(Received for publication, July 2, 1996, and in revised form, February 7, 1997)
From the Renal Pathophysiology Laboratory, Some major pathobiologic processes in renal
mesangial cells, elicited in response to immunoinflammatory stimuli,
are modulated via cAMP-protein kinase A (PKA) signaling pathways;
namely, generation of reactive oxygen metabolites (ROM) and accelerated
proliferation of mesangial cells. We investigated the role of cAMP
phosphodiesterase (PDE) isozymes in these regulatory mechanisms.
Generation of ROM in cultured rat mesangial cells was inhibited by
selective inhibitors of PDE4, rolipram and denbufylline, whereas PDE3
inhibitors, cilostamide and lixazinone, had no effect. Conversely,
cilostamide or lixazinone suppressed mitogenic synthesis of DNA in
mesangial cells, but 1 µM rolipram or 1 µM denbufylline showed no inhibitory effect. The efficacy
of PDE isozyme inhibitors (IC50) to suppress
[3H]thymidine incorporation or ROM generation paralleled
IC50 values for inhibition of cAMP PDE. Incubation of
mesangial cells with either rolipram alone or with cilostamide alone
increased significantly in situ activity of PKA in
mesangial cells, assessed by ( Mesangial cells in kidney glomeruli are specialized pericytes (1)
that are located in the intercapillary spaces of glomeruli and comprise
about 30% of glomerular cells (2). Mesangial cells have some
properties, such as contractility, reminiscent of smooth muscle cells,
and some that are common to monocytes and macrophages, i.e.
phagocytosis or the ability to generate reactive oxygen metabolites (ROM)1 (3, 4). According to the current
view, mesangial cells have a key role in maintaining the integrity of
renal glomerular structure and function mainly by regulating capillary
blood flow, uptake of macromolecules by phagocytosis, and synthesis of
the extracellular matrix (1, 3, 4). Pathobiologic responses of
mesangial cells to immunoinflammatory stimuli often determine and/or
contribute to pathogenic processes involved in the development of acute
or chronic glomerulonephritides, in particular, mesangial proliferative
glomerulonephritis (5, 6).
In our recent studies of rat mesangial cells grown in primary culture
we observed that inhibition of cyclic-3 Extracts of rat mesangial cells grown in primary culture (7) have the
capacity to catalyze hydrolysis of cAMP by PDE3 and, to a significantly
higher degree, by PDE4. These are the two major PDE isozymes with low
Km for cAMP present in rat mesangial cells (7). The
question arises as to which of these two PDE isozymes (or both), and to
what degree, metabolizes the presumed cAMP pool that specifically
modulates ROM generation in mesangial cells; we set out to investigate
this problem.
We also addressed a more general and largely unsolved question,
i.e. whether within one cell type, in this instance
mesangial cells, two distinct cellular functions that are both known to be modulated by cAMP-PKA signaling pathways are under separate control
of two distinct cellular pools of cAMP which are functionally linked
to, and thereby compartmentalized by, activity of specific PDE
isozymes. The findings reported herein support a hypothesis that in rat
mesangial cells, the ROM generation catalyzed by NADPH oxidase is
regulated by a cAMP pool functionally coupled to isozyme PDE4, whereas
regulation of mitogenesis in mesangial cells is regulated by another
cAMP pool that is regulated by activity of PDE3. The results thus
provide evidence for the intracellular compartmentalization of cAMP
signaling pathways effected by PDE isozymes within mesangial cells.
Cell Culture This was determined with the probe
2 ROM production was determined in mesangial cells stimulated with
serum-opsonized zymosan (SOZ) (zymosan opsonized by incubation with
fresh rat serum) added at a final concentration of 2 mg/ml. It has been
shown that SOZ stimulates both ROM generation and phagocytosis in
mesangial cells (20). The development of SOZ-stimulated fluorescence
was almost completely blocked (94 ± 2%; n = 10)
by the addition of superoxide dismutase (40 µg/ml) and also was
suppressed by other scavengers of ROM, i.e. catalase and
N-acetylcysteine (data not shown). The difference in
fluorescence without and with superoxide dismutase (Fig.
1), the "superoxide dismutase-suppressible value,"
was taken as a measure of ROM generation. In control experiments, we
determined that none of the test compounds employed in our study either
quenched fluorescence or emitted spurious fluorescence at the detection
wavelength under the experimental conditions. In preliminary
experiments, this method produced similar results in assessing ROM
generation in the murine macrophage cell line J774 (18). The superoxide
dismutase-suppressible fluorescence generated by SOZ-stimulated
mesangial cells accumulated linearly with time for at least 4 h
(Fig. 1). Inhibition by 10 µM rolipram (35%) or 10 µM forskolin (33%) was the same at 1, 2, or 3 h of incubation. Therefore, fluorescence readings were made after a 3-h
incubation to optimize precision of measurements.
PKA activation was assayed by the
method described by Corbin et al. (21), with minor
modifications (7, 10). Quiescent mesangial cell cultures in Petri
dishes (6-cm diameter) were incubated either without additions
(controls), with PDE inhibitors for 60 min, or with forskolin for 10 min. Incubations were terminated by chilling plates (0-4 °C) and
quickly scraping cells, which were then pelleted by centrifugation at
2,500 × g for 5 min at 0-4 °C. The supernatant was
discarded, and mesangial cells were homogenized in buffer containing
(final concentrations): 0.25 M sucrose, 0.5 mM
3-isobutyl-1-methylxanthine, 4 mM EDTA, and 20 mM MES-NaOH, pH 6.8. The homogenate was centrifuged at
27,000 × g for 30 min at 0-4 °C, protein content
was determined, and PKA activity was assayed in the supernatant using
Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) as a substrate (7, 22). PKA
activity was assayed without and with added 1 µM cAMP and
with 1 µM cAMP plus a maximal inhibitory concentration
(10 µM) of a specific PKA oligopeptide pseudosubstrate
inhibitor WIPTIDE (7, 10, 22). The difference between PKA activity
without and with 10 µM WIPTIDE was taken as specific PKA
activity and expressed as ( DNA synthesis was measured in
mesangial cells that were rendered quiescent by incubation in medium
with 0.5% fetal calf serum for 72 h. After overnight (16 h)
incubation with test agents, the cells were incubated with
[6-3H]thymidine for 4 h, and incorporation of
[3H]thymidine was determined as described previously (7,
10, 22).
This was determined by incubating
mesangial cells with test agents and terminating the incubation by
adding 5% trichloroacetic acid (final concentration) to wells (cells
were collected by scraping with a rubber policeman and the mixture
incubated on ice for 30 min). Trichloroacetic acid-precipitated protein
was extracted with water-saturated ether, and the cAMP content was
measured by radioimmunoassay as described (7, 10).
PDE activity was determined in total mesangial
cell extracts prepared by homogenization in buffer containing 0.1%
Triton X-100 (7, 10). Briefly, PDE activity was measured by incubating portions of homogenates in a reaction mixture (final volume 110 µl)
containing (final concentrations) 10 mM MgSO4,
2 mM EGTA, 0.1% bovine serum albumin, 15 mM
Tris-HCl adjusted to pH 7.4, and 0.5 µM
[3H]cAMP as substrate. Hydrolysis of cAMP was less than
20% and was linearly proportional to incubation time and enzyme
protein concentration (7, 10). In experiments examining the dose response to PDE inhibitors, maximal inhibition by rolipram or cilostamide was reached at 3 µM. Therefore, as in our
preceding studies (7, 10), we employed 3 µM rolipram and
3 µM cilostamide as maximally effective inhibitory doses.
The proportion of PDE3 and PDE4 activities in extracts from mesangial
cells was defined as cAMP PDE activity inhibited by 3 µM
cilostamide or 3 µM rolipram, respectively (7). In
current studies with cultured mesangial cells (passages 2-4), as in
previous studies (7) with cells of later passages, PDE3 accounted for
~30% and PDE4 for ~60% of total cAMP PDE activity in homogenates
assayed with 0.5 mM cAMP as substrate.
The effects of PDE inhibitors or
other agents upon responses of cultured mesangial cells were measured
during a 72-h incubation in a medium containing 0.5% fetal calf serum
(7) under "ambient conditions" without added exogenous growth
factors, hormones, cytokines, or agonists of adenylate cyclase,
i.e. under the autocrine/paracrine influence of autacoids
and other regulatory factors generated by mesangial cells themselves
(5, 6). Protein measurements were done by the Lowry method (23). Stock
solutions of all inhibitors were made in 100% dimethyl sulfoxide;
incubation media in all conditions, including controls, contained 0.1%
dimethy sulfoxide (final concentration), which did not interfere with
measured parameters. Values for IC50 (concentration of PDE
inhibitors required to inhibit by 50% cAMP PDE activity,
[3H]thymidine incorporation or ROM generation) were
determined from concentration-response curves in which inhibitor
concentrations ranged from 1 nM to 10 µM.
IC50 values are means ± S.E. from three or four
experiments.
The results were evaluated statistically with the use of two-tailed
Student's t test; values of p < 0.05 were
considered statistically significant. NS denotes no significant
difference.
Cilostamide,
N-cyclohexyl-N-methyl-4-(1,2-dihydro-2-oxo-6-quinolyloxy),
butyramide (OPE-3639), and cilostazol,
6-[4-1-cyclohexyl-1H-tetrazol-5-yl/butoxy]-3,4-dihydro-2 (IH)-quindinone (OPC-13013) were gifts from Otsuka Pharmaceutical Company (Osaka, Japan). Rolipram (racemic),
4-(3-cyclopentyloxy-4-methylphenyl)-2-pyrolidine (ZK 62711), was a gift
from Berlex Laboratories (Cedar Knolls, NJ). Denbufylline, BRL 30892 (1,3-di-n-butyl-7-(2 [ ROM generation and accumulation in cultured mesangial cells were
linearly proportional to time of the incubation (Fig. 1). Incubation
with dibutyryl cAMP, forskolin, or rolipram, a selective PDE4
inhibitor, all resulted in marked inhibition (30-40%) of ROM
generation, and the extent of inhibition was not significantly different among the three agents (Fig. 2). The
inhibitory effect of rolipram was dose-dependent with
maximal inhibition at 1 µM (Fig. 3 and
Table I). Denbufylline, an inhibitor of PDE4 which is
structurally unlike rolipram (24), inhibited ROM generation to a degree
similar to that of rolipram (Table I). In contrast, under the same
experimental conditions, two structurally dissimilar selective PDE3
inhibitors, cilostamide and lixazinone (25), had no effect on ROM
generation (Table I and Fig. 2). Incubation of mesangial cells with 10 µM rolipram and 10 µM cilostamide together did not inhibit ROM generation (45 ± 6%, mean ± S.E.;
n = 7) to a significantly greater extent than 10 µM rolipram alone (32 ± 4%, mean ± S.E.;
n = 7) (Fig. 2, lower panel). The
IC50 values for rolipram inhibition of cAMP PDE activity
and ROM generation were similar (Table II).
Comparison of the effects in mesangial cells of PDE4 antagonists,
rolipram and denbufylline, and PDE3 antagonists, cilostamide and
lixazinone, upon DNA synthesis and ROM generation, measured as
described under "Experimental Procedures."
The effects are expressed in relative terms as
IC50 values for inhibition of cAMP PDE activity,
[3H]thymidine incorporation, and ROM generation by selective
PDE isozyme inhibitors
For details, see "Experimental Procedures." Each value denotes
mean ± S.E. of n = 3-4 experiments.
Department of Laboratory
Medicine,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
cAMP/+cAMP) PKA activity ratio, and the
stimulatory effects were additive. Results indicate that in mesangial
cells a cAMP pool that is metabolized by PDE4 activates PKA and thereby
inhibits ROM generation; another cAMP pool that is metabolized by PDE3
activates another PKA (isozyme or pool) which suppresses proliferation
of mesangial cells. We propose that in mesangial cells, a cAMP-PKA
pathway that regulates mitogenesis is determined by activity of PDE3,
whereas another cAMP-PKA pathway is directed by activity of PDE4 and
controls ROM generation. Therefore, two PDE isozymes within one cell
type compartmentalize distinct cAMP signaling pathways.
,5
-nucleotide phosphodiesterase (PDE) isozyme type-3 (PDE3) by selective inhibitors causes activation of cAMP-protein kinase A (PKA) and suppresses mesangial cell proliferation, either basal or stimulated by addition of
growth factors such as epidermal growth factor or platelet-derived growth factor (7). Inhibitors of PDE isozyme type-4 (PDE4) had only
minor or no inhibitory effects upon mesangial cell mitogenesis (7). In
rats with experimentally induced mesangial proliferative glomerulonephritis, administration of a PDE3 inhibitor alone (8) or
with a PDE4 inhibitor (9) suppresses development of glomerulonephritis, including decreased proliferation of mesangial cells (8, 9). We also
observed that generation of ROM in freshly isolated rat glomeruli is
suppressed when the cAMP-PKA pathway is activated by incubating whole
glomeruli with rolipram, a selective inhibitor of PDE4, and to a lesser
degree with cilostamide, a selective inhibitor of PDE3 (10).
Interpretation of these results was limited since all three cell types
populating renal glomeruli, i.e. mesangial, epithelial, and
endothelial cells, are endowed with NADPH oxidase, an enzyme complex
that catalyzes ROM generation (11, 12). Therefore, all of these cell
types can contribute to ROM production by the whole glomeruli; it
remained unknown in which cells and to what extent inhibitors of PDE3
and/or PDE4s exert its inhibitory effects upon ROM generation (10).
Further, it cannot be excluded that some of blood cells trapped within glomeruli, namely neutrophils that have very active NADPH oxidase (13),
contribute to glomerular ROM generation. Studies of several types of
leukocytes, e.g. eosinophils (14), monocytes (15), and
neutrophils (16), all highly active in generation of ROM, have shown
that only PDE4 inhibitors suppress NADPH oxidase and ROM synthesis in
these cell types (14-16).
Studies were conducted on rat mesangial
cells isolated from freshly prepared glomeruli and grown in primary
culture as described in detail in our preceding papers (10, 17).
Specifically, mesangial cells (passages 2-18) were grown in an RPMI
medium with 20% fetal calf serum until reaching confluence; fetal calf
serum was withdrawn for 24-48 h prior to experiments. Mesangial cells were grown on multiple well plates until confluence, prior to measurement of ROM generation (12-well plate), the rate of mitogenesis or cAMP accumulation (24-well plate), or measurement of in
situ PKA activation (6-well plate), as in our preceding reports
(10, 17).
,7
-dichlorodihydrofluorescein diacetate (DCFH-DA), in a
microfluorometric assay originally designed for measurement of the ROM
burst in monocytes (18). The same probe was also employed for detection
of ROM in cultured vascular smooth muscle cells (19). DCFH-DA is a
compound that is membrane-permeant because of two esterified acetates,
which are cleaved by esterases in the cells (14). Both DCFH-DA and the
deesterified product 2,
7
-dichlorodihydrofluorescein (DCFH) are
nonfluorescent, but both are readily oxidized by ROM to a fluorescent
2
,7
-dichlorofluorescein derivatives (18, 19). This method detects
both ROM that remain within the cell and ROM in the extracellular
medium (18, 19). For measurement of ROM generation, mesangial cells
were incubated with additions, including the probe, as specified. The
development of fluorescence initiated by the added stimulus was
expressed as relative fluorescence units monitored at 485 nm excitation
and at 530 nm emission wavelength in microplate fluorometer interfaced
with a PC-compatible computer for data processing (18).
Fig. 1.
Time course of fluorescence due to generation
of ROM in cultured rat mesangial cells stimulated by SOZ, without ()
or with the addition of superoxide dismutase (SOD) to the
incubation medium (
). Ordinate, fluorescence (arbitrary
units); abscissa, elapsed time of incubation.
[View Larger Version of this Image (15K GIF file)]
cAMP/+cAMP) PKA activity ratio (7, 10,
22).
-oxoyprophyl)-xanthine, was donated by
Smith Kline Beecham Pharmaceutical (Worthing, West Sussex, United
Kingdom). Lixazinone,
-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one)butyramide (RS-82856), as a gift from Dr. R. Alvarez, Syntex
Research, Palo Alto, CA. Ro 20-1724, 4-(3-butoxy-4-methylbenzyl)-2-inidazolidinone was purchased from
BIOMOL. Oligopeptide WIPTIDE, inhibitor of PKA, as well as the PKA
substrate Kemptide were synthesized in the Mayo Peptide Core Laboratory
(Dr. D. T. McCormick).
-32P]ATP and [6-3H]thymidine were
purchased from Amersham Corp., and [2,8-3H]cAMP was from
DuPont NEN. DCFH-DA was purchased from Molecular Probes (Eugene, OR).
Catalase (Sigma) was purified prior to use by passing through a
Sephadex G-50 column. Other reagents, all of highest purity grades,
were purchased from standard suppliers.
Fig. 2.
Effect of PDE isozyme antagonists: 10 µM cilostamide (CS), 10 µM
rolipram (RP), alone or together, 10 µM
forskolin (FSK), or 1 mM dibutyryl cAMP
(DB cAMP) on in situ activation of PKA (upper panel) and inhibition of ROM generation (lower
panel) in mesangial cells, as described under "Experimental
Procedures." Upper panel, bars denote in
situ PKA activity after incubation without (control,
CONT.) or with the indicated agents. The black portions of the bars depict increases in PKA activity
in response to added agents compared with control values. The
asterisks denote statistically significant increases in
(cAMP/+cAMP) PKA activity ratio over control values (by paired
t test, p < 0.05, or higher degree of
significance). Each bar denotes mean ± S.E. of six or seven experiments. p values above the bars denote
level of statistical significances of differences (by two-tailed paired
t test). Lower panel, bars denote
relative (
%) inhibition of ROM generation by the same agents as in
the upper panel. Each bar is the mean ± S.E. of four to seven experiments. The asterisks denote
statistically significant inhibition (p < 0.01; paired
t test); NS, no statistically significant
difference.
[View Larger Version of this Image (24K GIF file)]
Fig. 3.
Inhibition of ROM generation by different
concentrations of rolipram. Each point denotes the mean ± S.E. of
three experiments. Maximally effective concentrations of rolipram
reduced ROM by 31-40%.
[View Larger Version of this Image (18K GIF file)]
% difference from
basal values (without addition of test compound) taken as 100%.
[3H]Thymidine
incorporationa
ROM generationa
%
p valueb
%
Denbufylline
Rolipram
1
µM
+5
± 4
NS
1
µM
27
± 7d
<0.05
Rolipram
10
µM
31 ± 7
<0.05
1
µM
+4
± 18
NS
Denbufylline
10
µM
29 ± 3
<0.02
1
µM
20 ± 4
<0.02
Lixazinone
10 µM
25
± 4
<0.05
1 µM
46
± 2c
<0.01
Cilostamide
Cilostamide
10
µM
+1 ± 2
NS
1
µM
42 ± 8
<0.05
Lixazinone
10
µM
70 ± 7c
<0.01
10
µM
+0.5 ± 1
NS
a
Each value denotes mean ± S.E. of
n = 3-4 experiments.
b
p values for statistical significance of changes
(± %) by paired t test. NS, not significant.
c
Significantly different (p < 0.05, t test) from value with 10 µM rolipram.
d
Not significantly different from values with 10 µM rolipram and 1 µM or 10 µM
denbufylline.
cAMP
phosphodiesterase
[3H]Thymidine
incorporation
ROM generation
n
IC50
n
IC50
n
IC50
Cilostamide
4
0.21 ± 0.04
× 10
6 M
4
0.89
± 0.10 × 10
6 M
Lixazinone
4
2.85 ± 0.46
× 10
9 M
3
4.2 ± 1.9
× 10
9 M
Rolipram
4
0.50 ± 0.12
× 10
6 M
3
0.68 ± 0.24
× 10
6 M
Effects of the PDE4 inhibitors, rolipram and denbufylline, and the PDE3 inhibitors, cilostamide and lixazinone, on mitogenesis in mesangial cells were opposite to their effects upon ROM generation. Incubation with 3 µM cilostamide inhibited [3H]thymidine incorporation into mesangial cells (67 ± 3%, mean ± S.E.; n = 7) four times greater (p < 0.005; t test) than that caused by 3 µM rolipram (17 ± 4%, mean ± S.E.; n = 7) or 3 µM denbufylline (14 ± 4%, mean ± S.E.; n = 4). At lower (1 µM) concentrations of PDE inhibitors, differential selective inhibition of ROM generation and mitogenesis was even more clear-cut. Both 1 µM cilostamide and 1 µM lixazinone suppressed (>42%) markedly [3H]thymidine incorporation, whereas 1 µM rolipram and 1 µM denbufylline were ineffective (Table I). Further, we found that IC50 values for inhibition of cAMP PDE by cilostamide and lixazinone correspond closely to their IC50 values for suppression of [3H]thymidine incorporation (Table II); the IC50 values for lixazinone were, for both parameters, about 2 orders lower than IC50 for cilostamide (Table II).
The effects of incubation of mesangial cells with PDE isozyme
inhibitors and forskolin upon in situ activation of PKA in
mesangial cells, as determined by measuring (cAMP/
cAMP) PKA
activity ratios, and upon inhibition of ROM generation in mesangial
cells are depicted in Fig. 2. Incubation with either rolipram alone or
cilostamide alone increased significantly (and to a similar extent) the
in situ PKA activity in mesangial cells (Fig. 2). However,
cilostamide exhibited no effect upon ROM, whereas rolipram caused
maximum inhibition of ROM generation (Fig. 2). The increase in in
situ PKA activation in response to incubation of mesangial cells
with 10 µM rolipram and 10 µM cilostamide
added together (
+ 338 ± 42; mean ± S.E.,
n = 9), expressed as the increment in (
cAMP/+cAMP) PKA ratio, was equal to or higher than the arithmetic sum of PKA responses to 10 µM rolipram added alone (
% + 97 ± 32; mean ± S.E., n = 9) and to 10 µM cilostamide added alone (
% + 129 ± 42;
mean ± S.E., n = 9). Yet, the extent (
%) of
inhibition of ROM generation either by rolipram alone, by rolipram and
cilostamide added together, or by forskolin was not significantly
different (Fig. 2). Interestingly, incubation with rolipram alone or
cilostamide alone resulted in in situ PKA activation (Fig.
2) without any measurable increase in cAMP content after incubation for
1 h (7) or 16 h. At the time of pulse labeling with
[3H]thymidine (16 h), the cAMP content (in pmol/well ± S.E.) was 1.23 ± 0.04 (controls), 1.17 ± 0.76 (with 10 µM rolipram), 1.11 ± 0.72 (with 10 µM
cilostamide), and 15.7 ± 3.5 (with 10 µM
forskolin).
The effects of PDE4 inhibitors or PDE3 inhibitors on cAMP accumulation
in mesangial cells were measured when cAMP synthesis was stimulated by
forskolin, a potent direct stimulator of adenylate cyclase. Under these
conditions PDE4 inhibitors, denbufylline and Ro-20-1714, caused a
200-fold increase in cAMP accumulation (Fig. 4). In
contrast, PDE3 inhibitors lixazinone and cilostazol caused only minor
elevation of cAMP (Fig. 4).
The observations presented herein argue in support of the hypothesis that PDE isozymes can, within a single cell type (i.e. mesangial cell), direct or compartmentalize the cAMP-PKA signaling pathways that regulate two distinct cellular functions. The results of the present study conducted on homogenous populations of mesangial cells grown in vitro indicate that generation of ROM in mesangial cells is regulated by a cAMP-PKA pathway that is uniquely linked to activity of PDE4 isozymes and, conversely, that mitogenesis in mesangial cells is regulated by a cAMP-PKA pathway specifically linked to activity of PDE3 isozymes (Figs. 2 and 3 and Table I).
The existence in mesangial cells of at least two functional and/or ultrastructural compartments of cAMP and related PKA which are determined by activities of PDE3 and PDE4, respectively, is supported by several lines of evidence. First, two structurally dissimilar inhibitors of PDE4 (rolipram and denbufylline) suppressed ROM generation, whereas two structurally dissimilar inhibitors of PDE3 (lixazinone and cilostamide) had no inhibitory effect (Table I). Conversely, 1 µM cilostamide or lixazinone, but not 1 µM rolipram or 1 µM denbufylline, inhibited [3H]thymidine incorporation in mesangial cells (Table I). Second, the efficacy of selective PDE isozyme inhibitors (IC50) for inhibition of cAMP PDE closely corresponded to their respective potencies (IC50) for suppression of either ROM generation or mitogenesis (Table II).
Third, the existence of PDE isozyme-determined cAMP-PKA compartments in
mesangial cells is indicated by observations on in situ
activation of PKA (Fig. 2). As documented by others (21) and our
previous studies (7, 22), measurements of the in situ
activity of PKA, determined as (cAMP/+cAMP) PKA activity ratios (7,
21) or by similar methods (25, 26), are much more sensitive indices of
the in situ activation of cAMP-PKA signaling pathways than
is determination of the total cellular (or tissue) content of cAMP (7,
26, 27). Results of our experiments examining in situ PKA
activation in mesangial cells (Fig. 2) argue for the existence of two
separate PKAs (PKA isozymes or pools) which are independently activated
by cAMP that is increased by selective inhibition of PDE3 or PDE4,
respectively. Although incubation with either rolipram alone or
cilostamide alone resulted in an increase of in situ PKA
activity (Fig. 2 and "Results"), only PKA activation elicited by
rolipram, but not cilostamide, was associated with decreased ROM
generation in mesangial cells (Fig. 2). In mesangial cells incubated
with rolipram and cilostamide together, the extent of in
situ PKA activation was at least additive, and perhaps even
synergistic (Fig. 2; see also "Results"); yet, the inhibition of
ROM generation by rolipram and cilostamide combined was not
significantly greater than by rolipram alone (Fig. 2). Therefore, it
appears that the portion of mesangial cell PKA which is activated in
response to cilostamide has no functional relationship to inhibition of
ROM generation (Fig. 2). Conversely, the inhibition of mitogenesis in
mesangial cells by cilostamide alone was not significantly different
from the effect of cilostamide and rolipram combined, whereas the
in situ activation of PKA in response to cilostamide and
rolipram added together was significantly higher than PKA activity
elicited by cilostamide alone or rolipram alone (Fig. 2; see
"Results"). Taken together, the additivity of in situ
PKA activation in response to incubation with rolipram and cilostamide,
both at maximally effective concentrations, suggests that increases of
intracellular cAMP content in mesangial cells elicited by inhibition of
PDE4 or that of PDE3 do activate different portions of cellular PKA.
Conceivably, the two cAMP pools regulated by different PDE isozymes may
activate two distinct PKA isozymes, i.e. PKA-I and PKA-II
(28), or alternatively, the same PKA type (e.g. PKA-II)
which within the same cell is attached via specific anchoring proteins to different cellular ultrastructures (29, 30). The
exact sequence of interactions between cAMP pools determined by PDE
isozymes PDE3 and PDE4, related PKA isozymes (or isoforms), and the
functional/structural targets within the mesangial cells remains to be
elucidated. However, the present experimental evidence indicates that
cAMP processing by specific PDE isozymes is a determining step that
targets cAMP signaling pathways to specific cell functions.
At higher molar concentrations (3 µM) PDE4 inhibitors
caused minor but distinct suppression of mitogenesis, although at lower concentrations (1 µM), neither rolipram nor denbufylline
inhibited mitogenesis (Table I and "Results"). On the other hand,
PDE3 inhibitors are potent blockers of proliferation (Tables I and II)
but even at 10 µM had no effect upon ROM generation
(Table I). The cause of this phenomenon is not yet clarified, but the following interpretation should be considered. When mesangial cells
were incubated with the direct stimulator of adenylate cyclase, forskolin, the addition of PDE4 inhibitors, denbufylline and Ro 20·1714, resulted in a huge (200 times) increase in cAMP, whereas addition of PDE3 antagonists, lixazinone and cilostazol, merely doubled
cAMP content (Fig. 4). These findings, which are in accord with
previously observed effects of rolipram and cilostamide (7), strongly
suggest that the intracellular pool of cAMP which is metabolized by
PDE4 in mesangial cells is considerably larger than the pool
metabolized by PDE3. Consequently, some of the cAMP accumulated in the
presence of 3-10 µM selective PDE4 inhibitors might have
"spilled over" into the compartment of cAMP which is specifically
linked to PDE3, thereby causing a minor suppression of mitogenesis in
mesangial cells (Table I). It should be stressed that at lower (1 µM) concentrations at which PDE isozyme inhibitors have
the highest discriminatory power (31, 32), neither rolipram nor
denbufylline had any inhibitory effect upon mitogenesis while having a
maximal suppressant effect upon ROM (Table I and Fig. 3).
Although the evidence presented herein is mostly correlative and indirect, it constitutes a sound basis for the working hypothesis that in rat mesangial cells the cAMP-PKA signaling pathway that controls ROM generation is coupled to cAMP metabolism by PDE4, whereas the cAMP-PKA signaling pathway coupled to cAMP metabolized by PDE3 regulates mitogenesis. In general terms, our findings support the proposition that PDE isozymes can compartmentalize and direct cAMP-mediated cell responses toward specific functions within the same cell (33-35). Such a novel function of PDE isozymes is particularly relevant in view that activities of both PDE3 and PDE4 are highly regulated (36, 37). Activity of PDE3 isozymes can be inhibited by cGMP, activated via phosphorylation by PKA or by insulin-stimulated protein kinase (38). Likewise, some isoforms of PDE4 are up-regulated via cAMP (37); cAMP-PKA phosphorylation increases activity of the "long" isoforms such as PDE4D3 (37, 39), or cAMP can induce transcription of cognate mRNA and de novo synthesis of "short" isoforms, such as PDE4D1 (37).
Finally, considering the central role of mesangial cells in response of glomeruli to various pathogenic stimuli (4-6), it is of potential importance that inhibitors of PDE3 and PDE4, which are effective both in vitro (7) and in vivo (8, 9), can independently suppress two major pathobiologic processes within mesangial cells. Based on this paradigm, novel "signal transduction" pharmacotherapies (40) of glomerulonephritis targeted to specific PDE isozymes may be designed and developed.
We thank Michael Thompson, Henry Walker, and Deborah C. Melder for expert technical assistance; and Carol A. Davidson and Mary E. Bennett for excellent secretarial assistance.