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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 MOmega 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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.

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.

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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Table 1.   Slope conductance of bombesin-induced current

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
E<SUB>rev</SUB><IT>=</IT>(<IT>RT/F</IT>) ln (<IT>P<SUB>x</SUB></IT>[<IT>X<SUP>+</SUP></IT>]<SUB>o</SUB><IT>/P</IT><SUB>K</SUB>[K<SUP>+</SUP>]<SUB>i</SUB>) (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.

Similarly, the permeabilities for divalent cations could be obtained by measuring Erev using Eqs. 2 and 3 in the presence of external divalent cations
E<SUB>rev</SUB><IT>=</IT>(<IT>RT/F</IT>) ln [(4<IT>P′<SUB>Y</SUB></IT>[<IT>Y</IT><SUP>2<IT>+</IT></SUP>]<SUB>o</SUB><IT>+P</IT><SUB>NMDG</SUB>[NMDG<SUP>+</SUP>]<SUB>o</SUB>]<IT>/</IT>[<IT>P</IT><SUB>K</SUB>[K<SUP>+</SUP>]<SUB>i</SUB><IT>+P</IT><SUB>Na</SUB>[Na<SUP>+</SUP>]<SUB>i</SUB>] (2)

P′<SUB>Y</SUB>=P<SUB>Y</SUB>×[1+exp(<IT>E</IT><SUB>rev</SUB><IT>×F/RT</IT>)]<SUP><IT>−</IT>1</SUP> (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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Table 2.   Erev and permeability ratio for monovalent and divalent cations

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.

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.

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 (+).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(2):G333-G341
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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