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Vol. 290, Issue 1, 76-82, July 1999
Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, Texas
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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We examined the effects of several protein kinase C (PKC) inhibitors on
the murine 5-hydroxytryptamine3 (5-HT3)
receptor to determine whether they acted directly on the receptor. The
5-HT-evoked currents in Xenopus laevis oocytes
expressing the recombinant 5-HT3 receptor were measured
with the two-electrode voltage-clamp technique. The PKC inhibitors
bisindolylmaleimide I (BIM, GF109203x) and staurosporine, but not
calphostin C or chelerythrine, decreased the 5-HT3
receptor-mediated currents when coapplied with 5-HT. BIM blocked 0.5 µM 5-HT-elicited currents with an IC50 value of 7 nM,
whereas in the presence of 5 µM staurosporine, 42% inhibition of 0.5 µM 5-HT-mediated currents was observed. Increasing concentrations of
BIM resulted in a rightward shift of the 5-HT concentration-response curve, without altering efficacy. A Schild plot was generated, which
had a slope of 
1.01, suggesting competitive antagonism. The
Ki value of BIM was determined to be 29 nM.
To confirm competitive antagonism, a competitive binding assay was
performed on Sf21 insect cells infected with the mouse
5-HT3 receptor cDNA in a baculovirus expression vector. BIM
completely displaced binding of the selective 5-HT3
receptor antagonist [3H]GR65630. BIM bound to the
5-HT3 receptor with a Ki value
of 61 nM, which was slightly less potent than that of the selective 5-HT3 receptor antagonist MDL72222 (27 nM). The PKC
inhibitor BIM is a potent competitive antagonist at the
5-HT3 receptor.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
5-hydroxytryptamine3
(5-HT3) receptor is a member of the superfamily
of ligand-gated ion channels (Maricq et al., 1991
). Members of this
family share considerable structural similarities. Receptors consist of
five subunits, each of which has four transmembrane-spanning domains
(Galzi and Changeux, 1994). To date, only one subunit of the
5-HT3 receptor has been cloned for any given
species, with the first being the murine receptor that was cloned from
the NCB-20 neuroblastoma cell line (Maricq et al., 1991). Analogous
subunits for the rat (Isenberg et al., 1993), human (Miyake et al.,
1995), and guinea pig (Lankiewick et al., 1998)
5-HT3 receptors have since been cloned and share
94%, 84%, and 81% identity with the mouse receptor, respectively.
The 5-HT3 receptor forms a
Na+/K+-permeable channel in
the plasma membrane (Jackson and Yakel, 1995). Activation of
5-HT3 receptors in the area postrema has been
shown to trigger emesis. Blockade of these receptors by ondansetron is
a clinically used treatment for nausea associated with chemotherapy and
general anesthesia (Greenshaw, 1993). Antagonists of the
5-HT3 receptor have been suggested to be useful
in inflammatory pain, anxiety, depression, schizophrenia, dementia, and
drug abuse (Greenshaw, 1993).
A wide variety of drugs have been found to possess
5-HT3 receptor antagonist action aside from their
primary action. The 5-HT3 receptor was originally
named the M-receptor because the action of 5-HT at this receptor was
blocked by morphine (Gaddum and Picarelli, 1957). More recently,
morphine was determined to be a competitive antagonist at
5-HT3 receptors in rat nodose ganglia (Fan,
1995a). Cocaine has long been known to block
5-HT3 receptors (Fozard et al., 1979). More
recently, cocaine, as well as the related local anesthetic agents
lidocaine and tricaine, were found to reduce currents of the cloned
5-HT3 receptor expressed in Xenopus
laevis oocytes (Fan et al., 1995). Cocaine likely acts as a
competitive antagonist at the 5-HT3 receptor (Fan
et al., 1995) Cannabinoid agonists, such as anandamide, have also been
shown to inhibit 5-HT3 receptor-mediated currents
(Fan, 1995b). The selective serotonin reuptake inhibitor fluoxetine
inhibits 5-HT3 receptor-mediated responses in the
low micromolar range and facilitates receptor desensitization (Fan,
1994a). Several of the antipsychotic phenothiazines, such as
chlorpromazine, displace binding of the selective
5-HT3 receptor antagonist
[3H]GR65630 with
Ki values in the low micromolar range
(Lummis and Baker, 1997).
Drugs used to study protein kinase activity have also been found to
block ligand-gated ion channels. Several cAMP analogs and the protein
kinase inhibitor H-7 displace binding of the 
-aminobutyric acidA receptor antagonist
[3H]SR 95531 from microsacs and inhibit
36Cl
uptake (Leidenheimer
et al., 1990). Forskolin, an activator of adenylyl cyclase, alters
nicotinic acetylcholine receptor function by a cAMP-independent
mechanism (Wagoner and Pallotta, 1988). H-7 acts directly on
N-methyl-D-aspartate receptor channels
(Amador and Dani, 1991). In addition, H-7 inhibits
5-HT3 receptor-mediated currents in rat nodose
ganglia independent of protein kinase activity (Fan, 1994b). A compound
commonly used to stimulate protein kinase C (PKC) activity,
4-
-phorbol-12,13-dibutyrate, acts directly on nicotinic
acetylcholine receptors (Nishizaki and Sumikawa, 1995). The protein
kinase inhibitor staurosporine inhibits 5-HT3 receptor function in rat nodose ganglia currents at concentrations as
low as 20 nM (Robertson and Bevan, 1991). However, Zhang et al. (1995)
reported that preincubation with 2 µM staurosporine was without
effect on 5-HT-elicited currents in X. laevis oocytes expressing the mouse 5-HT3 receptor. Previously,
it was reported that the selective PKC inhibitor bisindolylmaleimide
(BIM) inhibited 5-HT3 receptor-mediated currents
in X. laevis oocytes at nanomolar concentrations. This
action was attributed to PKC inhibition. However, the action was
instantaneous, which casts doubt on a kinase-dependent mechanism
(Glitsch et al., 1996).
In the current study, we examined several PKC inhibitors to evaluate whether kinase inhibition or antagonism of the receptor complex was responsible for 5-HT3 receptor modulation. We report that, indeed, 5-HT3 receptor-mediated currents are inhibited by BIM. However, this effect is a result of competition with 5-HT at the agonist binding site rather than a result of PKC inhibition. The PKC inhibitors calphostin C and chelerythrine had no effect on 5-HT3 receptor-mediated currents. Staurosporine inhibited 5-HT3 receptor function but was about 800-fold less potent than BIM.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of X. laevis Oocytes. X. laevis frogs were kept in tanks on a 12-h light/dark cycle at 19°C and fed a diet of dehydrated liver in Xenopus I chow three times per week. Frogs were anesthetized by immersion in cold 0.12% 3-aminobenzoic acid ethyl ester for 20 min. After removal, ovarian lobes were placed in modified Barth's solution (MBS) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, and 0.91 mM CaCl2, pH 7.5.
Before dissection, ovarian lobes were placed in a hypertonic isolation medium containing (in mM) NaCl 108 mM, KCl 2 mM, EDTA 1 mM, and HEPES 10 mM, pH 7.5. Stage V and VI oocytes were dissected with fine surgical forceps and placed in MBS. Dissected oocytes were incubated for 10 min in buffer containing 0.5 mg/ml collagenase Type IA and 83 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 10 mM HEPES, pH 7.5, to remove the follicular cell layer. After several rinses with MBS, oocytes were placed in sterile filtered incubation medium. Incubation medium was composed of either ND96, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.5, or MBS. Incubation medium was supplemented with 10 mg/l streptomycin, 50 mg/l gentamicin, 10,000 U/l penicillin, 96 mg/l sulfamethoxazole (Elkins-Sinn Inc., Cherry Hill, NJ), 19.2 mg/l trimethoprim (Elkins-Sinn Inc.), 0.5 mM theophylline, and 2 mM sodium pyruvate.Transcription of cDNA to cRNA. The mouse 5-HT3 receptor cDNA was linearized with NotI, extracted with phenol-chloroform, precipitated with sodium acetate and ethanol, and resuspended in diethyl pyrocarbonate-treated water. The cDNAs were then transcribed with T3 polymerase (mCAP RNA capping kit; Stratagene, La Jolla, CA; or mMESSAGE mMACHINE, Ambion, Austin, TX).
Microinjection of Oocytes with 5-HT3 Receptor cRNA. An aliquot of cRNA was centrifuged at 15,000g, and the ethanol was removed with a tuberculin syringe. After air drying, the pellet was resuspended in a volume of diethyl pyrocarbonate-treated water to yield a concentration of 5 to 30 ng of cRNA/50 nl. The cRNA was drawn up into a micropipette (10-20-µm tip size). Then, 50 nl of cRNA was injected into the animal/vegetal pole equator of each oocyte. Oocytes were stored in incubation medium in Corning cell well plates (Corning Glass Works, Corning, NY) at room temperature. Incubation medium was changed daily. Oocytes were recorded from days 2 through 7 after injection.
Two-Electrode Voltage-Clamp Recordings.
Oocytes were
perfused in a 100-µl volume chamber with MBS via a roller pump
(Cole-Parmer Instrument, Co., Chicago, IL). Oocytes were impaled with
two glass electrodes (1.2-mm outside diameter and 1-10 M
resistance) filled with 3 M KCl. Except where noted, oocytes were
voltage-clamped to 70 mV with a Warner Instruments model OC-725A,
OC-725B, or OC-725C oocyte clamp (Hamden, CT). Clamping currents were
plotted on a strip-chart recorder (Cole Parmer Instrument).
5%. Oocytes were then
perfused with 5-HT, and one of the following protein kinase inhibitors:
bisindolylmaleimide I (0.1-500 nM), calphostin C (2 µM),
chelerythrine (2 µM), or staurosporine (0.5, 2, or 5 µM). Current
responses in the presence of protein kinase inhibitor were reported as
percent of baseline response. To determine the competitive nature of
BIM, oocytes were perfused with 200 µM 5-HT, as mentioned, until two
consecutive responses varied by 5%. Oocytes were then perfused with
5-HT and 0, 25, 150, 300, or 550 nM BIM. Responses in the presence of
protein kinase inhibitor were reported as a percent of the 200 µM
5-HT baseline response.
Schild Plot. From the concentration-response curves of 5-HT in the presence of various concentrations of BIM, EC50 values were determined. A Schild plot was generated based on these values, and the slope and Ki were obtained. The negative logarithm of the Ki value is the intercept on the x-axis. Prism (GraphPAD Software, San Diego, CA) was used to generate the concentration-response curves and the Schild plot.
Preparation of Recombinant Baculovirus.
The cDNA encoding
the murine 5-HT3 receptor was excised from the
pBluescript KS()vector with the restriction
endonucleases NotI and XhoI and subcloned into
the pBacPAK-His3 (Clontech Laboratories Inc., Palo Alto, CA) vector at
the same sites. The production of recombinant baculovirus and viral
infections was conducted by using the media and protocols described in
the BacPAK Baculovirus Expression System of Clontech. Briefly, the host
cell line, Sf21, was cotransfected with BacPAK6 viral DNA
(Bsu36I digested) and pBacPAK-His3 containing murine 5-HT3 receptor cDNA. Recombinant baculovirus was
plaque purified, propagated, and then used to infect Sf21 insect cells
growing at 27°C in Grace's insect cell medium containing 10% FBS
(Sigma Chemical Co.).
Sf21 Cell Membrane Preparation. After harvesting, cells were centrifuged at 600g for 10 min at 4°C. The supernatant was discarded, and the cells were resuspended in 50 mM HEPES buffer, pH 7.4. The concentration of cells was adjusted to 3.5 to 4.5 × 106 with 50 mM HEPES buffer, pH 7.4. The cells were then disrupted with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) at position 6 for 20 s. The disrupted cells were then centrifuged at 600g for 15 min at 4°C. The plasma membrane-containing supernatant was saved for binding assays. The protein concentration of the membrane solution was determined with the bicinchoninic acid reagent (Pierce, Rockford, IL).
Competitive Binding Experiments.
All experiments were
carried out at room temperature in disposable glass tubes with a final
volume of 250 µl. Binding of 1 nM
[3H]GR65630, with a specific activity of 60 to
87 Ci/mmol (New England Nuclear Life Sciences Products, Boston, MA),
was measured in reactions containing 1, 10, 50, 100, 200, or 400 µg
of protein. The linear portion of the
[3H]GR65630 binding curve ranged from 10 to 200 µg. Competition assays were carried out with 140 to 150 µg
protein/reaction. The concentration of
[3H]GR65630 used in competition experiments was
either 0.3 or 0.6 nM. BIM (0.01 nM to 10 µM), MDL-72222 (0.01 nM to
10 µM), or chelerythrine (1 nM to 2 mM) was used to compete with
[3H]GR65630 for 5-HT3
receptor binding. The KD value of
[3H]GR65630 was previously determined to be 0.4 nM (unpublished observation). The incubation was started by the
addition of a 100-µl aliquot of Sf21 cell membranes, and the tubes
were incubated for 10 min. Nonspecific binding was measured in the
presence of 50 µM MDL-72222 (Hellevuo et al., 1991). All analyses
were done in duplicate.
). The plots of antagonist displacement were generated
with GraphPAD Prism.
One- or two-way ANOVA was used to assess differences in treatment
effects between groups of oocytes. Newman-Keuls analysis was used for
post hoc testing. Unpaired t test was used to assess differences in a single drug concentration between two groups of
oocytes. GraphPAD Prism or Instat software was used for all statistical analyses.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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BIM Inhibits 5-HT3 Receptor-Mediated Currents.
Drugs that inhibit protein kinases have been shown to act directly on
ligand-gated ion channels (Leidenheimer et al., 1990; Fan, 1994b). The
coapplication of nanomolar concentrations of BIM with 0.5 µM 5-HT was
found to significantly reduce the elicited current in the absence of
PKC stimulation. The representative tracing shown in Fig.
1 demonstrates the ability of 50 nM BIM to inhibit the 5-HT-evoked current. Inhibition of current was immediate; however, several applications of 5-HT alone were required for the current to return to baseline. This may be due to the fact that
some competitive antagonists are slow to wash off (Machu and Harris,
1994).
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), calphostin C (Gillette and
Dacheux,1996), chelerythrine (Green and Cottrell, 1997), and
staurosporine (Uchiyama et al., 1994; Green and Cottrell, 1997) were
used at concentrations previously determined to block PKC modulation of
ligand-gated ion channels. The coapplication of 0.5 µM 5-HT with 2 µM chelerythrine, 2 µM calphostin C, and 0.5 µM staurosporine
resulted in currents that were 101.8 ± 1.89% (n = 5), 95.2 ± 1.71% (n = 6), and 93.62 ± 4.39% (n = 5) of control, respectively (Fig.
2). Higher concentrations of
staurosporine (2 and 5 µM) were found to reduce receptor function to
80.22 ± 2.97% (n = 5) and 58.47 ± 1.61%
(n = 4) of control, respectively. Currents were reduced
to 8.54 ± 1.17% (n = 5) of control with the
coapplication of 0.5 µM 5-HT and 100 nM BIM. One-way ANOVA revealed
significant differences among the reported data (p < .0001). Dunnett's post hoc test was used to determine that only the
currents produced in the presence of 100 nM BIM and 2 and 5 µM
staurosporine were significantly less than control (*p < .01).
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0.9. Concentrations as low as 1 nM BIM significantly decreased the 5-HT elicited current to
83.97 ± 2.9% of control. In the presence of 500 nM BIM, only 1.14 ± 0.42% of the control current remained.
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1.01 ± 0.17, suggesting that
BIM is a competitive antagonist (Taylor and Insel, 1990). The
pA2 (x intercept) was 7.53, and the
Ki value
of BIM at the 5-HT3 receptor is 29.4 nM.
Further confirmation of the competitive antagonism of
5-HT3 receptors by the PKC inhibitor BIM was
provided by competitive binding studies. BIM and the selective
5-HT3 receptor antagonist MDL 72222, as well as
chelerythrine, were used to displace
[3H]GR65630 binding from membranes of Sf21
insect cells infected with the mouse 5-HT3
receptor cDNA. Preliminary saturation binding studies were performed to
confirm that [3H]GR65630 bound to the
5-HT3 receptors in Sf21 cells with a
Kd value (0.4 nM) similar to that seen
with NCB-20 cells. Competition binding experiments were performed with
0.01 nM to 10 µM BIM or MDL 72222 or 1 nM to 2 mM chelerythrine to
displace 0.3 to 0.6 nM [3H]GR65630 binding from
the receptors. Nonspecific binding was ~15% of total binding. BIM,
MDL 72222, and chelerythrine completely displaced
[3H]GR65630 binding. The
Ki value for displacement of
[3H]GR65630 binding by BIM was found to be
61 ± 6.3 nM. MDL 72222 displaced
[3H]GR65630 binding with a
Ki value of 27 ± 4.5 nM. The
Ki values were significantly different
by two-tailed t test (p = .005), making BIM
somewhat less potent than MDL 72222. In contrast, chelerythrine was an
extremely weak antagonist at the receptor, with a
Ki value of 535 ± 25 µM.
These results demonstrate that the selective PKC inhibitor BIM
(GF109203x) is a potent competitive antagonist at the
5-HT3 receptor. Staurosporine, another PKC
inhibitor structurally related to BIM, also inhibited
5-HT3 receptor-mediated currents. Because of the
structural similarities between BIM and staurosporine, it is likely
that staurosporine also acts directly on the
5-HT3 receptor. However, staurosporine is
approximately 800-fold less potent than BIM at the
5-HT3 receptor and was not tested further.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The actions of a variety of PKC inhibitors were examined on
5-HT3 receptor function. The inhibitory actions
of these compounds have been extensively tested against PKC purified
from tissues from numerous mammalian species, and PKC isoforms from
some of these species have been cloned (Newton, 1995). Staurosporine
and BIM are structurally related and act competitively at the ATP recognition site of PKC, which is located in the C3 region of the
enzyme and is common to the conventional, novel, and atypical isoforms
(Hofmann, 1997). Staurosporine blocks all except the 
isoform with
nanomolar efficacy, whereas BIM has been tested against the
conventional isoforms and blocks them all with
IC50 values of 16 to 20 nM (Hofmann, 1997). In
addition to the ATP binding domain, chelerythrine acts at the C3 region
of PKC, which controls phosphoryl transfer. Chelerythrine inhibits PKC
purified from rat brain with an IC50 value of 660 nM (Herbert et al., 1990). Calphostin C interacts with the C1 domain of
the enzyme, which contains the phorbol ester and diacylglycerol site;
its Ki value is ~50 nM (Kobayashi et
al., 1989). The C1 domain is highly conserved among the conventional
and novel classes of PKC isoforms, and slight modifications occur in
the atypical forms (Hofmann, 1997). Thus, based on their respective
sites of action and previously published work, the PKC inhibitors used
in this study would be predicted to inhibit most, if not all, of the
isoforms of PKC that have been cloned from mammalian species.
There has been limited identification of PKC isoforms in X. laevis. Sahara et al. (1992) performed Western blot
analyses of partially purified PKC from X. laevis oocytes.
Antibodies against the conserved region (C3 plus C4) of the rat isoform recognized an 80-kDa X. laevis protein, whereas
antibodies against variable regions of rat isoforms 
, I, II,
and failed to identify X. laevis proteins. A PKC-related
kinase 2 has been cloned from X. laevis oocytes; this group
of PKC-related kinases cloned from human tissues lacks the C1 domain of
the classic PKC isoforms (Cryns et al., 1997). Their C2 domain is
closely related to that of the novel PKC isoforms. Despite the lack of
identification of X. laevis PKC isoforms, PKC-dependent
modulation of the function of heterologously expressed proteins in
X. laevis oocytes has been studied extensively. Furthermore,
inhibitors such as calphostin C (Dildy-Mayfield and Harris, 1995),
staurosporine (Uchiyama et al., 1994; Zhang et al., 1995), and BIM
(Nishizaki and Ikeuchi, 1995) have been demonstrated to block
PKC-dependent actions in oocytes.
The present study shows that the PKC inhibitor BIM decreases 5-HT3 receptor-mediated currents in X. laevis oocytes in the absence of any treatments to enhance PKC activity. The inhibition of current by BIM is concentration dependent. The observations made here are consistent with BIM acting as a competitive antagonist at the 5-HT3 receptor rather than through a PKC-dependent mechanism. Several lines of evidence suggest that the inhibition of current is not PKC dependent. The inhibition of 5-HT-evoked current is immediate in onset, and the currents were reduced without preincubation of BIM. The enhancement of 5-HT3 receptor-mediated currents by the PKC-stimulating compound phorbol-12-myristate-13-acetate (PMA) in oocytes was not observed until at least 2 min of constant perfusion (unpublished observation). In this study, the reduction in current by BIM occurred instantaneously, which is not consistent with PKC involvement.
The lack of effect by other PKC inhibitors also is not consistent with
a PKC-dependent mechanism of action. Several structurally unrelated PKC
inhibitors were examined for effects on 5-HT3
receptor function. In this study, the PKC inhibitors were used in
concentrations previously reported to inhibit PKC alteration of
ligand-gated ion channel function. Nishizaki and Ikeuchi (1995) blocked
the PKC-dependent alteration of glycine-mediated currents in oocytes expressing the 1 or 2
receptors with 500 nM GF109203x (BIM). In the oocyte expression system,
staurosporine (1 µM) reversed decreases in glycine
1 receptor function after PMA treatment (Uchiyama et al., 1994). The PMA-induced reduction in
Cl current of GABAA
receptors in rabbit retinal rod bipolar cells was shown to be blocked
by 0.5 µM calphostin C (Gillette and Dacheux, 1996). The phorbol
ester-induced stimulation of dopamine-activated currents in
Helix neurons was inhibited by both 2 µM chelerythrine and
1 µM staurosporine (Green and Cottrell, 1997). In the present study,
both chelerythrine (2 µM) and calphostin C (2 µM) had no effect on
5-HT3 receptor-mediated currents. If BIM produced
its inhibition through a PKC-dependent mechanism, then other PKC
inhibitors should have a similar action.
In the present study, staurosporine was found to reduce 5-HT-evoked
currents at concentrations of 2 µM or greater. Zhang et al. (1995)
reported that preincubation with 2 µM staurosporine did not alter
murine 5-HT3 receptor-mediated currents in
oocytes; however, staurosporine was not coapplied with 5-HT but was
applied for 10 min, followed by a washout before 5-HT application. This staurosporine application method may have resulted in the washout of
any staurosporine from the agonist recognition site. In this same
study, however, 2 µM staurosporine did inhibit the PMA-induced enhancement of 5-HT-elicited currents. This concentration of
staurosporine is sufficient to block PKC activity. Furthermore, the
inhibition of basal PKC activity did not alter the
5-HT3 receptor-mediated currents (Zhang et al.,
1995). The inhibition of 5-HT3 receptor function
by staurosporine seen in the present study, in which 5-HT and
staurosporine are coapplied, probably is due to a direct action on the
receptor. In addition, 5-HT3 receptor-mediated
currents in rat dorsal root ganglion neurons were immediately reduced
after a 15-s application of 100 nM staurosporine (Robertson and Bevan, 1991). Again, it is likely that this effect is not due to inhibition of
PKC activity but rather a direct antagonist action on the
5-HT3 receptor. Staurosporine is structurally
related to BIM; both compounds contain indole rings, as does 5-HT.
The alteration of the 5-HT concentration-response curve by BIM provides
further evidence that this compound is a competitive antagonist at the
5-HT3 receptor. The 5-HT concentration-response curve was shifted to the right and the EC50 value
was increased by increasing concentrations of BIM. Furthermore, high
concentrations of 5-HT completely overcame the inhibitory effect of
BIM. Activation of PKC by PMA resulted in a great increase in the
efficacy of 5-HT (Coultrap and Machu, 1996). Therefore, it would be
logical to conclude that inhibition of tonic PKC activity, if it does in fact modulate 5-HT3 receptor function, would
reduce the efficacy of 5-HT. However, the efficacy of 5-HT was not
altered by BIM. A Schild plot was generated that had a slope of
1, as was expected for a competitive antagonist. These results fit
the classic model of a competitive antagonist.
Finally, competitive binding assays with the selective
5-HT3 receptor antagonist
[3H]GR65630 demonstrate that BIM acts at the
5-HT recognition site. The Ki value of
27 ± 4.5 nM for displacement of
[3H]GR65630 by MDL72222 is similar to published
Ki values for this compound. Green et
al. (1995) found MDL72222 to displace
[3H]granisetron binding with a
Ki value of 13 ± 3 nM from the
mouse 5-HT3 receptor in baculovirus-infected Sf9
insect cells. Ki values of 11 and 46 nM have also been reported for MDL72222 displacement of
[3H]GR65630 binding from N1E-115 membranes
(Lummis and Martin, 1992). BIM displacement of
[3H]GR65630 binding from Sf21 insect cells
yielded a Ki value of 61 ± 6.3 nM. Therefore, the PKC inhibitor BIM is only a slightly less potent
antagonist at the 5-HT3 receptor than the
selective 5-HT3 receptor antagonist MDL72222. The
PKC inhibitor chelerythrine also competitively inhibits
[3H]GR65630 binding but with a
Ki value of 535 ± 25 µM, which
is roughly three orders of magnitude greater than the
Ki value of 660 nM for the inhibition
of PKC. It is not surprising that chelerythrine has actions at the
5-HT3 receptor because it has inhibitory actions on other proteins that are independent of its effects on PKC. For
instance, chelerythrine inhibits rat retinal ATPase activity and
taurine uptake with IC50 values of 59 and 88 µM, respectively (Militante and Lombardini, 1998). It also inhibits
Na+,K+-ATPase with an
IC50 value of 30 to 50 µM (Cohen et al., 1978).
In summary, the PKC inhibitor BIM is a potent, competitive antagonist
at the murine 5-HT3 receptor. The PKC inhibitor
staurosporine is also likely an antagonist at the
5-HT3 receptor, whereas the inhibitor
chelerythrine competitively antagonizes the receptor at extremely high
concentrations. Drugs used to study protein kinase activity have
previously been shown to act directly on the
5-HT3 receptor, as well as on other ligand-gated
ion channels (Leidenheimer et al., 1990; Fan, 1994b). However, the
potency with which BIM blocks the 5-HT3 receptor
is notable. With a nanomolar Ki value,
BIM is nearly as potent as an antagonist at the
5-HT3 receptor as it is at PKC. We have
determined that BIM inhibits 5-HT3
receptor-mediated currents with a Ki
value of 29.5 nM and reduces currents produced by a low concentration
of 5-HT (0.5 µM) with an IC50 value of 7.6 nM.
BIM inhibits PKC activity with IC50 values of 10 to 20 nM (Toullec et al., 1991). Therefore, when protein kinase
modulators are used to study the effects of phosphorylation on receptor
function, care must be taken to ensure that the drug does not act
directly on the receptor. Because of its potent competitive antagonism
and structural similarity to the endogenous agonist 5-HT, BIM may be
useful in studying the agonist recognition site of the
5-HT3 receptor and in the development of new
selective antagonists.
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Footnotes |
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Accepted for publication February 26, 1999.
Received for publication November 3, 1998.
1 This work was supported by National Institute of Alcohol Abuse and Alcoholism Grant AA10561 (to T.K.M.).
Send reprint requests to: Dr. Tina K. Machu, Department of Pharmacology, Texas Tech University Health Sciences Center, Lubbock, TX 79430.
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Abbreviations |
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BIM, bisindolylmaleimide; 5-HT3, 5-hydroxytryptamine3; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; MBS, modified Barth's solution.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) cocaine and some related compounds.
Eur J Pharmacol
59:
195-210[Medline].-aminobutyric acidA receptor complex function.
Mol Pharmacol
38:
823-828[Abstract].1 and 2 glycine receptor channels.
Brain Res
687:
214-216[Medline].-phorbol-12,13-dibutyrate (PDBu) on nicotinic acetylcholine receptor channel independent of protein kinase C activation.
Neurosci Lett
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),
MDL72222 (
), and chelerythrine (
) (n = 3-4). The Ki values for displacement of
specific [3H]GR65630 binding are 61 ± 6.3 nM,
27 ± 4.5 nM, and 535 ± 25 µM for BIM, MDL 72222, and
chelerythrine, respectively.