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Journal of Physiology (2001), 535.2, pp. 349-358
© Copyright 2001 The Physiological Society

Regulation of slowly activating potassium current (I_Ks) by secretin in rat pancreatic acinar cells

Sung Joon Kim, Jin Kyoung Kim *, Hermann Pavenstädt †, Rainer Greger *, Martin J. Hug * and Markus Bleich ‡

	ABSTRACT

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1. The secretagogue-activated K⁺ conductance is indispensable for the electrogenic Cl^- secretion in exocrine tissue. In this study, we investigated the effect of secretin and other cAMP-mediated secretagogues on the slowly activating voltage-dependent K⁺ current (I_Ks) of rat pancreatic acinar cells (RPAs) with the whole-cell patch clamp technique.
2. Upon depolarization, RPAs showed I_Ks superimposed upon the instantaneous background outward current. Secretin (5 nM), vasoactive intestinal peptide (5 nM), forskolin (5 µM), isoprenaline (10 µM) or 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM) increased the amplitude of I_Ks two- to fourfold.
3. The physiological concentration of secretin (50 pM) had a relatively weak effect on I_Ks (160 % increase), which was significantly enhanced by transient co-stimulation with carbachol (CCh) (10 µM). However, the secretin-induced production of cAMP, which was measured by enzyme-linked immunosorbent assay, was not augmented by co-stimulation with CCh.
4. This study is the first to demonstrate the regulation of K⁺ channels in RPAs by cAMP-mediated agonists. The I_Ks channel is a common target for both Ca²⁺ and cAMP agonists. The vagal stimulation under the physiological concentration of secretin facilitates I_Ks, which provides an additional driving force for Cl^- secretion.

	INTRODUCTION

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The primary action of secretin in the exocrine pancreas is to induce the secretion of HCO₃^--rich fluid via the cAMP pathway while stimulation by ACh and cholecystokinin (CCK) induces Cl^--rich fluid secretion via a Ca²⁺ pathway. It is generally accepted that the HCO₃^--rich fluid comes from the pancreatic ducts while the Cl^--rich fluid is derived from the acini (Cook & Young, 1996). In NaCl-secreting tissues like pancreatic acini, the luminal Cl^- efflux is believed to be a secondary active process where the energy coupling is to the Na⁺,K⁺-ATPase (Petersen, 1992; Greger, 1996). To accomplish this mechanism, the presence of K⁺ channels in the basolateral membrane is indispensable. K⁺ channels would cause the membrane voltage to be set to a more negative value than the Nernst equilibrium potential for Cl^- (E_Cl), and thereby provide the driving force for Cl^- exit. Also, the efflux of K⁺ balances the K⁺ uptake by the Na⁺,K⁺-ATPase and other co-transporters (e.g. Na⁺-2Cl^--K⁺ co-transporter). Therefore, in many exocrine gland cells, the basolateral K⁺ channels are targets for the regulation of Cl^- secretion (Greger, 1996).

Recently, we found a new type of K⁺ channel current in rat pancreatic acinar cells (RPAs), namely the slowly activating, voltage-dependent K⁺ channel current I_Ks (Kim & Greger, 1999). The characteristic properties of I_Ks are a very slow activation upon depolarization to >-40 mV, no apparent inactivation during depolarization and slow deactivation after repolarization (Sanguinetti et al. 1996; Suessbrich & Busch, 1999). In RPAs, the stimulation of muscarinic receptors by CCh and the subsequent increase of the cytosolic Ca²⁺ activity ([Ca²⁺]_c) augments the amplitude of I_Ks, which indicates that I_Ks may play an important role in electrolyte secretion (Kim & Greger, 1999).

In RPAs, when a Ca²⁺-mediated secretagogue is combined with a cAMP-mediated secretagogue, the enzyme secretory response is greater than the additive response to the two individual secretagogues (Collen et al. 1982; Gardner & Jensen, 1986). Up to now, cellular models of electrolyte secretion in the pancreatic acini have dealt exclusively with channel opening mediated by intracellular Ca²⁺ and there has been little information about the role of cAMP in the regulation of ion channels of pancreatic acinar cells (Petersen, 1992). In the rodent pancreatic acini, evidence for secretin receptors was provided both from an immunofluorescence microscopic study and from functional studies assessing adenylyl cyclase activity or amylase release (Gardner & Jensen, 1986; Ulrich et al. 1998).

With this background, the aim of our study was to investigate whether cAMP agonists like secretin might regulate I_Ks of RPAs, and to test the proposal of a positive interaction between the effects of cAMP and Ca²⁺ signalling on I_Ks.

	METHODS

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Preparation of RPAs

Rats (100-150 g) were killed by decapitation after anaesthesia by thiopental sodium (100 mg kg^-1 body mass) and the pancreas was removed rapidly. All animal procedures were performed following the guidelines laid down by the animal welfare committee of our institution. A total of 38 rats were used for the whole experiment and 3-14 rats were used for each condition. The tissue was cut into small pieces (1-2 mm³) and digested in a bicarbonate-buffered Ringer solution containing (mM): NaCl, 130; KH₂PO₄, 0.4; K₂HPO₄, 1.6; MgSO₄, 1; NaHCO₃, 25; calcium gluconate, 1.3; sodium acetate, 10; D-glucose, 5; glycine, 2; and -ketoglutarate, 1; with collagenase type IV (800 mg l^-1, Serva, Heidelberg, Germany) and trypsin inhibitor (660 mg l^-1, Sigma, Deisenhofen, Germany), gassed with 94 % O₂-6 % CO₂. The tissue was incubated for 6 min at 37 °C and gently agitated using a fire-polished wide-bored (1-2 mm diameter) Pasteur pipette. After sedimentation, RPAs were washed twice with enzyme-free bicarbonate-buffered Ringer solution and finally filtered through a nylon mesh with a pore size of 150 µm. The entire preparation took about 30 min. Afterwards, the material was kept at 35 °C for up to 3 h in the same solution gassed with 94 % O₂-6 % CO₂.

Patch-clamp methods

Isolated acini were transferred into a bath chamber of volume 1 ml, mounted on the stage of an inverted microscope (Zeiss). A suction pipette held a cluster consisting of several acinar cells. The bath was perfused at a rate of 20 ml min^-1, ensuring rapid exchange and constant temperature (35 °C). Both cell poles were accessible to the bath solution. The individual cells were approached at the basal pole by patch pipettes with a mean resistance of 2-3 M. For whole-cell recordings, the patch pipettes were filled with the following solution (mM): potassium gluconate, 105; KCl, 30; NaH₂PO₄, 0.4; Na₂HPO₄, 1.6; calcium gluconate, 0.73; MgCl₂, 1; EGTA, 1; D-glucose, 5; and ATP, 1; pH 7.3, pCa 7. The standard bath solution, a phosphate-buffered Ringer solution, contained (mM): NaCl, 145; K₂HPO₄, 1.6; KH₂PO₄, 0.4; calcium gluconate, 1.3; MgCl₂, 1; and D-glucose, 5; pH 7.4. All chemicals used were of the highest grade of purity available and were obtained from Sigma and Merck (Darmstadt, Germany).

The reference electrode was of the flowing KCl (1 M) type in order to minimize liquid junction voltages. The pipette capacitance was cancelled by the compensation circuit of the amplifier (U. Fröbe & R. Busche, this institute) with the help of sine wave command voltage. The voltage and current data were low-pass filtered (3 kHz) and stored on DAT tape. Analysis was performed from the on-line chart recording or by computer analysis using the program F-patch written by U. Fröbe (this institute).

Measurement of [Ca²⁺]_c

Measurement of [Ca²⁺]_c was performed as described previously (Slawik et al. 1996). Briefly, RPAs were isolated as described above and loaded for 45 min at 4 °C with Fura-2 AM (10 µmol l^-1, Molecular Probes, Eugene, OR, USA) in the incubation solution to which 1 µmol l^-1 Pluronic F127 had been added. Cells were then transferred into the bath chamber mounted on the stage of an inverted microscope (Axiovert 10, Zeiss) and washed with normal bath solution. Dye excitation was performed at 340, 360 and 380 nm, respectively, using a filter wheel system. Fluorescence emission was collected at 480-520 nm using a photomultiplier system (Hamamatsu, Garching, Germany). As a measure of [Ca²⁺]_c, the fluorescence emission ratio at 340 nm/380 nm excitation is presented after subtraction of the autofluorescence.

Measurements of intracellular cAMP

RPAs were isolated by the same method as above, rinsed with phosphate-buffered Ringer solution and divided into six groups. Each group was composed of three to five samples of cell suspension in Eppendorf tubes (0.5 ml). After pre-incubation with 3-isobutyl-1-methylxanthine (IBMX, 50 µM) at 37 °C for 5 min, each group of RPAs was exposed to the added agents for 15 min, i.e. forskolin (5 µM), secretin (10 nM), low-secretin (50 pM), CCh (10 µM), low-secretin + CCh, and control (no further treatment). To terminate the assay, the supernatants were rapidly removed and cells were rinsed with ice-cold ethanol (70 %). After ethanol extraction, cAMP concentrations were measured with an enzyme-linked immunosorbent assay (Amersham Buchler, Braunschweig, Germany). The same experiment with a full series of test groups was carried out in three rats. Because the absolute number of RPAs analysed could be variable between experiments, we first calculated the mean cAMP concentration of the control condition for each experiment. The measured values of the other conditions were standardized to the mean control concentration of the corresponding experimental group and expressed as the percentage change.

Data presentation

The data are presented as original recordings, I-V curves and as bar graphs of mean values ± S.E.M. (n = number of tested RPAs). Except for the results of the cAMP measurement (Student's unpaired t test), Student's paired t test was applied since the control and the test experiments were done in the same acinus, and P < 0.05 was accepted for significance.

	RESULTS

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The averaged resting membrane voltage (V_m) of RPAs before any stimulation was -41 ± 0.6 mV (n = 76). This value is comparable with data from our previous whole-cell patch clamp experiments (Slawik et al. 1996; Kim & Greger, 1999). To activate voltage-dependent K⁺ channels, step-like depolarizing pulses were applied from a holding potential of -60 mV. Figure 1A shows the membrane current responses to depolarization (0 mV) for different pulse durations (see protocol in Fig. 1A). Slowly activating outward current (I_Ks) was superimposed upon the instantaneous outward current (I_ins). I_Ks of RPAs did not saturate even after 9 s of depolarization and showed a slowly deactivating outward tail current on repolarization, which are the known characteristics of I_Ks in cardiac myocytes or in the heterologous expression system (Suessbrich & Busch, 1999). The sum of I_Ks and I_ins was measured, and is presented as the total outward current (I_t) in this study. At the holding potential (-60 mV), a net inward current (I_inw) was usually observed since the resting membrane voltage was around -40 mV. In all further experiments, the duration of both holding (-60 mV) and test V_m (0 mV) was set to 2 s.

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Figure 1. Membrane current responses of a rat pancreatic acinar cell (RPA) to step-like depolarizations A, the RPA was clamped at -60 mV and depolarized to 0 mV for 0.3, 1, 2, 3, 4, 5, 7 and 9 s. Eight current traces from the same RPA are superimposed. Labels indicate measured components of current traces: Iins, instantaneous outward current; It, total outward current amplitude at the end of depolarizing pulse; IKs, slowly activating outward current; Iinw, inward current at -60 mV. B, effect of secretin (5 nM) on membrane current. Upper and lower traces show membrane current and voltage, respectively. Membrane voltage was repetitively switched between -60 and 0 mV (2 s for each voltage) except during the application of CCh (zero-current clamp condition). Note the sharp depolarization by CCh and slow repolarization on washout of CCh.

Figure 1B gives a representative original trace demonstrating the effect of secretin (5 nM) on the outward current of RPAs. The concentration of secretin used here is known to induce a maximal increase of cAMP production in the rat pancreas (Gardner & Jensen, 1986). The step-like depolarization of V_m (2 s) was repeated, which activated I_Ks upon I_ins. I_Ks and its deactivating tail current were increased by the application of secretin. Slight increases of I_ins and I_inw were also observed. I_Ks was abolished completely by the addition of chromanol 293B (30 µM, data not shown but see Fig. 3), a well-known blocker of I_Ks (Kim & Greger, 1999; Suessbruch & Busch, 1999). Similar results were obtained in 22 RPAs and summarized results are shown in Fig. 4.

When the effect of secretin reached a steady state, CCh (10 µM) was added for a short period (< 30 s), which induced a sharp depolarization (Fig. 1B). CCh alone normally induces a large increase of I_Ks and Ca²⁺-activated Cl^- current in RPAs (Kim & Greger, 1999). The augmented I_Ks can be more clearly observed immediately after the repolarization during the washout of CCh, since the overlapping Ca²⁺-activated Cl^- current decays much faster than I_Ks (Kim & Greger, 1999; see also Fig. 8A). However, under maximal stimulation by 5 nM secretin, CCh augmented the amplitude of I_Ks by only about 10 % (110 ± 4.5 % of control, n = 9).

In six RPAs, various levels of step-like depolarizations were applied to obtain the current-voltage relationships (I-V curves) in the absence and presence of secretin. Figure 2A shows the representative current traces. I_Ks activated at clamp voltages more depolarized than -50 mV and was increased by secretin. Figure 2B gives the I-V curves for mean I_ins (squares) and I_t (triangles) of control (open symbols) and secretin-treated (filled symbols) RPAs. The corresponding I-V curves of I_Ks are drawn in Fig. 2C (n = 6). The increase of I_Ks by secretin was significant from -30 mV.

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Figure 2. Increase of IKs by secretin in RPAs A, effect of secretin (5 nM) on outward currents induced by various levels of step-like depolarizations (see protocol above traces in control). B, I-V curves of Iins (squares) and It (triangles) of control (open symbols) and secretin-treated (filled symbols) RPAs (n = 6). C, I-V curve of IKs of control (open square) and secretin-treated (filled circle) RPAs (n = 6). Asterisks indicate a significant difference from control (P < 0.05).

In another series of test groups, we tested the effect of forskolin, a potent activator of adenylyl cyclase. Similar to the effect of secretin, I_Ks was greatly increased by forskolin (5 µM) in 11 RPAs (see Fig. 4 for summary). Figure 3A shows representative current traces obtained from a RPA where I_Ks was increased by forskolin and completely blocked by 293B (30 µM). In six RPAs, various step depolarizations were applied and the effect of forskolin was tested. I-V curves and the changes in them caused by forskolin (Fig. 3B and C) were very similar to those obtained with secretin (Fig. 2B and C). The open and filled symbols represent the control and forskolin-treated conditions, respectively.

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Figure 3. Increase of IKs by forskolin in RPAs A, effect of forskolin (5 µM) on outward currents induced by various levels of step-like depolarizations (see protocol above traces in control). The chromanol compound 293B (30 µM) completely inhibits time-dependent outward currents. B, I-V curves of Iins (squares) and It (triangles) of control (open symbols) and forskolin-treated (filled symbols) RPAs (n = 6). C, I-V curves of IKs of control (open circles) and forskolin-treated (filled circles) RPAs (n = 6). Asterisks indicate a significant difference from control (P < 0.05).

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Figure 4. Summarized effects of secretin and forskolin on the membrane current of RPAs Effects of secretin (n = 22; A) and forskolin (n = 12; B) on the membrane current at 0 mV (It, IKs) and at -60 mV (Iinw) of RPAs. Open and filled bars represent the control and treated conditions, respectively. Asterisks indicate a significant difference from control (P < 0.05). C, effects of secretin (left panel) and forskolin (right panel) on the membrane voltage. Each curve represents a recording from a single RPA.

In Fig. 4A and B, the averaged amplitudes of I_t, I_ins and I_Ks at 0 mV, and of I_inw at -60 mV are summarized for the whole series of test groups. I_Ks was increased from 161 ± 33.4 pA to 405 ± 44.6 pA (n = 22) by secretin (5 nM) and from 191 ± 51.0 pA to 533 ± 143 pA by forskolin (5 µM, n = 11). When we compare the increase of I_Ks in each acinus, the mean percentage increase (paired percentage increase) of I_Ks was 315 ± 24.1 % by secretin and 299 ± 42.8 % by forskolin. The strong augmentation of I_Ks by secretin or by forskolin could explain the increase of I_t. It is worth noting that I_ins and I_inw were also increased, although only slightly, by secretin, but were not increased by forskolin. The resting membrane potential under the zero-current clamp condition was measured before and during cAMP stimulation (Fig. 4C). We could not detect a statistically significant unidirectional effect. However, the summed results show that those RPAs with a relatively depolarized resting membrane potential tend to be hyperpolarized by the treatment with secretin (n = 22) or forskolin (n = 12). The slight increase of inward current raised the question whether secretin alone may increase the [Ca²⁺]_c, which can activate Ca²⁺-activated Cl^- or non-selective cation channels. To test this possibility, we directly measured [Ca²⁺]_c using Fura-2 microspectrofluorimetry. Secretin (10 nM) by itself had no effect on [Ca²⁺]_c of RPAs (n = 7), whereas the application of CCh (0.5 µM) induced a typical biphasic increase of [Ca²⁺]_c (Fig. 5).

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Figure 5. Effects of CCh and secretin on [Ca2+]c of RPAs The original trace (top panel) shows the fura-2 fluorescence ratio plotted against time as a measure of [Ca2+]c . CCh (0.5 µM) induces a fast increase in [Ca2+]c (peak) followed by a plateau-like phase reflecting a balance between Ca2+ influx and export. In some experiments oscillations of [Ca2+]c were observed in this plateau-like phase. Secretin (10 nM) has no detectable effect on [Ca2+]c. The bottom panel summarizes results from seven experiments. Asterisk indicates a significant difference from control (con; P < 0.05). w/o, washout.

Since cAMP produced by secretin receptor stimulation will activate protein kinase A (PKA), we tested whether a PKA inhibitor, H-89, could suppress the effect of secretin on I_Ks. After confirming the effect of secretin on I_Ks, the same RPA was treated with H-89 (0.5 µM) for 3 min and secretin (5 nM) was added. The bar graph in Fig. 6 shows a summary of data from seven different RPAs. The increase of I_Ks by secretin was significantly suppressed in the presence of H-89.

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Figure 6. Effect of H-89, a PKA inhibitor, on the modulation of IKs by secretin The bar graphs show the amplitude of IKs (mean ± S.E.M.) in control (con), and with 5 nM secretin, 0.5 µM H-89 and H-89 + secretin (n = 7). The asterisk indicates a significant difference from control (P < 0.05).

In another series of experiments, we tested the effects of vasoactive intestinal peptide (VIP), isoprenaline and IBMX. VIP (5 nM), a peptide released from intrinsic neurons of the pancreas, increased the amplitude of I_Ks from 188 ± 27.4 to 418 ± 53.9 pA (n = 11, Fig. 7). The mean of the paired percentage increase of I_Ks by VIP was 235 ± 18.6 %. -Adrenoceptors are linked to the G_s-type GTP-binding proteins and activate adenylyl cyclases. In RPAs, a stimulatory effect by a -adrenoceptor agonist on fluid and enzyme secretion was reported previously (Lingard & Young, 1983). Therefore, we tested whether RPAs also respond to -adrenergic stimulation by an increase in membrane current. Isoprenaline (10 µM), a -adrenoceptor agonist, significantly increased the amplitude of I_Ks from 70 ± 19.0 to 157 ± 42.2 pA (n = 6). The mean of the paired percentage increase of I_Ks by secretin was 234 ± 28.8 %. The effect of isoprenaline was completely blocked by pretreatment with propranolol (10 µM), a -adrenoceptor antagonist (n = 5, data not shown). As a next step, we tested whether the amplitude of I_Ks could be increased by treatment with IBMX, an inhibitor of cyclic nucleotide phosphodiesterase. In the presence of IBMX (0.1 mM), it was anticipated that the cAMP produced by the constitutive adenylyl cyclase activity would accumulate. With a relatively long treatment time (> 5 min) it was possible to observe a steady-state increase of I_Ks by IBMX (from 76 ± 22.3 to 289 ± 64.5 pA, n = 7, Fig. 7). The mean of the paired percentage increase of I_Ks by IBMX was 544 ± 157.3 %.

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Figure 7. Effects of VIP, isoprenaline and IBMX on IKs induced by the voltage pulse Summarized results are shown (for pulse protocol see Fig. 3). The number of tested RPAs is indicated under each bar graph. Asterisks indicate a significant difference from control (P < 0.05).

According to previous reports, the postprandial concentration of secretin in the plasma of experimental animals is between 10 and 50 pM (Kim et al. 1979; Chey & Konturek, 1982; Green et al. 1989), which is much lower than the concentration tested above (5 nM). Therefore, we examined whether a lower concentration of secretin (low-secretin, 50 pM) could also induce a change in the size of I_Ks in RPAs. I_Ks was significantly increased by low-secretin, from 162 ± 34 to 229 ± 37 pA, which corresponds to a paired percentage increase of 165 ± 21.0 % (n = 17, P < 0.01). In the physiological situation of pancreatic secretion, the hormonal stimulation by secretin could be accompanied by neuronal stimulation from vagal fibres or intrinsic cholinergic neurons (Cook & Young, 1996). Therefore, we tested whether the relatively small effect of low-secretin on I_Ks could be augmented by a transient co-stimulation of muscarinic receptors.

As recently shown by us (Kim & Greger, 1999), the muscarinic stimulation of RPAs augments the amplitude of I_Ks, which reverses completely within a minute after the washout of CCh. In Fig. 8, the same protocol of muscarinic stimulation was applied in the presence of low-secretin and compared with the response to CCh alone in the same RPA. The muscarinic stimulation markedly increased the size of I_Ks and the augmented I_Ks lasted significantly longer in the presence of low-secretin than in its absence (Fig. 8A). To compare the time course of I_Ks, we normalized the amplitude of I_Ks against the peak amplitude measured immediately after washout of CCh. The same experimental protocol was carried out in seven RPAs and the averaged results clearly demonstrate that the muscarinic stimulation interacts positively with the effect of secretin on I_Ks (Fig. 8B).

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Figure 8. Facilitation of low-secretin (50 pM) action on IKs by transient muscarinic stimulation (CCh, 10 µM) A, original traces of an experiment from a single RPA are shown. The pulse protocol is the same as that shown in Fig. 1. B, the amplitude of IKs was normalized against the maximal amplitude of IKs of each experiment and the mean values are plotted versus time (n = 7). Open and filled squares represent the control and low-secretin-treated conditions, respectively. The vertical hatched bar indicates the timing of CCh application where membrane voltage was not clamped.

As a next step, we measured the concentration of cAMP in RPAs to examine whether the secretin-induced cAMP production was facilitated by muscarinic stimulation (see Methods for the experimental procedure). The summed results show a potent effect of forskolin and secretin on the production of cAMP (Fig. 9). The concentration of cAMP was increased by 841 ± 127.3, 322 ± 58.3 and 168 ± 20.6 % by forskolin, secretin (10 nM) and low-secretin (50 pM), respectively (n = 13). There was no difference of cAMP production between low-secretin and the co-stimulation with low-secretin and CCh.

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Figure 9. Effects of the cAMP agonists and CCh on the production of cAMP Numbers of experiments are indicated in parentheses. All experiments were performed in the presence of IBMX (0.1 mM). Asterisks indicate a significant difference from control (P > 0.05). There was no significant difference between low-secretin and CCh + low-secretin (P > 0.05). con, control; fsk, forskolin (5 µM); secr, secretin (10 nM); low-secr, low-secretin (50 pM); and CCh, carbachol (10 µM).

	DISCUSSION

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In this study, we show the positive regulation of I_Ks by cAMP-producing secretagogues in RPAs. The positive effect of IBMX alone suggests a constitutive activity of adenylyl cyclase in RPAs. Together with our previous report (Kim & Greger, 1999), this result indicates that I_Ks would be a common target for both cAMP- and Ca²⁺-mediated secretory signalling pathways in the rat pancreas.

Recent findings from molecular biological and electrophysiological studies indicate that the ion channel for I_Ks in native tissues is a complex of the K⁺ channel subunit KvLQT1 and the IsK protein. The respective genes are KCNQ1 and KCNE1 (Sanguinetti et al. 1996; Suessbrich & Busch, 1999). I_Ks is unusually slow in its voltage-dependent activation and the deactivation is much slower than that of most other K⁺ channels. Our recordings in this and previous studies show the voltage dependence and typical slow kinetics of I_Ks (Kim & Greger, 1999). The presence of transcripts for both mRNAs has been shown in the rat pancreas (Köttgen et al. 1999; Suessbrich & Busch, 1999). The presence of KvLQT1 mRNA was also confirmed in the human pancreas (Sanguinetti et al. 1996), although the electrophysiological evidence for I_Ks is still lacking in human pancreatic acini.

Modulation of I_Ks by the cAMP pathway has been described for I_Ks in guinea-pig cardiac myocytes (Walsh & Kass, 1988), gerbil vestibular dark cells (Sunose et al. 1997) and in the Xenopus laevis oocyte expression system (Blumenthal & Kaczmarek, 1992). The positive modulation of I_Ks by cAMP seems to be mediated by PKA since it is mimicked by the catalytic subunit of PKA (Walsh et al. 1991) and is blocked by a specific protein inhibitor of PKA (Blumenthal & Kaczmarek, 1992). Our present study also suggests that the effect of secretin on I_Ks is mediated via the cAMP-PKA pathway (Fig. 6). Because there is no PKA consensus site on the IsK protein, the phosphorylation of KvLQT1 channel protein is supposed to be a mechanism of positive regulation of I_Ks (Blumenthal & Kaczmarek, 1992).

Although the major effect of secretin is to stimulate the secretion of HCO₃^--rich fluid through the pancreatic duct, it has been consistently reported that the activation of the cAMP pathway also stimulates amylase secretion in isolated pancreatic acini (Gardner & Jackson, 1977; Collen et al. 1982; Gardner & Jensen, 1986). For solubilization and washout of secreted enzymes into the gut, the process of enzyme release will be accompanied by the secretion of an isotonic NaCl-rich fluid from pancreatic acini where the parallel up-regulation of ion channels and transporters is indispensable.

Unlike other animal species (e.g. pig) where Ca²⁺-activated K⁺ channels dominate the response to Ca²⁺-agonists, rodent pancreatic acini show depolarization upon ACh or CCK stimulation as a result of the strong activation of a Ca²⁺-activated Cl^- conductance in the luminal membrane (Petersen, 1992). Therefore, a voltage-dependent K⁺ channel current which does not inactivate and is augmented by secretagogues would be an appropriate system to support electrogenic secretion of Cl^-. In RPAs, secretin increased the Na⁺ influx, which was inhibited by bumetamide, a well-known inhibitor of the Na⁺-2Cl^--K⁺ co-transporter (Zhao & Muallem, 1995). Other investigators have shown that secretin stimulates the initial rate of pump turnover in guinea-pig pancreatic acini using the rate of [³H]ouabain binding as an index of Na⁺/K⁺ pump turnover (Hootman et al. 1983). Since the enhanced transport rate of the above electrolyte transporters would accumulate K⁺ in the cytosol, the up-regulation of K⁺ efflux is a prerequisite for the K⁺ recycling and continuous operation of the whole mechanism (Greger, 1996). In this context, the positive regulation of I_Ks by secretin may contribute to K⁺ recycling, especially when the stimulation of secretin receptors is combined with muscarinic or CCK receptor stimulation. Another role of basolateral K⁺ channels is to provide a driving force for the Cl^- efflux by holding the membrane potential below E_Cl. In this context, it was expected that the augmented I_Ks would decrease the CCh-induced maximal depolarization in RPAs. In the present study, CCh alone depolarized the membrane from -39.1 ± 1.28 to -18.8 ± 1.05 mV (n = 19), and CCh with secretin pretreatment could depolarize the membrane from -40.7 ± 1.42 to -21.2 ± 1.20 mV (n = 14). However, the difference was not statistically significant. Probably a huge increase of the Cl^- conductance by muscarinic stimulation overwhelms the effect on the K⁺ conductance and prevents an accurate evaluation. Another explanation is that we could not clamp the intracellular concentration of Cl^- because of the size of cell cluster. When CCh was applied repetitively, we could frequently observe that the peak depolarization varies by 1-3 mV in the same acinus.

In the present study, we observed the effects of VIP and isoprenaline on I_Ks of RPAs. VIP is a 28-residue neuropeptide showing very close homology with secretin, and binds to VIP-specific receptors (VIP₁ and VIP₂) with high affinity. The activation of VIP receptors in the pancreatic acini induces cAMP production and amylase release (Gardner & Jensen, 1986; Cook & Young, 1996; Ito et al. 2000). VIP can also bind to secretin receptors, but only with low affinity (Cook & Young, 1996; Ito et al. 2000). Therefore, the effect of VIP that we observed here would have been mediated by the activation of VIP receptors in RPAs. The positive effect of isoprenaline on I_Ks supports the notion that RPAs have functional -adrenoceptors that are related to electrolyte movement. In RPAs, -adrenergic stimulation increases the amylase release while -adrenergic stimulation inhibits it (Varga et al. 1990).

In the present study, we did not try to dissect the underlying channels of time-independent current (I_inw and I_ins). Our recent patch clamp recordings of RPAs indicated the presence of background K⁺ conductance as well as inwardly rectifying K⁺ channels (Kim et al. 2000). Since the resting membrane voltage is positive to the K⁺ equilibrium potential, there might be a background Cl^- or non-selective cation conductance. Our data show that secretin also induced a slight increase in inward current at the chosen holding potential of -60 mV (Fig. 4A). Considering the electrolyte composition of RPAs and the Nernstian equilibrium potential for each ion, a slight increase of Cl^- conductance or non-selective cation conductance might be induced by secretin. However, secretin (10 nM) has no effect on the [Ca²⁺]_c of RPAs (Fig. 5). The presence of CFTR chloride channels in rat pancreatic acinar cells was suggested by a immunocytochemical study (Zeng et al. 1997). We did not further try to characterize the secretin-induced inward current since its change was too small and not consistently observed. Moreover, the increase of I_inw and I_ins was not confirmed by other cAMP agonists (e.g. forskolin) in this study.

Another interesting finding in our study is the facilitating action of muscarinic stimulation on the effect of low-secretin (50 pM), or vice versa, on I_Ks (Fig. 6). Cross-talk between signal transduction pathways, such as the cAMP pathway stimulated by secretin and the PLC-Ca²⁺ pathway stimulated by acetylcholine, appears to be necessary for integration of multiple stimuli involved in a single physiological response. Previous in vivo studies clearly showed that the physiological concentration of secretin is between 10 and 50 pM, a concentration that only weakly stimulates the exocrine pancreas. However, when combined with CCK or with vagal stimulation, there is a marked potentiation of pancreatic secretion (Gardner & Jackson, 1977; Chey et al. 1979; You et al. 1983). Under physiological conditions, the co-activation of both signal pathways would provide the K⁺ conductance that is necessary for an enhanced Cl^- secretion. Therefore, our findings may partially explain the synergistic effect of cAMP and Ca²⁺-mediated secretagogues on the function of exocrine pancreas. In the rat pancreatic duct, the interaction between secretin and acetylcholine (ACh) is more complex. Evans et al. (1996) have reported that applying secretin and ACh simultaneously to the rat pancreatic duct caused either stimulation or inhibition of fluid secretion depending on the applied concentration. According to the above report, the inhibitory effect was likely to be mediated by the protein kinase C (PKC) pathway (Evans et al. 1996). The effect of PKC activation was not tested in the present study.

To explain the synergistic effect of CCh and low-secretin, it could be proposed that the production of cAMP might be augmented by CCh because some subtypes of adenylyl cyclase can be activated by a Ca²⁺-dependent signalling pathway (Xia & Storm, 1997). However, we failed to detect any potentiation of low-secretin-induced cAMP production when combined with CCh, although we could observe a slight increase of cAMP by muscarinic stimulation alone. A previous report also did not find an increase of cAMP concentration by the muscarinic co-stimulation in RPAs (Collen et al. 1982). Because the increase of I_Ks by CCh stimulation is mediated by [Ca²⁺]_i (Kim & Greger, 1999), another explanation for the facilitation effect could be an enhanced Ca²⁺ signal of CCh in the presence of low-secretin. However, a sustained increase of [Ca²⁺]_i after the washout of CCh in the presence of low-secretin was not likely to be the case in our experimental condition since we could observe consistent repolarization after CCh washout with or without low-secretin in the same time frame. Since the depolarization by CCh is caused by the activation of Ca²⁺-dependent Cl^- channels (Petersen, 1992), the repolarization of RPAs indicates the recovery of [Ca²⁺]_i to its resting level. In the rat pancreatic cell line AR4-2J, secretin (1 nM) could augment the production of InsP₃ by CCK (Bold et al. 1995). However, the concentration of secretin used in the AR4-2J cell line (1 nM) was much higher than the one used in our study (50 pM). Moreover, another previous study indicates that the stimulation of the cAMP system partly suppress the ACh-evoked [Ca²⁺]_i increase (Camello et al. 1996). After excluding the above possibilities, it is supposed that the synergistic effect might occur at the channel protein level, and precise knowledge about the mechanism of I_Ks modulation by Ca²⁺ and by cAMP is necessary to understand the synergistic effect.

In conclusion, our patch clamp study on RPAs shows that the stimulation of secretin receptors alone as well as co-stimulation of muscarinic receptors can increase the potassium outward current I_Ks. The modulation of I_Ks by both cAMP- and Ca²⁺-mediated secretagogues suggests that the I_Ks channel might be an important target site that controls the electrolyte secretion in rat pancreatic acini.

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Acknowledgements

This study was supported by the Alexander von Humboldt Foundation, the Korean Science and Engineering Foundation (KOSEF, PD 981-47) and Deutsche Forschungsgemeinschaft (DFG) Gr 480/11-3. The technical assistance by A. Bausch, T. Kilic and Dipl.-Ing. R. Laufersweiler is gratefully acknowledged.

Corresponding author

M. Bleich: Aventis Pharma Deutschland GmbH, DG Cardiovascular, Bld. H821, D-65926 Frankfurt, Germany.

Email: markus.bleich{at}aventis.com

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