.55
containing 0.3 M NaCl and 0.3 Tween 20. The mem- Data were expressed as means6S.E. Statistical analysis
brane was blocked with dried milk 6, w v and incu- was performed using the Student’s t-test or paired t-test.
bated with specific polyclonal nNOS antibody 1:1000 Significance was accepted at the 5 level.
dilution and a horseradish peroxidase-conjugate of affinity column-purified goat antibody to rabbit IgG. The immune
complexes were detected on photographic film by H O
2 2
luminol chemiluminescence and the bands were measured 3. Results
by an imaging analyzer, as previously described [23,32]. 3.1. NOS catalytic activity in mid-colon preparations
2.6. Western blot analysis of PGP 9.5 from young and aged rats
3
It has been demonstrated that antisera raised against Basal
L
-[ H]citrulline formation was 61506925 cpm neuronal cytosolic protein, protein gene product PGP 9.5,
mg protein of the colonic tissues obtained from young rats.
3
provides excellent staining of the generic neurons in the Veratridine 5 mM significantly increased
L
-[ H]citrulline CNS and the myenteric plexus [8]. As previously de-
formation to 10 51062590 cpm mg protein of the colonic scribed [31,32], possible quantitative changes of the
tissues obtained from young rats Fig. 1; n57, P,0.05, by generic neurons in the colonic LMMP preparations in aged
Student’s t-test.
3
rats was studied by Western blot analysis of PGP 9.5. Basal
L
-[ H]citrulline release was decreased by 5866, Equal amounts of protein 20 mg in each sample were
and veratridine-induced release was reduced by 78612 separated by SDS–PAGE 15, w w, gel and transferred
in preparations from aged animals compared to young to a nitrocellulose membrane. After blocking with dried
animals Fig. 1; n57, P,0.05, by Student’s t-test.
3
milk 6, w v, the membrane was incubated with specific Veratridine-induced
L
-[ H]citrulline formation did not in- polyclonal PGP 9.5 antibody 1:10 000 dilution [8].
creased significantly in aged preparations Fig. 1; n57, P50.2.
2.7. Northern blot analysis of NOS mRNA expression For Northern blot analysis of NOS mRNA, the total
RNA was isolated from homogenized colonic LMMP preparations from young and aged rats, as previously
described [31,32]. Extraction and preparation of RNA was performed using TRIzol reagent. The amount of RNA was
estimated by measuring absorbance at 260 nm. Following isolation, the total RNA samples 20 mg each were
electrophoresed on an agarose gel and transferred onto a nylon membrane. As previously demonstrated by Huang et
al. [17], we used 2947 base rat neuronal NOS cDNA probe that extends 2.1 kb in the 39 direction beyond the first exon
244–3191 of the cloned cDNA. This probe was made from original cDNA provided by Dr. Solomon H. Snyder
cut by the restriction endonuclease, AatII. The cDNA
3
Fig. 1. Basal and veratridine-induced
L
-[ H]citrulline formation in
32
probe 2.9 kb was labeled with [ P]dCTP 111 TBq
colonic tissues obtained from young and aged rats. Basal and veratridine
3
mmol by the random primer labeling system. The samples
5 mM-stimulated
L
-[ H]citrulline formation were significantly reduced
were prehybridized for 3 h at 658C and hybridized with the
in aged rats P,0.05, n57.
18 T
3.2. NOS immunohistochemistry of the colonic myenteric 3.5. Northern blot analysis of NOS mRNA of the colonic
plexus tissue
In the young rats, nNOS-immunoreactive neuronal cell We examined the reduced NOS synthesis in the colonic
bodies were found throughout the myenteric plexus of the myenteric plexus in aged rats to determine if it was caused
mid-colon. Non-neuronal tissues e.g., muscle cells, endo- by down-regulation of nNOS mRNA expression. nNOS
thelium, mucosal cells, and macrophages were unstained. mRNA expression 9.5 kb was clearly observed in the
The average number of nNOS-immunoreactive cells of the colonic tissues from young rats. It has been demonstrated
mid-colon was 5.361.2 ganglion in young rats n54 that nNOS mRNA expression is abundant in the rat
Fig. 2A. The average number of nNOS-immunoreactive cerebellum [4,17]. We have previously shown that nNOS
cells was significantly less in the mid-colon of the aged mRNA bands observed in the myenteric plexus corres-
rats 2.360.5 ganglion, n54, P,0.01, by Student’s t-test ponded well with the bands observed in the rat cerebellum
Fig. 2B. [31]. The density of nNOS mRNA of colonic tissues
observed in aged rats was significantly reduced to 35615 of that observed in young rats n53, P,0.05, by
3.3. Western blot analysis of NOS synthesis paired t-test Fig. 4.
The molecular weight of neuronal NOS nNOS and endothelial NOS eNOS has been shown to be 155 and
4. Discussion
