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Vol. 292, Issue 1, 131-135, January 2000
Department of Pharmacology II, Graduate School of Medicine, Osaka University, Osaka, Japan
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The effects of a novel vasorelaxant agent, MCC-134 (1-[4-(1H-imidazol-1-yl)benzoyl]-N-methyl-cyclobutanecarbothioamide), were examined on reconstituted ATP-sensitive K+ (KATP) channels, which are composed of an inwardly rectifying K+ channel, Kir6.2, and three types of sulfonylurea receptors (SUR): SUR1, SUR2A, and SUR2B. Each type of KATP channel was heterologously expressed in human embryonic kidney 293T cells. The expressed KATP channel currents were measured with the whole-cell configuration of the patch-clamp method. MCC-134 activated the SUR2B/Kir6.2 channel, was a weak activator of the SUR2A/Kir6.2 channel, but did not activate the SUR1/Kir6.2 channel. MCC-134 suppressed SUR1/Kir6.2 channel currents that had been fully activated by either diazoxide or NaCN, whereas it did not affect the fully activated SUR2A/Kir6.2 or SUR2B/Kir6.2 channel currents. Thus, MCC-134, which is a relatively effective opener of the vascular smooth muscle type (SUR2B) of KATP channel, is an antagonist of the pancreatic type (SUR1) of KATP channel. Therefore, depending on the subtype of SUR, a pharmacological agent can cause either activation or inhibition of KATP channel activity.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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ATP-sensitive
K+ (KATP) channels are
inhibited by intracellular ATP and activated by NDPs and thus provide a
link between the metabolic state and cellular excitability in various
tissues (Ashcroft, 1988
; Terzic et al., 1995). These channels are
associated with various cellular functions, such as vasodilatation,
insulin secretion, cardiac preconditioning during ischemia,
neurotransmitter release, and oocyte maturation. It is established that
KATP channels are heteromultimers composed of an
ATP-binding cassette protein, known as the sulfonylurea receptor (SUR),
and an inwardly rectifying K+ channel (Kir)
subunit, Kir6.2 (Aguilar-Bryan et al., 1995; Inagaki et al., 1995;
Sakura et al., 1995). When expressed with Kir6.2, all three types of
SUR identified so far (i.e., SUR1, SUR2A, and SUR2B) form
KATP channels. The reconstituted
KATP channels all exhibit the same single-channel
characteristics; weak inward rectification and a unitary conductance of
~80 pS in the inward direction in the presence of 150 mM
extracellular K+. However, they show distinct
sensitivities to various vasorelaxant K+ channel
openers (KCOs; Inagaki et al., 1995, 1996; Isomoto et al., 1996). For
instance, the SUR1/Kir6.2 channel is activated by diazoxide but not by
pinacidil; the SUR2A/Kir6.2 channel is activated by pinacidil but not
by diazoxide; and the SUR2B/Kir6.2 channel is activated by both
pinacidil and diazoxide. Therefore, it is now widely accepted that SURs
are responsible for the differential effects of KCOs on each type of
KATP channel in various tissues.
Pharmacological and electrophysiological studies have reported that
SUR1/Kir6.2 represents the pancreatic 
-cell
KATP channel, whereas SUR2A/Kir6.2 is thought to
represent the cardiac KATP channel (Aguilar-Bryan
et al., 1995; Inagaki et al., 1995, 1996; Sakura et al., 1995).
However, the molecular composition of native vascular smooth muscle
cell K+ channels is controversial. Current data
strongly suggest that SUR2B/Kir6.1 may represent the vascular
NDP-sensitive K+ (KNDP)
channel, which is the main target of KCOs in vascular smooth muscle
(Beech et al., 1993; Quayle et al., 1997; Yamada et al., 1997; Satoh et
al., 1998). Because KNDP and
KATP channels differ in their single-channel
characteristics and intracellular nucleotide-mediated gating, it has
been difficult to electrophysiologically compare the affinities of
various KCOs for smooth muscle KNDP channels with
those for pancreatic and cardiac KATP channels in a quantitative manner. Therefore, for this purpose, we adopted a
strategy using the reconstituted KATP channels
with the same pore subunit, Kir6.2 and different SURs to examine the
effects of KCOs on these channels (Shindo et al., 1998).
In this study, we examined the effects of a newly synthesized vasorelaxant agent, 1-[4-(1H-imidazol-1-yl)benzoyl]-N-methyl-cyclobutanecarbothioamide (MCC-134), on the heterologously expressed SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels in human embryonic kidney (HEK) 293T cells. We found that this compound is most effective as an agonist for the SUR2B/Kir6.2 channel but is an antagonist for the SUR1/Kir6.2 channel. This study suggests that depending on the subtype of SUR, a single pharmacological agent can cause not only activation but also inhibition of KATP channel activity.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Functional Expression of SUR1/Kir6.2, SUR2A/Kir6.2, and
SUR2B/Kir6.2 Channels.
The cDNA clones of rat Kir6.2 and mouse
SURs (SUR1, SUR2A, and SUR2B) were used (Isomoto et al., 1996; Ohta et
al., 1998; Shindo et al., 1998). The coding region of each cDNA was
individually subcloned into an expression vector pcDNA3 (InVitrogen,
San Diego, CA). The plasmid containing Kir6.2 was cotransfected with
either SUR1, SUR2A, or SUR2B cDNA into HEK 293T cells with the use of LipofectAMINE (Life Technologies, Grand Island, NY) according to the
manufacturer's instruction. Electrophysiological measurements were
usually conducted 2 to 4 days after transfection.
Electrophysiological Recordings.
The channels expressed in
the HEK 293T cells were studied using the whole-cell configuration of
the patch-clamp method at room temperature as described previously
(Shindo et al., 1998). The agents used in these experiments were
diluted in the bathing solution and applied to the bath. The tip of the
electrodes had a resistance of 2 to 5 M
after being coated with
silicon and fire polished. The channel currents were measured with a
patch-clamp amplifier (Axopatch 200A; Axon Instruments, Foster City,
CA) and monitored throughout the experiments with an analog-storage
oscilloscope (Dual Beam Storage Oscilloscope; Tektronix, Inc.,
Beaverton, OR). For subsequent analyses, currents were recorded on
videocassette tapes by using a PCM recorder (VR-10B; Instrutech Corp.,
Great Neck, NY). For analysis, the data were reproduced, low-pass
filtered at 1.0 kHz (
3 dB) with an 8-pole Bessel filter (Frequency
Devices, Haverhill, MA) and digitized at 3 or 5 kHz with an AD
converter (ITC-16; Instrutech Corp.). The data were analyzed off-line
using a computer (Macintosh Quadra 700; Apple Computer Inc., Cupertino, CA) with commercially available programs: Pulse Program (HEKA Electronik, Lambrecht, Germany) and Patch Analyst Pro (MT Corporation, Hyogo, Japan).
85 mV with
extracellular K+ concentration
([K+]o) of 5.4 mM, as
reported previously (Okuyama et al., 1998). The
K+ currents activated by MCC-134 in the cells
expressing the SUR2A/Kir6.2 or SUR2B/Kir6.2 channels showed the same
properties (data not shown). For comparison of the potency of various
KCOs, the cells were held at either 60 or 30 mV. The whole-cell
current response to pinacidil or MCC-134 in the cells expressing
SUR2A/Kir6.2 or SUR2B/Kir6.2 channels was measured by subtracting the
basal current from that in the presence of these agents. The subtracted
current at each concentration of MCC-134 was normalized to that induced by 100 µM pinacidil in each cell, which was 58 ± 13 and 55 ± 9 pA/pF (mean ± S.E., n = 7 for each) at 60
mV in the coexpressed SUR2A/Kir6.2 or SUR2B/Kir6.2 channels,
respectively (5.4 mM extracellular K+). No
significant difference was detected between these two values (P = .837). The inhibitory response of the whole-cell
current in SUR1/Kir6.2 channels to MCC-134 was measured by subtracting the basal current from that in the presence of this agent. The subtracted current at each concentration of the agent was normalized to
that recorded in the presence of 300 µM diazoxide or 2 mM NaCN in
each cell (5.4 mM extracellular K+, and the cells
were held at 30 mV).
Data are expressed as mean ± S.E. The Student's unpaired
t test was used for statistical analysis. A value of
P < .05 was taken to be statistically significant.
Solutions and Chemicals.
In the whole-cell current recording
configuration, the bath was perfused with a control bathing solution
containing 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES-NaOH (pH 7.4). In the experiments
simulating "in vitro ischemia" with the use of cyanide, glucose was
omitted (Duchen, 1990). The pipette was filled with an internal
solution containing 140 mM KCl, 2 mM MgCl2, 5 mM
EGTA-KOH, and 5 mM HEPES-KOH (pH 7.3). ATP (3 mM) and GTP (100 µM)
were added to the internal solution with the concentration of free
Mg2+ adjusted to 1.4 mM with reference to the
stability constants of the Mg/nucleotide complexes (Dawson et al.,
1986; Sigel, 1987), except in the experiments with cyanide. Stock
solutions of compounds were prepared as follows: 200 mM MCC-134 in
glacial acetic acid, 100 mM pinacidil in 0.1 M HCl, 90 mM diazoxide in
0.1 M NaOH, 100 mM tolbutamide in 0.1 M NaOH, and 10 mM glibenclamide
in dimethyl sulfoxide. These vehicles by themselves did not have any
significant effect on the whole-cell current of the nontransfected or
transfected HEK 293T cells at the maximum vehicle concentrations used
in this study (n = 5 for each). Drugs were diluted to
the desired concentrations in the control bathing solution. The maximum
final concentration of MCC-134 that could be dissolved under these
conditions was 100 µM. MCC-134 was a gift from Mitsubishi Chemical
Corporation, Research and Development Division (Yokohama, Japan; Fig.
1A). Pinacidil was purchased from
Research Biochemicals, Inc. (Natick, MA). ATP and glibenclamide were
obtained from Sigma Chemical Co. (St. Louis, MO). GTP, diazoxide,
tolbutamide, and NaCN were obtained from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan). Other chemicals and materials were obtained from
commercial sources.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effects of MCC-134 on SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2
Channel Whole-Cell Currents.
Figure 1 (B-D) shows the effect of
different concentrations of MCC-134 on the whole-cell current in HEK
293T cells expressing SUR1/Kir6.2, SUR2A/Kir6.2, or SUR2B/Kir6.2
channels, respectively. In these experiments, the cells were held at
60 mV. In the cells expressing SUR1/Kir6.2 channels, the sequential
application of 1, 10, and 100 µM MCC-134 was without an obvious
effect, except for slight depression of the basal cell current.
85 mV with 5.4 mM
extracellular K+ and were inhibited completely by
3 µM glibenclamide (data not shown; Okuyama et al., 1998).
Figure 1E depicts the relationship between the concentration of MCC-134
and the whole-cell SUR1/Kir6.2, SUR2A/Kir6.2, or SUR2B/Kir6.2 channel
currents. The currents induced by each concentration of MCC-134 were
normalized to that evoked by 100 µM pinacidil (SUR2A/Kir6.2 and
SUR2B/Kir6.2) or 300 µM diazoxide (SUR1/Kir6.2) in each cell. The
SUR1/Kir6.2 channel was not activated by MCC-134. In both SUR2A/Kir6.2
and SUR2B/Kir6.2 channels, glibenclamide-sensitive outward
K+ currents were activated by MCC-134 in a
concentration-dependent manner. The threshold concentration of MCC-134
to activate the SUR2A/Kir6.2 channel was ~3 µM. In SUR2A/Kir6.2
channels, the maximum current evoked by 30 µM MCC-134 was only
22 ± 4% (n = 5) of that induced by 100 µM
pinacidil. On the other hand, the maximum current produced by
SUR2B/Kir6.2 channels in response to 100 µM MCC-134 was 108 ± 17% (n = 5) of that evoked by 100 µM pinacidil. The
concentration-response relationship for MCC-134-activation of
SUR2A/Kir6.2 or SUR2B/Kir6.2 channels obtained from five different cells was fitted with the following modified Hill equation: Relative current = A/{1 + (K/[MCC-134])n}
where the relative current is that normalized with
reference to the current induced by 100 µM pinacidil in the same
cells, A is the maximum relative current induced by MCC-134,
K is the apparent dissociation constant of MCC-134,
[MCC-134] is the concentration of MCC-134, and n is the
Hill coefficient. The values of A, K, and n were
0.21, 8.5 µM, and 2.23 for the SUR2A/Kir6.2 channel and 1.09, 5.2 µM, and 1.09 for the SUR2B/Kir6.2 channel, respectively. We recently
reported that these values for pinacidil were 1.05, 9.8 µM, and 1.24 for the SUR2A/Kir6.2 channel and 1.03, 1.4 µM, and 1.42 for the
SUR2B/Kir6.2 channel, respectively (Shindo et al., 1998). Therefore,
MCC-134 showed almost the same potency and efficacy as pinacidil in
activating SUR2B/Kir6.2 channels but was much less efficient than
pinacidil in activating SUR2A/Kir6.2 channels.
Effects of MCC-134 on KCO- or NaCN-Induced Currents of SUR1/Kir6.2
Channel in Whole-Cell Configuration.
In Fig.
2, we examined the effects of MCC-134 on
the diazoxide- or NaCN-activated SUR1/Kir6.2 channel recorded at 30
mV. SUR1/Kir6.2 channels were fully activated by 300 µM diazoxide. MCC-134 added to the bath inhibited the diazoxide-induced SUR1/Kir6.2 channel currents in a concentration-dependent and reversible manner (Fig. 2A). In Fig. 2B, the SUR1/Kir6.2 channel current was induced by
the addition of NaCN (2 mM) to the glucose-free bathing solution. MCC-134 also reversibly inhibited the NaCN-induced channel activity. We
further examined the effects of nicorandil and pinacidil on the
NaCN-induced SUR1/Kir6.2 channel currents. Neither 1 mM nicorandil nor
100 µM pinacidil inhibited the K+ current
induced by NaCN in these cells (n = 3 for each drug; data not shown). Therefore, inhibition of the SUR1/Kir6.2 channel current seems to be specific to MCC-134. Figure 2C depicts the relationship between the concentration of MCC-134 and the relative SUR1/Kir6.2 channel activity. MCC-134 inhibited the channel currents induced by either 300 µM diazoxide or 2 mM NaCN in a similar
concentration-dependent fashion at the concentrations of 3 to 100 µM
(n = 5, for each).
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Effects of MCC-134 on KCO- or NaCN-Induced Currents of SUR2A/Kir6.2
and SUR2B/Kir6.2 Channels in Whole-Cell Configuration.
We further
examined the effects of MCC-134 on SUR2/Kir6.2 channels that had been
activated by KCOs or NaCN recorded at 30 mV (Fig.
3). Because the SUR2A/Kir6.2 channel
could not be activated by diazoxide, we used pinacidil for its
activation (Fig. 3A, a). Pinacidil (30 µM, Fig. 3A, a) and NaCN (2 mM, Fig. 3A, b) added to the bath effectively activated the
SUR2A/Kir6.2 channel. MCC-134 (100 µM) did not inhibit the pinacidil
or NaCN-induced currents, whereas 3 µM glibenclamide completely
inhibited the K+ currents. Similarly, MCC-134 did
not inhibit the diazoxide- or NaCN-induced SUR2B/Kir6.2 channels
current as shown in Fig. 3B, a and b.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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MCC-134 is a newly synthesized vasorelaxant agent (Seino et al.,
1996). We found that this agent effectively activates the KATP channel with smooth muscle type of SUR
(SUR2B/Kir6.2 channel) but inhibits the pancreatic type of
KATP channel (SUR1/Kir6.2 channel). Therefore,
this study clearly shows that a single compound can cause not only
activation but also inhibition of SUR/Kir6.2 channel activity in a SUR
subtype-dependent manner.
Because MCC-134 inhibited specifically the SUR1/Kir6.2 channel currents
induced by either diazoxide or NaCN but did not inhibit the fully
activated SUR2A or SUR2B/Kir6.2 channel currents, the inhibitory effect
of this agent may be mediated through its action on SUR1 and not on the
Kir6.2 pore. MCC-134 did not affect the NaCN-induced
K+ currents of HEK 293T cells expressing the
Kir6.2
C26 mutant alone, which show significant currents (Tucker et
al., 1997; data not shown). This is consistent with current theories
for the mechanism of KCO action, which suggest that the drugs interact
primarily with the SURs and not the Kir subunit of the
KATP channel (Hambrock et al., 1998;
Schwanstecher et al., 1998). Because the inhibitory effect of MCC-134
on the SUR1/Kir6.2 channel was the same when tested against
diazoxide-induced K+ currents and NaCN-induced
K+ currents, the inhibitory effect may not be due
to specific inhibition of the action of diazoxide. Therefore, MCC-134
seems to be directly affecting the function of SUR receptors and acts
as an inverse agonist for the SUR1/Kir6.2 channel (Milligan et al.,
1995), whereas it is a full agonist for the SUR2B/Kir6.2 channel and a
partial agonist for the SUR2A/Kir6.2 channel. Previously, it was shown that diazoxide, which activates native pancreatic
KATP channels, inhibits cardiac
KATP channels (Faivre and Findlay, 1989), which indicates that diazoxide is an agonist for SUR1 but an inverse agonist
for SUR2A.
This study also indicates the possibility of developing drugs whose
profiles are optimal to treat patients with certain diseases. For
example, because MCC-134 may possess hypoglycemic and vasodilatating actions, it may be useful to treat patients with noninsulin-dependent diabetes mellitus and hypertension. Because MCC-134 does not inhibit the cardiac type of KATP channel, it might also
be beneficial for the treatment of patients with noninsulin-dependent
diabetes mellitus by avoiding the cardiovascular complications
associated with sulfonylurea derivatives (Cleveland et al., 1997;
Garratt et al., 1999). Thus, MCC-134 may be a prototype of new drugs
acting selectively on different types of KATP channels.
SUR2A and SUR2B are splice variants generated from the same gene, and
they are both composed of 1546 amino acids differing only in the last
42 amino acid residues at their carboxyl-terminal ends (amino acids
1505-1546; Isomoto et al., 1996). It was recently shown that these 42 amino acid residues are important for determining the affinities of
KCOs in binding to SUR2 subtypes (Schwanstecher et al., 1998). In the
present study, half-activation of the whole-cell current by MCC-134
occurred at an ~10 µM concentration of the drug in both
SUR2A/Kir6.2 and SUR2B/Kir6.2 channels, whereas the evoked maximum
current of the SUR2A/Kir6.2 channel was ~20% of that of the
SUR2B/Kir6.2 channel. Therefore, like the case of nicorandil (Shindo et
al., 1998), it may be reasonable to suggest that the carboxyl-terminal
regions of SUR2A and SUR2B may play a critical role in regulating the
efficacy of MCC-134 to activate SUR2A/Kir6.2 and SUR2B/Kir6.2 channels.
We do not know whether the 42 amino acid residues of the carboxyl-terminal end of SUR1 are important for MCC-134 inhibition of the SUR1/Kir6.2 channel current. This region of SUR1 showed 74% identity with that of SUR2B but only 33% identity with that of SUR2A. Because MCC-134 activated both SUR2A/Kir6.2 and SUR2B/Kir6.2 channels, the carboxyl-terminal region may not be responsible for the inhibitory action of this agent. Further studies that include the construction and expression of various chimeras of SUR1 and SUR2s are needed to clarify the sites of interaction between MCC-134 and subtypes of SUR and the mechanisms by which these interactions regulate channel activity in positive as well as in negative ways.
In conclusion, MCC-134 may be useful to describe the molecular mechanism underlying the tissue specificity of various KCOs and regulatory mechanisms of KATP channels through distinct SURs. The specific properties of this agent also suggest that it may lead to the development of novel nonsulfonylurea-derivative hypoglycemic agents.
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Acknowledgments |
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We thank Dr. Ian Findlay (Tours, France) for critical reading of the manuscript. We also thank Mari Imanishi for technical assistance and Keiko Tsuji for secretarial support.
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Footnotes |
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Accepted for publication September 14, 1999.
Received for publication May 13, 1999.
1 This work was supported by the Research Grant for Cardiovascular Disease (IIC-I) from the Ministry of Health and Welfare; grants from the Ministry of Education, Science, Sports and Culture of Japan; the "Research for the Future" Program from The Japan Society for the Promotion of Science (JSPS-RFTF96L00302); and the Human Frontier Science Program (RG0158/1997-B).
Send reprint requests to: Dr. Yoshihisa Kurachi, Department of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ykurachi{at}pharma2.med.osaka-u.ac.jp
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Abbreviations |
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KATP channel, ATP-sensitive K+ channel; KNDP, NDP-sensitive K+ channel; KCO, K+ channel opener; HEK, human embryonic kidney; SUR, sulfonylurea receptor; Kir, inwardly rectifying K+ channel.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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cell high-affinity sulfonylurea receptor: A regulator of insulin secretion.
Science (Wash DC)
268:
423-426-cells, brain, heart and skeletal muscle.
FEBS Lett
377:
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), SUR2A/Kir6.2 (
), and SUR2B/Kir6.2 (
) channels. In cells
containing SUR2/Kir6.2 channels, the current amplitude induced by each
concentration of MCC-134 was normalized to the pinacidil (100 µM)-induced current in the same cells. Symbols and bars indicate the
mean and S.E. values, respectively. The number of the observations at
each point was four or five. The lines are the fit of the data with the
equation (see text) at concentrations between 0.1 and 100 µM.
