Chem. Senses 27: 643-652,
2002
© Oxford University Press 2002
Characterization of cAMP Degradation by Phosphodiesterases in the Accessory Olfactory System
Department of Psychology and Laboratory of Molecular Neurobiology and Behavior, Boston University, Boston, MA 02215, USA
Correspondence to be sent to: James A. Cherry, Department of Psychology, 64 Cummington Street, Boston University, Boston, MA 02215, USA. e-mail: jcherry{at}bu.edu
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Abstract |
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To characterize the potential role of cAMP in pheromone transduction, we have examined the occurrence of cyclic nucleotide phosphodiesterases (PDEs) in the mouse vomeronasal organ (VNO). We show that the cAMP-specific isoforms PDE4A and PDE4D are found preferentially in the apical and basal layers, respectively, of the VNO neuroepithelium and in the rostral (PDE4A) and caudal (PDE4D) portions of the accessory olfactory bulb glomerular layer. Assays for cAMP hydrolysis showed that PDE activity in VNO homogenates was about half that measured in the cerebral cortex and olfactory epithelium, and the proportion of total activity inhibited by rolipram, a PDE4-specific inhibitor, was
40%. Activity in the VNO was enhanced 60% by Ca2+ and
calmodulin (CaM), implicating the presence of Ca2+/CaM-dependent
PDE1. Zaprinast, which is known to inhibit PDE1C isoforms, completely
suppressed Ca2+/CaM-stimulated activity and, together, zaprinast
and rolipram inhibited cAMP hydrolysis by 70%. Our results suggest that
PDE1 and PDE4 isoforms are the primary source of cAMP degradation in the
VNO. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Responses to pheromones are mediated in part by an accessory olfactory pathway that begins in receptor neurons of the vomeronasal organ (VNO). For rodents in particular, these neurons appear to be anatomically and neurochemically divided into apical and basal layers, from which axons project to non-overlapping rostral and caudal glomeruli in the accessory olfactory bulb—the AOB (Halpern et al., 1998
). From the AOB, projections are then made to
subcortical targets in the amygdala and hypothalamus. In contrast to the
neuroanatomical organization, far less is known about how pheromones are
processed in the mammalian VNO.
In the main olfactory system, generation of the cyclic nucleotide cAMP is
an essential component of the pathway by which odors lead to the
depolarization of olfactory sensory neurons
(Pace et al., 1985;
Sklar et al., 1986).
However, whether cAMP plays a similar (or any) role in the transduction of
pheromones in the mammalian VNO is unclear. Adenylyl cyclases ACII
(Berghard and Buck, 1996) and
ACVI (Rossler et al.,
2000), as well as the cAMP hydrolyzing enzyme PDE4A
(Lau and Cherry, 2000) are
present in the mammalian VNO, but studies in which cAMP levels have been
measured in the VNO after exposing animals or isolated membranes to pheromones
have been conflicting (Zhou and Moss,
1997; Sasaki et al.,
1999; Rossler et al.,
2000). Although a cyclic-nucleotide-gated channel subunit is
expressed in VNO neurons, cyclic-nucleotide-gated channel conductance has not
been detected (Liman and Corey,
1996). Rather, most current evidence points to a primary role of
an inositol 1,4,5-triphosphate (IP3) pathway in pheromonal
signaling in mammals (Inamura et
al., 1997; Wekesa and
Anholt, 1997; Liman et
al., 1999).
To understand better the role of cAMP in the accessory olfactory system, it
will be important to identify the primary regulators of cAMP levels in the
VNO. In particular, there has been little attention focused on the enzymes
responsible for the breakdown of cAMP—the cyclic nucleotide
phosphodiesterases (PDEs). These proteins consist of 11 distinct families
grouped according to biochemical and pharmacological properties, although only
eight families are known to hydrolyze cAMP
(Francis et al.,
2001). For example, the Type I PDEs (PDE1) consist of enzymes that
can hydrolyze both cAMP and cGMP and are activated by Ca2+ and
calmodulin (CaM). Meanwhile, PDE4s are characterized by high affinity and
specificity for cAMP as a substrate, as well as sensitivity to inhibition by
the PDE4-specific inhibitor rolipram. In olfactory sensory neurons, the
predominant PDE activity is generated by Ca2+-dependent PDE1
subtype(s) and the Ca2+-independent PDE4s
(Borisy et al.,
1992). In addition, an olfactory-specific member of the
cGMP-stimulated PDE2 family has been localized to a restricted group of
sensory neurons in the olfactory epithelium
(Juilfs et al.,
1997).
Previously, we showed that the PDE4 isoform, PDE4A, is selectively found in
apical VNO neurons and their targets in the rostral AOB glomeruli
(Lau and Cherry, 2000). We now
report that a second PDE4 isoform, PDE4D, is present in the accessory
olfactory system. Using isoform-specific antibodies, we show that PDE4D occurs
primarily in basal VNO neurons and caudal AOB glomeruli. In addition, we have
used an assay for cAMP hydrolysis to measure PDE activity in the VNO and found
that PDE4 enzymes make up 40% of overall cAMP hydrolysis under basal
conditions. Elevated PDE activity measured in the presence of Ca2+
and CaM suggests that PDE1 enzymes also contribute significantly to overall
cAMP hydrolysis in the VNO. Our results indicate that two differentially
localized PDE4 isoforms and one or more PDE1 enzymes are the principal
regulators of cAMP breakdown in the VNO.
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Materials and methods |
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Materials
Radiochemicals 2,8-[3H]cAMP and 2,8-[3H]adenosine were obtained from NEN (Boston, MA). Rolipram, milrinone, 3-isobutyl-1-methylxanthine (IBMX) and zaprinast were obtained from RBI (Natick, MA). Diethyl-(2-hydroxypropyl)aminoethyl (QAE) Sephadex A-25, protease inhibitors [phenylmethylsulfonyl fluoride (PMSF), aprotinin, pepstatin A, antipain, chymostatin and leupeptin], snake venom (Crotalus atrox) and all reagents and chemicals not otherwise identified were from Sigma (St Louis, MO).
Tissue preparation
Adult, Swiss Webster mice of both sexes were used in all experiments.
Tissues were prepared for immunohisto-chemistry as described previously
(Cherry and Davis, 1999). For
PDE assays and immunoblot analysis, animals were killed by decapitation and
tissues, including brain, VNO, olfactory epithelium, heart, liver and kidney,
were rapidly dissected, frozen on dry ice and placed at -80°C. Just prior
to use, tissues were homogenized in ice-cold buffer (20 mM Tris, pH 7.5, 0.2
mM MgCl2, 0.25 M sucrose, 1% Triton X-100, 0.5 mM PMSF, 3 µM
pepstatin, 3 µM leupeptin, 2 µg/ml aprotinin, 1 µg/ml antipain, 2
µg/ml chymostatin). Protein concentrations of homogenates were determined
by Bradford analysis (Bio-Rad, Hercules, CA). Except where indicated,
separately prepared homogenates consisted of tissues provided by at least
three different animals. The use of animals followed National Institutes of
Health guidelines and was approved by the Boston University Animal Care and
Use Committee.
Olfactory bulbectomy
Eight adult mice were anesthetized with xylazine and ketamine and a small incision was made in the scalp overlying the olfactory bulbs. The skin was retracted and a small hole was drilled through the skull to expose the olfactory bulbs. Suction was applied through a glass pipette and the olfactory bulbs were removed by aspiration. Gel-foam was packed into the cavity and the skin was sutured shut. Mice were allowed to recover for several hours under heat lamps before returning to the colony rooms.
Immunohistochemistry
VNOs from four animals were cryosectioned coronally at 20 µm onto
poly-L-lysine-coated glass slides and dried overnight. Frozen sections of the
AOB were cut sagittally at 30 µm and placed free-floating into
phosphate-buffered saline (PBS). Sections were processed for
immunohistochemistry with affinity-purified, rabbit polyclonal anti-PDE4A and
anti-PDE4D antibodies (Cherry and Davis,
1999) and a 1:200 (VNO) or 1:500 (AOB) dilution of secondary
antibody (biotinylated goat anti-rabbit immunoglobulin; Vector Laboratory,
Burlingame, CA). Control sections in which the primary antibodies were omitted
were also included. Tissue was then reacted with Vector Elite ABC reagent and
product was visualized with 1 mg/ml 3',3'-diaminobenzidine and
0.03% H2O2. Photomicrographs of selected regions were
taken with a Nikon DXM 1200 digital camera mounted on an Olympus BX-60
microscope. Final images were cropped and assembled in Adobe Photoshop, v. 6.0
(Adobe Systems Inc., San Jose, CA). Brightness and contrast were adjusted for
each photomicrograph to achieve uniform image quality.
Immunoblotting
Aliquots of tissue homogenates containing 10-20 µg of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS—PAGE) and transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA). Membranes were then processed for immunoblotting with anti-PDE4A, anti-PDE4B, or anti-PDE4D antibodies. The secondary antibody was a 1:2500 dilution of horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin (Bio-Rad) and immunoreactive proteins were visualized using chemiluminescence reagent (NEN, Boston, MA). Signals were recorded on BioMax Imaging film (Eastman Kodak, Rochester, NY) and scanned into Adobe Photoshop. To examine the effects of olfactory bulbectomy (BX) on PDE4 levels, band intensities were determined for sham-treated and BX animals on adjacent blots processed at the same time on the same piece of film. Optical densities were obtained using MultiAnalyst (BioRad) and signal density differences between groups were compared statistically using Student's t-tests.
PDE assay
Activity of cyclic nucleotide PDEs was determined using a modification of
the Thompson and Appleman (Thompson and
Appleman, 1971) two-step procedure
(Bauer and Schwabe, 1980;
Cherry et al., 2001).
Briefly, tubes were prepared consisting of 20 µl of diluted tissue
homogenate in 40 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 40 000 c.p.m.
[3H]cAMP and cold cAMP. Final sample volumes were 200 µl and,
except for assays in which substrate concentration was specifically varied,
all reactions were conducted at 1 µM cAMP. Tubes were incubated at 30°C
for 10 min, then transferred briefly into an ethanol/dry ice bath before
placing in boiling water for 45 s. Snake venom (25 µl of 2 mg/ml) was added
for an additional 10 min incubation before applying the reaction mixture to
equilibrated chromatography columns (BioRad) containing QAE Sephadex A-25.
Cyclic AMP PDE activity was computed from the eluates and expressed as nmol
cAMP hydrolyzed/mg protein/min.
Stock solutions of 625 µg/ml CaM and 1 M CaCl2 were prepared in 40 mM Tris-HCl, pH 8.0. Rolipram, milrinone, zaprinast and IBMX were prepared as 10 mM solutions in ethanol and diluted as necessary before adding to tissue tubes in 2 µl aliquots. When these reagents were used, 2 µl of ethanol were also added to control tubes with no apparent effect on PDE activity.
Samples were run in duplicate or triplicate and averaged. For the determination of cAMP hydrolysis in different tissues, two separate preparations of homogenates derived from at least three animals were assayed at least twice each on different days. Assay results were averaged for each homogenate, and final data presented as the mean (±SEM) of the averages for the two homogenates (n = 2). For the olfactory BX experiment, samples were obtained from individual animals and differences in PDE activity between control and BX animals were examined statistically with Student's t-tests. Statistical tests comparing percentages were performed on data after arcsin transformation.
For the experiment comparing the effects of various inhibitors and Ca2+ environment on cAMP hydrolysis in the VNO, six VNO homogenates containing VNOs from at least three animals were prepared. Each homogenate was assayed in duplicate under each drug and Ca2+ condition, and the mean PDE activities in different groups were compared statistically with Student's t-tests for paired samples. For the rolipram + zaprinast + IBMX condition, only two homogenates were used.
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Results |
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Immunolocalization of PDE4 isoforms
Labeling within the VNO neuroepithelium after staining with anti-PDE4A
antibody was robust and confined primarily to sensory neurons in the apical
layer, as shown in Figure 1A
(Lau and Cherry, 2000).
However, a different pattern of immunoreactivity was observed with the
PDE4D-specific antibody (Figure
1B). Overall, anti-PDE4D labeling was weaker in comparison to
anti-PDE4A, but nevertheless appeared limited to sensory neurons and not
supporting cells. Basal VNO neurons seemed more darkly stained by the PDE4D
antibody than apical cells, but labeling intensity between these populations
was not as distinct as that observed with anti-PDE4A antibody. At higher
magnification (Figure 1C),
labeling was seen in dendrites and perikarya of PDE4D-stained neurons, but
there was no staining in the neuronal microvilli that line the lumen of the
VNO, where chemosignal transduction is believed to initiate. Although staining
in the neuro-epithelium was relatively weak, the most significant labeling
appeared to be in clumps of cells along the basal layer. Axon bundles (not
seen here) were also labeled by both antibodies.
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In the rodent, projections from apical and basal VNO neurons terminate in
rostral and caudal glomeruli of the AOB, respectively
(Jia and Halpern, 1996;
Belluscio et al.,
1999; Rodriguez et
al., 1999). Consistent with this anatomical arrangement,
brain sections exposed to anti-PDE4A antibody were stained in the rostral half
of AOB glomerular layer, as shown in Figure
1D (Lau and Cherry,
2000). Conversely, we found that PDE4D immunoreactivity in the AOB
was restricted to the caudal half of the glomerular layer
(Figure 1E). The relative
difference between rostral/caudal PDE4D staining intensity in the AOB was
considerably greater than the staining differences observed between apical and
basal VNO neurons. A control VNO section in which the primary antibody was
omitted during processing was not stained
(Figure 1F).
Immunoblots prepared from tissue homogenates of the VNO, olfactory
epithelium and cerebral cortex were next examined using antibodies that
specifically recognize either PDE4A, PDE4B, or PDE4D subtypes
(Cherry and Davis, 1999). For
these blots, equal amounts (20 µg) of protein were loaded per lane to show
the relative levels of PDE4 enzyme in each of the tissues. Single proteins of
105 and 100 kDa were revealed in the VNO by the anti-PDE4A and anti-PDE4D
antibodies, respectively, whereas no signal was observed with anti-PDE4B
(Figure 2). All three
antibodies recognized PDE4 proteins in the cortex and PDE4A was most abundant
in olfactory epithelium. In contrast, only trace PDE4B and PDE4D
immunoreactivity was observed in olfactory epithelium, suggesting that PDE4A
is the predominant PDE4 enzyme in this tissue. Control blots in which the
primary antibody was omitted were completely blank (data not shown).
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Finally, to verify that PDE4 expression in the VNO occurs primarily in
neurons, olfactory bulbs were removed bilaterally. This procedure results in
the selective degeneration of most VNO neurons in response to the removal of
their target sites in the AOB, whereas nonsensory components of the VNO remain
largely unaffected (Barber and Raisman,
1978). Examination of immunoblots of VNOs from BX and sham-treated
animals indicated that mean PDE4A expression was significantly reduced after
BX [t(6) = 4.44, P < 0.005] and that mean PDE4D levels
were also reduced in such animals compared to controls
(Figure 3). For PDE4D, this
reduction was of borderline significance [t(6) = 2.21, P
< 0.07]. Thus, PDE4A and PDE4D levels after BX were on average only 10 and
40% of control values, respectively, providing additional evidence that both
PDE4 subtypes occur in sensory neurons.
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cAMP PDE activity
In addition to the PDE4 enzymes, members of at least seven other PDE families can hydrolyze cAMP. Therefore, the total level of PDE activity measured in our assay reflects the combined contribution of all cAMP-hydrolyzing enzymes present in the tissue. We first examined and compared the overall level of PDE activity in the VNO with other tissues. As shown in Figure 4, total PDE activity was greatest in cerebral cortex, olfactory bulb and olfactory epithelium, lowest in heart and liver, and intermediate in the VNO and kidney.
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To determine the proportion of activity in the VNO that was attributable to different PDEs, the effects of various PDE inhibitors (all used at 100 µM) were examined both in the presence and absence of 100 µM Ca2+ and 4 µg/ml CaM (Figure 5). In the absence of inhibitors, addition of Ca2+/CaM increased cAMP hydrolysis significantly from 0.12 ± 0.014 to 0.19 ± 1.7 nmol/mg protein/min, an increase of 61% [t(5) = 9.9, P < 0.001], suggesting that a Ca2+-dependent PDE is present in the VNO. The PDE4-specific inhibitor rolipram reduced cAMP hydrolysis by 40% in the absence of Ca2+/CaM and 27% in the presence of Ca2+/CaM. Although both reductions are significant [t(4) = 6.6, P < 0.005; t(4) = 8.4, P < 0.001, respectively], these results indicate that PDE4s account for less than half of the total VNO PDE activity, even under basal conditions.
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Zaprinast, which is generally considered a PDE5/6 inhibitor
(Beavo, 1995), has also more
recently been shown to inhibit several isoforms of the PDE1 family
(Loughney et al.,
1996). While completely suppressing the increase in PDE activity
observed with Ca2+/CaM [zap - Ca2+ versus zap +
Ca2+, t(4) = 1.7, P = 0.16], zaprinast inhibited
cAMP hydrolysis by 48% versus basal controls in the absence of
Ca2+/CaM [t(4) = 7.4, P = 0.002;
Figure 5]. We have shown
elsewhere, however, that zaprinast can also significantly reduce PDE4
activity: cAMP hydrolysis by a PDE4A subtype (mouse PDE4A1) transiently
transfected in HEK293 cells was 45% inhibited by 100 µM zaprinast versus
95% inhibition by 100 µM rolipram [unpublished observations and Cherry
et al. (Cherry et al.,
2001)]. This suggests that zaprinast in the present study
inhibited both PDE1 and PDE4 activity in VNO homogenates, a conclusion
supported by the observation that zaprinast and rolipram inhibition was not
additive (68% inhibition by zaprinast + rolipram compared to 40 and 48% by
rolipram or zaprinast alone, respectively).
The generic PDE inhibitor IBMX alone reduced PDE activity by 85% and at a
slightly higher level (92%) when added to samples in combination with rolipram
and zaprinast (Figure 5). This
indicates that only a small proportion of cAMP PDE activity in the VNO can be
accounted for by IBMX-insensitive PDEs, such as PDE8
(Fisher et al.,
1998). As for other IBMX-sensitive PDEs, there is evidence from
the rat that isoforms of the cGMP-stimulated PDE2 and cGMP-inhibited PDE3
families may be present in the olfactory epithelium
(Borisy et al.,
1992). However, in data not shown, we failed to find any effect of
adding either 1 µm cGMP or 100 µM of the PDE3 inhibitor milrinone on
cAMP PDE activity in homogenates from the mouse VNO.
Finally, we examined PDE activity in VNOs from animals given bilateral BX. Olfactory BX significantly reduced overall mean PDE activity by about half in comparison to control animals that received sham operations [Figure 6; t(19) = 3.8, P < 0.002]. Interestingly, the mean proportion of rolipram-inhibitable PDE activity from VNOs of BX animals was significantly greater than for controls [Figure 6; 47 ± 5.3% versus 32 ± 4.8%, t(19) = 2.12, P < 0.05]. This indicates that despite the loss of neurons and the reduction in the levels of PDE4A and PDE4D that occurs following BX (Figure 3), a relatively higher proportion of remaining total cAMP PDE activity is due to PDE4 enzymes in comparison to controls.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Activation of cyclic nucleotides by G-protein-coupled receptors is a feature of transduction in virtually all sensory systems. In olfaction, taste, vision and mechanosensation, generation of either cAMP or cGMP occurs in direct response to a sensory stimulus, implicating PDEs as important regulators of cyclic nucleotide levels. Although the role of cAMP in the VNO remains to be determined, our data indicate that multiple PDE enzymes are present in this tissue. Moreover, the segregated distribution of two PDE4 isoforms that we have described in the accessory olfactory system suggests that the regulation of cAMP may differ in apical and basal VNO neurons and their projections.
Our study provides anatomical evidence that PDE4s occur in different
subpopulations of VNO neurons. As shown previously
(Lau and Cherry, 2000), PDE4A
was found predominantly in apical VNO neurons and in rostral AOB glomeruli. In
contrast, several observations support the contention that PDE4D is expressed
in VNO neurons but more so in basal than apical neurons. Despite relatively
poor resolution, PDE4D appeared by immunocytochemical methods to occur mostly
in basal neurons. In addition, immunoblot analysis indicated that 7 days after
BX, PDE4D levels were reduced in accord with the dramatic loss of neurons that
occurs after lesion. Finally, we showed that caudal AOB glomeruli, which
receive projections exclusively from basal VNO neurons, were more heavily
stained by PDE4D antibody than the adjacent rostral glomeruli.
There are several explanations for why two closely related PDE4 isoforms
would be preferentially expressed in different sets of VNO neurons. In
general, proteins encoded by PDE4A and PDE4D genes display similar biochemical
characteristics: all isoforms examined to date are low Km
enzymes inhibited by micromolar concentrations of rolipram. However,
comparison of individual proteins expressed from alternative splice forms of
each gene has revealed differences in rolipram sensitivity, subcellular
distribution and interaction with other proteins. For example, the RD1 splice
variant of PDE4A is exclusively membrane associated, whereas PDE4A5, which
differs only in the N-terminal domain, is distributed in both the cytosolic
and particulate fractions of both brain and transfected COS cell extracts
(McPhee et al.,
1995). Similarly distinct patterns of subcellular distribution
have been found for other PDE4 isoforms found in the brain
(Lobban et al., 1994;
Bolger et al., 1997).
In addition, differences in binding of PDE4 subtypes to other proteins have
been described. The PKC-binding scaffold protein RACK1 selectively binds to
the N-terminal region of the PDE4D5 isoform
(Yarwood et al.,
1999) and only the PDE4D4 and PDE4A5 subtypes have been shown to
interact with SH3 domains found in various kinases
(Beard et al., 1999).
Functionally, such interactions could alter the catalytic activity of the
enzyme, or, alternatively, increase local concentrations of PDE4s by
recruitment to protein complexes involved in specific signaling pathways
(Pawson and Scott, 1997;
Houslay et al.,
1998). Thus, the regulation of cAMP by PDE4s within a cell may
depend not only on the kinetic properties of individual subtypes, but also
upon their subcellular distribution and the presence of specific interacting
proteins.
Our results indicate that only single PDE4A and PDE4D proteins occur in the
VNO. On immunoblots labeled with PDE4D antibody, a 100 kDa protein was
identified from VNO homogenates that likely represents the product of the
splice form PDE4D3. This enyzme is the smallest of three `long' PDE4D isoforms
that have been characterized (Bolger et
al., 1997). Little, however, is known about the 105 kDa PDE4A
protein that was found in both the VNO and olfactory epithelium. In brain,
this protein is found only in the olfactory bulbs
(Cherry and Davis, 1995),
although it is not yet known whether it is expressed intrinsically in
olfactory bulb cells or is localized only in axons from chemosensory cells.
This unique isoform may therefore represent a protein encoded by a novel PDE4A
splice form that is specific to chemosensory tissue. A third PDE4 examined in
this study, PDE4B, was not localized in the VNO and the other member of this
class, PDE4C, is virtually absent from the rodent nervous system
(Bolger et al., 1994;
Engels et al.,
1995).
The presence of PDE4 proteins in the VNO was confirmed by examination of
cAMP hydrolysis in VNO homogenates. Overall, the level of cAMP PDE activity we
measured in the mouse VNO was about half that measured in the olfactory
epithelium. Thus, despite the differences in signal transduction components
that exist between the olfactory epithelium and VNO
(Berghard et al.,
1996), cAMP PDE activity was represented in both tissues. This
included a significant proportion of both rolipram-inhibited as well as
Ca2+/CaM-activated PDE activity
(Borisy et al.,
1992). A prospective candidate to account for the
Ca2+-activated PDE activity in the VNO is PDE1C2. This protein is a
PDE1 isoform found in olfactory sensory neurons and is particularly abundant
in olfactory cilia (Yan et al.,
1995). The presence of PDE1C would also be consistent with the
finding that PDE activity in the VNO was markedly decreased in the presence of
zaprinast, which effectively inhibits PDE1C subtypes
(Loughney et al.,
1996; Han et al.,
1999).
Our data suggest that most cAMP hydrolysis in the VNO is attributable to
PDE1 and PDE4 isoforms, but that other PDEs may be present at lower levels.
Overall, IBMX inhibited cAMP hydrolysis by 85%, leaving only 15% of activity
that could be due to IBMX-insensitive isoforms. Of the two families of
IBMX-insensitive PDEs so far documented, only PDE8 enzymes use cAMP as a
substrate. However, PDE8 isoforms are also insensitive to rolipram and
zaprinast (Fisher et al.,
1998; Hayashi et al.,
1998) and in VNO homogenates, only 8% of activity remained in
samples containing IBMX + zaprinast + rolipram versus controls. Thus, cAMP
hydrolysis in the VNO due to PDE8 activity is probably minor.
In contrast, 68% of cAMP hydrolysis was inhibited in the VNO by zaprinast +
rolipram, which implicates PDE1 and PDE4 isoforms as the primary regulators of
cAMP breakdown in the VNO. The contributions of all other IBMX-sensitive PDEs
in the VNO are represented in the additional 17% of cAMP PDE activity that was
inhibited by IBMX but not by rolipram and/or zaprinast. Enzymes fitting these
criteria have been described in the PDE2, PDE7 and PDE10 families
(Beavo, 1995;
Soderling et al.,
1999). Further studies will be needed to determine if particular
isoforms from any of these families contribute to cAMP hydrolysis in the
VNO.
After BX, reductions in levels of both PDE4A and PDE4D were observed.
However, despite these decreases in PDE4 protein levels, the proportion of
rolipram-inhibitable PDE activity that remained after BX actually increased.
In rats, levels of olfactory marker protein (OMP) increase in neurons
remaining in the olfactory epithelium following BX
(Carr et al., 1998)
and we have observed similar increases in PDE4A immunoreactivity in olfactory
sensory neurons of the mouse (Cherry, unpublished observations). Although it
is not known why either OMP or PDE4A would be up-regulated in olfactory
neurons that survive removal of target sites in the olfactory bulb, a
BX-induced increase in the relative proportion of rolipram-inhibitable (PDE4)
PDE activity seems to occur in VNO neurons as well.
There is now strong evidence that PDE4 enzymes are present in sensory
neurons of both the main and accessory olfactory systems
(Borisy et al., 1992;
Cherry and Davis, 1995;
Lau and Cherry, 2000). What
remains to be determined is the specific functional role of each enzyme. In
addition to its localization in apical neurons of the VNO, PDE4A is also found
in mature receptor cells of the olfactory epithelium. Lack of PDE4A in the
olfactory cilia of these neurons, however, suggests that rather than
participating directly in the early steps of the transduction process, its
function may be somewhere downstream. Similarly, both PDE4A and PDE4D seem to
be absent from microvilli, the receptor-bearing structures of the VNO. A
possible role of dendritic and somatic PDEs has been suggested by studies on
deciliated olfactory receptor cells of the salamander. In dissociated cells,
application of adrenaline increased both the threshold for firing and the
number of spikes generated in response to strong stimuli, responses which were
dependent on phosphorylation by cAMP-dependent protein kinase
(Kawai et al., 1999).
Thus, dendrosomatically localized PDEs could affect sensory perception by
increasing the contrast between chemosignals of different concentrations.
The role of different second messenger systems in chemosignal transduction
by the vertebrate VNO is currently unresolved. Studies in a variety of species
have provided evidence that IP3 may be involved in the VNO response
to chemosignals (Luo et al.,
1994; Inamura et al.,
1997; Wekesa and Anholt,
1997; Krieger et al.,
1999; Sasaki et al.,
1999). In addition, a novel member of the transient receptor
potential family of ion channels (TRP2) has been cloned and found to be
exclusively localized in VNO microvilli
(Liman et al., 1999).
Evidence that TRP channels can be activated by diacylglycerol
(Hofmann et al.,
1999) provides a possible mechanism by which IP3
induction might lead to opening of TRP2 in the VNO after pheromonal
stimulation.
However, the role that cAMP plays in VNO chemosignal transduction is less
clear, particularly for mammalian species. There are reports that cAMP levels
are unchanged in the male hamster VNO following stimulation by female
chemosignals (Kroner et al.,
1996), but in the mouse, exposure to urine-derived compounds
decreases levels of cAMP generated in the VNO
(Zhou and Moss, 1997). A
recent study in the rat has examined the time course of both IP3
and cAMP production during application of urinary components to preparations
of microvilli from VNO neurons (Rossler
et al., 2000). Whereas these stimuli induced a rapid
IP3 signal, they also generated a delayed and prolonged decrease in
cAMP. This indicates that both IP3- and cAMP-mediated
pathways are activated by exposure to pheromones, but with unique
temporal—and perhaps spatial—characteristics. Thus, a scenario can
be envisioned whereby IP3-dependent events could be induced by
pheromones initially in the sensory microvilli and, later, modulatory
influences on transduction by cyclic nucleotides located in other neuronal
compartments could occur. More studies will be needed to test this
possibility.
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Acknowledgments |
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We appreciate the assistance of Yanny Lau with immuno-histochemistry and thank Drs M.J. Baum and J.-W. Lin for reviewing earlier versions of the manuscript. This study was supported by NIH grant DC03019.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Accepted June 13, 2000
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