Nonselective cation channel as a Ca2+ influx
pathway in pepsinogen-secreting cells of bullfrog esophagus
Seiichiro
Kimura1,
Hiroshi
Mieno2,
Kenji
Tamaki3,
Masaki
Inoue4, and
Kazuaki
Chayama5
1 Saiseikai Kure Hospital, Kure City, Hiroshima 737-0821;
2 Hiroshima General Hospital of the West Railroad Company,
Hiroshima City, Hiroshima 732-0057; 3 Chugoku Rosai
Hospital, Kure City, Hiroshima 737-0193; and 4 Department of
Geriatric Medicine and First 5 Department of Internal Medicine,
Institute of Health Sciences, Hiroshima University School of
Medicine, Hiroshima 734-8551, Japan
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ABSTRACT |
In
pepsinogen-secreting cells of bullfrog (Rana catesbeiana),
recent evidence suggests that Ca2+ release from internal
stores followed by Ca2+ influx across the plasma membrane
elicits pepsinogen secretion. Such a Ca2+ influx could be
carried by a background current, potentiated by bombesin, that was
found in these cells using the whole cell patch-clamp technique. The
permeability ratio of
Cs+-Rb+-K+-Na+-Li+-N-methyl-D-glucamine+-Ca2+
was 1.01:1:1:0.86:0.72:0.54:0.34. The current was almost totally blocked by the nonselective cation channel blockers La3+
(0.1 mM) and Gd3+ (0.1 mM) and was activated by
intracellular Ca2+. These properties demonstrated that the
current, which was activated by bombesin, was a nonselective cation
current. At the same time, Gd3+ suppressed pepsinogen
secretion by 29 ± 5.6% in isolated pepsinogen-secreting glands.
These results are in accord with the idea that a nonselective cation
channel in pepsinogen-secreting cells plays a role as a Ca2+ influx pathway leading to secretion of pepsinogen in
bullfrog esophageal mucosa.
patch clamp; bombesin; nifedipine; lanthanum; gadolinium; calcium
ion
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INTRODUCTION |
IT HAS BEEN REPORTED
THAT pepsinogen secretion was induced by three known second
messengers, such as intracellular Ca2+, cAMP, and
diacylglycerol, in isolated guinea pig gastric glands (21). Of the three, the role of Ca2+ was the
predominant focus. For instance, bombesin, one of the peptidergic
agonists, was reported to stimulate the pepsinogen secretion of
bullfrog (Rana catesbeiana) esophageal mucosa by increasing
the intracellular free Ca2+ (6). That group
reported that the early phase of Ca2+ elevation was
relatively independent of external Ca2+ and that the
sustained phase of Ca2+ was eliminated by adding 0.5-1
mM EGTA. Many reports have demonstrated that this increase in the
intracellular free Ca2+ concentration
([Ca2+]i) derives from the following two
Ca2+ sources: a release from intracellular Ca2+
stores and an influx from extracellular space (7, 16, 23, 28). Until now, however, the mechanism of Ca2+
influx has not been established in pepsinogen-secreting cells.
On the other hand, in pancreas acinar cells, an increase in the
[Ca2+]i by agonist stimulation was reported
along with the existence of a nonselective cation channel. In human
keratinocytes and guinea pig endocardial endothelial cells, a route for
Ca2+ influx from the extracellular side was attributed to
nonselective cation channels (2, 11).
In the present study, we have identified a pathway of
Ca2+ influx in frog pepsinogen-secreting cells for the
first time, using the whole cell patch-clamp technique
(5).
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MATERIALS AND METHODS |
Cell preparations.
Pepsinogen-secreting cells were prepared from bullfrog (Rana
catesbeiana) esophageal mucosa by the procedure previously
described (17). Briefly, the cells were isolated by
digestion with 0.01% collagenase (collagenase A; Boehringer Mannheim,
Mannheim, Germany) in a Ca2+-free solution for 12 min at
37.5°C. The cell suspension was then passed through a 120-µm nylon
mesh. The filtrate was centrifuged at 200 g, and the
sediments were washed three times. The final cell suspension was stored
in a normal amphibian solution (specified below) at room temperature.
Measurement of pepsinogen secretion.
Pepsinogen secretion was measured by using a perifusion chamber, as
previously described (15). The isolated
pepsinogen-secreting glands (multicellular preparation) were also
obtained by 0.1% collagenase (collagenase 1; Funakoshi, Tokyo, Japan)
digestion. The isolated peptic glands were collected on Teflon mesh
(4.75 µm) that was mounted in a perifusion chamber. The normal
amphibian solution at 25°C was perfused across the surface of the
glands, and the perfusate was collected at 1-min intervals. In
addition, an aliquot of the supernatant was assayed for pepsinogen
activity. Pepsinogen was measured using acid-denatured Hb at pH 2.0 as
the substrate by the modified Anson-Mirsky method. After 18 h of
incubation, pepsinogen activity was measured as tyrosine concentration
(15). The cell pellet was homogenized with a
Potter-Elvehjem homogenizer (Polytron) in 2 ml water and was used to
estimate the total cellular pepsinogen content (16).
Pepsinogen secretion was expressed as a percentage of the total
pepsinogen initially present in the cells.
Solution and chemicals.
The normal amphibian solution contained (in mM) 110 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES-NaOH, and pH was
adjusted to 7.3. The Ca2+-free solution contained (in mM)
111.5 NaCl, 2 KCl, 1 MgCl2, and 10 HEPES-NaOH, and pH was
7.3. In several pilot experiments with a pipette solution containing no
ATP, the currents were quiescent and the secretagogue could not
activate any currents in these cells. Therefore, we decided to use a
pipette solution containing 1 mM ATP. The standard pipette solution
contained (in mM) 110 KCl, 1 MgCl2, 0.354 CaCl2, 1 EGTA , 1 Tris-ATP, and 10 HEPES-NaOH; pH was
adjusted to 7.3 and pCa (
log Ca2+ concentration) was 7, unless otherwise stated. The free Ca2+ concentrations of
the solutions were estimated using the Chelator program
(22).
Tris-ATP, LaCl3, GdCl3,
N-methyl-D-glucamine (NMDG)-Cl, EGTA,
anthracene-9-carboxylic acid (9-AC), and Rb were purchased from Sigma
(St. Louis, MO). Bombesin was obtained from the Peptide Institute
(Osaka, Japan). All other chemicals were of the highest purity available.
Recording methods.
The whole cell currents were recorded using a patch-clamp amplifier
(Axopatch 200A; Axon Instruments, Foster City, CA). Records were
filtered through a 4-pole Bessel low-pass filter with a cutoff frequency at 2 kHz and were digitized at a rate of 1-2 kHz. Data were analyzed with the "pClamp" program (Axon Instruments). The patch pipettes had resistances of 4-5 M
when filled with the standard pipette solution. All experiments were carried out at room
temperature (22-24°C).
Ramp pulse and square pulse experiments.
Current-voltage (I-V) relationships were obtained either by
applying ramp pulses or square pulse sequences. Ramp pulses ranging between
120 and +30 mV were given from a holding potential of 0 mV at
a ramp speed of 1 V/s. Square pulses were given in 20-mV step
increments between
140 and +40 mV from a holding potential of
60
mV. The slope conductance was determined by calculating a line over an
appropriate short segment, about
60 mV, of the I-V curve.
The reversal potentials of the currents were measured as the potentials
where the I-V curves of the control (without stimulation)
crossed over those either stimulated by bombesin or blocked by
La3+ or Gd3+. Cell capacitance, measured by the
test-pulse mode of the patch-clamp amplifier under the whole cell
configuration, was 6.4 ± 0.8 pF (n = 90).
Data are expressed as means ± SE. Statistical significance
between groups was determined by Mann-Whitney U-test, with
P < 0.05 considered statistically significant.
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RESULTS |
Bombesin-induced currents.
Bombesin, which is a peptidergic agonist, is known to stimulate
pepsinogen secretion by increasing [Ca2+]i in
bullfrog esophageal mucosa. The ability of bombesin to increase [Ca2+]i is most prominent compared with other
agonists in these cells. The effect of bombesin is shown in Fig.
1. An inactivated sustained current in
the normal amphibian solution without bombesin was elicited by applying
square pulses in 20-mV steps in the range between
140 and +40 mV from
a holding potential of
60 mV (Fig. 1Ab). Bombesin
(3.2 × 10
7 M) induced a significant increase in the
conductance of the current (Fig. 1Ac). The resultant
I-V curves are displayed in Fig. 1B. The slope
conductance increased significantly (P < 0.05) from 69.3 ± 3.9 to 194.4 ± 15.1 pS/pF (n = 3).
The reversal potential for the bombesin-sensitive current was 6 ± 0.8 mV. The I-V curves obtained by the currents elicited by
the ramp pulse protocol (Fig. 1C) exhibited a result similar
to that elicited by square pulse sequences (Fig. 1D). The
slope conductance of the current was increased significantly
(P < 0.05) by bombesin from 47.2 ± 13.7 to
213.1 ± 9.6 pS/pF (n = 3). Bethanechol (3.2 × 10
5 mM), a cholinergic agonist that is known to
increase [Ca2+]i in pepsinogen-secreting
cells, also exhibited an enhancing effect on this current. The slope
conductance increased significantly from 35.9 ± 10.5 to
124.3 ± 29.4 pS/pF (n = 4; data not shown).

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Fig. 1.
Current-voltage relationships of the bombesin-induced
current. A: after establishment of whole cell configuration,
square pulse protocol was applied by pulses from a holding potential of
60 mV to different potentials between 140 and +40 mV in 20-mV steps
of 800-ms duration. a: Current traces in normal amphibian
solution (in mM: 110 NaCl , 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES-NaOH, pH = 7.3, adjusted with
NaOH) in the absence (b) and presence (c) of
3.2 × 10 7 M bombesin. Horizontal lines indicate the
0 current level. B: current-voltage relationships in normal
amphibian solution before ( ) and after
( ) the addition of 3.2 × 10 7 M
bombesin. The pipette was filled with standard pipette solution [in
mM: 110 KCl, 0.35 CaCl2, 1 EGTA, 1 MgCl2, 1 ATP-Tris, and 10 HEPES-NaOH, pH = 7.3, adjusted with KOH,
intracellular Ca2+ concentration
([Ca2+]i) = 10 7 M]. These
are representative data. C: after establishment of whole
cell configuration, a ramp pulse protocol was applied from a holding
potential of 0 mV to different potentials between 120 and +30 mV of
150-ms duration. The bath contained normal amphibian solution. The
pipette contained standard pipette solution. D:
representative data for the current-voltage relationships in normal
amphibian solution before (a) and after (b) the
addition of 3.2 × 10 7 M bombesin. The reversal
potential of the agonist-induced current was 0 mV.
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With the use of the ramp pulse protocol, the time-dependent development
of the bombesin (3.2 × 10
7 M) effect was observed.
As shown in Fig. 2, the slope conductance started to increase within 10 s after bombesin application
(time 0) and reached a maximum between 30 and 50 s.
Thereafter, the current gradually decreased, and the slope conductance
at 60 and 70 s was significantly lower than that from 30 to
50 s.

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Fig. 2.
Time course of the slope conductance after bombesin
stimulation. A: after establishment of whole cell
configuration, the ramp pulse protocol shown in Fig. 1C was
applied after 3.2 × 10 7 M bombesin stimulation in
normal amphibian solution. The time course of the slope conductance
after bombesin stimulation is displayed. The pipette contained standard
pipette solution. +Values were significantly different
(P < 0.05) from respective values of 30, 40, and
50 s. *Values were significantly different (P < 0.05) from respective values of 10 and 20 s (Mann-Whitney
U-test). B: current-voltage relationships after
bombesin stimulation. Left: relation at 10-40 s;
right, 50-70 s.
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Selectivity for monovalent and divalent cations.
To determine the ion selectivity of the channel, reversal potentials
(Erev) for various conditions of external
Na+ and K+ concentrations were obtained while
the internal K+ concentration was maintained at 100 mM.
NMDG+ was used as a replacement for the external monovalent
cations. Measurements of the Erev of
bombesin-induced currents were carried out to find potentials where
I-V curves in the presence of bombesin (3.2 × 10
7 M) crossed those in the absence of bombesin, as shown
in Fig. 3. The resultant
Erev values should be accurate, assuming that bombesin only activated this channel and leakage and other currents were not affected by the application of bombesin. Data are summarized in Table 1.

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Fig. 3.
Bombesin-induced inward current depends on the concentrations of
external K+ ([K+]o) and
Na+ ([Na+]o). After establishment
of whole cell configuration, ramp pulses, such as shown in Fig.
1C, were applied. Current-voltage relationships in the
external solution of 100 mM KCl and 10 mM
N-methyl-D-glucamine (NMDG)-Cl (A);
30 mM KCl and 80 mM NMDG-Cl (B); 10 mM KCl and 100 mM
NMDG-Cl (C); 100 mM NaCl and 10 mM NMDG-Cl (D);
30 mM NaCl and 80 mM NMDG-Cl (E); and 10 mM NaCl and 100 mM
NMDG-Cl (F) are compared before (a) and after
(b) stimulation by 3.2 × 10 7 M bombesin.
The pipette was filled with standard pipette solution.
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Changes in Erev corresponding to changes in the
extracellular K+ concentration or the extracellular
Na+ concentration did not obey the Nernst equation,
indicating that they were not selective permeant ions. These results
suggest that permeant ions other than K+ or Na+
were present. Because anions do not permeate, the only possible permeant ion under this condition is NMDG+, which will be
described as follows. A permeability (P) ratio of
PNMDG/PK = 0.58 was
calculated from the measured Erev values according to the following equation
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(1)
|
where R is the gas constant, T is the
absolute temperature, and F is Faraday's constant.
X is any monovalent cation that was employed in the
experiments. Px is a permeability ratio of x ion.
The permeability ratios of other ion species like Cs+,
Rb+, and Li+ against K+ were
measured as in Fig. 3, and these experiments are shown in Fig.
4. The averaged
Erev values were 0.28 ± 0.81 (Cs+), 0.16 ± 1.08 (Rb+), 0 ± 0 (K+),
3.63 ± 0.56 (Na+),
8.16 ± 0.67 (Li+), and
15.6 ± 0.4 (NMDG+) mV.
These data yielded permeability ratios of
PCs+-PRb+-PK+-PNa+-PLi+-PNMDG+ = 1.01:1:1:0.86:0.72:0.54. The permeability ratio of
PNMDG/PK (0.58)
separately obtained in Fig. 3 is well in accord with that (0.54)
obtained here. This nonselective nature toward monovalent cations fits
the definition of nonselective cation channels.

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Fig. 4.
Inward current carried by Cs+ (A),
Rb+ (B), K+ (C),
Na+ (D), Li+ (E), and
NMDG+ (F) After establishment of the whole cell
configuration, ramp pulses, such as shown in Fig. 1C, were
applied. Current-voltage relationships in a 110 mM chloride solution of
each cation in the absence (a) and presence (b)
of 3.2 × 10 7 M bombesin. The pipette was filled
with standard pipette solution. These are representative data.
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Similarly, the permeabilities for divalent cations could be obtained by
measuring Erev using Eqs. 2 and 3 in the presence of external divalent cations
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(2)
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(3)
|
where Y denotes any divalent cation, brackets
denote concentration, subscript i denotes intracellular, and subscript
o denotes extracellular.
As shown in Fig. 5A, in the
presence of 20 mM Ca2+ and 80 mM NMDG+,
bombesin (3.2 × 10
7 M) stimulated the current,
leading to an increase in the slope conductance (data are shown in
Table 1). The resultant Erev gives a
permeability ratio of
PCa/PK = 0.34. Data
are summarized in Table 2.

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Fig. 5.
A: inward current carried by Ca2+.
After establishment of whole cell configuration, ramp pulses, such as
shown in Fig. 1C, were applied. Current-voltage
relationships in 20 mM CaCl2 and 80 mM NMDG-Cl solution in
the absence (a) and presence (b) of 3.2 × 10 7 M bombesin. The pipette was filled with standard
pipette solution. B: dependence of the nonselective cation
current on the extracellular Ca2+ concentration. After
establishment of whole cell configuration, ramp pulses, such as shown
in Fig. 1C, were applied. The current-voltage relationships
after stimulation with 3.2 × 10 7 M bombesin were
obtained at different external Ca2+ concentrations. The
external bath solutions contained 1 mM CaCl2 and 227 mM
mannitol (a; n = 3), 3 mM CaCl2
and 220 mM mannitol (b; n = 3), or 10 mM
CaCl2 and 200 mM mannitol (c; n = 3). The pipettes were filled with standard pipette solution.
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Other evidence for Ca2+ permeation through the channel is
provided by the concentration dependence of the relevant ions on the conductance. As shown in Fig. 5B,
[Ca2+]o was increased from 1 to 3 mM and to
10 mM while other cations were substituted with mannitol in the
presence of bombesin (3.2 × 10
7 M). I-V
curves were obtained at the maximum effect of bombesin. The slope
conductances for the current were increased significantly (P < 0.05), from 41.7 ± 0.9 pS/pF (1 mM
Ca2+; n = 3) to 53.9 ± 4.6 pS/pF (3 mM Ca2+; n = 4) and to 181.2 ± 21.4 pS/pF (10 mM Ca2+; n = 3). These findings
produced the important result that this nonselective cation channel is
permeable to divalent cations and monovalent cations.
Ca2+ sensitivity of the nonselective
cation channel.
Another property specific to nonselective cation channels is their
Ca2+ sensitivity. There are many reports regarding this
property (4, 12). An increase in
[Ca2+]i activated the background current of
the cells bathed in an external solution containing 110 mM
Na+ without bombesin. As shown in Fig.
6, the slope conductances obtained in
three conditions of [Ca2+]i were 32.7 ± 4.0 pS/pF (n = 4) for 0.01 µM, 133.7 ± 7.6 pS/pF (n = 4) for 0.1 µM, and 167.4 ± 17.9 pS/pF (n = 4) for 1 µM. The elevation of
intracellular Ca2+ activated these currents in a
dose-dependent manner. The values for 0.1 µM
[Ca2+]i and 1 µM
[Ca2+]i were significantly larger than those
for 0.01 µM [Ca2+]i. These results indicate
that at least 0.1 µM [Ca2+]i is necessary
for the channel to be in an activated state.

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Fig. 6.
Dependence of the nonselective cation current on
[Ca2+]i. Current-voltage relationships where
the internal solution contained 1.0 × 10 6 M
[Ca2+]i (a; n = 4)
, 1.0 × 10 7 [Ca2+]i
(b; n = 3), and 1.0 × 10 8 M [Ca2+]i (c;
n = 4). The external solution used was normal amphibian
solution.
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Cl
conductance through the nonselective cation
channel.
The data presented so far do not exclude Cl
permeation
through the channel. We observed the currents with total replacement of
Cl
by aspartate
. Irrespective of the
presence or the absence of Cl
, the cation permeability
was not altered (data not shown). Erev were
3.7 ± 1.5 mV (potassium aspartate) and
1 ± 0 mV (KCl).
These results indicate that this channel is not permeable to
Cl
. Another source of evidence is provided by the
experiments with the Cl
-channel blocker 9-AC. Application
of 0.5 mM 9-AC did not suppress the conductance of the current of cells
bathed in an external solution containing 110 mM NaCl (data not shown),
nor was Erev changed by the application of 9-AC.
Insensitivity of bombesin-sensitive current to the dihydropyridine
blocker nifedipine.
One possible route for Ca2+ influx through the
plasma membrane is the L-type Ca2+ channel found in a
variety of excitable cells. We therefore examined the effects of
nifedipine on bombesin-induced currents. The enhanced currents induced
by bombesin (3.2 × 10
7 M) were unaltered by
nifedipine (5 × 10
6 M; data not shown). The slope
conductances of the application of bombesin alone (3.2 × 10
7 M) and both bombesin (3.2 × 10
7
M) and nifedipine (5 × 10
6 M) were 181.6 ± 11.9 pS/pF (n = 3) and 185.0 ± 7.2 pS/pF
(n = 4). These data suggest that the bombesin-induced
current was not through voltage-gated L-type Ca2+ channels.
Blockers for the nonselective cation current.
It is known that La3+ and Gd3+ are effective
blockers of nonselective cation channels (9, 11, 25). We
therefore tested the effects of 100 µM Gd3+
(n = 3) and 100 µM La3+
(n = 4) on bombesin-induced currents (Fig.
7). Bombesin-induced currents were
suppressed both by La3+ and Gd3+ to a level
lower than that without bombesin, indicating that channel activation by
bombesin is completely suppressed with La3+ and
Gd3+. Gd3+ (100 µM) reduced the membrane
conductance by 93.5 ± 0.5% and La3+ (100 µM) by
95.0 ± 0.3% with respect to current maximally activated by
bombesin (3.2 × 10
7 M). It should also be noted
that all of the I-V curves in the three conditions crossed
at one point (Fig. 7, points a, b, and c). This confirmed that the Erev
values had been measured accurately because the assumption that only
the nonselective cation channel was influenced by bombesin was
supported. Based on the Erev obtained here,
5.20 ± 1.37 mV (n = 3) with Gd3+
and
3.18 ± 1.06 mV (n = 4) with
La3+, PNa/PK
ratios were calculated to be 0.81 and 0.88, respectively. These are
consistent with data obtained earlier in this study.

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Fig. 7.
Blockade of bombesin-induced current by Gd3+ and
La3+. Current-voltage relationships were obtained in 110 mM
NaCl solution (a) without bombesin stimulation. In the
presence of 3.2 × 10 7 M bombesin (b),
100 µM GdCl3 and 100 µM LaCl3
(c) inhibited the current. The pipette was filled with
standard pipette solution.
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Effect of nonselective cation channel blockers on pepsinogen
secretion.
If Ca2+, via the nonselective cation channel, plays a role
in pepsinogen secretion, Gd3+ should suppress that
secretion. That was found to be the case, and the evidence is shown in
Fig. 8. Pepsinogen secretion was expressed as a percentage of total pepsinogen per hour and was monitored every few minutes. Bombesin (Fig. 8) stimulated pepsinogen secretion by 3.4 ± 0.07-fold without Gd3+
(n = 5). Gd3+ (100 µM) significantly
suppressed bombesin-induced pepsinogen secretion by 25.1 ± 0.3%
(n = 5) with respect to data without Gd3+.
These data indicate that nonselective cation channels are indeed involved in pepsinogen secretion in bullfrog esophageal mucosa.

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Fig. 8.
Effect of GdCl3 on pepsinogen secretion.
After a 4-min stabilizing period, pepsinogen-secreting cells were
exposed (arrow) to either 3.2 × 10 7 M bombesin
alone(n = 4) or to 3.2 × 10 7 M
bombesin + 100 µM Gd3+ (n = 4). Data
represent means ± SE. Significant differences (P < 0.05, Mann-Whitney U-test) compared with the values of
bombesin + Gd3+ (*) and compared with the basal value
at 0 min (+).
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DISCUSSION |
Using the whole cell patch-clamp technique, we revealed the
presence of a nonselective cation channel in pepsinogen-secreting cells
of bullfrog during agonist stimulation. The channel was activated by
bombesin or bethanechol, a secretagogue that elevates intracellular
Ca2+ and stimulates pepsinogen secretion.
The permeabilities to monovalent cations in the agonist-sensitive
channel of frog pepsinogen-secreting cells found in this study were in
the order
PCs+-PRb+-PK+-PNa+-PLi+ = 1.01:1:1:0.86:0.72. With regard to monovalent cation permeability, it
was reported that the nonselective cation channels were equally permeable to Na+ and K+ but impermeable to
Cl
. Other studies have reported permeability ratios
(PNa/PK) of nonselective
cation channels of 0.89 in a cultured secretory epithelial cell line
(4), 1.0 in pig coronary artery endothelial cells (1), 0.67 in guinea pig endocardial endothelial cells
(11), and 1.0 in mouse neuroblastoma cells
(30). Among these cell types, the following properties are
common in nonselective cation channels: poor discrimination between
monovalent cations, activation by intracellular Ca2+
(1, 3, 4, 10, 30), and considerable permeability to
Ca2+ (1, 4, 10, 11, 14, 19). All of these
characteristics were found in our newly discovered nonselective cation channels.
Previous studies reported that Ca2+ was one of the major
intracellular messengers of pepsinogen secretion. The concentration of
intracellular Ca2+ was elevated by the stimulation by ACh
and/or bombesin (24, 26). It was also reported that a
transient increase in [Ca2+]i depends on the
intracellular Ca2+ store, and a sustained plateau of
[Ca2+]i depends on extracellular
Ca2+ (23, 27). Furthermore, as we have
previously reported, when the extracellular Ca2+ was
chelated by EGTA, the sustained plateau of intracellular Ca2+ was abolished, and, as a result, pepsinogen secretion
was also abolished (6). There are many other reports
concerning the role of intracellular Ca2+ in pepsinogen
secretion (16, 23, 28). All of these studies predicted the
existence of a Ca2+ entry pathway from the extracellular
space, although the nature of such a pathway had not been elucidated.
In these experiments, we demonstrated the existence of nonselective
cation channels in these cells. It has been shown that nonselective
cation channels have a relative permeability to Ca2+ in
many cells. In exocrine cells, there are some reports that these
channels can provide a route for Ca2+ entry (4, 13,
20). In this study, we demonstrated that the nonselective cation
channel was permeable to Ca2+ in a dose-dependent manner on
the extracellular Ca2+ concentration.
There have been a few reports concerning a possible Ca2+
influx pathway in pepsinogen-secreting cells. In guinea pig gastric chief cells, Konda et al. (8) reported that ethanol
stimulated pepsinogen secretion by enhancing Ca2+ influx
through Ca2+ channels. Because La3+ blocked the
increase in [Ca2+]i by ethanol, whereas
neither nifedipine nor verapamil could inhibit it, they concluded that
the extracellular Ca2+ passed through
La3+-sensitive Ca2+ channels but not through
L-type Ca2+ channels. However, it has been reported that
La3+ blocks L-type Ca2+ channels and
nonselective cation channels (18). Therefore, the blockage
by La3+ is not conclusive evidence that the influx pathway
by ethanol is via Ca2+ channels. Similarly, in frog
esophageal pepsinogen-secreting cells, we have now shown that the
nonselective cation channels were blocked by La3+ but were
not blocked by nifedipine. In these cells, La3+ blocked
both bombesin-stimulated Ca2+ influx and pepsinogen
secretion (29). These data suggest that guinea pig gastric
cells probably also have nonselective cation channels.
Most nonselective cation channels are sensitive to intracellular
Ca2+. In pepsinogen-secreting cells, the channels were
activated by intracellular Ca2+ in the range of
10
7 to 10
6 M. We demonstrated that the
channel was activated by secretagogues that elevate
[Ca2+]i. However, it has not been clarified
whether such activation was attributable to a direct receptor-coupled
mechanism or secondary to intracellular Ca2+ elevation. In
this study, we successfully demonstrated that a prerequisite for the
activation of nonselective cation channels was an increase in
[Ca2+]i rather than the receptor stimulation
by bombesin or other secretagogues. Nevertheless, the mechanisms that
activate the channel by Ca2+ and the signal pathways
leading to pepsinogen secretion after an increase in
[Ca2+]i remain to be clarified. Because the
channels could be seen only in the circumstances of a few millimolar
intracellular ATP if the Ca2+ concentration of the bathing
solution was in the physiological range, the channel activation would
seem to depend on the phosphorylation pathway.
The evidence for the involvement of nonselective cation channels in
secretion is thought to be compatible with many other reports on peptic
cells. First, the nonselective cation channel was activated by
secretagogues, which stimulated pepsinogen secretion by intracellular
Ca2+ elevation. Second, this channel was activated by
intracellular Ca2+ elevation in a dose-dependent manner.
Third, La3+ and Gd3+ blocked both pepsinogen
secretion and channel activity. Fourth, La3+ blocked the
intracellular Ca2+ elevation by agonist stimulation. These
data also suggested that the nonselective cation channels might
participate as a physiological function of peptic cells. It is
speculated that, at least in part, the nonselective cation channel is
the physiological pathway of Ca2+ influx in peptic cells.
In pepsinogen-secreting cells of the bullfrog, Uemura et al.
(29) previously described that pepsinogen secretion was
suppressed by La3+. As demonstrated in this study,
in these cell types, La3+ blocked the nonselective cation
channel. It was also shown that Gd3+ blocked both channel
activities and pepsinogen secretion. These data suggest that the
activation of nonselective cation channels is certainly an important
step in pepsinogen secretion. However, there remains the discrepancy
that, although La3+ or Gd3+ reduced pepsinogen
secretion by only 20-50%, these blockers suppressed the
nonselective cation channels almost totally. This discrepancy can be
explained in part by differences in the final dose of these blockers
because of differences in the preparations used; the former was
multicellular, whereas the latter was a single cell preparation.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Issei Seyama and Dr. Kaoru Yamaoka for helpful advice
and Hidemichi Miyahara and Nobue Harada for technical assistance. We
are also very grateful to Dr. Goro Kajiyama for useful suggestions and support.
 |
FOOTNOTES |
This work was supported by a Grant-in-Aid 62480196 for Scientific
Research from the Ministry of Education, Science, and Culture, Japan.
Address for reprint requests and other correspondence: S. Kimura, Nigata Miyagami-cho 1-25, Kure 737-0146, Japan.
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 25 October 2000; accepted in final form 7 April 2001.
 |
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