Characterization of inhibitors of phosphodiesterase 1C on
a human cellular system
Torsten R. Dunkern and Armin Hatzelmann
Biochemistry 2 Inflammation, ALTANA Pharma AG, Member of the Nycomed Group, Konstanz, Germany
Keywords
glioblastoma cell line; 8-methoxymethyl-
3-isoabutyl-1-methylxanthine;
phosphodiesterase 1C; SCH51866;
vinpocetine
Correspondence
T. R. Dunkern, Department of Biochemistry
Inflammation RPR ⁄ B2, ALTANA Pharma
AG, Member of the Nycomed Group,
Byk-Gulden-Str. 2, 78467 Konstanz,
Germany
Fax: +49 7531 84 2712
Tel: +49 7531 84 3121
E-mail:
(Received 25 April 2007, revised 17 July
2007, accepted 19 July 2007)
doi:10.1111/j.1742-4658.2007.06001.x
Different inhibitors of the Ca
2+
⁄ calmodulin-stimulated phosphodiester-
ase 1 family have been described and used for the examination of phospho-
diesterase 1 in cellular, organ or animal models. However, the inhibitors
described differ in potency and selectivity for the different phosphodiester-
ase family enzymes, and in part exhibit additional pharmacodynamic
actions. In this study, we demonstrate that phosphodiesterase 1C is
expressed in the human glioblastoma cell line A172 with regard to mRNA,
protein and activity level, and that lower activities of phosphodiesterase 2,

esterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited
the ionomycin-induced decline of forskolin-induced cAMP at nanomolar
concentrations. Thus, our data indicate that SCH51866 and compounds 31
and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in
contrast to the weakly selective and low-potency phosphodiesterase inhibi-
tors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172
cells therefore represent a suitable system in which to study the cellular
effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-
1-methylxanthine and vinpocetine seem not to be suitable for the study of
phosphodiesterase 1-mediated functions.
Abbreviations
IL, interleukin; 8MM-IBMX, 8-methoxymethyl-3-isobutyl-1-methylxanthine; PDE, phosphodiesterase; si, small interfering.
4812 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS
The superfamily of cyclic nucleotide-hydrolyzing phos-
phodiesterase (PDE) enzymes comprises 11 family
members (PDE1–PDE11). Within each family, differ-
ent genes encode different PDE subtypes, and alterna-
tive splicing of the messenger RNAs generates multiple
isoforms. The entire PDE superfamily consists of
about 50 proteins [1]. The historical classification into
different PDE families is based on their substrate spec-
ificity for cAMP, cGMP or both, and mechanisms of
activation and inhibitor sensitivity. The PDE family
PDE1, which consists of the subtypes PDE1A, PDE1B
and PDE1C, each encoded by one gene, becomes acti-
vated upon Ca
2+
⁄ calmodulin binding. Thus, PDE1
enzymes enable signaling crosstalk between the two
important cellular second messengers Ca

entiation (PDE1B [15]), and gall bladder relaxation
[16].
Until now, knowledge on the function of PDE1 in
different cell types, organs or even animal models has
come from the experimental use of more or less weak
inhibitors with relatively low PDE1 selectivity, such as
vinpocetine and 8-methoxymethyl-3-isobutyl-1-methyl-
xanthine (8MM-IBMX) [with the exception of some
PDE1-targeting small interfering (si)RNA approaches]
[17]. Unfortunately, changes in cyclic nucleotide con-
centrations, which are indicative of PDE-inhibitory
action, were often not investigated in such studies.
This gives rise to the question of the validity of inter-
pretations based on results obtained with the use of
these compounds, especially as other modes of action
besides PDE inhibition have been documented for the
nootropic drug vinpocetine as well as for 8MM-IBMX,
such as modulation of ion currents [18–20] and adeno-
sine receptor inhibition [21], respectively.
Because of the lack of a cellular model that allows
separate monitoring of PDE1 activity, no comparison
of the cellular potency of PDE1 inhibitors described in
the literature has been performed up to now. This is
important, because the cellular efficiency of PDE
inhibitors does not only depend on their potency. It is
also affected by their physicochemical properties, such
as lipophilicity, which influences membrane permeabil-
ity, and the subcellular localization. Furthermore, cel-
lular potency is influenced by the concentrations of the
substrates in the subcellular local environment, due to

rate of cAMP and cGMP hydrolysis was a first indica-
tion that this might be PDE1C activity. This enzyme is
well known to hydrolyze both cyclic nucleotides with
similar efficiency, in contrast to PDE1A and PDE1B.
Our assumption was confirmed by determining the
corresponding K
m
values, which were very similar
for both cAMP (1.7 lm) and cGMP (1.3 lm) hydro-
lysis (published data for the other PDE1 sub-
types: PDE1A2, K
m
[cAMP ⁄ cGMP] ¼ 112.7 ⁄ 5.0 lm;
PDE1B1, K
m
[cAMP ⁄ cGMP] ¼ 24.3 ⁄ 2.7 lm) and were
also similar to the published data for PDE1C [4]. In
addition, lower activities of PDE5 and PDE4 and
minor activities for PDE2 and PDE3 were measured.
In accordance with the data for PDE activity, we
T. R. Dunkern and A. Hatzelmann Characterization of inhibitors of PDE1C
FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4813
confirmed the expression of PDE1C protein in A172
cells by western blotting (Fig. 3A), in which recombi-
nant PDE1C protein served as a molecular mass con-
trol. More precisely, we detected two protein bands
that probably reflected the PDE1C splice variants ⁄ iso-
forms PDE1C1 and presumably PDE1C3 (based on
the determined molecular masses of 72 and 75 kDa).
The polyclonal antibody used has been reported to

It was important to assess the concentrations of iono-
mycin that cells could tolerate over a 30 min period.
Cells were therefore preincubated for 30 min with a
range of ionomycin concentrations up to 10 lm, and
then with 10 lm ionomycin plus 5 mm Ca
2+
to elicit a
maximum Ca
2+
response (indicated by a dashed arrow
in Fig. 4A). This response no longer occurred when the
ionomycin concentration in the 30 min preincubation
N
N
N
N
N
N
O
O
O
H
H
Vinpocetin
8MM-IBMX
N
N
N
N
N

PDE5 [cGMP]
0
10000
20000
30000
40000
PDE activity
pmol x min
-1
x 10
8
cells
-1
Fig. 2. Expression of family-specific PDE activities in A172 cells.
Cells were analyzed for PDE1 (cAMP and cGMP), PDE2, PDE3,
PDE4 and PDE5 hydrolysis activity. The mean values and standard
deviations of 10 independent experiments are shown. The sub-
strates (cAMP ⁄ cGMP) used to determine each PDE activity are
shown in parentheses.
Characterization of inhibitors of PDE1C T. R. Dunkern and A. Hatzelmann
4814 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS
phase was higher than 6 lm, which suggests a cyto-
toxic effect of ionomycin at these higher concentra-
tions. The addition of 50 mm EGTA (as a control) at
the end of the experiment (indicated by a solid arrow
in Fig. 4A) decreased the Ca
2+
signal. On the basis of
these results, 6 lm ionomycin (in the presence of 5 mm
Ca

cells under these conditions.
Inhibitors of PDE1 block the ionomycin-induced
decrease in forskolin-induced cAMP
concentrations
Various inhibitors of PDE1 are described in the litera-
ture, including vinpocetine, 8MM-IBMX, SCH51866,
and the Schering-Plough compounds 31 and 30 [23].
PDE1C
Lipid
ERK2
Control
PDE1C
siRNA
Lipid
Negative control siRNA
PDE1C siRNA
0
5000
10000
PDE1C protein expression
(arbitrary densitometric units)
PDE1C
A
BC
2010
30
1 2.5
A172 cells
Sf21/PDE1C1
63.2 kDa

lipid-treated cells (B). The mean values and
standard deviations of three experiments
are shown. Otherwise, cells were harvested
72 h after transfection, and analyzed for
PDE1C protein expression by western blot-
ting and densitometric analysis (C). To con-
firm equal protein loading, the blot was
additionally incubated with an antibody to
ERK2.
T. R. Dunkern and A. Hatzelmann Characterization of inhibitors of PDE1C
FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4815
We investigated these compounds with regard to
potency against PDE1A3, PDE1B1 and PDE1C1 and
overall PDE selectivity (Table 1). The rank order of
potency of these compounds against PDE1C1 is
compound 31 > compound 30 > SCH51866 > 8MM-
IBMX > vinpocetine. Indeed, the frequently used
PDE1 inhibitors vinpocetine and 8MM-IBMX have low
potency (IC
50
values 100 lm and 10 lm, respectively)
and selectivity vs. other PDEs. The IC
50
values that we
have determined for inhibition of the PDE1 enzymes by
these compounds agree with those in the literature
[23–26].
Next, we analyzed the effect of these inhibitors on
the ionomycin-induced decrease in forskolin-stimulated
cAMP concentrations in A172 cells. Figure 6A shows

36000
Control
Ionomycin
5
10
15
Time (min)
A
B
0.005%
0.010%
0.015%
0.020%
Ionomycin
0
50
100
Triton X-100
Death cells (%)
C
RFU
Time (min)
10000
20000
30000
40000
50000
Control
2 µM
4 µM

signal and (b)
determine the maximal possible Ca
2+
signal
intensity. Thereafter, EGTA (solid arrow)
was added; this decreased the signal. As
shown in (B), the addition of ionomycin
(straight arrow) and 5 m
M Ca
2+
(dotted
arrow) induced a rapid increase in intracellu-
lar Ca
2+
concentrations (the mean values
and standard deviations of three experi-
ments are shown). (C) As measured by lac-
tate dehydrogenase assays, treatment of
A172 cells with 6 l
M ionomycin plus 5 mM
Ca
2+
for 15 min did not induce any cytotox-
icity, whereas Triton X-100, which served as
a positive control, induced cytotoxicity in a
concentration-dependent manner.
Characterization of inhibitors of PDE1C T. R. Dunkern and A. Hatzelmann
4816 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS
SCH51866) also enhanced the forskolin-induced
increase in intracellular cAMP concentrations, which

hypertension and chronic obstructive pulmonary dis-
ease, inhibitors of other PDE subtypes are mostly in
preclinical development phases. This is due to the lim-
ited information on the function of these enzymes, a
lack of target-specific cellular test systems for drug
screening purposes, and their drugability. The cellular
1 5 20
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Control
Ionomycin
Forskolin
Forskolin +
Ionomycin
Time (min)
cAMP (pmol/1x10
6
cells)
Fig. 5. Pretreatment of A172 cells with ionomycin dampens a fors-
kolin-induced increase in intracellular cAMP concentrations. A172
cells pretreated with ionomycin (6 l
M) or untreated were stimu-
lated for adenylyl cyclase activation with forskolin (10 l
M) or vehicle
(control). cAMP was measured 1 min, 5 min and 20 min after fors-

charide-induced IL-6 release from alveolar epithelial
cells [12], or gall bladder relaxation [16], and might
thus give rise to false interpretations.
In this article, we show that, in comparison to newer
compounds such as SCH51866 [24], compound 31 and
compound 30 [23], the ‘classic’ inhibitors vinpocetine
and 8MM-IBMX (Fig. 1) have low enzymatic potency
and low PDE subtype specificity. We asked: (a) what
the effective cellular concentration of these inhibitors
is; and (b) whether the difference in enzymatic potency
would also transfer to differing cellular potency. This
need not necessarily be true, as cellular potency also
depends on the physicochemical properties of inhibi-
tors, such as lipid ⁄ liquid distribution and membrane
permeability. Thus, we searched for a cellular testing
system that would allow us to measure a functional
outcome, which is explicitly due and directly linked to
the inhibition of PDE1.
We showed that the human glioblastoma cell line
A172 expresses high levels of Ca
2+
⁄ calmodulin-stimu-
lated PDE1C activity and protein. The identity of
PDE1C was confirmed by K
m
value determination,
demonstrating that the Ca
2+
⁄ calmodulin-stimulated
PDE1 activity can hydrolyze cAMP and cGMP with

SCH 51866 -8 log M
SCH 51866 -7 log M
SCH 51866 -6 log M
0
5
10
15
B
A
Forskolin
cAMP (fold of control)
*
*
*
*
Fig. 6. The PDE1 inhibitor SCH51866 concentration-dependently
reverses the ionomycin effect on the forskolin-induced increase in
intracellular cAMP concentrations. (A) A172 cells pretreated with
ionomycin (6 l
M) and SCH51866 () 8to) 5 log M final concentra-
tion) or untreated were stimulated for adenylyl cyclase activation
with forskolin (10 l
M) or vehicle (control). cAMP was measured
1 min after forskolin stimulation in the corresponding cellular
extracts by cAMP-ELISA. The mean values and standard deviations
of three independent experiments are shown. A statistical analysis
(Student’s paired t-test) was performed to compare the forskolin
plus ionomycin-treated group with those treated in addition with
SCH51866. (B) A172 cells were treated only with SCH51866 () 8
to ) 6 log

influx
within a few minutes (as measured by Ca
2+
imaging
scanning fluorometry), which in theory should lead to
an activation of PDE1C. We determined an optimal
ionomycin concentration for our experiments, which
induced no cytotoxicity and increased Ca
2+
concentra-
tions by about 40% of the maximal inducible Ca
2+
response in the time frame used for subsequent investi-
gations.
Cells pretreated with ionomycin in such a way
responded to forskolin with an attenuated or even
abolished increase in intracellular cAMP concentra-
tions. This demonstrates that the ionomycin-induced
increase in intracellular Ca
2+
concentration leads to
activation of the PDE1C enzyme, which then hydro-
lyzes cAMP generated by the forskolin-stimulated ade-
nylyl cyclase. Activation of PDE1C was seen 1 min
after ionomycin treatment of the cells. This cellular
system proved to be highly useful for the cellular
potency screening of PDE1 inhibitors. Pretreatment of
A172 cells with the three recently described PDE1
inhibitors SCH51866 [24], compound 31 and com-
pound 30 [23] reversed the attenuating effect of

***
***
Control
Forskolin
Forskolin + Ionomycin
31 -10 log M
31 -9 log M
31 -8 log M
31 -7 log M
31 -6 log M
31 -5 log M
30 -10 log M
30 -9 log M
30 -8 log M
30 -7 log M
30 -6 log M
30 -5 log M
0
1
2
3
4
5
6
7
8
Forskolin + Ionomycin
*
***
**

human PDE1C enzyme. Within this concentration
range, or even higher, up to 100 nm, these three com-
pounds do not inhibit any of the PDEs expressed in
A172 cells except for PDE1C. Thus we can be sure to
observe effects of these inhibitors on cAMP concentra-
tions controlled by PDE1C. At higher, nonselective
concentrations, SCH51866 (at 1–10 lm), compound 31
or compound 30 (at 100 nm to 10 lm) caused a greater
increase in cAMP concentrations (in the presence of
ionomycin and forskolin) than that induced by forsko-
lin. This increase must be attributed to the inhibition
of other PDEs in addition to PDE1C at these concen-
trations by these compounds.
As expected on the basis of their low inhibitory
potency against PDE1C, vinpocetine and 8MM-IBMX
exhibited lower cellular potency. Even up to 10 lm,no
effects of these compounds were observed. This is quite
surprising, because there are several publications dem-
onstrating effects of these drugs in this concentration
range. For example, Haddad et al. [12] demonstrated
on alveolar epithelial cells that 8MM-IBMX inhibits
lipopolysaccharide-mediated IL-6 biosynthesis with an
IC
50
of 7.08 lm. Vinpocetine has been shown to
strengthen the contraction of smooth muscle cells and
shorten action potentials at 1 lm [13], and inhibit plate-
let-derived growth factor-induced proliferation of pul-
monary artery smooth muscle cells at 4 lm [14]. Even at
30 nm, vinpocetine was shown to be effective in relaxing

Vinpocetin -5 log M
0
1
2
3
Forskolin + Ionomycin
cAMP (fold of control)
Control
Forskolin
8MM-IBMX -8 log M
8MM-IBMX -7 log M
8MM-IBMX -6 log M
8MM-IBMX -5 log M
Vinpocetin -8 log M
Vinpocetin -7 log M
Vinpocetin -6 log M
Vinpocetin -5 log M
0
2
4
6
8
10
B
A
Forskolin
cAMP (fold of control)
Fig. 8. The low selective PDE1 inhibitors vinpocetine and 8MM-
IBMX do not reverse the ionomycin effect on the forskolin-induced
increase in intracellular cAMP up to concentrations of 10 l

pizone [28] was a generous gift from Sanofi-Aventis
(formerly Rhone-Poulenc Rorer, Ko
¨
ln, Germany).
The polyclonal rabbit antibody to PDE1C is a product of
Fabgennix (Taufkirchen, Germany), and has been reported
to be not cross-reactive with PDE1A, PDE1B or other PDE
family members. The polyclonal rabbit antibody to ERK2
was purchased from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA, USA). Peroxidase-coupled secondary antibodies
used for western blotting are a product of Jackson Immuno-
Research Laboratories Inc. (West Grove, PA, USA).
Cell culture
The human glioblastoma cell line A172 was cultured in
DMEM plus 4500 mgÆL
)1
glucose (Gibco, Invitrogen Life
Technologies, Grand Island, NY, USA), 2 mml-glutamine,
1mm sodium pyruvate and 10% heat-inactivated fetal
bovine serum at 37 °C and 5% CO
2
.
Measurements of PDE isoenzyme activities and
preparation of cellular extracts
Washed cells were sonicated in homogenization buffer
(137 mm NaCl, 2.7 mm KCl, 8.1 mm Na
2
HPO
4
, 1.5 mm

previously [30], with modifications. Briefly, PDE4 was
calculated as the difference in PDE activities at 0.5 lm
cAMP in the presence and absence of 1 lm piclamilast
(RP73401), and PDE3 as the difference between RP73401-
inhibited cAMP hydrolysis in the presence and absence of
10 lm motapizone. The fraction of cGMP (0.5 lm) hydro-
lysis in the presence of 10 lm motapizone that was inhib-
ited by 100 nm sildenafil represented PDE5. The increase
in cAMP (0.5 lm) hydrolysis (in the presence of 1 lm
RP73401 and 10 lm motapizone) induced by 5 lm cGMP
represented PDE2. PDE1 was defined as the increment of
cAMP hydrolysis (in the presence of 1 lm RP73401 and
10 lm motapizone) or cGMP hydrolysis induced by 1 m m
Ca
2+
and 100 nm calmodulin.
The K
m
values of the PDE1 activity were determined by
measuring the specific activity of Ca
2+
⁄ calmodulin-stimu-
lated cAMP or cGMP hydrolysis under different substrate
concentrations and by transferring the data into a linear
Lineweaver–Burk diagram.
PDE assay
⁄
inhibitor testing
Inhibition of PDE activity by the inhibitors was measured
on human PDE enzymes in a modified scintillation proxim-

6
cells
were seeded into 10 cm dishes. The day after seeding, cells
were transfected with 10 nm pan-PDE1C siRNA smart pool
(Ambion Inc., Austin, TX, USA) [pool number M-007643–
00, sequences CCAAGGAGATTGAAGAATT (1), GAT
CATGCACTGAAATTTA (2), GATGAAACCTCTCAA
ACTG (3), and CATCATCGCTGGACAATGT (4)], using
T. R. Dunkern and A. Hatzelmann Characterization of inhibitors of PDE1C
FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4821
1 lgÆmL
)1
argfectin-50 lipid as the tranfection agent (Atu-
gen, Berlin-Buch, Germany). For western blotting experi-
ments, 72 h after transfection, cells were washed with
NaCl ⁄ P
i
, trypsinized, shock-frozen as a cell pellet in liquid
nitrogen, and stored at ) 80 °C. For mRNA analysis, 48 h
after transfection, the cells were lysed with b-mercaptoetha-
nol-supplemented RLT buffer provided with the RNeasy-
Quiagen RNA-preparation kit. Thereafter, RNA isolation,
quantification and cDNA synthesis from 1 lg of RNA was
performed according to the manufacturer’s standard proto-
cols. Successful cDNA synthesis was controlled by gel elec-
trophoresis of an aliquot of the cDNA samples. cDNA was
used for subsequent real-time Taq-Man PCR analysis for
PDE1C (Applied Biosystems, Foster City, CA, USA). The
sense and antisense primer sequences were 5¢-TGTGAGT
CCATTAATCGATGAAACC-3¢ and 5¢-ACCTGATCG

(Lumi Light Plus; Roche GmbH, Mannheim, Germany)
and a luminescent image analyzer (Fujifilm LAS1000 Pro,
Tokyo, Japan). To control for equal protein loading, blots
were additionally incubated with a primary antibody to
extracellular signal-related protein kinase 2 (ERK2)
(1 : 3000).
cAMP measurements
A172 cells (1.5 · 10
5
cells in 5 mm Ca
2+
-containing med-
ium) were preincubated for 5 min in tubes in the presence
or absence of the various PDE1 inhibitors. Then, 6 lm
ionomycin (or Ca
2+
-containing medium in controls) was
added, and the samples were incubated for a further
5 min at 37 °C. Then, the adenylyl cyclase activator
forskolin (final concentration 10 lm) was added (or
Ca
2+
-containing medium in controls). At this time, the
final assay volume of 300 lL was reached. One minute
later, the incubations were stopped by adding 15 lLof
3.3 m HCl. After a further 15 min of incubation at room
temperature, the samples were centrifuged for 5 min at
10 000 g to pellet the cell debris using an Eppendorf cen-
trifuge 5417R and rotor F45-30-11. The supernatant was
diluted 1 : 10, and analyzed for cAMP content using a

2+
signal was investi-
gated by adding 10 lm ionomycin and 5 mm Ca
2+
.To
reach a subsequent decrease in Ca
2+
therafter at 35 min,
50 mm EGTA was added. All measurements were per-
formed in triplicate.
Cytotoxicity assay (lactate dehydrogenase assay)
A172 cells, 1 · 10
4
, were seeded into 96-well plates and
cultured for 24 h. Ionomycin (6 lm) or different concen-
trations of Triton X-100 (positive controls) were then
added for 15 min. For the generation of a standard curve,
different cell numbers (1 · 10
3
up to 1.3 · 10
4
cells per
well in triplicate) were seeded and treated 24 h later for
Characterization of inhibitors of PDE1C T. R. Dunkern and A. Hatzelmann
4822 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS
1 h with a supracytotoxic concentration of 1% Triton
X-100, known to lyse all cells within this time. After the
various treatments, 50 lL of the cell culture medium
supernatant was transferred to a 96-well plate, followed
by 50 lL of a substrate solution (Promega, Madison, WI,

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Characterization of inhibitors of PDE1C T. R. Dunkern and A. Hatzelmann