Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effects of intracellular nucleotide triphosphates on time-dependent changes in muscarinic receptor cation currents (Icat) were investigated using the whole cell patch-clamp technique in guinea pig ileal muscle. In the absence of nucleotide phosphates in the patch pipette, Icat evoked every 10 min decayed progressively. This decay was slowed dose dependently by inclusion of millimolar concentrations of ATP in the pipette. This required a comparable concentration of Mg2+, was mimicked by UTP and CTP, and was attenuated by simultaneous application of alkaline phosphatase or inhibitors of tyrosine kinase. In contrast, a sudden photolytic release of millimolar ATP (probably in the free form) caused a marked suppression of Icat. Submillimolar concentrations of GTP dose dependently increased the amplitude of Icat as long as ATP and Mg2+ were in the pipette, but, in their absence, GTP was ineffective at preventing Icat decay. The decay of Icat was paralleled by altered voltage-dependent gating, i.e., a positive shift in the activation curve and reduction in the maximal conductance. It is thus likely that ATP exerts two reciprocal actions on Icat, through Mg2+-dependent and -independent mechanisms, and that the enhancing effect of GTP on Icat is essentially different from that of ATP.
nucleotide phosphate; cation channels; muscarinic receptor; rundown
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATP IS INVOLVED IN A VARIETY of fundamental cellular reactions associated with, e.g., synthesis of many bioactive molecules, performance of mechanical work, and active and passive transport of molecules and ions. Modification of ionic channel activity by ATP is a widespread mode of regulating cellular functions. For instance, phosphorylation of channel protein and associated regulatory subunits by protein kinases leads to altered kinetics of activation, inactivation, or desensitization in many different types of channel (24). In some, ATP is required through its hydrolysis, whereas in others it inhibits the channel activity through direct interaction with intracellular sites [ATP-sensitive K+ channels (31, 34); voltage-dependent Ca2+ channels, (17, 28, 41); Ca2+-activated nonselective cation channels (33, 35); ion transporters and exchangers (10)].
The muscarinic receptor-activated cation channel (or current; Icat) has been found ubiquitously over the whole gastrointestinal tract and in chromaffin cells and some neuronal tissues (for review see Refs. 4 and 23). This channel is activated through a pertussis toxin-sensitive G protein (Gi/Go or Go) (14, 20, 37, 42) and undergoes effective regulation by the membrane potential (2, 12, 42), intracellular Ca2+ concentration ([Ca2+]i) (13, 29), and mechanical distortion (38).
The functional significance of Icat regulation by intracellular ATP and other high-energy phosphates has recently been investigated in some detail. In the guinea pig gastric muscle, phosphorylation of the muscarinic receptor by protein kinase C, Ca2+/calmodulin, and myosin light chain kinase has been implicated in desensitization and maintenance of Icat activity (18, 19, 21). In guinea pig ileal muscle, a strong enhancing effect of phosphocreatine and an inhibitory effect of ATP were observed on Icat, and the sites of these actions appeared to be downstream of the receptor (1). However, whether protein phosphorylation is involved in these effects remains equivocal, and, more importantly, the results of this study seem inconsistent in several critical points with those of previous studies including our own, particularly as to the actions of ATP, GTP, and Mg2+ (15, 42; for details see DISCUSSION). We reasoned that this may be due to problems with the protocol for evaluating Icat activity and the efficacy of internal perfusion, which depends critically on the properties of intracellular perfusates as well as the accessibility between the patch pipette and the cell interior (see below). Failure to allow for the complex interactions of metals (e.g., Mg2+, Ca2+) with their chelators [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ATP, GTP] in different concentrations and combinations might have also affected the results, since divalent cations are potentially effective regulators of Icat activity.
The goal of the present study was therefore twofold. First, we sought appropriate conditions for assessing the efficacy of intracellular perfusion on Icat. This was essential, since no single channel recording of Icat channels in the inside-out patch configuration has yet been published, due to their rapid rundown on patch membrane excision. Second, we reevaluated the roles of nucleotide phosphates such as ATP and GTP under these conditions. The results of our work indicate 1) that intracellular ATP has dual actions on Icat in a Mg2+-dependent and independent fashion and 2) that the intracellular GTP concentration is a critical determinant of Icat activity in a manner independent of ATP, although both nucleotide phosphates can modify the gating properties of Icat channels in a similar way.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Isolation
The procedures used for cell isolation are essentially the same as described previously (38). Guinea pigs of either sex (500-1,000 g) were and killed quickly by exsanguination. After opening the abdominal cavity, an ~5-cm-long segment of intestine (5-10 cm proximal from the ileocecal valve) was rapidly excised. A glass pole (10 cm in length and ~2 mm in diameter) was inserted into the segment, which was tied onto it at both ends with a silk thread, and incubated successively in nominally Ca2+-free Krebs solution and a solution containing 1.5 mg/ml collagenase (Sigma type I) at 35°C for 5 and 20 min. Thereafter, the digested segment was cut open and thoroughly rinsed with Krebs solution (1 mM Ca2+ added) and stored in a refrigerator until use. Just before each experiment, the longitudinal muscle layer was peeled off from the remainder of ileum using two fine forceps and minced into small pieces. Single cells were mechanically dispersed by agitating these pieces using a blunt tipped Pasteur pipette.The temperature of the superfusing solution was strictly controlled
using a commercial warmer unit (TC-344B; Warner Instruments; accuracy
±0.5°C). This was necessary because the activity of
Icat is profoundly affected by the ambient
temperature, as shown in Fig.
1C. All experiments
were performed at 25°C, unless otherwise stated.
|
Electrophysiology
The system used for patch-clamp experiments was essentially the same as described previously (38). Cell capacitance (56.7 ± 0.7 pF, n = 270) and 50-80% of series resistance (or access resistance Ra; 10.7 ± 0.2 M[Ca2+]i was clamped close to a resting value
typical for gut smooth muscle cells (~100 nM; e.g., Ref. 29) to
minimize the influence of changes in [Ca2+]i
on Icat (13, 29). A mixture of 10 mM BAPTA and 5 mM Ca2+ was used for this purpose, since
BAPTA is superior to EGTA in buffering
[Ca2+]i to an almost constant level with
varying concentrations of nucleotide phosphates and Mg2+
(see Table
1).
|
To minimize the desensitization of Icat, the time of carbachol (CCh) application was limited to 20 s (which was, however, long enough to evoke the maximum response) and a recovery interval of 10 min was used, which were empirically determined according to the results of nystatin-perforated recordings. The amplitude of Icat was normalized by cell capacitance to eliminate variations arising from different cell sizes. Care was also taken to minimize the tissue-to-tissue variation in Icat activity, by comparing data between cells from the same batch for the same series of experiments.
Flash Photolysis
Caged ATP (8 mM) was introduced into the cell via a patch pipette, and brief control applications of CCh (100 µM) were made twice in the dark. A high-pressure mercury lamp (intensity 320 mW/10 mm2 at 360 nm) was used to generate ultraviolet (UV) light, which was applied through an electronic shutter (HB-10103AF; Nikon, Tokyo, Japan) controlled by a voltage pulse generator (SEN-7103; Nihon Kohden). Because the flash intensity of 20 mJ/10 mm2 at 347 nm causes ~40% photolysis of 5 mM caged ATP in vitro, we assumed that 200 ms flash duration would be sufficiently long to degrade 8 mM caged ATP almost completely.Solution
Table 1 gives the detailed composition of internal solutions used in the present study, where free and bound concentrations of nucleotides, Mg2+, and Ca2+, except for test substances, are kept as constant as possible (3, 9). Bath solution [physiological salt solution (PSS)] contained (in mM) 140 Na+, 6 K+, 1.2 Mg2+, 2 Ca2+, 151.4 ClChemicals
ATP, GTP, UTP, CTP, ADP, AMP, and caged ATP {P3-[1-(2-nitrophenyl)ethyl]adenosine-5'-trisphosphate, trisodium salt} were purchased from Dojin, 5'-adenylylimidodiphosphate (AMP-PNP), alkaline phosphatase, calphostin C, H-7, KN-62, and okadaic acid were from Calbiochem, and FK506 was a kind gift of Dr. H. Onoue (Department of Pharmacology, Kyushu University, Fukuoka, Japan).Statistics
All results are expressed as means ± SE. Statistical significance of differences between given sets of data were evaluated by Student's t-test for single comparison and one-way ANOVA or Dunnett's tests for multiple comparison. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Factors Affecting Icat Activity
Efficacy of internal perfusion.
When single guinea pig ileal smooth muscle cells were intracellularly
perfused with Cs+ via a patch pipette, full development of
voltage-dependent Ca2+ current was observed within a few
minutes after establishment of the whole cell configuration (data not
shown; cf. Ref. 22; ~2 min in urinary bladder). Similarly,
Ca2+ chelators such as EGTA and BAPTA included in the patch
pipette rapidly abolished a Ca2+-dependent component of
outward rectifying K+ current (data not shown). These
results strongly suggest that, despite the spindle-shaped geometry of
ileal smooth muscle cells, equilibration of these substances between
the pipette and cytosol was fast and comparable to simple diffusion
(30). In contrast, when nucleotide phosphates were omitted
from the pipette solution, the amplitude of Icat
changed with a much slower time course. For example, without ATP in the
pipette, >20 min were needed for Icat to decay
almost completely after the start of whole cell configuration (Fig.
1A; see also Fig.
2Ab). The rate of
Icat decay was significantly slower in cells
having large Ra values (>20 M), compared
with those with smaller Ra values (<15 M
; Fig. 1A). Accordingly, the Icat density 30 min after internal perfusion was larger as the value of
Ra increased (closed circles and dotted line in
Fig. 1B). In contrast, no clear correlation was found between Ra and Icat
density for the first application of CCh, when the cell had already
been dialyzed for 10 min (open circles and solid line in Fig.
1B). Essentially, the same observations were obtained when
Mg2+ instead of ATP was eliminated from the pipette
solution (data not shown).
|
Influence of ambient temperature.
The temperature of the bathing solution also critically affected
Icat activity. As shown in Fig. 1C, a
fall in temperature from 25 to 15°C caused an about threefold
reduction (i.e., Q10 3.0) in
Icat amplitude. This means that
Icat activity is highly susceptible to changes
in the ambient temperature, which could fluctuate by >10°C within 1 day or between different seasons.
MgATP Complex Maintains Icat in Guinea Pig Ileal Smooth Muscle Cells
Figure 2A shows representative time courses of Icat evoked every 10 min by 100 µM CCh, with (a) and without (b) 10 mM ATP and a comparable concentration of Mg2+ in the patch pipette. Although the initial amplitude of Icat was similar, it decayed rapidly when there was no ATP in the pipette, but, in its presence, the amplitude of Icat remained almost constant over a period of 30 min. The rapid decay of Icat in the absence of ATP was usually accompanied by progressive cell contracture, even though the cell was initially relaxed and dialyzed with a high concentration of Ca2+ buffer (10 mM BAPTA).Figure 2B summarizes data pooled from 8-10 similar
experiments, with respect to 10, 1, and 0 mM ATP included in the
pipette (Mg2+ present). Single exponential fitting of data
revealed that the time course of Icat decay was
significantly retarded by increasing the concentration of ATP in the
pipette ([ATP]i). The apparent time constant estimated
from the averaged Icat decay was 7.6 min for 0 mM ATP, and this value was increased to 24.5, 32.8, and >50 min for 1, 2, and 10 mM ATP, respectively. Correspondingly, Icat density 30 min after the start of internal
perfusion became significantly larger with higher [ATP]i
(Fig. 3A; P < 0.05 with one-way ANOVA).
|
The retarding effect of intracellular ATP on Icat decay is likely to be associated with the concentration of the MgATP complex rather than that of free ATP. First, under conditions in which the concentrations of Mg2+, Ca2+, and the free and bound forms of GTP were kept almost constant, the calculated [MgATP] is well correlated with Icat density 30 min after internal perfusion (Fig. 3A and Table 1). Second, reducing the Mg/ATP ratio while keeping the concentration of the MgGTP complex at a similar level (10ATP/2Mg) or rigorous chelation of Mg2+ by 2 mM EDTA (10ATP/2EDTA) resulted in a marked reduction in Icat density (Fig. 3B; P < 0.05 with Dunnett's test).
We also tested the ability of other nucleotide phosphates such as CTP, UTP, GTP, GDP, ADP, and AMP to maintain Icat. Among them, CTP and UTP exhibited a comparable efficacy to ATP (Mg2+ required) in prolonging the time course of Icat decay (time constants >50 and 49.0 min, respectively; Fig. 3C). In contrast, GTP, GDP (not shown), ADP, and AMP were all virtually ineffective at preventing Icat decay when added alone in the pipette (Fig. 3C; P < 0.01 with Dunnett's test). These results indicate that the effect of ATP is not mediated by its metabolites such as ADP and AMP or through its conversion to GTP by adenylyl transferase.
ATP Hydrolysis Is a Prerequisite
The requirement of the MgATP complex for maintaining Icat activity suggests that some cellular process(es) that utilizes the hydrolytic energy of ATP may be involved. To test this, we performed the next series of experiments and obtained the following results (Fig. 3D).First, the poorly hydrolyzable analog of ATP, AMP-PNP (2 mM), was
totally ineffective at preventing Icat decay.
Second, internal perfusion of the thiophosphate analog of ATP,
adenosine 5'-O-(3-thiotriphosphate) (ATPS; 2 mM),
resulted in induction of a small inward current by itself and sustained
activation of Icat in response to subsequently applied CCh (Fig. 3D; see also the
inset). Third, simultaneous inclusion of alkaline
phosphatase (100 µg/ml) with ATP in the pipette significantly reduced
Icat density 30 min after the start of internal
perfusion. Fourth, no significant difference in
Icat density was observed between control cells
and those perfused with a mixture of inhibitors for A, C, and G kinases
and calmodulin-dependent kinase II (30 µM H-7, 5 µM KN-62, and 1 µM calphostin C). Fifth, two mechanistically distinct tyrosine kinase
inhibitors, genistein (50 µM; Fig. 3D) and tyrphostin A-25
(not shown), greatly reduced Icat density
(16). Sixth, okadaic acid and FK506, at concentrations known to block types 1, 2A, and 2B phosphatases (2.5 and 3 µM), or 30 µM calmodulin, which has been reported to prevent the rundown of
Icat in guinea pig stomach (18),
did not significantly alter the effects of 2 mM ATP alone.
These results collectively suggest that ATP hydrolysis is essential for maintaining Icat activity and that tyrosine phosphorylation may play an integral role.
Inhibitory Effect of ATP on Icat
It has previously been reported that the Icat channel is subject to ATP-mediated inhibition (1). This is consistent with our present observation that Icat density, evaluated on a statistical basis, was significantly smaller with 10 mM ATP and 2 mM Mg2+ (10ATP/2Mg) than with 2 mM ATP and 2.5 mM Mg2+ (2ATP; Fig. 3, A and B; P < 0.05 with unpaired t-test), where the concentration of free ATP is ~13 times higher in the former than the latter, while [MgATP] and [MgGTP] are almost the same (Table 1). To confirm this possible inhibitory effect of free ATP on Icat more directly, we examined the effects of photolytically released ATP on the time course of Icat in the same cell.As demonstrated in Fig. 4, UV flashes of
a maximally effective duration (200 ms; see METHODS)
produced little effect on Icat evoked by the
continued application of CCh (100 µM), when caged ATP was not
included in the pipette (2ATP*, Fig. 4A). In contrast, when
8 mM caged ATP was present, a single 200-ms flash, which is expected to
increase free ATP concentration from 0.68 to ~4 mM (compare 2ATP* and
10ATP/2Mg in Table 1 for an estimated change in [ATP] before and
after photolysis), caused a marked reduction in
Icat amplitude (Fig. 4B; time
constant = 10.0 ± 2.0 s, n = 15). A
subsequently applied flash was no longer effective. The effect of
photolysis is unlikely to be due to the release of photoproducts other
than ATP (i.e., iminodiacetic acid), since uncaging of caged inositol
trisphosphate, which had the same caging moiety
(nitrophenylethyl ester), did not affect Icat
(data not shown). As summarized in Fig. 4D, an ~50%
inhibition of Icat occurred on photolysis of 8 mM caged ATP (2ATP* + 8 caged ATP). Remarkably, the inhibitory effect
of photolysis was almost abolished, when free ATP concentration had
already been raised to ~4 mM (10ATP/2Mg + 8 caged ATP; Fig. 4D; see also Table 1). These results strongly suggest that
free ATP rather than MgATP is responsible for this inhibition, which would saturate at free ATP concentrations of several millimolar. The
degree of inhibitory effect of photolytically released ATP was not
significantly changed when Icat was activated by
internally applied guanosine 5'-O-(3-thiotriphosphate)
(GTP
S; 50 µM) with inhibitors of protein kinases A, C, and G and
calmodulin kinase II, H-7 (30 µM), calphostin C (1 µM),
and KN-62 (5 µM) (Fig. 4, C and D). These
results suggest that the inhibitory effect of ATP may be exerted in a
nonenzymatic fashion, presumably by acting directly on the
Icat channel protein.
|
Intracellular GTP Potentiates Icat But Does Not Affect Its Slow Time-Dependent Decay
It has been suggested that persistent stimulation of muscarinic receptor in the presence of CCh causes progressive desensitization of Icat due mainly to depletion of intracellular GTP (43). We therefore investigated how intracellular GTP concentration ([GTP]i) affects the time-dependent decay of Icat evoked intermittently by brief application of CCh, while keeping [MgATP] high enough to maintain Icat (note that MgGTP itself was ineffective; see Fig. 3C) and free ATP concentration, [Mg2+], and [Ca2+] as constant as possible (see Table 1) to exclude their indirect influence on Icat activity. When [GTP]i was lowered to <1 mM (open circles and squares), the amplitude (or density) of Icat decreased rapidly toward a steady level, although the initial Icat density was similar regardless of [GTP]i (Fig. 5A; P > 0.05 with one-way ANOVA). The time course of this decrease was not slowed by increasing [GTP]i (time constant of decay: 4.3 and 5.8 min for 0.01 and 0.1 mM GTP, respectively) and appeared to be faster than that of Icat decay observed with low [ATP]i (7.6 and 24.5 min for 0 and 1 mM ATP, respectively; Fig. 2B), suggesting that different mechanisms may be involved. At the steady phase (30 min after internal perfusion), Icat density was an incremental function of [GTP]i, with an apparent EC50 value of ~100 µM (Fig. 5B; P < 0.05 with one-way ANOVA). No further significant increase or decrease was observed at GTP concentrations >1.2 mM and <0.01 mM, respectively (P > 0.05 with pooled variance t-test).
|
These results clearly indicate essential differences in the actions of ATP and GTP on Icat; intracellular GTP critically controls the steady-state activity of Icat but cannot prevent the time-dependent decay of Icat (see Fig. 2B), and vice versa for ATP.
Altered Receptor Sensitivity May Only in a Minor Way Contribute to Icat Decay
Time-dependent decay of agonist-induced responses has often been ascribed to reduced receptor sensitivity (25). However, as illustrated and summarized in Fig. 6, A and B, respectively, the relationship between CCh concentration and Icat amplitude was only modestly changed during the course of Icat decay. The apparent dissociation constant for CCh evaluated empirically by Hill analysis increased only slightly during the progression of internal perfusion (5 and 15 µM at 10 and 50 min, respectively). These results suggest that reduced receptor sensitivity, albeit present, would contribute only in a minor way to the time-dependent decay of Icat.
|
Icat Decay Parallels the Negative Shift in Half-Activation Voltage and Reduction in Maximal Conductance
In the next series of experiments, we examined what changes occur in the properties of voltage-dependent gating of Icat during the course of Icat decay. Typical examples of current-voltage (I-V) relationships and steady-state activation curves at 10, 20, 40, and 60 min are displayed for a case in which 2 mM ATP was present in the pipette (2ATP; Fig. 7, A and B). As the decay proceeded, the peak of the U-shaped I-V curve was shifted positively, and correspondingly the half-activation voltage (Vh) and maximal conductance (Gmax) became more positive and smaller, respectively. In contrast, the reversal potential and the slope factor of the Boltzmann curve remained almost unchanged (Fig. 7, A and D). Figure 7, C and D, summarizes this type of experiment from eight different cells. Forty minutes after the onset of internal perfusion, Vh increased by ~25 mV and Gmax decreased to ~0.4 of the initial value (open symbols in Fig. 7C). The extent of reduction in Icat density expected from these changes (about one-fourth or one-third of the initial value at 40 min) was comparable to the observed decrease in Icat density during the decay (see, e.g., 1ATP in Fig. 2B). In contrast, only slight changes occurred in Vh and Gmax when 10 mM ATP was present in the pipette (10ATP; closed symbols in Fig. 7C). These results strongly indicate that the main part of Icat decay can be accounted for by altered voltage-dependent gating and reduced maximal conductance of the channels underlying Icat.
|
Icat Activated Bypassing the Muscarinic Receptor Shows Similar Characteristics
Finally, we investigated how the time course of Icat decay would be affected, if the current is activated bypassing the receptor. This was achieved by internal perfusion of 100 µM GTP
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study has clearly indicated that, in addition to
Ra, the perfusates and the protocol of receptor
stimulation critically affect the behavior of
Icat when recorded via a single patch electrode. This is supported by the findings that 1) the effects of
internally perfused ATP and GTP on Icat occurred
with a considerably slower time course than those of Cs+
loading on voltage-dependent Ca2+ current or
Ca2+ chelation on Ca2+-dependent K+
current, despite a similar rate of aqueous diffusion (time constant: 1-2 min) for ATP, GTP, Cs+, and BAPTA [a cell volume
of 4.1 pl (13) and Ra of 10.7 M were assumed; refer to the equation in Ref. 30]; and 2) the effects of internally applied ATP or GTP on Icat
became evident only after repeated receptor stimulation and were not
recognizable at the first application of CCh (Figs. 1, 2, and
5A), while internal perfusion of Cs+ and
Ca2+ buffers were effective. One possible explanation for
such slowly appearing and stimulation-dependent effects of ATP and GTP
(and presumably other nucleotides too) would be an extremely slow
equilibration between the patch pipette and the subsarcolemmal space
underneath Icat channels. This might be due to
the presence of intervening ATPases, kinases, high-affinity binding
proteins (e.g., actin filaments), and intracellular pools sequestrating
nucleotide phosphates (6). Endogenous nucleotide
phosphates tightly bound to intracellular proteins and vigorously
produced in the vicinity of the Icat channel might account for the requirement of repeated receptor stimulation to
uncover the effects of exogenously applied nucleotide phosphates. Whatever mechanisms are involved, it seems reasonable to say that data
based on a single agonist application after a short internal perfusion
time should be interpreted with great caution, since they may not
faithfully reflect the real effects of internally applied nucleotide phosphates.
There are indeed some essential differences found in the effects of
nucleotide phosphates between our results and those of a similar study
in the same preparation where only the first response to CCh 5 min
after internal perfusion was evaluated (1). According to
this study 1) the stimulatory effect of internally applied high-energy phosphates on Icat was exclusively
specific for phosphocreatine (ATP was almost ineffective) and did not
require Mg2+ (but creatine together with a few millimolar
ATP and Mg2+ is effective); 2) internal
perfusion of GTP or total elimination of nucleotide phosphates and
Mg2+ from the patch pipette did not affect
Icat activity; 3) the inhibitory effect of ATP seemed inconsistent and the involvement of hydrolysis and
the need of Mg2+ equivocal. These results are not easily
interpretable using the standard knowledge about ATP, GTP, and other
high energy phosphates. In contrast, our present data clearly indicate
that ATP exerts (and also phosphocreatine; Ref. 40) a stimulatory or
maintaining effect on Icat in a manner requiring
Mg2+. This effect was dose dependent on [MgATP] and also
occurred with CTP or UTP, which are convertible to ATP by the action of nucleotide diphosphate kinase. ATPS, which thiophosphorylates protein targets, prolonged activation of Icat,
while a nonhyrdolyzable ATP analog AMP-PNP was totally ineffective.
Furthermore, alkaline phosphatase, which nonspecifically
dephosphorylates proteins, greatly attenuated the maintaining effect of
ATP. All these data are compatible with the idea that ATP hydrolysis is
a prerequisite for maintaining Icat activity,
and the observed notable dependence of Icat on
temperature also supports this idea.
The pharmacology of kinase and phosphatase inhibitors (Fig.
3D) suggests that the main part of the maintaining effect of
MgATP on Icat may be mediated by phosphorylation
of tyrosine residues tightly associated with the
Icat activity (16). Protein kinases A, G, or C, and calmodulin kinase II are unlikely to be involved in
this (Fig. 3D), although they are frequently found as
effective modulators of ion channels and receptors coupling to them
(10, 21, 24). The type of tyrosine kinase/phosphatase
responsible for maintaining Icat activity
remains elusive. However, one plausible candidate is the nonreceptor
type tyrosine kinase Src, which is abundantly expressed in various
types of smooth muscle and is thought to play a pivotal role in
regulating the Ca2+ sensitivity of the contractile
machinery (7, 32). Interestingly, Src has been implicated
in the G protein/protein kinase C-mediated regulation of
N-methyl-D-aspartate cation channels by
muscarine and lysophosphatidic acids in some neuronal tissues, where
coimmunoprecipitation of the channel protein and Src has also been
demonstrated (27, 36). It is also interesting to note that
a similar dependence on intracellular MgATP is reported for the cardiac
and islet -cell ATP-sensitive K+ channels as well as
L-type voltage-dependent Ca2+ channels in cardiac and
smooth muscle cells (see Introduction), both of which exhibit a
time-dependent decline without MgATP that can preferentially be
reversed by glycolytic production of ATP (26, 39). Because
these properties appear to be shared by Icat
channels (40), it may be profitable to investigate the roles of cytoskeletal elements and phosphatidylinosities on
Icat activity, both of which are known to alter
the rate of rundown or the availability of ATP-sensitive K+
and voltage-dependent Ca2+ channels (11, 31).
Comparison of whole cell data with different free ATP concentration
values has suggested that the free form of ATP may inhibit Icat (2ATP vs. 10ATP/2Mg in Fig. 3). This was
further corroborated by the experiments using flash photolysis of caged
ATP (Fig. 4). The degree of ATP-mediated Icat
inhibition was not significantly affected when the current was
activated by internally applied GTPS in the presence of various
protein kinase inhibitors. This suggests that enzymatic reactions may
not be involved in the inhibitory effect of ATP and that its target
site is located downstream of the muscarinic receptor, presumably on
the Icat channel protein. However, the observed
time course of Icat inhibition by photolytically released ATP seems rather slow (time constant
10 s) when
considering direct actions. One likely explanation is limited diffusion
of ATP through the mechanisms discussed above, which may be valid even
in the close proximity of the Icat channel. Thus
the most likely mechanism involved in the inhibitory effect of ATP is
direct inhibition of Icat channels, as has been
suggested for the ATP-sensitive K+ channels and
Ca2+-activated nonselective cation channels (31, 33,
34). However, more robust evidence should be provided under
experimentally simpler conditions (e.g., single channel recordings).
The dependence of Icat on intracellular GTP concentration has been investigated in some detail previously (42, 43). In these studies, the concentration of activated G protein, which is subject to the intensity of receptor stimulation or the extent of desensitization, has been implicated as a critical determinant of voltage-dependent gating and maximal conductance of Icat channels. We quantified more strictly the enhancing effect of GTP on Icat under the conditions in which [MgATP], [ATP], [Mg2+], and [Ca2+] were kept almost constant, and we found that activation of the Icat channel by GTP occurred most noticeably in its hundred micromolar range (apparent Kd = ~100 µM; Fig. 5B).
Kinetic changes induced by GTP (43) are unexpectedly very similar to those observed for MgATP in the present study (Fig. 7). Both nucleotide phosphates cause a negative shift in the activation curve and an increase in the maximal conductance of Icat (Fig. 7) (43). Nevertheless, the effects of GTP and ATP are clearly distinguishable. GTP itself failed to slow the decay of Icat (Fig. 5A) but increased Icat density after a sufficiently long period of internal perfusion, if millimolar concentrations of MgATP were present (Fig. 5B). In contrast, ATP dose-dependently slowed Icat decay (Fig. 2B) but was not able to enhance steady-state Icat density if the concentration of coexisting GTP was low (Fig. 5B). It is thus likely that the main effect of GTP is to determine the extent of activation of Icat channels by changing the activated concentration of a G protein during receptor stimulation (42), whereas that of MgATP is to prime Icat channels for opening, presumably through a phosphorylation-dependent but G protein-independent mechanism. Such effects of ATP are highly analogous to those for the ATP-sensitive K+ channels (8) and thus will deserve further investigation at a single-channel level.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. A. F. Brading, Department of Pharmacology, University of Oxford, UK, for English editing.
![]() |
FOOTNOTES |
---|
This work was supported in part by a grant-in-aid to R. Inoue from the Ministry of Education and Culture, Japan.
Part of the present work was presented at an International Smooth Muscle Symposium held in Korea (16).
Address for reprint requests and other correspondence: R. Inoue, Dept. of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan (E-mail: inouery{at}pharmaco.med.kyushu-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 November 1999; accepted in final form 17 May 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bakhramov, A.
Effects of high-energy phosphates on carbachol-evoked cationic current in single smooth muscle cells from guinea-pig ileum.
J Physiol (Lond)
485:
659-669,
1995[Abstract].
2.
Benham, CD,
Bolton TB,
and
Lang RJ.
Acetylcholine activates an inward current in single mammalian smooth muscle cells.
Nature
328:
275-278,
1985.
3.
Brooks, SPJ,
and
Storey KB.
Bound and determined: a computer program for making buffers of defined ion concentrations.
Anal Biochem
201:
119-126,
1992[ISI][Medline].
4.
Carl, A,
Lee HA,
and
Sanders KM.
Regulation of ion channels in smooth muscles by calcium.
Am J Physiol Cell Physiol
271:
C9-C34,
1996
5.
Chen, S,
Inoue R,
and
Ito Y.
Pharmacological characterization of muscarinic receptor-activated cation channels in guinea-pig ileum.
Br J Pharmacol
109:
793-801,
1993[Abstract].
6.
Detimary, P,
Jonas JC,
and
Henquin JC.
Stable and diffusible pools of nucleotides in pancreatic islet cells.
Endocrinology
13:
4671-4676,
1996.
7.
Di Salvo, J,
Pfitzer G,
and
Semenchuk LA.
Protein tyrosine phosphorylation, cellular Ca2+, and Ca2+ sensitivity for contraction of smooth muscle.
Can J Physiol Pharmacol
7:
1434-1439,
1994.
8.
Edwards, G,
and
Weston AH.
The pharmacology of ATP-sensitive potassium channels.
Annu Rev Physiol
33:
597-637,
1993.
9.
Fabiato, A,
and
Fabiato F.
Calculation programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Paris)
75:
463-505,
1979[Medline].
10.
Hilgenmann, DW.
Cytoplasmic ATP-dependent regulation of ion transporters and channels.
Annu Rev Physiol
59:
193-220,
1997[ISI][Medline].
11.
Huang, C-L,
Feng S,
and
Hilgemann DW.
Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by G.
Nature
391:
803-806,
1998[ISI][Medline].
12.
Inoue, R,
and
Isenberg G.
Effects of membrane potential on acetylcholine-induced inward currents in guinea-pig ileum.
J Physiol (Lond)
424:
57-71,
1990[Abstract].
13.
Inoue, R,
and
Isenberg G.
Intracellular calcium ions modulate acetylcholine-induced inward current in guinea-pig ileum.
J Physiol (Lond)
424:
73-92,
1990[Abstract].
14.
Inoue, R,
and
Isenberg G.
Acetylcholine activates nonselective cation channels in guinea-pig ileum through a G-protein.
Am J Physiol Cell Physiol
258:
C1173-C1178,
1990
15.
Inoue R and Waniishi Y. Critical requirement of phosphorylation
for the regulation of receptor-operated nonselective cation channels in
smooth muscle. Proc Int Symp Smooth Muscle Seoul, Korea
1995, p. 46-51.
16.
Inoue, R,
Waniishi Y,
Yamada K,
and
Ito Y.
A possible role of tyrosine kinases in the regulation of muscarinic receptor-activated cation channels in guinea-pig ileum.
Biochem Biophys Res Commun
203:
1392-1397,
1994[ISI][Medline].
17.
Irisawa, H,
and
Kokubun S.
Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea-pig.
J Physiol (Lond)
338:
321-337,
1983[Abstract].
18.
Kim, SJ,
Ahn SC,
So I,
and
Kim KW.
Role of calmodulin in the activation of carbachol-activated cationic current in guinea-pig gastric antral myocytes.
Pflügers Arch
430:
757-762,
1995[ISI][Medline].
19.
Kim, YC,
Kim SJ,
Kang TM,
Suh SH,
So I,
and
Kim KW.
Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes.
Pflügers Arch
434:
346-353,
1997[ISI][Medline].
20.
Kim, YC,
Kim SJ,
Sim JH,
Cho CH,
Juhann Y-S,
Suh SH,
So I,
and
Kim KW.
Suppression of the carbachol-activated nonselective cationic current by antibody against alpha subunit of Go protein in guinea-pig gastric myocytes.
Pflügers Arch
436:
494-496,
1998[ISI][Medline].
21.
Kim, YC,
Kim SJ,
Sim JH,
Jun JY,
Kang TM,
Suh SH,
So I,
and
Kim KW.
Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes.
Pflügers Arch
436:
1-8,
1998[ISI][Medline].
22.
Klöckner, U,
and
Isenberg G.
Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea-pig).
Pflügers Arch
405:
340-348,
1985[ISI][Medline].
23.
Kuriyama, H,
Kitamura K,
Itoh T,
and
Inoue R.
Physiological features of visceral smooth muscle cells, with special reference to receptors and ion channels.
Physiol Rev
78:
811-920,
1998
24.
Levitan, IB.
Modulation of ion channels by protein phosphorylation and dephosphorylation.
Annu Rev Physiol
56:
193-212,
1994[ISI][Medline].
25.
Lohrse, MJ.
Molecular mechanisms of membrane receptor desensitization.
Biochim Biophys Acta
1179:
171-188,
1993[ISI][Medline].
26.
Lorenz, JN,
and
Paul RJ.
Dependence of Ca2+ channel currents on endogenous and exogenous sources of ATP in portal vein smooth muscle.
Am J Physiol Heart Circ Physiol
272:
H987-H994,
1997
27.
Lu, WY,
Xiong ZG,
Lei S,
Orser BA,
Dudek E,
Browning MD,
and
MacDonald JF.
G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors.
Nat Neurosci
2:
331-338,
1999[ISI][Medline].
28.
O'Rouke, B,
Backx PH,
and
Marban E.
Phosphorylation-independent modulation of L-type calcium channels by magnesium-nucleotide complexes.
Science
257:
245-248,
1992[ISI][Medline].
29.
Pacaud, P,
and
Bolton TB.
Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells.
J Physiol (Lond)
441:
477-499,
1991[Abstract].
30.
Pusch, M,
and
Neher E.
Rates of diffusional exchange between small cells and a measuring patch pipette.
Pflügers Arch
411:
204-211,
1988[ISI][Medline].
31.
Quayle, JM,
Nelson MT,
and
Standen NB.
ATP-sensitive and inwardly rectifying potassium channels in smooth muscle.
Physiol Rev
77:
1165-1232,
1997
32.
Steusloff, A,
Paul E,
Semenchunk LA,
Di Salvo J,
and
Pfitzer G.
Modulation of Ca2+ sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation.
Arch Biochem Biophys
320:
236-242,
1995[ISI][Medline].
33.
Sturgess, NC,
Hales CN,
and
Ashford MLJ
Inhibition of a Ca-activated, nonselective cation channel, in rat insulinoma cell line, by adenine derivatives.
FEBS Lett
208:
397-400,
1986[ISI][Medline].
34.
Terzic, A,
Jahangir A,
and
Kurachi Y.
Cardiac ATP-sensitive K+ channels: regulation by intracellular nucleotides and K+ channel-opening drugs.
Am J Physiol Cell Physiol
269:
C525-C545,
1995[Abstract].
35.
Thorn, P,
and
Petersen OH.
Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.
J Gen Physiol
100:
11-25,
1992[Abstract].
36.
Wang, YT,
and
Salter MW.
Regulation of NMDA receptors by tyrosine kinases and phosphatases.
Nature
369:
233-235,
1994[ISI][Medline].
37.
Wang, Y-X,
Fleischmann BK,
and
Kotlikoff MI.
M2 receptor activation of nonselective cation channels in smooth muscle cells: calcium and Gi/Go requirements.
Am J Physiol Cell Physiol
273:
C500-C508,
1997
38.
Waniishi, Y,
Inoue R,
and
Ito Y.
Preferential potentiation by hypotonic cell swelling of muscarinic cation current in guinea-pig ileal smooth muscle.
Am J Physiol Cell Physiol
272:
C240-C253,
1997
39.
Weiss, JN,
and
Lamp ST.
Glycolysis preferentially inhibits ATP-sensitive K+ channels in isolated guinea-pig myocytes.
Science
238:
67-69,
1987[ISI][Medline].
40.
Yanagida, H,
Inoue R,
and
Ito Y.
Glycolytic production of ATP rather than phosphocreatine-mediated energy delivery is a critical determinant of cationic channel activity of the muscarinic receptor in guinea-pig ileal smooth muscle.
Jpn J Pharmacol
73, Suppl I:
54P,
1997.
41.
Yazawa, K,
Kameyama A,
Yasui K,
Li J-M,
and
Kameyama M.
ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation.
Pflügers Arch
433:
557-562,
1997[ISI][Medline].
42.
Zholos, AV,
and
Bolton TB.
G-protein control of voltage dependence as well as gating of muscarinic metabotropic channels in guinea-pig ileum.
J Physiol (Lond)
478:
195-202,
1994[Abstract].
43.
Zholos, AV,
and
Bolton TB.
A novel GTP-dependent mechanism of ileal muscarinic metabotropic channel desensitization.
Br J Pharmacol
119:
997-1005,
1996[Abstract].