GENERAL AND COMPARATIVE
ENDOCRINOLOGY
www.elsevier.com/locate/ygcen
General and Comparative Endocrinology 137 (2004) 166–176
Expression of natriuretic peptides, nitric oxide synthase,and guanylate cyclase activity in frog mesonephros during
the annual cycle
Carla Fenoglio,a,* Livia Visai,b Concetta Addario,a Giuseppe Gerzeli,a Gloria Milanesi,a
Rita Vaccarone,a and Sergio Barnia
a Dipartimento di Biologia Animale, Universit�a di Pavia, Piazza Botta 10, 27100 Pavia, Italyb Dipartimento di Biochimica—Sezione di Medicina, Universit�a di Pavia, Via Taramelli, 3/B, 27100 Pavia, Italy
Received 28 February 2003; revised 17 February 2004; accepted 10 March 2004
Available online 24 April 2004
Abstract
Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase
(NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation
of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the
activation of guanylate cyclase (GC) and the generation of guanosine 30,50-cyclic monophosphate (cGMP), which is considered a
common messenger for the action of these factors. The present study was designed to investigate—by histochemical methods—the
expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the
mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their
expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NAD-
PHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during
active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and
some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the
presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at
light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the
immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or
expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided
interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in the frog
kidney extract was evidenced by dose-dependent response in the ELISA. The data suggest that both ANP and NO are intra-renal
paracrine and/or autocrine factors which may modulate the adaptations of frog renal functions to seasonal changes through the
action of the cGMP generated from GC activity.
� 2004 Elsevier Inc. All rights reserved.
Keywords: Natriuretic peptides; Nitric oxide; Amphibians; Mesonephros; Hibernation
1. Introduction
The regulation of ions and water balance in different
vertebrate organs and principally in the kidney is greatly
influenced by hormones and hormone-like substances.
Among these, molecules like natriuretic peptides (NPs)
* Corresponding author. Fax: +39-0382-506406.
E-mail address: [email protected] (C. Fenoglio).
0016-6480/$ - see front matter � 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygcen.2004.03.008
and nitric oxide (NO) are attracting an increasing
interest.
NPs constitute, in different species of each vertebrate
class, a hormonal system that includes three types of
peptides: atrial NP (ANP), brain NP (BNP), and C-type
NP (CNP). Both ANP and BNP are synthesized mainly
in the heart and in the brain, and stored in granule formfor release into the bloodstream, whereas CNP is
expressed principally in the brain (for reviews see
C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176 167
Beltowski and Wojcicka, 2002; Forssmann et al., 1998;Takei, 2000, 2001). Moreover, a peptide closely related
to ANP—urodilatin (URO)—has been localized within
the distal tubules of human kidney and urine (Beltowski
and Wojcicka, 2002; Forssmann et al., 1998; Kone,
2001). URO and ANP derive from the same gene and a
common peptide but, unlike ANP, urodilatin is syn-
thesized, processed, and secreted by the kidney, and
does not circulate in the blood.Nitric oxide (NO) is a highly diffusible gas generated
from LL-arginine by nitric oxide synthase (NOS, EC
1.14.13.39) exerting a variety of functions in different
tissues and organs, including the kidney (for reviews see
Aoki et al., 1995; Moncada and Higgs, 1993). In mam-
mals, multiple isoforms of NOS have been identified,
i.e., neuronal NOS (nNOS), inducible NOS (iNOS) and
endothelial NOS (eNOS)—also termed NOS1, NOS2and NOS3, respectively—which vary considerably in
their intracellular location, structure, and function.
Moreover, variants of these isoforms are now beginning
to be identified (Alderton et al., 2001).
Regarding the kidney, compelling evidence indicates
the presence of nNOS in rat kidney macula densa,
whereas existing data concerning the presence of iNOS
in rat renal tubules are controversial (Bachmann et al.,1995; Morrissey et al., 1994; Terada et al., 1992); eNOS
has been detected mainly in kidney vascular components
(Bachmann et al., 1995; Kone, 2001). All three isoforms
possess NADPH diaphorase activity which, unlike other
NADPH diaphorases, survive fixation. Therefore,
NADPHd activity can be specifically used as a marker
for NOS in different paraformaldehyde-fixed tissues
(Nakos and Gossrau, 1994).A number of reports conducted both in vivo and in
vitro support the assumption that both ANP and NO
play an important role in fluid and electrolyte homeo-
stasis by modulating ion transport pathways and blood
pressure. It is generally believed that they inhibit
transepithelial Naþ and water resorption in tubule cells
mainly through the action of Naþ, Kþ-ATPase (for re-
views see Beltowski and Wojcicka, 2002; Kourie andRive, 1999).
In previous immunohistochemical studies, antibodies
against both mammalian ANP and NOS have been
successfully employed in different tissues of non-mam-
malian vertebrates, including amphibians (Bodegas
et al., 1995; Chapeau et al., 1985; De Falco et al., 2002;
Feuilloley et al., 1993; Netchitailo et al., 1988; Reinecke
et al., 1989). In particular, specific research has dem-onstrated that the structure of frog proANP and ANP
shows a structural homology with the mammalian
peptides (Gilles et al., 1990; Lazure et al., 1988; Sakata
et al., 1988).
The biological effects of NPs and NO on the target
cells are mediated by the second messenger guanosine
30,50-cyclic monophosphate (cGMP) through the action
of guanylate cyclase (GC, EC 4.6.1.2). Numerous stud-ies have documented the presence of GC-coupled na-
triuretic peptide receptors (NPR) in different segments
of the rat renal tubule (Grandclement and Morel, 1998;
Hirsch et al., 2001; Ritter et al., 1995; Terada et al.,
1991). Moreover, studies have shown the presence of
ANP binding sites also in the kidney of some species of
amphibians (Rana temporaria, Kloas and Hanke, 1992a;
Xenopus laevis Kloas and Hanke, 1992b; Ambystoma
mexicanus, Kloas and Hanke, 1993; Bufo marinus, Meier
et al., 1999).
It is a well known fact that, in most amphibians,
environmental conditions induce a series of complex
physiological adaptations. Moreover, during hiberna-
tion many amphibians drastically reduce most of their
metabolic processes, including osmotic and ionic regu-
latory processes. Therefore, amphibians are of particu-lar interest with respect to osmotic regulation and the
endocrine mechanisms they utilize for this process
(Bentley, 1998).
In a previous study performed on the mesonephros of
active and hibernating frogs, we evidenced some struc-
tural and cytochemical modifications of this organ, in-
cluding a variation of Naþ, Kþ-ATPase activity
(Fenoglio et al., 1996).To extend our knowledge on the different mecha-
nisms behind body fluid homeostasis, we studied the
distribution of ANP and NOS in the frog kidney
throughout active life and hibernation. The ELISA was
also used to demonstrate immunoreactive NPs in kidney
extracts. We also tested the expression of the ANP-
propeptide (proANP) to verify the synthetic activity of
the organ. For this purpose, immunoreactions for pro-ANP, ANP, and NOS and enzyme cytochemical studies
for NOS/NADPHd and GC were performed in the
mesonephros of the Rana esculenta during the different
periods of the natural annual cycle.
2. Materials and methods
Adult samples of edible Rana esculenta L. of both
sexes were caught in their natural environment near
Pavia for two consecutive years. Active frogs were col-
lected from rice-field drains in June and September
(environmental temperature ranging from 16 to 25 �C);frogs hibernating underground were collected in Janu-
ary (environmental temperature about 0 �C). Five ani-
mals for each period were used immediately aftercapture. To avoid circadian variations, all samplings
were conducted between 15.00 and 16.00 h. The experi-
ments were performed in compliance with the Italian
law. The animals were stunned and killed by decapita-
tion and the kidneys were removed, excised, and pro-
cessed for the following studies: (a) recognition of ANP
in frog kidney extract by ELISA, (b) proANP and ANP
168 C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176
immunohistochemistry, (c) NOS immunohistochemis-try, (d) NOS/NADPH-diaphorase histochemistry, and
(e) guanylate cyclase cytochemistry.
We compared the relative intensity of both immu-
nostaining and enzyme reactions between hibernating
and active samples on the same slide to avoid possible
discrepancies resulting from different processing times.
The relative expression levels of immunodeposits and
the enzyme reaction product of different specimens wereevaluated by two independent observers. Different val-
ues of positivity were scored as weak (+), moderate
(++), and intense (+++), respectively. Sections lacking
reactivity were graded as negative ()).
2.1. Frog kidney extract
Fresh frog renal tissue samples were homogenizedunder liquid nitrogen using mortar and pestle and
suspended in 0.1M hydrochloric acid and 0.6% Triton
X-100. After sonication (6� 20 s) the material was cen-
trifuged at 60,000g for 20min and the supernatant was
filtered and kept at )20 �C. The protein contents of the
extracts were determined by the bicinchoninic acid assay
(BCA) following the manufacturer’s specifications
(Pierce).
2.2. Frog NPs ELISA
ELISAs were performed as previously described (Visai
et al., 2000). Briefly, microtitre wells were coated with
100 ll of 2 lg/ml of commercial available human ANP
(HANP), human urodilatin (HURO) or frog ANP
(FANP) (Sigma) in coating buffer (50mM sodiumcarbonate, pH 9.5) overnight at 4 �C. To control for non-
specific binding, some of the wells were coated with
bovine serum albumin (BSA), laminin, fibronectin, and
fibrinogen as negative controls. After three washes with
PBS (pH 7.4) containing 0.1% (v/v) Tween 20 (PBST), the
wells were blocked by incubating them with 200 ll PBScontaining 2% (w/v) BSA for 1 h at 22 �C. Diluted (1:600)
rabbit anti-HANP (Biogenesis) in PBST containing 1%BSA was added to each well and incubated for 2 h at
37 �C. The wells were subsequently washed with PBST
and incubated for 1 h at 22 �C with 100 ll horseradishperoxidase (HRP)-conjugated goat anti-rabbit IgG
(1:500 dilution in 1% BSA). The wells were finally
incubated with 100 ll of development solution (phos-
phate–citrate buffer containing o-phenylenediamine
dihydrochloride substrate, as prepared according tomanufacturer’s specifications). The reaction was stopped
with 100 ll of 0.5M H2SO4 and absorbance values
(490 nm) were measured using a microplate reader
(Bio-Rad).
Similarly, microtitre wells were also coated with
100 ll of increasing concentrations of the frog kidney
extract prepared as previously described and diluted in
coating buffer overnight at 4 �C. After washing, blockedwells were incubated with rabbit anti-HANP or rabbit
pre-immune antibodies (dilution 1:600 in 1% BSA for
both antibodies) for 2 h at 37 �C. The pre-immune an-
tibodies used did not recognize wells coated with
commercial HANP or FANP, indicating the absence of
anti-ANP antibodies. The wells were washed and then
incubated with for 1 h at 22 �C with HRP-conjugated
goat anti-rabbit IgG (1:500 dilution in 1% BSA). Thecolor development reaction was performed and mea-
sured as previously described.
2.3. ANP immunohistochemistry
Pieces of kidney were fixed in 2% (w/v) paraformal-
dehyde in 0.1M phosphate buffer, embedded in parap-
last-wax and sectioned at 6 lm. Sections were dewaxedin xylene, rehydrated, and treated with 3% H2O2 in 10%
methanol for 15min to block endogenous peroxidase
activity. They were then briefly (15min) incubated with
0.5% Triton X, rinsed and then treated with 10% bovine
serum albumin (BSA) in PBS for 45min to prevent non-
specific staining. For immunohistochemical staining the
following primary antisera were applied: anti-human
proANP 26–92, anti-human or anti-rat aANP, pur-chased from Peninsula. All antisera were raised in rab-
bits and diluted 1:200. The anti-HANP antibody from
Biogenesis was also tested, diluted 1:100. The sections
were incubated overnight in a moist chamber at 4 �C.After rinsing, the sections were treated with the sec-
ondary peroxidase-conjugated swine anti-rabbit anti-
body (Dako), diluted 1:100 for 45min, and afterwards
incubated for 5min for peroxidase reaction, in a me-dium containing 0.05% 3,30-diaminobenzidine tetrahy-
drochloride (DAB) and 0.01% H2O2 in 0.05M Tris–HCl
buffer. Slides were dehydrated and mounted.
The specificity of the immunoreaction was controlled
by replacing the primary antisera with PBS or normal
serum; no staining was observed under these conditions.
2.4. NOS immunohistochemistry
Paraformaldehyde-fixed, paraplast-embedded sec-
tions (6 lm thick) were dewaxed and rehydrated in
graded ethanol. After washing in PBS, endogenous
peroxidase activity was blocked by incubation in 3%
H2O2 in 10% methanol for 15min. Sections were incu-
bated in 0.5% Triton X and then treated with 10%
normal swine serum in PBS (45min) to block non-spe-cific staining. For immunohistochemical staining the
sections were incubated overnight at 4 �C in a humid
chamber with the respective rabbit anti-rat nNOS, 1:500
dilution (Chemicon) or rabbit anti-mouse iNOS, 1:100
dilution (Serotec). After washing in PBS the sections
were incubated for 45min at room temperature with
peroxidase-conjugated swine anti-rabbit antibody
Fig. 1. Binding of anti-HANP antibody to HANP, FANP, and HURO
in the ELISA. Human and frog atrial natriuretic peptides, human
urodilatin, and BSA were immobilized (2lg/well) to microtitre wells,
incubated with rabbit anti-human ANP antibody (1:600), and the
bound antibody was detected with HRP-conjugated goat anti-rabbit
IgG. Data represent means�SD of triplicate measurements.
C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176 169
(Dako), diluted 1:100. After rinsing off the secondaryantibody with PBS the peroxidase reaction was per-
formed with 0.05% DAB in 0.05M Tris–HCl buffer, pH
7.6, containing 0.01% H2O2, for 5min. Finally, the
slides were dehydrated and mounted for examination.
For the negative controls, primary antibodies were
replaced by normal rabbit serum or PBS; they did not
show any immunoreactivity.
2.5. NOS/NADPH-diaphorase histochemistry
Kidneys were frozen in liquid nitrogen in closed vials.
Cryostat sections, 8 lm thick, were cut on a cryostat at a
cabinet temperature of )21 �C. Sections from both ac-
tive and hibernating samples were picked up on poly-LL-
lysine coated slides; several slides were stored in the
cryostat cabinet until used.To demonstrate the catalytic activity of NOS specif-
ically, cryostat sections were prefixed in 4% parafor-
maldehyde, buffered with 0.1M phosphate (pH 7.4) for
15min at 4 �C, washed in phosphate buffer for 10min,
and then incubated in a medium according to Nakos
and Gossrau (1994), with our modifications (Fenoglio
et al., 1997). The standard medium consisted of poly-
vinyl alcohol (PVA) 15% in 100mM phosphate buffer,pH 7.4, 5mM levamisole, 2mM bNADPH (Sigma),
3mM NBT (Sigma), 0.2% Triton X, and 0.2% para-
formaldehyde. Incubation was performed for 50min at
37 �C in the dark.
Controls were carried out with incubation media: (a)
free of bNADPDH and (b) with NADH instead of
NADPH. In both cases no reaction product was
observed in the tissue sections.
2.6. Guanylate cyclase cytochemistry
Small sections from kidneys were prefixed in 2%
paraformaldehyde and 0.25% glutaraldehyde in 0.05M
cacodylate buffer (pH 7.4) for 1 h at 4 �C. After fixation,
they were washed overnight in cacodylate buffer at 4 �C.GC activity was localized ultracytochemically by us-
ing cerium as capture agent. The incubation medium
consisted of 0.05M Tris–maleate buffer, pH 7.4, with
5% sucrose, 1mM guanylyl-imidodiphosphate sodium
salt (Gpp(NH)p, Sigma) 10mM theophylline (as inhib-
itor of phosphodiesterase), 10mM levamisole (as in-
hibitor of alkaline phosphatase), 6mM MnCl2, and
2mM CeCl3. The incubation lasted for 45min at 37 �C;the original medium was replaced with a freshly pre-pared medium after 20min. Some specimens were in-
cubated in a standard medium plus URO 1 lM (Sigma)
as enzyme activator. Control samples were incubated in
a substrate-free medium.
After incubation, the samples were washed several
times in Tris–maleate buffer and in cacodylate buffer,
refixed for 60min at 4 �C in 1.5% glutaraldehyde, and
washed again before postfixation with OsO4 for 2 h at4 �C. Samples were dehydrated in a graded ethanol se-
ries and embedded in Epon 812. Some ultrathin sections
were left unstained while others were stained with uranyl
acetate and lead citrate, and observed in a Zeiss EM 900.
Controls incubated in the absence of substrate did
not show any specific deposits of the enzyme reaction
product.
3. Results
3.1. Morphological aspect
Sections of frog mesonephros stained with conven-
tional H&E showed that the frog nephron consists of the
following parts: glomerulus, proximal tubule, neck andintermediate segment, distal tubule, collecting tubule,
and duct. No macula densa was observed in any of the
samples.
The distal tubules occupy the ventromedial region of
the mesonephros while the rest are located in the dor-
solateral region. The glomeruli are present between the
two regions.
3.2. Recognition of frog kidney NPs through ELISA
The ELISA—a sensitive biochemical technique—was
performed to determine the presence of natriuretic
peptides in the frog kidney extract before proceeding to
Fig. 2. Percentage of binding of anti-ANP antibody to frog kidney
extract by ELISA. Increasing concentrations (from 50 ng to 5lg) offrog kidney extract prepared as described in ‘‘Section 2’’ were immo-
bilized to microtitre wells, incubated with rabbit anti-HANP (1:600) or
rabbit pre-immune antibody (1:600), and the bound antibody was
detected with HRP-conjugated goat anti-rabbit IgG. Data represent
means� SD of triplicate measurements.
170 C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176
other immunohistochemical analyses. With this same
technique, the immunoreactivity of the rabbit anti-
HANP was then tested on HANP, FANP, and HURO.
Fig. 1 shows that the antibody preparation used reactsstrongly not only with immobilized HANP as expected
but also with FANP and HURO. As can be seen, rabbit
anti-HANP (the negative control) did not bind to BSA.
The ELISA test further indicated that the anti-HANP
antibody did not bind to other immobilized proteins
such as ovalbumin, laminin, fibronectin, and fibrinogen
Fig. 3. Immunohistochemical stainings for ANP in active (A) and hibernati
most distal and proximal tubules. (B) Dense immunodeposits of ANP can be
labelled. gm, glomeruli; dt, distal tubules; pt, proximal tubules, 130�.
(data not shown), a fact which underscores the speci-ficity of the antibody. Fig. 2 shows the percentage of
binding of the rabbit anti-HANP antibody to frog kid-
ney extract. The anti-HANP antibody reactivity to in-
creasing concentrations of immobilized frog kidney
extract indicates the presence of NPs in the frog kidney
extract. The rabbit pre-immune antibody showed no
binding whatsoever to the frog kidney extract.
3.3. ANP immunohistochemistry
The immunohistochemical experiment with the anti-
sera against NPs resulted in a generally positive labelling
in several tubule segments. Essentially, similar staining
patterns were obtained with different antisera. No sig-
nificant differences in the distribution of immunoreac-
tive deposits were noted in any samples using eitherhuman or rat ANP antiserum.
Interestingly, some differences in immunostaining
were observed when comparing active and hibernating
frogs. In active frogs, moderate to intense ANP-like
immunostaining was detected in some tracts of proxi-
mal, distal, and collecting tubules (Fig. 3A). Moreover,
within the tubule segments some cells showed different
degrees of positivity. In hibernating specimens ANP-likeimmunoreactivity was mainly observed as dense depos-
its in some distal tubules, whereas other segments of
renal tubule—mainly proximal ones—showed only weak
immunostaining (Fig. 3B). Some cells slightly positive to
ANP-like immunostaining were observed within the
glomeruli of both active and hibernating frogs (Figs. 3A
and B).
The application of the proANP resulted in a weak tomoderate immunoreactive labelling of some proximal
ng frog mesonephros (B). (A) Anti-ANP immunolabeling is intense in
observed mainly in distal tubules (arrows); proximal tubules are faintly
C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176 171
and distal tubule segments both during the active andhibernating periods, being more intense in active frogs
(data not shown).
3.4. NOS immunohistochemistry and histochemistry
In active frogs, iNOS immunoreactivity was weak to
moderate and it was mainly localized in cells consti-
tuting the proximal segments of the mesonephros tu-bules (Fig. 4A). In hibernating samples, moderate
immunohistochemical staining for iNOS was mainly
found in most proximal tubules. A dense rim of im-
munodeposits labelled the apical portion of these tu-
bules (Fig. 4B).
Fig. 4. Immunolocalization of iNOS (A and B). (A). Light to moderate imm
sonephros. (B). Moderate to intense immunostaining (arrows) can be obs
NADPHd activity. (C) Most proximal tubules of the active frog mesonephro
proximal tubule cells during hibernation. pt, proximal tubules, 200�.
In all samples considered, no immunostaining couldbe detected by using an antiserum against nNOS (data
not shown).
Following NOS/NADPHd histochemistry, the epi-
thelial cells lining the proximal tracts of the renal tubules
showed a different intensity of reaction, depending on
the seasonal periods we considered. In particular, during
the active life, the cells constituting proximal tubules
were particularly positive to the NOS/NADPHd reac-tion (Fig. 4C), whereas distal and collecting tubules
showed little, if any, NOS/NADPHd staining. Hiber-
nating specimens displayed a decrease in reaction in-
tensity at the same sites (Fig. 4D). Glomeruli were not
stained in any of the samples.
unopositivity is noted in proximal tubules (arrow) in active frog me-
erved in proximal tubules of the hibernating frog. (C and D) NOS/
s show moderate to intense reactivity. (D). Low staining is observed in
172 C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176
3.5. Guanylate cyclase cytochemistry
In all samples, most epithelial cells constituting the
proximal tubules showed granules of the reaction
product on the apical plasma membranes; fine reaction
product granules were detected also on the lateral
membranes of these cells (Fig. 5A). Fine precipitates
were also evidenced on the cell membrane of the col-
lecting tubule cells (Fig. 5B). Granules of the reactionproduct were also detected within the cytoplasm of the
distal tubule cells (Fig. 5C). The localization of the re-
action product was the same in both active and hiber-
nating frogs, although the granules of the reaction
product were less numerous in the latter (Fig. 5D) than
in the former (Fig. 5A). In the glomeruli, mainly of the
active frogs, the podocytes and their foot processes ex-
hibited some reactivity. The reaction product was de-tected within the cytoplasm or lining some podocyte
processes (Figs. 6A and B).
The addition of URO in the basal medium caused an
increase of the granules mainly on the apical side of the
plasma membrane of the cells constituting the proximal
tubules (data not shown).
Fig. 5. Ultracytochemical demonstration of GC activity in active frogs (A–C
brane and brush border (bb) of proximal tubule cells (arrows), 9000�. (B). R
cells of the collecting tubule (arrow), 12,000�. (C). Cytoplasmic localization
8500�. (D). Hibernating frog. Granules of the reaction product are localize
detected on the brush border (bb) in comparison with active sample (A), 12
All the results are summarized in Tables 1 and 2.
4. Discussion
The present data show that the mesonephros of R.
esculenta is a site of synthesis and/or storage of ANP.
From the comparative point of view, and taking into
account the existing differences between amphibianmesonephros and mammalian metanephros, the distri-
bution of the immunodeposits in the frog kidney showed
some interesting similarities with mammalian species.
Immunoreactive sites were visualized by using anti-
sera against mammalian molecules (proANP, ANP). In
fact, literature data indicate that antigenic determinants
of frog ANP are related structurally to rat or human
ANP. Moreover, as concerns ANP the results we ob-tained with the ELISA performed on the frog extract
indicates the presence of NPs in the frog kidney. With
the ELISA technique we cannot distinguish between
proANP, mature ANP or URO but we may have evi-
dences of their effective presence in frog kidney extract.
It is of note that, in a study recently conducted on the
). (A) Abundant reaction product is detected on apical plasma mem-
eaction product granules can be observed on the plasma membranes of
of reaction product granules can be observed within distal tubule cells,
d on the luminal side of the proximal tubule cells; fewer granules are
,000�.
Fig. 6. Ultrastructural localization of GC activity in active frogs (A and B). Details of two glomeruli showing fine precipitates of the reaction product
in the cytoplasm of a podocyte (po) and also within and around some podocyte processes (arrows). (A) 14,000� and (B) 18,000�.
Table 1
Evaluation of immunohistochemical expression of natriuretic peptides
and NOS in active and hibernating frogs
Samples proANP ANP nNOS iNOS
Active frogs ++ +++ ) +/++
Hibernating
Frogs
+ ++ ) ++
+++, intense staining; ++, moderate staining; +, weak staining;
and ), no staining.
Table 2
Evaluation of enzyme activities in active and hibernating frogs
Samples NOS/NADPHd GC
Active frogs ++/+++ +++
Hibernating frogs +/++ ++
+++, intense staining; ++, moderate staining; and +, weak stain-
ing.
C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176 173
kidney of two species of anurans—R. esculenta and Rana
italica—De Falco et al. (2002) have demonstrated the
presence of ANP by using a mammalian antibody.
However, the authors found that the immunoreaction
was mainly present in the kidney of the R. esculenta, a
species living in an aquatic habitat. The authors suggest
that different patterns of ANP positivity could be related
to the different physiologies of the two frog species.In the two conditions we considered—activity and
hibernation—some interesting differences in the ANP-
like immunostaining distribution along the renal tubule
were evidenced. In both conditions the distal tubules
were the most reactive sites, being sometimes more
intense in hibernating animals.
These dense ANP immunodeposits in the hibernating
samples could represent a storage form of natriuretic
peptide, probably released in response to appropriate
stimuli when frogs awake. Consistent with this hypoth-
esis, Lenz et al. (1999) have demonstrated that HEK 293
cells (which display several characteristics of distal tu-
bule cells)—unless stimulated—are able to store a certain
amount of natriuretic peptide in the cytoplasm.As concerns NOS, in this study, no immunoreaction
resulted after incubation of kidney samples with nNOS
antibody probably due to the absence of a structure
similar to macula densa. It is of interest that in a review
of Sokabe and Ogawa (1974) have questioned the
presence of such a structure in different amphibian
species, suggesting that a macula densa is absent in
amphibian kidney.Apart from macula densa, the presence of NOS in
other nephron segments is still a controversial subject.
However, some authors have identified iNOS expression
in some segments of the rat renal tubule, including the
proximal (Liang and Knox, 2000; Mohaupt et al., 1994;
Morrissey et al., 1994; Shin et al., 1999; Yu, 1997). It has
also been demonstrated that iNOS mRNA is constitu-
tively expressed in rat proximal-tubule cells (Liang andKnox, 2000) and that the proximal convoluted tubule is
the primary site in mammal kidney to synthesize argi-
nine from citrulline (Levillain et al., 1993; Morel et al.,
1996). Moreover, the proximal tubule is able to produce
large quantities of NO upon a variety of stimuli (Liang
and Knox, 2000; McLay et al., 1994).
In frog mesonephros, the immunoreaction for iNOS
and the histochemical reaction for NOS/NADPHd
174 C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176
activity were both expressed mainly in the proximaltubules. Although the localization for the two reactions
was similar in both active and inactive frogs, their in-
tensity varied during the annual cycle and demonstrated
opposite results. In particular, active frog specimens
showed an intense NOS/NADPHd activity as compared
to the hibernating animals. The seasonal differences in
the NOS/NADPHd-staining intensity observed in the
proximal tubules could reflect different iNOS activation.In fact, some studies have shown that in mammals the
proximal tubule is able to produce large quantities of
NO after appropriate stimuli (i.e., LPS, cytokines, and
hypoxia) even after a few minutes (McLay et al., 1994;
Yaqoob et al., 1996; Yu et al., 1994). Consistent with
these findings, upregulation of iNOS could occur inde-
pendently of a de novo synthesis of the enzyme. In this
regard, some tyrosine kinases and phosphatases andANP are believed to modulate the functional activity of
this enzyme (Kiemer and Vollmar, 1998; Pan et al.,
1996).
There is an assumption based on experimental
findings that ANP and NO can interact in the control
of renal functions (Chatterjee et al., 1999; Eitle et al.,
1998; Kone, 2001; McLay et al., 1995). These studies,
using different methods and experimental models,consider cGMP the common messenger for both the
diuretic and natriuretic actions of ANP and NO on the
renal tubule. In particular, the modulation of tubular
sodium transport by ANP is mediated through acti-
vation of GC and generation of cGMP after they bind
to specific natriuretic peptide receptors, notably NPR-
A for both ANP and urodilatin. Therefore, the
presence of receptors for natriuretic peptides may beindirectly demonstrated through the cytochemical lo-
calization of ANP-stimulated GC activity.
As concerns NPRs Sekiguchi et al. (2001) have per-
formed a comparative study by cloning the NPR-A re-
ceptor subtype from the brain of the bullfrog Rana
catesbeiana. Interestingly, they found that the bullfrog
receptor is related to its mammalian counterpart,
showing a very high similarity (�92%) in the catalyticdomain where GC region resides. They also demon-
strated a dose-dependent stimulatory effect by ANP on
specific receptors in eliciting the cGMP production.
In mammal renal tubules ANP receptors or ANPR-
mRNA have been found in different segments of rat
nephron (Grandclement and Morel, 1998; Ritter et al.,
1995; Terada et al., 1991). Although the proximal tubule
is not considered the main target of ANP, there is evi-dence that ANP receptors also in the cells of this seg-
ment mainly occur in response to an increase in the
expression of ANP (Mistry et al., 2001; Terada et al.,
1991). In amphibians, some studies have found ANP-
binding sites on the glomeruli and renal tubules by au-
toradiography (Kloas and Hanke, 1993; Meier et al.,
1999).
In the present paper we evidenced the presence of GCultrastructurally, in particular in proximal and distal
tubules, its activity being more relevant in samples of
active frogs. Some reactivity was observed within some
podocytes and their processes of the glomeruli, mainly
in the active frogs. Discrepancies concerning the detec-
tion of ANP binding sites in the kidney of amphibians
between previous researches and the present study may
be due to differences in methodological approaches and/or amphibian species studied.
However, the seasonal differences we observed in the
GC activity can be reasonably associated to variations
in diuresis and natriuresis. In fact, NPs are believed to
act via cGMP to control different mechanisms involved
in ion and water balance, including the modulation of
the Naþ, Kþ ATPase activity (Beltowski and Wojcicka,
2002; Kourie and Rive, 1999; McLay et al., 1995;Scavone et al., 1995).
Naþ/Kþ ATPase plays a major role for sodium re-
absorption in different tubule segments. Evidence exists
that some hormones, including ANP, regulate the cat-
alytic activity of the renal Naþ/Kþ ATPase through
phosphorylation and dephosphorylation by second
messengers and protein kinases (Bertorello et al., 1991;
Ewart and Klip, 1995; F�eraille et al., 1999; Ominatoet al., 1996).
Our previous research provided some information
regarding the changes in the activity of the Naþ/Kþ
ATPase-pump in frog kidney during the annual cycle
(Fenoglio et al., 1996), whereas immunostaining for the
subunit a of the Naþ/Kþ ATPase remained practically
unchanged (Fenoglio et al., 2000). Therefore, we believe
that, in our experimental model, changes in enzymeactivity could be related to different ANP and also NO
production which, acting as paracrine and/or autocrine
factors, may modulate frog nephron functions directly.
In addition to the mesonephros, the skin and the
urinary bladder of R. esculenta both seem to be involved
in osmoregulatory processes, as we had previously de-
duced from cytochemical studies of the Naþ/Kþ ATPase
activity (De Piceis Polver et al., 1988, 1999). Some in-teresting studies have also investigated the possible in-
fluence of ANP on both skin and urinary bladder of
some amphibians (Coviello et al., 1989; Meier and
Donald, 2002; Uchiyama et al., 1998; Vagnetti et al.,
2001).
Lastly, we observed some seasonal variations in ANP
production in the heart of R. esculenta (Addario et al.,
2001). In this respect, we would like to mention amorphometrical study conducted by Aoki et al. (1989)
on the heart of Bufo arenarum showing seasonal changes
in this species regarding the production of ANP granules
by myocardiocytes.
In conclusion, it is possible to assume that integrated
stimuli of ANP and NO acting locally within the me-
sonephros or on different anatomical structures in
C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176 175
combination with other hormonal factors, play a sub-stantial role in controlling osmoregulation in frog.
Acknowledgments
The authors wish to express their thanks to Prof.
M.A. Masini for her suggestions. This research was
supported by a FAR grant from the Italian Ministry of
the University and Scientific Research.
References
Addario, C., Fenoglio, C., Vaccarone, R., Gerzeli, G., 2001. Immu-
nohistochemical localization of atrial natriuretic peptide (ANP) in
the heart and in the mesonephros of Rana esculenta during
hibernation and active life. Eur. J. Histochem. 45 (Suppl.), 36.
Alderton, W.K., Cooper, C.E., Knowles, R.G., 2001. Nitric oxide
synthase: structure, function and inhibition. Biochem. J. 357, 593–
615.
Aoki, E., Takeuchi, I.K., Shoji, R., 1995. Nitric oxide: an attractive
signaling molecule. Acta Histochem. Cytochem. 28, 97–106.
Aoki, A., Maldonado, W., Forssmann, W.G., 1989. Seasonal changes
of the endocrine heart. In: Forssmann, W.G., Scheuermann, D.W.,
Alt, J. (Eds.), Functional Morphology of the Endocrine Heart.
Steinkopff Verlag Darmstadt, Springer, New York, pp. 61–68.
Bachmann, S., Bosse, H.M., Mundel, P., 1995. Topography of nitric
oxide synthesis by localizing constitutive NO synthases in mam-
malian kidney. Am. J. Physiol. 268, F885–F898 (Renal Fluid
Electrolyte Physiol. 37).
Beltowski, J., Wojcicka, G., 2002. Regulation of renal tubular sodium
transport by cardiac natriuretic peptides: two decades of research.
Med. Sci. Monit. 8, RA39–RA52.
Bentley, P.J., 1998. Hormones and osmoregulation. In: Comparative
Vertebrate Endocrinology, third ed. University of Cambridge
Press, Cambridge, pp. 337–378.
Bertorello, A.M., Aperia, A., Walaas, S.I., Nairn, A.C., Greengard, P.,
1991. Phosphorylation of the catalytic subunit of Naþ, Kþ ATPase
inhibits the activity of the enzyme. Proc. Natl. Acad. Sci. USA 88,
11359–11362.
Bodegas, M.E., Villaro, A.C., Montuenga, L.M., Moncada, S.,
Riveros-Moreno, V., Sesma, P., 1995. Neuronal nitric oxide
synthase immunoreactivity in the respiratory tract of the frog,
Rana temporaria. Histochem. J. 27, 812–818.
Chapeau, C., Gultowska, J., Schiller, P.W., Milne, R.W., Thibault, G.,
Garcia, R., Genest, J., Cantin, M., 1985. Localization of immu-
noreactive synthetic atrial natriuretic factors (ANF) in the heart of
various animal species. J. Histochem. Cytochem. 33, 541–550.
Chatterjee, P.K., Hawksworth, G.M., McLay, J.S., 1999. Cytokine-
stimulated nitric oxide production in the human renal proximal
tubule and its modulation by natriuretic peptides: a novel immu-
nomodulatory mechanism? Exp. Nephrol. 7, 438–448.
Coviello, A., Soria, M.O., Proto, M.C., Berman, D.M., Gamundi,
S.S., Alonso, C.E., de Bold, A., 1989. Effect of rat cardionatrin I
(rat ANF 99–126) on the response of toad skin to angiotensin II.
Can. J. Physiol. Pharmacol. 67, 362–365.
De Falco, M., Laforgia, V., Valiante, S., Virgilio, F., Varano, L., De
Luca, A., 2002. Different patterns of expression of five neuropep-
tides in the adrenal gland and kidney of two species of frog.
Histochem. J. 34, 21–26.
De Piceis Polver, P., Fenoglio, C., Gerzeli, G., Rapuzzi, G., Barni, S.,
1988. Potassium-dependent p-nitrophenyl phosphatase and aden-
ylate cyclase activity in Rana esculenta skin during natural
hibernation and active life: a cytochemical study. Arch. Biol. 99,
183–195.
De Piceis Polver, P., Fenoglio, C., Barni, S., Gerzeli, G., 1999.
Comparison of Kþ-p-nitrophenyl phosphatase activity in the
urinary bladder of the frog Rana esculenta during hibernation
and active life. Eur. J. Histochem. 43, 55–62.
Eitle, E., Hiranyachattada, S., Wang, H., Harris, P.J., 1998. Inhibition
of proximal tubular fluid absorption by nitric oxide and atrial
natriuretic peptide in rat kidney. Am. J. Physiol. 274, C1075–
C1080.
Ewart, H.S., Klip, A., 1995. Hormonal regulation of the Naþ–Kþ-
ATPase: mechanisms underlying rapid and sustained changes in
pump activity. Am. J. Physiol. 269, C295–C311 (Cell Physiol.
38).
Fenoglio, C., Vaccarone, R., Chiari, P., Gervaso, M.V., 1996. An
ultrastructural and cytochemical study of the mesonephros of Rana
esculenta during activity and hibernation. Eur. J. Morphol. 34,
107–121.
Fenoglio, C., Necchi, D., Civallero, M., Ceroni, M., Nano, R., 1997.
Cytochemical demonstration of nitric oxide synthase and 50
nucleotidase in human glioblastoma. Anticancer Res. 17, 2507–
2511.
Fenoglio, C., Vaccarone, R., Addario, C., De Piceis Polver, P., Barni,
S., Gerzeli, G., 2000. La regolazione idrosalina nel mesonefro di
Rana esculenta durante il ciclo annuale: aspetti morfofunzionali.
Atti 61� Congresso UZI, p. 108.
F�eraille, E., Carranza, M.L., Gonin, S., Beguin, P., Pedemonte, C.,
Rousselot, M., Caverzasio, J., Geering, K., Martin, P.Y., Favre,
H., 1999. Insulin-induced stimulation of Naþ, Kþ-ATPase activity
in kidney proximal tubule cells depends on phosphorylation of the
a-subunit at Tyr-10. Mol. Biol. Cell 10, 2847–2859.
Feuilloley, M., Yon, L., Kawamura, K., Kikuyama, S., Gutowska, J.,
Vaundry, H., 1993. Immunocytochemical localization of atrial
natriuretic factor (ANF)-like peptides in the brain and hearth of
the treefrog Hyla japonica: effect of weightlessness on the distribu-
tion of immunoreactive neurons and cardiocytes. J. Comp. Neurol.
330, 32–47.
Forssmann, W.G., Richter, R., Meyer, M., 1998. The endocrine heart
and natriuretic peptides: histochemistry, cell biology, and func-
tional aspects of the renal urodilatin system. Histochem. Cell Biol.
110, 335–357.
Gilles, J., Netchitailo, P., Leboulenger, F., Cantin, M., Pelletier, G.,
Vaudry, H., 1990. Localization and characterization of the N-
terminal fragment of atrial natriuretic factor (ANF) precursor in
the frog heart. Peptides 11, 199–204.
Grandclement, B., Morel, G., 1998. Ultrastructural characterization of
atrial natriuretic peptide receptors (ANP-R) mRNA expression in
rat kidney cortex. Biol. Cell 90, 213–222.
Hirsch, J.R., Kruhoffer, M., Adermann, K., Heitland, A., Maronde,
E., Meyer, M., Forssman, W.G., Herter, P., Plenz, G., Schlatter,
E., 2001. Cellular localization, membrane distribution, and possible
function of guanylyl cyclase A and 1 in collecting ducts of rat.
Cardiovasc. Res. 51, 553–561.
Kiemer, A.K., Vollmar, A.M., 1998. Autocrine regulation of inducible
nitric-oxide synthase in macrophages by atrial natriuretic peptide.
J. Biol. Chem. 273, 13444–13451.
Kloas, W., Hanke, W., 1992a. Atrial natriuretic factor (ANF) binding
sites in frog kidney and adrenal. Peptides 13, 297–303.
Kloas, W., Hanke, W., 1992b. Localization and quantification of
angiotensin II (A II) binding sites in the kidney of Xenopus laevis.
Gen. Comp. Endocrinol. 85, 173–183.
Kloas, W., Hanke, W., 1993. Receptors or atrial natriuretic actor
(ANF) in kidney and adrenal tissue of urodeles-lack of angiotensin
II (A II) receptors in these tissues. Gen. Comp. Endocrinol. 91,
235–249.
Kone, B.C., 2001. Molecular biology of natriuretic peptides and nitric
oxide synthase. Cardiovasc. Res. 15, 429–441.
176 C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176
Kourie, J.I., Rive, M.J., 1999. Role of natriuretic peptides in ion
transport mechanisms. Med. Res. Rev. 19, 75–94.
Lazure, C., Ong, H., Mc Nicoll, N., Netchitailo, P., Chr�etien, M., De
L�ean, A., Vaudry, H., 1988. The amino acid sequences of frog
heart atrial natriuretic-like peptide and mammalian ANF are
closely related. FEBS Lett. 238, 300–306.
Lenz, W., Herten, M., Gerzer, R., Drummer, C., 1999. Regulation of
natriuretic peptide (urodilatin) release in a human kidney cell line.
Kidney Int. 55, 91–99.
Levillain, O., Hus-Citharel, A., Morel, F., Bankir, L., 1993. Arginine
synthesis in mouse and rabbit nephron: localization and functional
significance. Am. J. Physiol. 264, F1038–F1045.
Liang, M., Knox, F.G., 2000. Production and functional roles of nitric
oxide in the proximal tubule. Am. J. Physiol. 278, R1117–
R1124.
McLay, J.S., Chatterjee, P., Nicolson, A.G., Jardine, A.G., McKay,
N.G., Ralston, S.H., Grabowski, P., Haites, N.E., MacLeod,
A.M., Hawksworth, G.M., 1994. Nitric oxide production by
human proximal tubular cells: a novel immunomodulatory mech-
anism. Kidney Int. 46, 1043–1049.
McLay, J.S., Chatterjee, P.K., Jardine, A.G., Hawksworth, G.M.,
1995. Atrial natriuretic factor modulates nitric oxide production;
an ANF-C receptor mediated effect. J. Hypertens. 13, 625–630.
Meier, S.K., Toop, T., Donald, J.A., 1999. Distribution and charac-
terization of natriuretic peptide receptors in the kidney of the toad,
Bufo marinus. Gen. Comp. Endocrinol. 115, 244–253.
Meier, S.K., Donald, J.A., 2002. Functional analysis of natriuretic
peptide receptors in the bladder of the toad, Bufo marinus. Gen.
Comp. Endocrinol. 125, 207–217.
Mistry, S.K., Hawksworth, G.M., Struthers, A.D., McLay, J.S., 2001.
Differential expression and synthesis of natriuretic peptides deter-
mines natriuretic peptide receptor expression in primary culture of
human proximal tubular cells. J. Hypertens. 19, 255–262.
Mohaupt, M.G., Elzie, J.L., Ahn, K.Y., Clapp, W.L., Wilcox, C.S.,
Kone, B.C., 1994. Differential expression and induction of mRNA
encoding two inducible nitric oxide synthase in rat kidney. Kidney
Int. 46, 653–665.
Moncada, S., Higgs, A., 1993. The LL-arginine–nitric oxide pathway. N.
Engl. J. Med. 329, 2002–2012.
Morel, F., Hus-Citharel, A., Levillan, O., 1996. Biochemical hetero-
geneity of arginine metabolism along kidney proximal tubules.
Kidney Int. 49, 1608–1610.
Morrissey, J.J., McCracken, J.R., Kaneto, H., Vehaskari, M., Mon-
tani, D., Klar, S., 1994. Location of an inducible nitric oxide
synthase mRNA in the normal kidney. Kidney Int. 45, 998–1005.
Nakos, G., Gossrau, R., 1994. When NADPH diaphorase (NADPHd)
works in the presence of formaldehyde, the enzyme appears to
visualize selectively cells with constitutive nitric oxide synthase
(NOS). Acta Histochem. 96, 335–343.
Netchitailo, P., Feuilloley, L.M., Pelletier, G., De Lean, A., Ong, H.,
Cantin, M., Gutkowska, J., Leboulenger, F., Vaudry, H., 1988.
Localization and identification of immunoreactive atrial natriuretic
factor (ANF) in the frog ventricle. Peptides 9, 1–6.
Ominato, M., Satoh, T., Katz, A.I., 1996. Regulation of Na–K-
ATPase activity in the proximal tubule: role of the protein kinase C
pathway and of eicosanoids. J. Membr. Biol. 152, 235–242.
Pan, J., Burgher, K.L., Szczepanik, A.M., Ringheim, G.E., 1996.
Tyrosine phosphorilation of inducile nitric oxide synthase: impli-
cation for potential post-translational regulation. Biochem. J. 314,
889–894.
Reinecke, M., Betzler, D., Segner, H., Forssmann, W.G., 1989. Dual
distribution of cardiac hormones (CDD/ANP) in the heart and
brain of vertebrates. In: Forssmann, W.G., Scheuermann, D.W.,
Alt, J. (Eds.), Functional Morphology of the Endocrine Heart.
Steinkopff Verlag Darmstadt, Springer, New York, pp. 87–93.
Ritter, D., Dean, A.D., Gluck, S.L., Greenwald, J.E., 1995. Natriuretic
peptide receptors A and B have different cellular distributions in rat
kidney. Kidney Int. 48, 1758–1766.
Sakata, J., Kangawa, K., Matsuo, H., 1988. Identification of new atrial
natriuretic peptides in frog heart. Biochem. Biophys. Res. Com-
mun. 155, 1338–1345.
Scavone, C., Scanlon, C., McKee, M., Nathanson, J.A., 1995. Atrial
natriuretic peptide modulates sodium and potassium-activated
adenosine triphosphatase through a mechanism involving cyclic
GMP and cyclic GMP-dependent protein kinase. J. Pharmacol.
Exp. Ther. 272, 1036–1042.
Sekiguchi, T., Miyamoto, K., Minutami, T., Yamada, K., Yazawa, T.,
Yoshino, M., Minegishi, T., Takei, Y., Kangawa, K., Minamino,
N., Saito, Y., Kojima, M., 2001. Molecular cloning of natriuretic
peptide receptor A from bullfrog (Rana catesbeiana) brain and its
functional expression. Gene 273, 251–257.
Shin, S.J., Lai, F.J., Wen, J.D., Lin, S.R., Hsieh, M.C., Hsiao, P.J.,
Tsai, J.H., 1999. Increased nitric oxide synthase mRNA expression
in the renal medulla of water-deprived rats. Kidney Int. 56, 2191–
2202.
Sokabe, H., Ogawa, M., 1974. Comparative studies of the juxtaglo-
merular apparatus. Int. Rev. Cytol. 37, 271–327.
Takei, Y., 2000. Structural and functional evolution of the natriuretic
peptide system in vertebrates. Int. Rev. Cytol. 194, 1–66.
Takei, Y., 2001. Does the natriuretic peptide system exist throughout
the animal and plant kingdom? Comp. Biochem. Physiol. B 129,
559–573.
Terada, Y., Moriyama, T., Martin, B.M., Knepper, M.A., Garcia-
Perez, A., 1991. RT-PCR microlocalization of mRNA for guanylyl
cyclase-coupled ANF receptor in rat kidney. Am. J. Physiol. 261,
F1080–F1087.
Terada, Y., Tomita, K., Nonoguchi, H., Marmo, F., 1992. Polymerase
chain reaction localization of constitutive nitric oxide synthase and
soluble guanylate cyclase messenger RNAs in microdissected rat
nephron segments. J. Clin. Invest. 90, 659–665.
Uchiyama, M., Takeuchi, T., Matsuda, K., 1998. Effects of homolo-
gous natriuretic peptides in isolated skin of the bullfrog, Rana
catesbeiana. Comp. Biochem. Physiol. C 120, 37–42.
Vagnetti, D., Santarella, B., Di Rosa, I., Cardellicchio, R., Tei, S.,
2001. Atrial natriuretic peptide (ANP) system in frog skin. Eur. J.
Morphol. 39, 215–221.
Visai, L., Xu, Y., Casolini, F., Rindi, S., H€o€ok, M., Speziale, P., 2000.
Monoclonal antibodies to CNA, a collagen-binding microbial
surface component recognizing adhesive matrix molecules, detach
Staphylococcus aureus from a collagen substrate. J. Biol. Chem.
275, 39837–39845.
Yaqoob, M., Edelstein, C.L., Wieder, E.D., Alkhunaizi, A.M.,
Gengaro, P.E., Nemenoff, R.A., Schrier, R.W., 1996. Nitric oxide
kinetics during hypoxia in proximal tubules: effects of acidosis and
glycine. Kidney Int. 49, 1314–1319.
Yu, L., Gengaro, P.E., Niederberger, M., Burke, T.J., Schrier, R.W.,
1994. Nitric oxide a mediator in rat tubular hypoxia/reoxygenation
injury. Proc. Natl. Acad. Sci. USA 91, 1691–1695.
Yu, L., 1997. Role of nitric oxide in acute renal failure. Ren. Fail. 19,
213–216.
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