0022-3565/99/2882-0428$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:428 –437, 1999
Vol. 288, No. 2
Printed in U.S.A.
Pharmacological Inhibition of Protein Kinases in Intact Cells:
Antagonism of Beta Adrenergic Receptor Ligand Binding by
H-89 Reveals Limitations of Usefulness1
RAYMOND B. PENN, JEAN-LUC PARENT, ALEXEY N. PRONIN, REYNOLD A. PANETTIERI, JR. and
JEFFREY L. BENOVIC
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania
(R.B.P., J.-L.P., A.N.P., J.L.B.); and Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (R.A.P.)
Accepted for publication August 25, 1998
This paper is available online at http://www.jpet.org
Signal transduction through G protein-coupled receptors
(GPRs) is a highly regulated process in which numerous
protein kinases have been shown to play significant roles. By
virtue of their potential to phosphorylate not only receptors
but also downstream signaling molecules, the cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and
family of G protein-coupled receptor kinases (GRKs) all represent putative regulators of GPR signaling. Although much
of the initial discovery and characterization of protein kinase
regulatory features were identified using classic biochemical
in vitro analyses, investigation has naturally evolved into
Received for publication May 5, 1998.
1
J.-L.P. is the recipient of a postdoctoral fellowship from the Medical
Research Council of Canada. J.L.B. is the recipient of an American Heart
Association Established Investigator Award. R.A.P. is the recipient of a Career
Investigator Award from the ALA. This work was supported in part by National Institutes of Health Grants HL58506, GM44944, and HL55301.
acute addition of H-89 significantly attenuated isoproterenolstimulated cAMP accumulation. In cells pretreated with H-89
and then washed extensively, the subsequent dose-dependent
response to isoproterenol suggested beta-2 AR antagonism by
retained H-89. Competition binding of [125I]iodopindolol established Ki values of ;180 nM and 350 nM for H-89 antagonism
of beta-2 AR and beta-1 AR, respectively. Additional receptor
binding studies suggest selectivity of H-89 for the beta-2 AR
and beta-1 AR, although a weak antagonism (Ki values of ;10
mM or greater) of other G protein-coupled receptors was observed. Results from additional pharmacological and biochemical analyses of various protein kinase inhibitors further established the need for careful characterization of pharmacological
inhibitors when used in intact cell models.
studies using physiologically relevant systems. A picture has
emerged in which the relative importance of a given protein
kinase in regulating GPR function is dependent not only on
the specific GPR pathway involved but also on cell type and
stage of development (Penn and Benovic, 1998).
Numerous approaches have been used in intact cell models
to manipulate kinase function. Among such strategies, pharmacological agents with demonstrated ability to selectively
inhibit kinase function in vitro represent attractive tools.
However, their application to intact cell models may be limited by the necessity to use significantly higher concentrations (frequently 100 –1000-fold higher) than those used in
cell-free assays. Such concentrations are often obligated by
limitations of cell permeability, the need to achieve a favorable stoichiometry among inhibitor and all potential targets
in a given cell type, and the fact that most kinase inhibitors
are competitive with ATP, which exists in millimolar levels
ABBREVIATIONS: AR, adrenergic receptor; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; FSK, forskolin; GPR, G
protein-coupled receptor; GRK, G protein-coupled receptor kinase; HASM, human airway smooth muscle; HEK, human embryonic kidney; IBMX,
isobutylmethylxanthine; IPIN, iodopindolol; ISO, isoproterenol; mAchR, muscarinic acetylcholine receptor; NMS, scopolamine methylchloride;
PBS, phosphate-buffered saline; PGE2, prostaglandin E2; PKA, cAMP-dependent protein kinase; RT, reverse transcription; PCR, polymerase chain
reaction; PAGE, polyacrylamide gel electrophoresis; TXA2aR, thromboxane A2a receptor.
428
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ABSTRACT
The use of pharmacological inhibitors of protein kinases represents a potentially powerful tool in dissecting the regulatory
features of intracellular signaling pathways. However, although
the in vitro potency, selectivity, and efficacy of numerous kinase inhibitors have been characterized, little is known regarding the usefulness of these compounds as inhibitors in intact
cells. In attempting to characterize the role of protein kinase A
(PKA) in regulating the beta-2 adrenergic receptor (AR) in human airway cells, we observed a seemingly profound capacity
of the isoquinoline H-89, a potent and widely used PKA inhibitor, to attenuate agonist-mediated desensitization of the beta-2 AR. Although additional experiments identified H-89 as an
effective inhibitor of intracellular PKA, extended analysis of the
compound determined the principal effect of H-89 was via its
action as a beta-2 AR antagonist. Pretreatment with or the
1999
H-89 Is a Beta AR Antagonist
Experimental Procedures
Materials. H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidez2HCl) was obtained from Alexis Corp. (San Diego, CA),
BIOMOL (Plymouth Meeting, PA), and Calbiochem (La Jolla, CA).
H-85 (N-[2-(N-formyl-p-chlorocinnamylamino)ethyl]-5-isoquinolinesulfonamide) was obtained from Seikagaka Corp. (Rockville, MD). H-7
[(6)-1-(5-isoquinolinesulfonyl)-2-methylpiperazinez2HCl], staurosporine (ST), bisindolylmaleimide IX [Ro 31-8220; 2-[1-[3-(amidinothio)propyl]-1H-indol-3-yl]-3-(1-methylindol-3-yl)-maleimidezCH4O3S] (Bis
IX), and KT5720 were obtained from Alexis Corp. [125I]Iodopindolol
(IPIN) (2200 Ci/mmol), [125I]adenosine-39,59-cyclic phosphoric acid
(2200 Ci/mmol), [3H]dihydroalprenolol hydrochloride (82 Ci/mmol),
[32P]orthophosphoric acid (10,000 Ci/mmol), [g-32P]ATP, tetra(triethylammonium) salt (6000 Ci/mmol), [3H]scopolamine methylchloride (NMS) (84 Ci/mmol), [5,6-3H]SQ 29548 (51 Ci/mmol), and myo[1,2- 3H(N)]inositol (47 Ci/mmol) were obtained from DuPont-NEN
(Boston, MA). [0-methyl-3H]Yohimbine (95 Ci/mmol) was obtained
from Amersham (Arlington Heights, IL). FuGENE was obtained
from Boehringer-Mannheim (Indianapolis, IN). The rat mast cell line
RBL-2H3 stably expressing the human m1 muscarinic acetylcholine
receptor (m1 mAchR; 40 fmol/mg whole cell protein) was provided by
F. Santini. BEAS-2B human airway epithelial cells were provided by
C. Harris. Phosphospecific antibody against p42/p44 MAP kinase
was obtained from New England Biolabs (Beverly, MA). U46619 was
obtained from Cayman Chemical Co. (Ann Arbor, MI). All other
chemical reagents were obtained from Sigma Chemical (St. Louis,
MO).
Cell Culture. HASM cultures were established as described previously (Panettieri et al., 1989) from human tracheae obtained from
lung transplant donors, in accordance with procedures approved by
the University of Pennsylvania Committee on Studies Involving
Human Beings. BEAS-2B (Penn et al., 1994), COS-1 (Goodman et al.,
1996), and human embryonic kidney (HEK ) 293 (Krupnick et al.,
1997) cultures were maintained as previously described. RBL-2H3
cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 14% fetal bovine serum. Serum starvation of HASM cells consisted of 24-h incubation of cultures in DMEM
supplemented with 5 mg/ml transferrin and 5 mg/ml insulin.
Spodoptera frugiperda (Sf9) membranes expressing either the human beta-2 AR or beta-1 AR were prepared as described previously
(Penn et al., 1996).
Transfection Procedures. pcDNA3a2ARC10, encoding the human alpha-2C10 AR, was provided by A. Gagnon. pcDNA3 TXA2a
receptor (TXA2aR) was generated by reverse transcription-polymerase chain reaction (RT-PCR) of U937 (a human monocyte line) total
mRNA using primers corresponding to the 59 and 39 flanking sequences of the published human TXA2aR (Raychowdhury et al.,
1994) and cloning of the ;1300-bp product into the EcoRI/XhoI sites
in pcDNA3. COS-1 and HEK 293 cells were transfected using FuGENE and 0.1 to 0.5 mg/ml pcDNA3a2ARC10 (COS-1) or
pcDNA3TXA2aR (HEK 293) as per the manufacturer’s instructions.
In studies designated for phosphoinositide analysis, HEK 293 cells
were transfected in 6-well plates with FuGENE and 0.5 mg/ml
pcDNA3TXA2aR. For receptor binding assays, cells were harvested 2
days after transfection, and membranes were prepared as described
previously (Penn et al., 1994).
Effects of Kinase Inhibitors on Receptor-Mediated cAMP
Accumulation. Experiments examining the effects of various protein kinase inhibitors on beta-2 AR, prostaglandin E2 (PGE2) receptor, and adenylyl cyclase responsiveness were performed on HASM
and BEAS-2B cells in a manner similar to that described previously
(Penn et al., 1998). Briefly, serum-starved, confluent cells grown in
24-well dishes were pretreated with or without various concentrations of either vehicle [0.1% dimethyl sulfoxide (DMSO)], H-89, H-85,
H-7, staurosporine, KT5720, or Bis IX for up to 1 h, followed by a
30-min pretreatment with or without 1 mM isoproterenol (ISO). Cells
were then washed four times in cold phosphate-buffered saline
(PBS), and individual wells were stimulated with 500 ml of PBS
containing 300 mM ascorbic acid, 1 mM isobutylmethylxanthine
(IBMX), and either vehicle (basal), (2)-ISO, PGE2, or forskolin (FSK)
for 10 min (except time course studies). cAMP was isolated and
quantified by radioimmunoassay as described previously (Penn et
al., 1998).
Assessment of In Vivo PKA-Mediated Phosphorylation.
HASM cells grown to confluence in 6-well plates were serum starved
for 24 h and then loaded for 1 h with 0.3 mCi of [32P]orthophosphate
(in 1.2 ml of phosphate-free DMEM) per well. Cells were subsequently treated with various kinase inhibitors for 1 h and then
stimulated with either vehicle (0.01% ethanol) or 10 mM FSK for 30
min. Cells were then washed three times with cold Tris-buffered
saline and harvested in buffer containing 20 mM TriszHCl, pH 8.0, 5
mM EDTA, 5 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 10
mg/ml pepstatin, 20 mg/ml leupeptin, 20 mg/ml benzamidine, 10 mM
sodium pyrophosphate, 10 mM sodium fluoride, and 1% Triton
X-100. Then, 20 mg of protein of the Triton-soluble extract was
subjected to electrophoresis on a 10% SDS-polyacrylamide gel, and
the gel was stained with Coomassie blue, dried, and visualized by
exposure to Fuji RX film.
Analysis of MAPK Activity. HASM cells were grown in 6-well
plates to 80 to 90% confluence and then serum-starved for 48 h. Cells
were pretreated with the various protein kinase inhibitors, followed
by 30-min stimulation with either 10 ng/ml EGF or 100 nM PMA.
Cells were then washed with cold PBS and lysed by the direct
addition of 125 ml of SDS sample buffer. Harvested lysates were
subjected to Western analysis using a rabbit polyclonal IgG antibody
(NEB) that specifically recognizes the phosphorylated forms
(Thr202/Thr204) of p42 and p44 MAP kinase, goat anti-rabbit horseradish peroxidase-conjugated secondary antibody, and visualization
by enhanced chemiluminescence (ECL; Amersham). Equal loading of
samples was assessed by staining of blot with Ponceau S or by
subsequent probing of stripped blots with a polyclonal antibody
(NEB) that recognizes total p42/p44 MAPK (phosphorylation-state
independent).
Effects of Kinase Inhibitors on GRK Activity. Bovine GRK2
and human GRK5 were overexpressed and purified from Sf9 cells
(Kim et al., 1993; Kunapuli et al., 1994). GRK-mediated phosphorylation was assayed by incubating 0.8 pmol of GRK with urea-treated
rod outer segments membranes (120 pmol of rhodopsin) in 20 ml of 60
mM TriszHCl, pH 8.0, 4 mM MgCl2, 0.5 mM EDTA, 0.1 mM
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in the intact cell (MacKintosh and MacKintosh, 1994). Accordingly, the use of high levels of inhibitors in intact cells
may compromise the selectivity of these agents and increase
the likelihood of incurring nonspecific effects.
During the course of our investigation into the role of PKA
in regulating beta-2 adrenergic receptor (AR) function in
human airway smooth muscle (HASM) cells, we observed a
seemingly profound effect of the isoquinoline H-89, a potent
and widely used inhibitor of PKA (Engh et al., 1996) to
attenuate agonist-mediated desensitization of the beta-2 AR.
More extensive analysis of H-89 led us to discover that the
principal effect of H-89 on beta-2 AR responsiveness was not
through its capacity to inhibit intracellular PKA but rather
through its ability to antagonize beta AR binding of agonist
with high affinity. Receptor antagonism by H-89 was found
selective for beta-2 AR and beta-1 AR, although antagonism
of other GPRs was observed at significantly higher concentrations. Results from additional studies examining the effects of various other kinase inhibitors on GPR function and
binding properties underscore the need to carefully characterize protein kinase inhibitors when used in the analysis of
GPR signal regulation in intact cell models.
429
430
Penn et al.
Vol. 288
Results
Our initial inquiry into kinase inhibition in intact cells
arose from studies examining the role of PKA in the desensitization of the beta-2 AR in HASM. Several different kinase
inhibitors were tested for their ability to attenuate the agonist-specific desensitization of the beta-2 AR invoked by pre-
treating cells for 30 min with 1 mM ISO. Typically, the loss of
beta-2 AR responsiveness after beta-agonist exposure, characterized by a loss of ISO-stimulated cAMP with subsequent
rechallenge, is mediated by beta-2 AR phosphorylation by
GRKs and, in some cell types, by PKA as well (Penn and
Benovic, 1998). Figure 1 depicts the results of experiments in
which cells were pretreated for 1 h with 10 mM H-89, 500 mM
H-7, 10 mM KT5720, or 1 mM staurosporine before 30-min
pretreatment with 1 mM ISO. H-85 (10 mM), an analog of
H-89 that does not inhibit PKA, and Bis IX (1 mM), a PKCspecific inhibitor, were used as negative controls and to assess possible contributions of PKC. Among the suspected
PKA inhibitors, concentrations used were ;200 to 500 times
the Ki values reported to inhibit PKA activity in in vitro
assays. Interestingly, H-89 and, to a lesser extent, staurosporine were able to attenuate ISO-induced beta-2 AR desensitization in HASM. H-85 also had a small, significant effect,
whereas KT5720 and H-7 exhibited no effect. Bis IX also had
no effect, suggesting that PKC does not contribute to agonistspecific beta-2 AR desensitization in HASM and that the
effect of staurosporine is likely mediated via inhibition of
PKA, which we have reported previously (Penn et al., 1998).
The disparate effects of the various kinase inhibitors
prompted us to more directly examine the effectiveness and
specificity of these agents. The effects of inhibitors on PKAmediated whole-cell phosphorylation were examined in
HASM cells loaded with [32P]orthophosphate and then challenged with 10 mM FSK for 30 min. Analysis of cell extracts
by SDS-polyacrylamide gel electrophoresis (PAGE) (Fig. 2A)
demonstrates that stimulation with FSK induces the appearance of bands at ;40 and 20 kDa, whereas pretreatment with
Fig. 1. Effects of protein kinase inhibitors on beta-2 AR responsiveness in
HASM. Cultured HASM cells were pretreated for 1 h with either 0.1%
DMSO vehicle (Veh), 10 mM H-89, 1 mM staurosporine (ST), 10 mM
KT5720, 500 mM H-7, 1 mM Bis IX, or 10 mM H-85, followed by 30-min
pretreatment with either vehicle (CON) or 1 mM (2)-ISO. Cells were then
washed four times with cold PBS and rechallenged with 1 mM ISO and 1
mM IBMX for 10 min. cAMP was isolated and quantified by radioimmunoassay as described in Experimental Procedures. Data represent
mean 6 S.D. from three to eight experiments. *p ,.05 by paired t test,
Veh versus H-89, staurosporine, or H-85.
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[g- P]ATP, and varying concentrations of protein kinase inhibitors
for 8 min at 30°C in room light. Reactions were stopped with SDS
buffer and electrophoresed on a 10% SDS-polyacrylamide gel. Gels
were stained with Coomassie Blue, dried, and visualized. 32P-labeled
bands were excised and counted.
Receptor Binding Studies. Competition of radioligand binding
by various protein kinase inhibitors was performed using membranes prepared from cells expressing either the human beta-2 AR
(Sf9 and BEAS-2B), human beta-1 AR (Sf9), human m1 mAchR
(RBL-2H3), human m2 mAchR (HASM), human alpha2ARC10
(COS-1), or human TXA2aR (HEK 293). Typically, membranes were
incubated with a 1- to 3-fold concentration of the established or
empirically determined Kd for [125I]IPIN (beta-1 AR and beta-2 AR),
[3H]dihydroalprenolol (beta-1 AR and beta-2 AR), [3H]NMS (m1 and
m2 mAchR), [0-methyl-3H]yohimbine (alpha2ARC10), or [5,6-3H]SQ
29548 (TXA2aR ), in the presence of various concentrations of protein
kinase inhibitors. For saturation binding analysis, BEAS-2B cells
were pretreated with or without 10 mM H-89 for 30 min at 22°C in
PBS containing 5 mM EDTA, 1 mM ethylene glycol bis(b-aminoethyl
ether)-N,N,N9,N9-tetraacetic acid, 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin, 20 mg/ml leupeptin, and 20 mg/ml benzamidine and then washed four times in cold PBS. Cells were resuspended and incubated in PBS containing ;8 to 200 pM [125I]IPIN for
1 h at 22°C. All competition binding reactions were performed in 25
mM TriszHCl, pH 7.5, and 2 mM MgCl2, except [5,6-3H]SQ 29548
binding, which was performed as described previously (Raychowdhury
et al., 1994) using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidbuffered DMEM. The addition of compounds did not alter buffer pH,
except H-7 at 500 mM, which was adjusted to pH 7.5 with NaOH.
Nonspecific binding was determined using 1 mM alprenolol (beta-1 AR
and beta-2 AR), 1 mM atropine (m1 and m2 mAchR), 10 mM phentolamine (alpha2ARC10), or 1 mM SQ 29548 (TXA2aR ). All binding reactions (except [5,6-3H]SQ 29548) were terminated by the addition of 53
4 ml of ice-cold 25 mM TriszHCl, pH 7.5, and 2 mM MgCl2 and filtration
through Whatman GF/C filters using a Brandel Cell Harvester. IC50
values were estimated by interpolation, and Ki values were calculated
using the method of Cheng and Prusoff (1973).
Accumulation of Total [3H]Inositol Phosphates. [3H]Inositol
phosphate formation was determined as reported previously with
minor modifications (Widdop et al., 1993). Near-confluent cell monolayers in 12-well plates were incubated for 24 h at 37°C with 500 ml
of inositol-free DMEM containing myo-[3H]inositol (47 Ci/mmol) at a
concentration of 4 mCi/ml. After loading, cells were washed once with
PBS. Inositol-free DMEM containing 10 mM LiCl, with or without
kinase inhibitors, was added to each well, and the cells were incubated for 10 min at 37°C. Cells were then stimulated for 10 min with
20 mM (final concentration) histamine (HASM and BEAS-2B cells),
50 mM carbachol (RBL-2H3), or 20 nM U46619 (HEK 293). Reactions
were stopped by aspiration of medium and the addition of 0.8 ml of
ice-cold 0.4 M perchloric acid. One-half volume of 0.72 N KOH/0.6 M
KHCO3 was added, and the sample was centrifuged to settle the
precipitate. The supernatant was applied to 1 ml AG1-X8 (BioRad)
columns (100 –200 mesh, formate form), columns were washed with
10 ml of 0.1 N formic acid, and total inositol phosphates were eluted
with 1.5 M ammonium formate/0.1 N formic acid and counted.
Data Analysis. Except where noted, values are reported as
mean 6 S.D. cAMP accumulation was calculated by subtracting the
matched basal value from that determined with agent stimulation,
as described previously (Penn et al., 1994).
32
1999
H-89 Is a Beta AR Antagonist
431
H-89 or staurosporine, but not KT5720, H-7, Bis IX, or H-85
(data not shown), eliminates this induction. Thus, only H-89
and staurosporine appear to be effective PKA inhibitors in
HASM cells, a result consistent with the results of Fig. 1 and
our observation that H-89 and staurosporine are able to
attenuate the cAMP-mediated desensitization of the beta-2
AR in HASM elicited by pretreatment with either FSK or
PGE2 (a ;25% loss of beta-2 AR responsiveness) (Penn et al.,
1998, and data not shown). To examine compound selectivity,
the capacity of these agents to inhibit p44/p42 MAPK phosphorylation by EGF (through tyrosine kinase activation) or
the phorbol ester PMA (through PKC) was also tested (Fig.
2B). None of the agents inhibited EGF-stimulated p44/p42
phosphorylation. Staurosporine, H-7, and Bis IX effectively
inhibited PMA-stimulated MAPK activation, whereas H-89
and KT5720 had no effect. Interestingly, despite a similar
potency for inhibiting PKA and PKC in vitro (3– 6 mM), H-7
appears to preferentially inhibit PKC (Fig. 2B) and not PKA
(Figs. 1 and 2A) in HASM cells.
None of the agents demonstrated significant potency (i.e.,
were effective at concentrations that might be used to inhibit
kinase activity in intact cells) in inhibiting either GRK2 or
GRK5 when GRK activity was assessed by phosphorylation
of rhodopsin in vitro (Table 1). A slightly greater tendency of
compounds to inhibit GRK5- versus GRK2-mediated phosphorylation was observed, with staurosporine being the most
potent. Although methodology for direct assessment of GRK
activity in intact cells has yet to be developed, the low potencies demonstrated in an in vitro assay conducted using a low
concentration of ATP (0.1 mM) suggest that the examined
compounds are unlikely inhibitors of GRKs in intact cells.
Collectively, these studies suggest that of the compounds
TABLE 1
Effects of kinase inhibitors on GRK2 and GRK5 activity
Inhibitor
GRK2
GRK5
H-89
H-85
H-7
.100a
40 6 5
..100
.100
.1000
.1000
KT5720
..10
10 6 1
ST
.10
461
BisIX
40 6 5
12 6 2
a
1027 to 1023 M final concentrations of inhibitors were tested for their ability to inhibit GRK2- or GRK5-mediated phosphorylation of rhodopsin as described in
Experimental Procedures. Values reported are mean 6 S.D. (micromolar concentrations) in which 50% inhibition was determined.
., 20 to 40% inhibition observed at this (maximal) concentration.
.., ,20% inhibition observed.
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Fig. 2. Analysis of kinase inhibitor
effectiveness, specificity. A, inhibition
of PKA-mediated phosphorylation in
HASM cells. HASM cells cultured in
6-well plates were loaded for 2 h with
0.3 mCi of [32P]orthophosphate, pretreated for 1 h with either 0.1%
DMSO vehicle (CON), 10 mM KT5720
(KT), 10 mM H-89, 500 mM H-7, 1 mM
staurosporine (ST) or 1 mM Bis IX and
then stimulated for 30 min with either
vehicle (0.01% ethanol) or 10 mM
FSK. Cells lysates were subject to
SDS-PAGE, and the dried gel was
subsequently exposed to Fuji RX Film
overnight. Arrows indicate positions
of ;40- and 20-kDa bands induced by
FSK stimulation and inhibited by
prior treatment with H-89 or staurosporine. Data shown are representative of three independent experiments. B, inhibition of PKC-mediated
p44/p42 MAPK phosphorylation in
HASM cells. HASM were cultured in
6-well plates and serum-starved overnight. Cells were pretreated with
the indicated inhibitors or vehicle
(VEH 5 0.1% DMSO) as described
above for 30 min and then stimulated
with either 10 ng/ml EGF or 100 nM
PMA for 30 min. Immunoblots of cellular lysates were probed with an antibody that specifically recognizes the
phosphorylated forms of p44/p42
MAPK as described in Experimental
Procedures. Data are representative
of duplicate experiments.
432
Penn et al.
Fig. 3. Profile of ISO-stimulated cAMP accumulation in the presence of
kinase inhibitors. HASM cultures were pretreated for 1 h with either
vehicle (CON), 10 mM H-89, or 1 mM staurosporine (ST), after which 1 mM
ISO (final concentration) was added to culture media. Reactions were
stopped at the indicated time points 0 to 30 min after ISO addition. Data
represent mean 6 S.D. of three experiments.
inhibition was observed in the presence of 1 mM IBMX (data
not shown). Next, we examined the dose-dependent response
to ISO after H-89 and ISO pretreatment and extensive washing of cells. Oddly, H-89 pretreatment resulted in a reduction
in cAMP accumulation at low ISO concentrations, whether
cells were pretreated with or without ISO (Fig. 4A), and
significantly increased the EC50 for ISO. Similar results were
obtained using BEAS-2B cells (Fig. 4B).
These results were perplexing in light of early studies by
Clark and coworkers (1987, 1988), demonstrating that PKA
preferentially inhibits beta-2 AR responsiveness at low ISO
concentrations; thus inhibition of PKA should enhance cAMP
accumulation at low ISO concentrations and effectively decrease the EC50 for ISO. Indeed, this is the case in HASM
cells treated with staurosporine (Penn et al., 1998). The
observed effect of H-89 in HASM and BEAS-2B cells (Fig. 4,
A and B) was more consistent with classic pharmacological
receptor antagonism against a backdrop of intracellular PKA
inhibition (the latter suggested by the enhancement of maximal response in ISO-pretreated cells, consistent with Fig. 1
results). When HASM cells were treated with H-89 for 1 h
and then directly stimulated with ISO (no washing before
ISO addition), a dose-dependent effect of H-89 antagonism of
beta-2 AR activation is observed that appears competitive
with ISO (Fig. 4C). Moreover, antagonism of ISO-stimulated
cAMP accumulation is observed, albeit to a lesser extent,
when H-89 is added to culture media immediately before
stimulation with ISO (Fig. 4D). Basal levels of cAMP were
not significantly altered by the acute addition of H-89 in
either HASM or BEAS-2B cells (data not shown). These
results suggest that H-89 antagonizes ISO binding to beta-2
AR, and the effects of H-89 in attenuating beta-2 AR desensitization can be explained in part as a result of the compound being retained despite extensive washing.
Accordingly, receptor binding analyses demonstrate that
H-89 acts as a beta-2 AR antagonist of surprisingly high
affinity (180 nM) (Fig. 5A and Table 2). Sf9 membranes
overexpressing the human beta-2 AR (;20 pmol/mg protein)
were incubated with 40 pM of [125I]IPIN (;2 Kd) (Penn et al.,
1996) and increasing concentrations of various kinase inhibitors. Competition binding using membranes prepared from
BEAS-2B cells (;100 fmol beta AR/mg protein, .90% beta-2
AR) yielded nearly identical results (Table 2). Similar results
were obtained in binding studies using intact BEAS-2B cells
(data not shown). The addition of H-89 did not alter buffer
pH, and results were similar whether binding was performed
using 25 mM Tris, pH 7.5, 2 mM MgCl2, PBS, pH 7.5, or
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered
DMEM, pH 7.4 (data not shown). Interestingly, H-85, which
is identical in structure to H-89 except for an N-formylamine
substitution and a parachlorocinnamyl (replacing the parabromocinnamyl) group, inhibited beta-2 AR binding with approximately one-10th the potency (Ki 5 1.5 mM). Much
higher concentrations of H-7 (Ki . 500 mM) were required to
significantly inhibit [125I]IPIN binding to beta-2 AR, suggesting that the hydrophobic parahalocinnamyl moiety present
in H-89 and H-85 is a key determinant of affinity.
Analysis of the effect of 10 mM H-89 on ISO-stimulated
cAMP generation from Fig. 4C using the Schild equation
predicts a significantly higher (;7 fold) Ki value than that
determined by radioligand binding. However, the value of the
slope of the Schild regression (0.57, r 5 0.99) suggests that
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examined, only H-89 and staurosporine are effective inhibitors of PKA in HASM cells, that H-89 appears to be specific
for PKA inhibition, and that the effects of H-89 and staurosporine on beta-2 AR responsiveness in HASM are likely via
inhibition of PKA.
However, several observations caused us to question the
relatively large effect of H-89 in reversing ISO-mediated
beta-2 AR desensitization in HASM. One concern was that
although 10 mM H-89 appeared equally effective as 1 mM
staurosporine in inhibiting PKA, H-89 was much more effective than staurosporine in reversing agonist-specific desensitization (Fig. 1). Second, this reversal (from ;40 to 80% of
control values) far exceeded the magnitude of cAMP-mediated desensitization invoked by FSK or PGE2 treatment [a
;25% loss (Penn et al., 1998)], even though FSK/PGE2 stimulate higher levels of cAMP accumulation than does ISO.
Third, the small effect of H-85 in attenuating beta-2 AR
desensitization suggests the contribution of a nonspecific
component mediating the effect of H-89. Last, H-89 had a
similar effect in reversing agonist-specific beta-2 AR desensitization in BEAS-2B human airway epithelial cells (see
below), a cell line that exhibits little, if any, cAMP-mediated
desensitization (Penn et al., 1994).
A more thorough analysis of the effects of H-89 on beta-2
AR responsiveness was therefore undertaken in both HASM
and BEAS-2B cells. First, we examined the profile of cAMP
production in HASM that occurs during the pretreatment
phase with 1 mM ISO in the continued presence of H-89 or
staurosporine after incubation with these agents for 1 h.
Figure 3 reveals that although 1 mM staurosporine enhances
1 mM ISO-stimulated cAMP accumulation, 10 mM H-89
causes a slight inhibition, a result seemingly paradoxical
with data from Fig. 1. This effect could not be attributed to
alterations in phosphodiesterase activity because a similar
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1999
H-89 Is a Beta AR Antagonist
433
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Fig. 4. H-89 alters dose-dependent response to ISO in HASM and BEAS-2B cells. A, HASM cells were pretreated with 10 mM H-89 for 1 h followed
by 30 min 6 1 mM ISO, washed four times with cold PBS, and then challenged with 1029 to 1024 M ISO. cAMP was isolated and quantified as described
in Experimental Procedures. B, results from the same experiment performed using cultured BEAS-2B cells. Data represent mean 6 S.D. from two
(BEAS-2B) to four (HASM) experiments. C, H-89 antagonizes ISO-stimulated cAMP accumulation in a dose-dependent manner. HASM cells were
treated with 1027, 1026, or 1025 M H-89 for 1 h and then directly challenged with 1029 to 1024 M ISO (final concentration) for 10 min in the presence
of 1 mM IBMX. Data represent mean 6 S.D. of two experiments. D, acute addition of H-89 antagonizes ISO-stimulated cAMP accumulation. H-89 (10
mM final) was added to culture media either 60 min before (H-89 1 h before) or immediately before (H-89 0 min before) challenge of BEAS-2B cells
with 1029 to 1024 M ISO and 1 mM IBMX. Data represent mean 6 S.D. of duplicate experiments.
the effect of H-89 on the beta-2 AR-adenylyl cyclase signaling
cascade is not via strict competitive antagonism of the beta-2
AR and likely involves an effect occurring distinct from the
beta-2 AR binding site. To investigate the extent to which
H-89 binding to beta-2 AR is reversible and competitive,
saturation binding isotherms were performed using
BEAS-2B cells (Fig. 5B). These experiments demonstrate
that H-89 binding is largely surmountable, although binding
at submaximal concentrations of [125I]IPIN is still significantly inhibited in the pretreated and washed cells (a finding consistent with data in Fig. 4, A and B), suggesting
retention of H-89 despite extensive washing. The collective
findings from Figs. 2 through 5 suggest a multifactorial effect
of H-89 on beta-2 AR signaling that involves competitive
antagonism of agonist binding (inhibiting beta-2 AR activation) combined with inhibition of intracellular PKA (serving
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Discussion
For several reasons, the use of pharmacological inhibitors
of PKA (and H-89 in particular) represented an attractive
means by which to address an a priori hypothesis questioning
the role of PKA in beta-2 AR desensitization in human airway smooth muscle. Most importantly, pharmacological
agents could affect the entire population of cells, whereas the
effectiveness of transfection-based strategies would be limited by the level of transfection efficiency (;50% using a
Fig. 5. Beta AR ligand binding is antagonized by H-89. A, Sf9 membranes
expressing human beta-2 AR were incubated with 40 pM of [125I]IPIN in
the presence of varying concentrations of H-89, H-85, or H-7 for 1 h at
22°C followed by filtration and washing on GF/C filters as described
previously (Penn et al., 1994). Data from a single experiment representative of five experiments performed under similar conditions. Calculated
mean Ki values are presented in Table 2. B, reversibility of H-89 antagonism of [125I]IPIN binding to BEAS-2B cells. BEAS-2B cells were pretreated in PBS with (Pretreated) or without 1025 M H-89 followed by
extensive washing as described in Experimental Procedures. Saturation
binding isotherms were subsequently performed by incubating cells in
PBS with 8 to 200 pM of [125I]IPIN in the absence (CON and Pretreated)
or presence (1 1025 M H-89) of 1025 M H-89 for 1 h at 22°C. Calculated
Bmax and Kd values: CON, Bmax 5 32 fmol/mg whole cell protein, Kd 5 24
pM; Pretreated, Bmax 5 30 fmol/mg, Kd 5 56 pM. C, experiment described
in A performed using human beta-1 AR expressed in Sf9 membranes.
Data are from a single experiment representative of five experiments,
with mean values presented in Table 2.
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to enhance beta-2 AR responsiveness). Additional mechanisms underlying H-89 effects might also include PKA-independent alterations on the receptor environment, perhaps
influenced by the lipophilic nature of the compound.
Additional studies examining the selectivity of kinase inhibitors were performed. H-89 (Ki 5 350 nM) and H-85 (Ki 5
1.7 mM) also exhibited the ability to antagonize beta-1 AR
binding (Fig. 5C), albeit with a slightly lower affinity than
that observed for the beta-2 AR. Similar inhibition of [3H]dihydroalprenolol binding by H-89 to both beta-2 AR and beta-1
AR was observed (Ki values of ;240 nM and 500 nM, respectively). H-89 and H-85 appeared to be fairly selective for beta
ARs, exhibiting inhibition of other receptors at much higher
concentrations (Table 2). None of the other kinase inhibitors
tested significantly inhibited beta AR binding at concentrations that might be reasonably used to inhibit kinase activity
in intact cells (i.e., up to 1000-fold the Ki established in in
vitro assays), although it should be noted that some inhibition by H-7 and Bis IX of beta ARs and other receptors
occurred at high concentrations.
To further investigate the specificity of H-89 for antagonizing beta AR, we examined the effects of H-89 on the functional responses of different GPRs linked to either Gs and
adenylyl cyclase (beta-2 AR and PGE2 receptor) or Gq and
phospholipase C (m1 mAchR, H1 histamine receptor, and
TXA2aR). Various cells were treated with 10 mM H-89 before
stimulation with submaximal concentrations of agonists approximating 1 to 3 times the EC50 of activation. Figure 6A
demonstrates the lack of effect of H-89 on PGE2-stimulated
adenylyl cyclase in BEAS-2B cells. In a similar manner, total
inositol phosphate generation by either H1 histamine receptor activation in HASM and BEAS-2B cells, m1 mAchR activation in RBL-2H3 cells, or TXA2a activation in HEK 293
cells was not significantly influenced by H-89. Thus, analyses
of various Gs- and Gq-coupled receptor function suggest that
receptor antagonism by H-89 appears selective for beta ARs.
1999
435
H-89 Is a Beta AR Antagonist
TABLE 2
Ki values for kinase inhibitors determined from displacement curves of radioligand binding from various GPRs
Inhibitor
Receptor
beta-2AR
beta-2AR
beta-2AR (BEAS-2B)
m1 mAchR
m2 mAchR
alpha-2 ARC10
TXA2aR
a
b
c
H-89
H-85
H-7
KT5720
ST
BisIX
0.18a
0.35
0.17
.10b
.10
6
.10
1.5
1.7
1.8
..10c
.10
..10
..10
.500
.500
.500
200
200
..500
..500
..10
..10
..10
..10
..10
..10
..10
..10
..10
..10
..10
..10
..10
..10
2.2
2.3
2.3
2.0
1.0
5
.10
Values reported are micromolar concentrations calculated as the mean of two to five experiments.
.20 to 40% inhibition observed at this concentration.
..,,20% inhibition observed at this concentration.
tization we had observed in these cells (Penn et al., 1998).
The surprisingly large effect of H-89, relative to that observed for staurosporine, raised questions regarding potential nonspecific effects of H-89, which were also suggested by
the small effect of H-85 in attenuating beta-2 AR desensitization (Fig. 1).
The subsequent finding that pretreatment with H-89
caused a significant inhibition of cAMP accumulation in
HASM stimulated with submaximal concentrations of ISO
strongly implied that H-89 antagonized beta-2 AR responsiveness to agonist at some level upstream of adenylyl cyclase. Indeed, a very recent study by Clark and coworkers
(January et al., 1997) noted that HA6 cells pretreated with 5
mM H-89 alone exhibited a 4- to 5-fold increase in the EC50
for epinephrine activation of adenylyl cyclase, whereas the
addition of 5 mM H-89 directly to assays of adenylyl cyclase
caused a 20- to 50-fold increase in the EC50 of epinephrine
stimulation; these data led the authors to conclude that H-89
was unsuitable for use in desensitization experiments because of its uncoupling of beta-2 AR activation.
Our studies extend this observation to define H-89 as a
potent and selective inhibitor of beta-2 AR and beta-1 AR
ligand binding. A reproducible inhibition of both [125I]IPIN
and [3H]dihydroalprenolol binding to beta ARs was observed,
with similar results obtained in binding to both overexpressed recombinant beta-2 AR (in Sf9 membranes) and endogenously expressed beta-2 AR (in BEAS-2B cells). The
inhibition of binding by H-89 was not caused by alterations in
binding buffer pH (10 mM H-89 had no effect on pH) and was
reproducible using different types of binding buffers. Results
were independent of supplier or lot number of H-89.
Based on findings from the present study and those of
others, the following characteristics can be ascribed to H-89:
for PKA inhibition, an in vitro Ki of 48 nM and an in vivo Ki
of ;10 mM (see discussion above); Ki values of 180 and 350
nM for inhibition of beta-2 AR and beta-1 AR binding, respectively; and a variable Ki value for beta-2 AR antagonism
predicted by Schild analysis of H-89-mediated inhibition of
cAMP generation (Fig. 4C) that varies directly with H-89
concentration (e.g., a predicted Ki of ;200 nM with 0.1 mM
H-89 but a Ki of ;1.3 mM with 10 mM H-89).
The receptor binding properties of H-89 could possibly be
inferred from our understanding of the structurally related
tetrahydroisoquinolines and their analogs, which have been
shown to be effective agonists/antagonists for both beta-adrenergic and TXA2 receptors (Christoff et al., 1997; Fraundorfer et al., 1994; Shams et al., 1997). Although the structural characteristics of H-89 that confer its affinity to beta
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replication-deficient adenovirus) obtainable in HASM (Penn
et al., 1998). This was an important consideration because
the alterations in maximal beta-2 AR responsiveness induced
by PKA are frequently small (Clark, 1986) and the identification of an experimental effect caused by inhibition of PKA
would likely require significant PKA inhibition in all cells.
H-89 was among the most attractive compounds considered,
having been characterized in vitro as a highly potent and
selective inhibitor of PKA (Chijiwa et al., 1990). Moreover,
H-89 has been used in numerous studies to date examining
the role of PKA in the regulation of cellular signaling and
physiological effects induced by activation of a wide range of
GPRs.
As mentioned, the principal drawback in using most pharmacological kinase inhibitors in intact cells is the apparent
requirement for 100- to 1000-fold higher levels than those
determined effective in cell-free assays. Effective inhibition
of a target kinase in a given cell type is dependent in part on
1) the permeability of the inhibitor; 2) the relative stoichiometry of inhibitor, the intended target kinase, and any competing target enzymes; 3) the relative potencies of the inhibitor for all potential enzyme targets; and 4) the competitive
nature of the inhibitor with intracellular ATP (MacKintosh
and MacKintosh, 1994). This last consideration alone dictates that among those kinase inhibitors used in this study,
intracellular concentrations of inhibitor must be orders of
magnitude higher than those determined for kinases in vitro,
where reported Ki values are determined under conditions of
low micromolar levels of ATP. One of the first investigations
into the effect of H-89 in intact cells demonstrates that despite an in vitro Ki of H-89 for PKA of 48 nM, inhibition of
cAMP-induced neurite outgrowth in PC12D cells was only
observable at micromolar concentrations of H-89, with a Ki of
;10 mM (Chijiwa et al., 1990).
With such caveats in mind, we examined the effects of
various kinase inhibitors in a frequently used paradigm for
characterizing agonist-specific beta-2 AR desensitization.
HASM cells were treated with a saturating concentration of
beta agonist, washed, and then challenged again with a saturating concentration of beta agonist, with the diminution of
cAMP accumulation that occurs due to beta agonist pretreatment serving as an index of beta-2 AR desensitization. Pretreatment with high levels of various kinase inhibitors before
and throughout pretreatment with ISO might presumably
provide insight into the role of PKA in agonist-specific desensitization. We hypothesized that effective inhibition of PKA
would attenuate agonist-specific desensitization to a degree
commensurate with the cAMP-mediated beta-2 AR desensi-
436
Penn et al.
Vol. 288
Fig. 6. Antagonism of receptor-mediated function by H-89. A, BEAS-2B
cells were treated with increasing concentrations of H-89 and then immediately stimulated with 30 nM ISO or 30 nM PGE2, and 1 mM IBMX.
cAMP was isolated and quantified as described in Experimental Procedures. Data represent mean 6 S.D. of three experiments. B, H-89 effects
on receptor-mediated phosphoinositide metabolism. RBL cells expressing
the m1 mAchR, HASM and BEAS-2B cells expressing the H1 histamine
receptor (H1 R), and HEK 293 cells expressing the TXA2aR were loaded
with myo-[3H]inositol and stimulated with agonists at concentrations
approximating one to three times the EC50 of activation. Total [3H]inositol phosphates were isolated and quantified as described in Experimental
Procedures. Data represent mean 6 S.D. values (fold activation relative
to paired basal levels) of two or three experiments. Basal levels of
[3H]inositol phosphates were not affected by H-89 (data not shown).
ARs are not obvious, one might predict, based on the analysis
of ligand/beta-2 AR interactions described by Jasper and
Insel (1992), that the sulfonamide or amine group could
interact with Asp113 of the beta-2 AR. It should also be noted
Fig. 7. Chemical structures of H-89, H-85, and H-7.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
that H-89 lacks the hydroxyl groups on the aromatic ring
(isoquinoline group in H-89) that appear to be required for
beta-2 AR activation by ligand. Based on the relative affinities of H-7, H-85, and H-89 for beta ARs, it is also plausible to
suggest that the large hydrophobic side chain possessed by
H-89 and H-85 (Fig. 7) may also serve to anchor these molecules in the plasma membrane (or to a region of the beta AR
distinct from the ligand binding domain) and thus increase
their effective affinities for the beta ARs, perhaps in a manner not unlike that demonstrated for the beta agonist salmeterol (Clark et al., 1996; Green et al., 1996).
Although experiments examining the effects of H-89 on
both receptor binding and receptor-mediated signaling suggest that the action of H-89 at the receptor level are selective
for beta ARs, we did observe that binding to other receptors
could be slightly inhibited by concentrations of H-89 that
might be used to inhibit PKA in intact cells. Although this
may not be problematic under many experimental conditions, it does suggest that H-89 as well as other inhibitors
(e.g., Bis IX and H-7) used at high concentration in intact cell
or organ system models may inhibit the function of other
receptors, particularly those activated by low levels of agonist (e.g., by circulating hormones, or in a paracrine or autocrine manner). Thus, it would appear prudent in such circumstances not only to establish the selectivity of agents
with respect to intracellular kinases but also to consider
potential effects on receptors that are relative to the system.
An additional concern associated with the use of high levels of kinase inhibitors is cellular toxicity. Whether enhanced
cell morbidity or mortality represents a nonspecific side effect or is necessarily a consequence of effective inhibition of
target kinases is unclear. In both HASM and BEAS-2B cells,
prolonged incubation (.2 h) with 10 mM H-89 leads to a
reduction in FSK-stimulated cAMP accumulation (R. B. Penn
and J. L. Benovic, unpublished observations). A 16-h incubation of both BEAS-2B and HASM cells (but not COS-1 cells)
with 1 mM H-89 causes an ;20% loss of cells in culture by
detachment compared with DMSO-treated control, whereas
16-h treatment with 500 mM H-7 causes a small loss (;20%)
in COS-1 cells and a larger loss (;30%) in HASM.
In summary, the present study demonstrates that the significant attenuation of agonist-specific desensitization of the
beta-2 AR by the PKA inhibitor H-89 in two different physiologically relevant cell types is primarily a result of beta-2 AR
antagonism. Two other inhibitors, KT5720 and H-7, shown
effective in inhibiting PKA-mediated functions in numerous
1999
other cell types, failed to influence beta-2 AR desensitization
or inhibit PKA-induced protein phosphorylation in HASM,
thus suggesting the cell-specific nature of inhibitors. Additional studies examining the pharmacological and functional
properties of various kinase inhibitors demonstrate the need
to think beyond the obvious requirements for kinase selectivity and potency and consider the numerous nonspecific
effects that may occur with use of such agents in investigation using intact cells.
Acknowledgments
We acknowledge Kristin DiMezzes, Andrew Eszterhas, and Pascale Labrecque for technical assistance and Mike Orsini, Chris Carman, and Joanne Holland for critical discussion of the manuscript.
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