(Received for publication, March 1, 1995; and in revised form, June 16, 1995)
From the
Previous studies with U937 cells, a human monocyte cell line,
have shown that the activity of cyclic nucleotide phosphodiesterase 4
(PDE4) is increased by agents that elevate cyclic AMP content. The
present experiments were conducted to determine 1) whether an increase
in PDE4 steady-state message and/or protein accompanies the
up-regulation of PDE4 activity and 2) whether the up-regulation changes
the functional responses of U937 cells to activators of adenylyl
cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h
with a combination of 1 µM salbutamol, a
-adrenoceptor agonist, and 30 µM rolipram, a PDE4
inhibitor. Cells were washed extensively to remove drugs and used
immediately in various experimental protocols. Reverse
transcriptase-polymerase chain reactions conducted with primers
specific for the four PDE4 subtypes suggested that pretreatment with
salbutamol and rolipram increased steady-state mRNA levels of PDE4A and
PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit
polyclonal antibodies, one directed against human recombinant PDE4A and
PDE4D and a second directed against human recombinant PDE4B, revealed
bands of immunoreactivity corresponding to
125 kDa (PDE4A) and
70 kDa (PDE4B), respectively, that increased in intensity after
cells were treated with salbutamol and rolipram. As demonstrated in
both time course and concentration-response studies with prostaglandin
E
(PGE
), an agent that activates adenylyl
cyclase by a non-
-adrenoceptor-mediated mechanism, cAMP
accumulation was substantially decreased in cells in which PDE4
activity had been up-regulated. The difference in
PGE
-stimulated cAMP accumulation between control and PDE4
up-regulated cells was greatly reduced in the presence of rolipram,
consistent with the notion that an increase in PDE4 activity was
responsible for the heterologous desensitization. Functionally,
up-regulation of PDE4 markedly decreased the ability of PGE
to inhibit LTD
-induced Ca
mobilization in intact cells. A hypothetical implication of these
results is that increasing PDE4 activity in vivo by
administering
-adrenoceptor agonists could exacerbate inflammatory
processes by decreasing the activity of endogenous anti-inflammatory
agents such as PGE
.
Cyclic nucleotide phosphodiesterases (PDEs) ()are a
family of isozymes that catalyze the hydrolysis of the 3`-phosphoester
bond on adenosine cyclic 3`,5`-monophosphate (cAMP) and guanosine
cyclic 3`,5`-monophosphate to form the inactive 5`-monophosphate
products. Consequently, PDEs have a major role in regulating cellular
cyclic nucleotide content. It is now recognized that PDEs represent a
diverse family of isozymes, each with different kinetic and physical
characteristics, tissue distribution, and sensitivity to endogenous
regulators (Beavo, 1988). At least seven classes of PDE isozymes exist,
some of which contain multiple subtypes (Conti et al. 1991;
Beavo et al., 1994). All of these isozymes as well as many of
the subtypes are encoded by distinct genes (Beavo et al.,
1994).
The cAMP-specific PDE, designated PDE4 (for nomenclature see Beavo et al., 1994), is the predominant cAMP hydrolyzing isozyme class found in most, if not all, immune and inflammatory cells (Torphy and Undem, 1991; Giembycz and Dent, 1992). This, coupled with the well defined role of cAMP as a second messenger mediating a generalized suppression of immune and inflammatory cell activity (Bourne et al., 1973, 1974; Kammer 1988;), has led to the recognition that PDE4 plays a critical role in regulating the function of these cells (Torphy and Undem, 1991). Consequently, this isozyme has received considerable attention as a target for new antiinflammatory and immunomodulator drugs.
The PDE4 isozyme class is comprised of four subtypes, PDE4A through PDE4D (Beavo et al., 1994). The activity of certain subtypes can be increased in situ by either a short term regulatory process involving protein phosphorylation or by a long term regulatory process involving increased gene expression (Conti et al., 1991; Sette et al., 1994a, 1994b). Both of these regulatory mechanisms are cAMP-dependent and can be triggered by a variety of activators of adenylyl cyclase (Bourne et al., 1973; Conti et al., 1991; Torphy et al., 1992). This raises the possibility that PDE4 activity can be regulated in vivo by various hormones, drugs, and growth factors (Conti et al., 1991). Because of the predominance of PDE4 in immune and inflammatory cells, these regulatory pathways may have a substantial influence on the responsiveness of these cells to a variety of hormones, autacoids, and drugs.
We
previously reported that treatment of undifferentiated U937 cells, a
human monocytic cell line, with salbutamol, a -adrenoceptor
agonist, or prostaglandin (PG) E
produces a 2-4-fold
increase in the activity of PDE4, the major PDE in these cells (Torphy et al., 1992). This increase in activity develops after
2-4 h of agonist exposure and is: 1) preceded by an increase in
cAMP content and cAMP-dependent protein kinase activity; 2) potentiated
by cotreatment with rolipram, a PDE4 inhibitor; 3) mimicked by
8-bromo-cAMP; 4) marked by an increase in V
with
no change in the cAMP K
; 5) abolished by
inhibitors of mRNA or protein synthesis; and 6) reversible within 3 h
of agonist removal. In light of these results, the present experiments
were conducted to determine whether treatment of U937 cells with
-adrenoceptor agonists increases the steady-state levels of PDE4
protein or transcript and, if so, whether this phenomenon changes the
functional responsiveness of these cells to activators of adenylyl
cyclase.
To prepare human monocytes,
heparinized whole blood was centrifuged at 350 g for
30 min over Ficoll-Hypaque to remove erythrocytes and granulocytes. The
mononuclear cell layer was removed, washed twice with
Ca
- and Mg
-free Hanks'
balanced salt solution containing 1 mM EGTA, and resuspended
in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine
serum, 2.5 mM HEPES, 20 mML-glutamine, 100
U penicillin/ml, and 100 mg streptomycin/ml (RPMI). The suspension was
underlaid with iso-osmotic Percoll adjusted to a density of 1.062
g/cm
with RPMI and centrifuged at 560
g for 30 min. Monocytes were harvested from the interface, washed
twice with HBS, and resuspended in RPMI. Cells were incubated overnight
at a density of 0.5-1
10
cells/ml in
175-cm
flasks in a humidified, 37 °C atmosphere of 95%
air, 5% CO
.
When PGE-induced cAMP
accumulation was determined, control and ``PDE4-induced''
cells were centrifuged at 500
g for 5 min and washed
two times with cold Krebs-Ringer-Henseleit buffer composed of 118
mM NaCl; 4.6 mM KCl, 24.9 mM
NaHCO
, 1 mM KH
PO
, 11.1
mMD-glucose, 1 mM CaCl
, 1.1
mM MgCl
, and 5 mM HEPES, pH 7.4. Cells
were then resuspended at a final concentration of 10
cells/ml in Krebs-Ringer-Henseleit buffer containing 0.1% bovine
serum albumin (radioimmunoassay grade) and treated for the indicated
times with different concentrations of PGE
in the absence
or presence of 30 µM rolipram.
Protein concentrations were determined using
the Bio-Rad (modified Bradford) protein assay. Proteins (100
µg/lane) were separated via electrophoresis (Bio-Rad) on SDS-8%
polyacrylamide gels and electrophoretically transferred to
nitrocellulose membrane (Amersham, Buckinghamshire, United Kingdom)
using a tank electroblotter. Blots were briefly washed in PBS, 0.1%
Tween-20 and then blocked overnight in PBS/Tween-20/5% nonfat dry milk.
Blocked membranes were washed three times with PBS/Tween-20 before
incubation with the primary antibody.
Blots were incubated for 1 h with a 1:2000 dilution of polyclonal serum produced in New Zealand White rabbits. One antibody was raised against a galK-hPDE-1 fusion protein containing a major fragment of PDE4A, which included the conserved PDE4 catalytic domain (Livi et al., 1990). Preliminary characterization of this antibody indicated cross-reactivity with PDE4D, but not PDE4B (recombinant PDE4C is not available). A second antibody was raised against a peptide representing the unique carboxyl terminus of PDE4B2 (CDIDIATEDKSPVDT). For blocked antibody experiments, the antibody was diluted 1:100 into a lysate of either the nontransfected PDE-deficient Saccharomyces cerevisiae strain GL62 (control) or the same strain engineered to express a 686-residue fragment of human recombinant PDE4A (hrPDE4A) containing the conserved catalytic domain of PDE4. The mixtures were incubated overnight at 4 °C with gentle agitation. The antibody was used at a final dilution of 1:2000 in PBS/Tween-20, 1% nonfat dry milk.
After three washes with PBS/Tween-20 blots were then incubated for 1 h with horseradish peroxidase-linked anti-rabbit Ig whole antibody from donkey (Amersham) and washed five times with PBS/Tween-20. Immunoreactive proteins were detected by chemiluminescence (Amersham ECL reagents).
RT-PCR was carried out using a commercial RNA PCR kit
(Saiki et al., 1988). First strand cDNA was generated from
total RNA using random hexamers to prime the reverse transcription and
was directly amplified by PCR following the addition of specific primer
pairs (0.36 µg/tube) and Ampli-taq DNA polymerase. Oligonucleotide
primers were: PDE4A5, 5`-AACAGCCTGAACAACTCTAAC-3` and
3`-TCAGAGTCCACCCAAAATAAC-5`, defining a 907-bp product containing a XhoI site (Bolger et al., 1993); PDE4B2,
5`-AGCTCATGACCCAGATAAGTG-3` and 3`-CTGTGAGTCCTTCTACCAATA-5`, defining a
625-bp product containing a SalI site (McLaughlin et
al., 1993; Obernolte et al., 1993); PDE4C1,
5`-TCGACAACCAGAGGACTTAGG-3` and 3`-GAAAGAGGACCCGAAGATAGG-5`, defining a
289-bp product containing an SstI site (Bolger et
al., 1993); and PDE4D3, 5`-CGGAGATGACTTGATTGTGAC-3` and
3`-CGTGTGGTAAAAAGTCCTTGC-5`, defining a 641-bp product containing a StuI site (Bolger et al., 1993; Baecker et
al., 1994). A human -actin primer set was used in the
presence and absence of reverse transcriptase as a control for each RNA
sample. Reactions were 1 min at 95 °C, 30 s at 52 °C, and 1 min
at 72 °C for a subsaturating cycle number (35 or 40 cycles).
Products were electrophoresed on 3% agarose gels and visualized by
ethidium bromide staining.
Figure 1:
RT-PCR of transcripts for PDE4 subtypes
in U937 cells. U937 cells were untreated or exposed to 1 µM salbutamol and 30 µM rolipram for 4 h. After the 4-h
treatment period, RNA was prepared, reverse transcribed into cDNA, and
amplified using primer sets specific for the four subtypes of PDE4. The
PCR products were electrophoresed on 3% agarose gels and visualized
with ethidium bromide. RNA normalization for control and treated
samples was confirmed by conducting RT-PCR reactions using primers for
-actin RNA (+). Lack of DNA contamination was confirmed by
conducting the
-actin reaction in the absence of RT(-). DNA
molecular mass standards appear in the lane on the far left. The
results are representative of three experiments using material from
three different cell preparations.
Figure 2:
Western blot analysis of PDE4
immunoreactivity in U937 cells and human monocytes using antibody
raised against GalK-hrPDE4A. Panel A, U937 cells were
untreated or exposed to 1 µM salbutamol and 30 µM rolipram for 4 h. After the treatment period cells were washed
extensively and lysed. Supernatant fractions were then prepared and
identical amounts of protein (100 µg) were subjected to
SDS-polyacrylamide gel electrophoresis. Also run in these studies were
untreated human monocyte supernatants and lysates from yeast engineered
to express a truncated form of hrPDE4A that lacked 265 amino acids on
the N terminus. The data are representative of results from four (U937
cells) or three (monocytes) experiments. Panel B, additional
experiments were conducted to confirm that the antibody was detecting
PDE4 protein in U937 cells. In these studies, the antibody was diluted
1:100 into a lysate of either nontransfected yeast (control)
or yeast that had been engineered to produce the truncated form of
hrPDE4A (blocked). The following day, hrPDE4A (panel
B, left) and treated (1 µM salbutamol plus
30 µM rolipram) U937 cell supernatants (panel B, right) were run on SDS-polyacrylamide gels which were then
transferred to nitrocellulose membranes and probed with the control or
preadsorbed antibody.
The antibody raised against the GalK-hrPDE4 also detected hrPDE4D,
but not hrPDE4B (Fig. 3A). Although a faint band of
immunoreactivity in U937 cells was observed that corresponded to the
appropriate molecular mass for PDE4D (92 kDa), its intensity was
not increased in PDE4-up-regulated cells (Fig. 3A).
Figure 3:
Western blot analysis of PDE4A, PDE4B, and
PDE4D immunoreactivity in U937 cells. Panel A, as in Fig. 2A, U937 cells were untreated or exposed to 1
µM salbutamol and 30 µM rolipram for 4 h.
After the treatment period cells were washed extensively and lysed.
Supernatant fractions were then prepared and identical amounts of
protein (100 µg) were subjected to SDS-polyacrylamide gel
electrophoresis. Immunoreactivity was determined using antibody raised
against GalK-hrPDE4A. Also run in these studies were lysates from yeast
engineered to express hrPDE4A (full-length), PDE4B, or PDE4D. The data
are representative of results from four (U937 cell lysates) or two
(hrPDE4 subtypes) experiments. Panel B, additional experiments
were conducted to detect PDE4B immunoreactivity in control U937 cells
and cells treated for 4 h with salbutamol (1 µM) and
rolipram (30 µM). For reference purposes, hrPDE4B was also
run. The antibody used in these studies was raised against a unique
carboxyl-terminal PDE4B peptide. The data are representative of two
experiments.
An immunoblot produced with the antiserum raised against the PDE4B
peptide is shown in Fig. 3B. Two bands of
immunoreactivity were detected with hrPDE4B, a major band at 70
kDa and a minor band at
52 kDa. Several bands were detected in
U937 cells, but only those corresponding to 70 and 52 kDa were
increased in intensity after treating cells with a combination of
rolipram and salbutamol. Preabsorbing the antiserum with PDE4B
eliminated these two bands, but not the others.
Figure 4:
Effect of up-regulating PDE4 activity on
the time course for PGE-stimulated cAMP accumulation in
U937 cells. Control cells (solid symbols) and cells treated for 4 h
with 1 µM salbutamol and 30 µM rolipram (open
symbols) were washed extensively and treated with 1 µM
PGE
in the absence (circles) or presence (squares) of 30
µM (±)-rolipram. When present, rolipram was
reintroduced into the incubation medium 1 min before the addition of
PGE
. Cyclic AMP content was determined at the times
indicated. Basal cAMP content was 1.7 ± 0.2 pmol/10
cells in control cells and 2.4 ± 0.3 pmol/10
cells in PDE4 up-regulated cells. The values represent the mean
± S.E. of five experiments. *Significantly less than the value
in cells not pretreated with rolipram and salbutamol (p <
0.05).
PGE-induced cAMP accumulation in control and PDE4
up-regulated cells was also assessed in the presence of 30 µM rolipram (Fig. 4). We reasoned that if the decrease in
PGE
-stimulated cAMP accumulation observed in
salbutamol-pretreated cells was due, at least in part, to an
up-regulation of PDE4 activity, then inhibiting PDE4 in these cells
with rolipram would tend to normalize their responsiveness to
PGE
. Indeed, in the presence of rolipram,
PGE
-stimulated cAMP accumulation over the 15-min time
course was virtually identical in control versus PDE4-induced
cells (Fig. 4). Only after 15 min of exposure to PGE
was cAMP content slightly less in PDE4 up-regulated cells (68.2
± 11.2 pmol/10
cells) than in control cells (89.4
± 9.6 pmol/10
cells).
The ability of a range of
PGE concentrations (1 nM-10 µM) to
elevate cAMP content in control and PDE4-up-regulated U937 cells is
shown in Fig. 5. The conditions of these experiments were
identical to those of the time course studies, except that cells were
treated with various concentrations of PGE
for a single
fixed time (15 min) before cAMP content was determined. In control
cells PGE
produced a large, concentration-related increase
in cAMP accumulation. For example, basal cAMP content (0.99 ±
0.14 pmol/10
cells) was increased to 52.9 ± 2.9
pmol/10
cells by 10 µM PGE
,
greater than 50-fold over the basal level. In contrast, PGE
had much less effect on cAMP content in cells in which PDE4
activity had been up-regulated. In fact, cAMP accumulation stimulated
by 10 µM PGE
was only 5.4 ± 0.5
pmol/10
cells, nearly 10-fold less than in control cells
and only 5-fold above basal cAMP content (0.97 ± 0.12
pmol/10
cells).
Figure 5:
Effect of up-regulating PDE4 activity on
concentration-response curves for PGE-stimulated cAMP
accumulation in U937 cells. Control cells (solid symbols) and
cells treated for 4 h with 1 µM salbutamol and 30
(±) µM rolipram (open symbols) were washed
extensively and treated with PGE
in the absence (circles) or presence (squares) of 10 µM (±) rolipram. When used, rolipram was reintroduced into the
incubation medium 1 min before the addition of PGE
. The
cells were then exposed to various concentrations of PGE
(1-10,000 nM). Cyclic AMP content was determined
15 min after the addition of PGE
. Basal cAMP content was
0.99 ± 0.14 pmol/10
cells in control cells and 0.97
± 0.12 pmol/10
cells in PDE4 up-regulated cells. The
values represent the mean ± S.E. of five experiments.
*Significantly less than value in cells not pretreated with rolipram
and salbutamol (p < 0.05).
In the presence of 10 µM rolipram, PGE-induced cAMP accumulation was greater in
both control cells and, even more impressively, cells in which PDE4
activity had been up-regulated. Overall, inhibiting PDE4 activity with
rolipram increased PGE
-stimulated cAMP accumulation by
2-fold in control cells and 12-fold in PDE4 up-regulated cells. Thus,
in the presence of rolipram, maximal PGE
-stimulated cAMP
accumulation in cells in which PDE4 activity had been up-regulated was
only 2-fold less than in control cells. This contrasts with the 10-fold
difference detected in the absence of rolipram.
Figure 6:
Effect of up-regulating PDE4 activity on
LTD-induced Ca
mobilization in U937
cells. Cells were treated with vehicle (
) or 1 µM salbutamol and 30 µM (±) rolipram (O) for 4 h.
Cells were washed extensively to remove drugs before being treated with
various concentrations of LTD
(0.1-3300 nM).
Ca
mobilization was assessed via fura-2 fluorescence.
The data are representative of 3 experiments and reflect maximal
cytosolic free Ca
concentrations obtained in response
to the indicated concentrations of
LTD
.
Although up-regulation of PDE4 activity had no
direct effect on LTD-stimulated Ca
mobilization, it had a substantial effect on the ability of
PGE
to inhibit this response (Fig. 7). In control
cells PGE
suppressed maximal Ca
mobilization induced by 3.3 nM LTD
with an
IC
= 30 nM and a maximal inhibitory effect
of 70 ± 1% (Fig. 7A). Prior exposure of U937
cells to salbutamol (1 µM) and rolipram (30
µM) for 4 h to induce PDE4 resulted in a substantial
reduction in the inhibitory effect of PGE
. In these cells,
PGE
suppressed Ca
mobilization with an
IC
= 150 nM, 5-fold greater than in
untreated cells, and had a maximal inhibitory effect of only 27
± 2%.
Figure 7:
The effect of up-regulating PDE4 activity
on the ability of PGE to inhibit LTD
-induced
Ca
mobilization in U937 cells. Cells were treated
with vehicle (
) or 1 µM salbutamol and 30
µM (±) rolipram (
) for 4 h. The cells were
then washed extensively to remove drugs before being treated with
various concentrations of PGE
(1-10,000 nM)
in the absence (panel A) or presence (panel B) of 10
µM (±) rolipram. When rolipram was used in
combination with PGE
in the Ca
mobilization experiments, it was reintroduced into the incubation
medium 1 min before the addition of PGE
. Cells were
challenged with LTD
(3.3 nM) 5 min after the
addition of PGE
, and Ca
mobilization was
monitored via fura-2 fluorescence. The data represent the mean ±
S.E. of four experiments and reflect maximal cytosolic free
Ca
concentrations achieved in response to
LTD
. *Significantly less in PDE4-up-regulated
cells.
The heterologous desensitization to the inhibitory
effect of PGE in PDE4 up-regulated cells was largely
reversed in the presence of 10 µM rolipram (Fig. 7B). For example, although the maximal inhibitory
effect of PGE
on Ca
mobilization was
statistically less in PDE4 up-regulated cells even in the presence of
10 µM rolipram, the difference from control cells was
extremely small (70 ± 3%, control versus 65 ±
1%, up-regulated). Qualitatively, similar results were obtained in
studies in which PGE
-stimulated cAMP accumulation was
assessed in the presence of 100 µM rolipram, although a
significant diminution in the ability of PGE
to inhibit
Ca
mobilization in PDE4 up-regulated cells under
these conditions was observed only at one concentration of PGE
(10 nM PGE
; data not shown). No
statistically significant difference was observed with any of the other
concentrations of PGE
. Thus, the ability of PGE
to inhibit Ca
mobilization in cells that have
increased PDE4 activity is largely recovered in the presence of a PDE4
inhibitor.
PDE4 is the predominant cAMP-metabolizing enzyme family in inflammatory cells and has been identified as an important new molecular target for novel antiasthmatic and anti-inflammatory drugs (Torphy and Undem, 1991; Giembycz and Dent, 1992). Based upon the results of recent studies in which Sertoli cells were used as a model system (Sette et al., 1994a, 1994b), considerable attention has been focused on two general mechanisms by which the activity of PDE4 is regulated by hormones, particularly those that stimulate adenylyl cyclase activity. One regulatory mechanism, designated ``short term activation,'' involves a protein kinase A-mediated phosphorylation of a specific splice variant PDE4D. This phosphorylation results in an increase in catalytic activity, perhaps by allosteric modification of the catalytic domain (Sette et al., 1994b). A second regulatory mechanism, designated ``long term activation,'' occurs with two other splice variants of PDE4D (Swinnen et al., 1989, 1991). Activation of protein kinase A in intact cells increases the expression of these latter forms by enhancing mRNA synthesis or increasing mRNA stability.
Although
indirect evidence suggests that activators of adenylyl cyclase can
regulate PDE4 activity in immune and inflammatory cells (Chan et
al., 1982; Holden et al., 1987, Bourne et al.,
1973; Torphy et al., 1992), definitive information on the
precise nature of this phenomenon in these cells is not available.
Moreover, despite the growing body of evidence suggesting that the
activity of PDE4 can be up-regulated by hormonal stimulation, little is
known about the biological importance of this regulation. We have begun
to address these deficiencies by examining the nature and functional
consequences of PDE4 up-regulation in U937 cells, a human monocytic
cell line. As previously reported (Torphy et al., 1992),
activation of the protein kinase A cascade in these cells by
-adrenoceptor agonists increases PDE4 catalytic activity. The
magnitude of this up-regulation is enhanced if rolipram is included in
the incubation medium, presumably because inclusion of a PDE inhibitor
both heightens and prolongs the increase in cAMP content produced by
-adrenoceptor agonists. The increase is prevented by actinomycin D
or cycloheximide, indicating that the up-regulation of PDE4 activity
depends upon the synthesis of both mRNA and protein. The results of the
present experiments are consistent with this conclusion. Treatment of
U937 cells for 4 h with a combination of salbutamol and rolipram
increased the amount of immunoreactive PDE4A detected in cell
supernatants. The results were particularly striking in that PDE4A was
virtually undetectable in untreated cells but clearly evident in the
induced cells. An increase in PDE4B immunoreactivity also occurred in
response to 4-h exposure to salbutamol and rolipram. In contrast, this
treatment regimen had no effect on PDE4D3 levels. These studies do not,
however, eliminate the possibility that PDE4D activity can be regulated
directly by a protein kinase A-mediated phosphorylation pathway.
Moreover, since our focus was on PDE4D3, we cannot exclude the
possibility that
-adrenoceptor agonists regulate the expression of
other mRNA splice variants (i.e., PDE4D1 and PDE4D2).
mRNA transcripts encoding PDE4 subtypes were identified through RT-PCR methodology using subtype-specific oligonucleotide primers. In untreated U937 cells, the only PCR products detected were those corresponding to PDE4B and PDE4D. Although the technique utilized was not designed to be quantitative, distinct changes in the pattern of PCR products were detected after treatment with rolipram and salbutamol. Specifically, PCR product for PDE4A transcript was barely detectable in untreated cells, but a PCR product of the appropriate length was observed clearly and consistently in stimulated cells. In addition, the amount of PCR product corresponding to PDE4B appeared to increase, whereas that corresponding to PDE4D appeared to decrease. The functional consequences of these apparent changes in steady-state transcript levels as they relate to PDE4B and PDE4D protein expression are unknown. Consistent with the results of this study, Engels and colleagues(1994) also detected an increase in message for PDE4A and PDE4B in response to 18-h treatment of U937 cells with 0.5 mM dibutyryl cAMP.
Up-regulation of PDE4 activity in U937 cells
had a substantial impact on their responsiveness to PGE, an
activator of adenylyl cyclase. As demonstrated in both time course and
concentration-response studies, pretreatment of cells with a
combination of salbutamol and rolipram substantially decreased the
ability of PGE
to elevate cAMP content. This loss of
activity was mirrored by a decrease in the ability of PGE
to inhibit LTD
-induced Ca
mobilization. We reasoned that if an increase in PDE4 activity
had a role in producing the heterologous desensitization in pretreated
U937 cells, then inhibiting PDE4 activity in these cells would tend to
normalize their sensitivity to PGE
. This was indeed the
case. Whereas there was a substantial difference in the ability of
PGE
to stimulate cAMP accumulation and suppress
Ca
mobilization in desensitized versus control U937 cells, the difference was markedly reduced or, in
some instances, virtually abolished when functional studies were
carried in the presence of rolipram (10 µM).
It is not
yet known whether regulation of PDE4 activity represents a general
homeostatic mechanism by which all target cells, particularly
inflammatory cells, modulate their responsiveness to hormones and
autacoids that activate adenylyl cyclase. However, in support of the
broad applicability of this phenomenon, we recently have demonstrated
that PDE4 activity and steady-state protein levels in human monocytes
is up-regulated by -adrenoceptor agonists in a manner similar to
that seen in U937 cells. (
)
The results of these studies
have implications regarding the use of -adrenoceptor agonists as
bronchodilators in the treatment of asthma. Normally, endogenous
activators of adenylyl cyclase such as epinephrine, PGE
,
and prostacyclin act as natural anti-inflammatory and bronchodilator
agents (Barnes, 1986; Kuehl et al., 1987), presumably by
elevating cAMP content in the appropriate target tissues. In theory,
the beneficial actions of these agents would be compromised if chronic
treatment of asthmatic individuals with
-adrenoceptor agonists
resulted in an up-regulation of PDE4 activity in inflammatory cells and
airway smooth muscle. This would allow inflammatory processes and
bronchoconstriction to proceed unchecked. Indeed, chronic use of
inhaled salbutamol increases airway responsiveness to allergen and
causes tolerance to the protective effect of the
-adrenoceptor
agonist against allergen challenge (Cockcroft et al., 1993).
Presumably, this occurs as a result of a diminished ability of
-adrenoceptor agonists to inhibit mast cell mediator release
(Cockcroft et al., 1993).
In the long term, a generalized
induction of tolerance in inflammatory cells could lead to a worsening
of disease status. Regarding this proposal, several clinical reports
have linked a deterioration of disease status and increased mortality
with the excessive use of -adrenoceptor agonists (Barnes and
Chung, 1992; Sears et al., 1990; Spitzer et al.,
1992). The results of these studies, the interpretation(s) of which are
controversial and often conflicting, have led to the proposal of
several hypotheses to explain the apparent detrimental effect of
sympathomimetic bronchodilators. These include the possibility that the
overuse of
-adrenoceptor agonists: 1) down-regulate
-adrenoceptors, 2) mask a worsening of disease status, 3) increase
antigen load to the distal airway, or 4) compromise the
``protective'' role of lung mast cells (Barnes and Chung,
1992; Nelson et al., 1991; Page, 1991). It is tempting to
speculate that an up-regulation of PDE4 activity represents an
additional factor that contributes to the ``bronchodilator
paradox.''
An issue that remains to be addressed is whether the concentrations of salbutamol that increase the expression of PDE4 in U937 cells are similar to those required to produce bronchodilation in the clinic. In a previous study (Torphy et al., 1992), we demonstrated that PDE4 activity is up-regulated by salbutamol, used alone or in combination with rolipram, over a concentration range of 10 nM to 10 µM. This range is identical to that required to relax human airway smooth muscle in vitro (Goldie et al. 1986; Nally et al., 1994). While it is virtually impossible to determine the local concentrations of inhaled salbutamol within the airway, this information strongly suggests that salbutamol up-regulates PDE4 activity at clinically relevant concentrations.
In conclusion, treatment of U937 cells with agents
that activate the cAMP/protein kinase A cascade results in an increase
in PDE4 activity. Coincident with the elevation in total cellular PDE4
catalytic activity is an increase in steady-state message and protein
for PDE4A. From a functional standpoint, the up-regulation of PDE4
activity results in a heterologous desensitization of U937 cells to the
actions of PGE and, presumably, other adenylyl cyclase
activators. Conceivably, this regulatory pathway could compromise the
long term anti-asthmatic efficacy of
-adrenoceptor agonists, the
most commonly used class of bronchodilators.
Note Added in Proof-Cyclic AMP-elevating agents produce a similar, but not identical pattern of PDE4 subtype up-regulation in human monocytes and Mono Mac 6 cells, a human monocytic cell line (Verghese et al., 1995).