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Italy; and ‡Department of Experimental Pathology and Oncology, University of Florence, I-50134. Firenze, Italy. ABSTRACT. The human ether-a-go-go-related ...
Glucose- and arginine-induced insulin secretion by human pancreatic ␤-cells: the role of HERG Kⴙ channels in firing and release BARBARA ROSATI,* PIERO MARCHETTI,† OLIVIA CROCIANI,‡ MARZIA LECCHI,* ROBERTO LUPI,† ANNAROSA ARCANGELI,‡ MASSIMO OLIVOTTO,‡ AND ENZO WANKE*,1 *Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milano, Italy; † Department of Endocrinology and Metabolism, Metabolic Unit, Ospedale Cisanello, I-56100 Pisa, Italy; and ‡Department of Experimental Pathology and Oncology, University of Florence, I-50134 Firenze, Italy ABSTRACT The human ether-a-go-go-related genes (herg) are expressed in tissues other than heart and brain where the HERG Kⴙ channels are known to regulate the repolarization of the heart action potential and the neuronal spike-frequency accommodation. We provide evidence that herg1 transcripts are present in human pancreatic islets that were used to study both insulin secretion and electrical activity with radioimmunoassay and single cell perforated patch-clamp techniques, respectively. Glucose- and arginine-induced islets insulin secretion data suggested a net increase of release under perfusion with antiarrhythmic drugs known to selectively block HERG channels. Indeed we could routinely isolate a Kⴙ current that was recognized as biophysically and pharmacologically similar to the HERG current. An analysis of the glucose- and arginine-induced electrical activity (several applications during 30 min) in terms of firing frequency and putative insulin release was done in control and in the presence of selective blockers of HERG channels: the firing frequency and the release increased by 32% and 77%, respectively. It is concluded that HERG channels have a crucial role in regulating insulin secretion and firing of human ␤-cells. This raises the possibility that some genetically characterized hyperinsulinemic diseases of unknown origin might involve mutations in the HERG channels.—Rosati, B., Marchetti, P., Crociani, O., Lecchi, M., Lupi, R., Arcangeli, A., Olivotto, M., Wanke, E. Glucose- and arginine-induced insulin secretion by human pancreatic ␤-cells: the role of HERG Kⴙ channels in firing and release. FASEB J. 14, 2601–2610 (2000)

Key Words: pancreas 䡠 eag gene family 䡠 erg gene 䡠 nesidioblastosis

It is well established that glucose metabolism in pancreatic ␤-cells induces the closure of ATP-dependent K⫹ channels (K(ATP)), thus causing cell depolarization followed by bursts of action potential ac0892-6638/00/0014-2601/$02.25 © FASEB

tivity (1). The action potentials produce pulses of Ca2⫹ entry through voltage-dependent Ca2⫹ channels that ultimately lead to [Ca2⫹]i-dependent exocytosis (2). Ca2⫹ and K⫹ currents have been recognized and characterized in human ␤-cells (3, 4), and some electrical activity has been recorded using perforated patch techniques (4 –7). Like rodent islets, human islets respond to a prolonged glucose step of 3–15 mM, with biphasic insulin secretion characterized by a spike-like first phase (5–10 min), followed by a long-lasting plateau second phase (7). The initial rise is characterized by latency and a long duration before the beginning of the oscillatory pattern. Secretion can be negatively modulated by Ca2⫹ channel blockers and induced in resting ␤-cells by tolbutamide, a reversible oral hypoglycemic sulfonylurea. The correlation between electrical activity and [Ca2⫹]i has been demonstrated in mouse ␤-cells (8). The use of simultaneous microfluorimetric and amperometric techniques in the same type of preparation has shown (9 –11) that a close correlation exists between [Ca2⫹]i and insulin release. HERG potassium channels (12, 13) are known to regulate the duration of the heart action potential (14).The slowly decreasing action potential (AP) plateau potential shifts these channels to a less inactivated state that leads to an increasing outward K⫹ current, and this sustains further repolarization until complete repolarization is attained (15). In addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation and thus contribute to the control of burst duration. Chiesa et al. (16) have shown that ERG channels in a differentiated neuroblastoma cell sustain spike-frequency adaptation during long trains of spikes because, at rest, 1 Correspondence: Dipartimento di Biotecnologie e Bioscienze, Universita` di Milano-Bicocca, Piazza della Scienza, 2, 20126 Milano, Italy. E-mail: [email protected]

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an outward ERG current (IERG) develops that is sufficient to inhibit firing; Scho¨nherr et al. (17) found that the molecular determinants of this process are the specific properties of the activation gate. This suggests that HERG channels may also play a crucial role in other tissues in which excitability is of primary significance for neurotransmitter and hormone release. We provide evidence that RNAase protection assay experiments performed in human pancreatic islets show the presence of herg1 transcripts. Moreover, in voltage-clamped human ␤-cells, we found a K⫹ current that has all of the biophysical and pharmacological properties of the current sustained by HERG channels (14, 18). We characterized the properties of the electrical activity of brief (3–5 min) and repeated applications of high glucose and arginine in single patch-clamped ␤-cells with and without HERG channel blocker, and found the presence of hyperexcitability during the blockade of these K⫹ channels, which suggests an increased insulin release. By means of radioimmunoassay measurements of insulin in islets under the same pharmacological conditions as those used during the electrical recordings, we confirmed the predicted insulin hypersecretion.

MATERIALS AND METHODS Islet preparation Isolated islets were prepared from the pancreas of eight (cadaver) multiple organ donors, in whom the cold ischemia time had been less than 12 h. The preparation procedures have been reported in detail elsewhere (19, 20). Briefly, the enzyme collagenase (type XI, Sigma Chemical Co., St. Louis, Mo.) was used to digest the gland. The pancreatic duct was cannulated and the digestion solution [collagenase, 2 mg/ml, dissolved in 300 ml Hanks’ balanced salt solution (HBSS), a volume equivalent to approximately three times the weight of the pancreas] was slowly injected in order to distend the tissue. After distension, the gland was placed into a 500 ml glass beaker to which was added what remained of the distension solution, and the beaker was loaded into a shaking 37°C water bath, activated at 120 revolutions/min. After ⬃10 min, the pancreas was shaken with forceps for 60 s; the digestate was filtered first through a 300 ␮m, then through a 90 ␮m mesh stainless steel filter. The filtered solution and the tissue remaining in the 300 ␮m mesh filter were returned to the water bath for further digestion; the tissue remaining in the 90 ␮m filter was washed with HBSS and 10% bovine serum (BS). The same procedure of filtration, washing, and settling in HBSS solution was repeated 8 –10 times for up to 40 –50 min. For the purification, 3 ml of tissue was loaded into 220 ml conical plastic containers and resuspended in 60 ml of 80% Histopaque (Sigma) 1.077 in HBSS, topped up with 40 ml of HBSS. After centrifugation at 800 g for 5 min at 4°C, the islets were recovered at the interface between the two layers, washed with HBSS 2% BS by centrifugation at 800 g for 2 min at 4°C, and resuspended in HBSS. After being put in 15 ml of M199 culture medium (supplemented with 10% serum and antibiotics), the islets were loaded into 25 cm2 uncoated 2602

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plastic flasks (⬃2500 islets per flask, Biobraun, Milan, Italy) and cultured at 37°C in a CO2 incubator for 7–10 days, at which time the electrophysiological and secretion studies were performed. Secretion experiments The insulin secretory function of the islets was evaluated by means of static incubation as described previously (19, 20). After a 30⬘ preincubation period at 37°C in Krebs-Ringer bicarbonate solution, 0.5% albumin, pH 7.4, containing 3.3 mM glucose, they were incubated for 45 min in Krebs-Ringer solution with either (mM) 1.1 glucose, 1.1 glucose plus 20 arginine, 5.5 glucose, 5.5 glucose plus 20 arginine, or 11.1 glucose with or without the addition of 1, 5, or 10 ␮M WAY-123,398 (Wyeth-Ayerst Research, Princeton, N.J.). At the end of the incubation time, aliquots of the incubation medium were taken for insulin immunoassay measurements (Medgenix Diagnostics, Fleurs, Belgium). Molecular biology The RNAase Protection Assays were performed essentially according to Dixon and McKinnon (21). Briefly, 5–10 ␮g of total RNA from either human brain or human pancreatic islets was hybridized overnight at 48°C with 32P-UTP-labeled RNA probes. The herg probe was a fragment (240 bp long) of the herg1 clone (accession number NM000238.1KCNH2) in pBluescript SK⫹, linearized with HindIII, and transcribed with T7 polymerase. Human cyclophillin (Ambion, Austin, Tex.) was used as an internal loading control. Digestion was then performed for 1 h at room temperature with RNAase A (40 ␮g/ml) and T1 (2 ␮g/ml). Five micrograms of yeast tRNA was used as a negative control for the probe selfprotection bands. The samples were then run on a 6.6% polyacrylamide gel and exposed for 3 days. Patch-clamp solutions The standard extracellular solution contained (mM) NaCl 130, KCl 5, CaCl2 2, MgCl2 2, HEPES-NaOH 10, D-glucose 3, and pH 7.40. In the high K⫹ external solution ([K⫹]o⫽ 40 mM), NaCl was replaced by an equimolar amount of KCl. During the glucose-induced responses, 12 mM glucose was added to the standard solution; during the arginine-induced responses, 20 mM arginine was added in a 1 mM glucose extracellular solution. The standard pipette solution at [Ca2⫹]i ⫽ 10⫺7 M (pCa 7) contained (mM) K⫹-aspartate 130, NaCl 10, MgCl2 2, CaCl2 1.3, EGTA-KOH 10, HEPES-KOH 10, ATP (Mg2⫹ salt) 1, pH ⫽ 7.30. For perforated patch, pipettes were immersed briefly in the following internal solution (mM): K⫹-aspartate 140, NaCl 10, MgCl2 2, HEPES-KOH 10, pH ⫽ 7.30; they were backfilled with the same solution containing amphotericin B (150 ␮g/ml) kept at 4°C and made fresh every 2 h from a stock solution (20 mg/ml in dimethylsulfoxide, prepared before each experiment). In some experiments (data presented in Figs. 2 and 3), the control recordings were repeated (with the same protocol) in the presence of 1–2 ␮M WAY-123,398 (WAY; 22); the difference between these recordings (called WAY-sensitive currents) was used for analysis under the hypothesis that it represents the current flowing in the HERG channels. WAY (received from Dr. W. Spinelli, Wyeth-Ayerst Research) was dissolved in distilled water in order to make 10 mM stock solutions.

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Patch-clamp recordings The currents were recorded at room temperature as described previously (18, 23). Pipette resistance (3–5 M⍀), cell capacitance, and series resistance errors were carefully compensated (85–95%) before each voltage-clamp protocol run. The APs and firing were recorded (10 kHz sampling rate) in perforated patch current-clamp by means of a particular patch-clamp amplifier developed in our laboratory (24) or an Axopatch 200A in Ifast mode (Axon Instruments, Foster City, Calif.). The extracellular solutions were delivered through a 9 hole (0.6 mm), remote-controlled linear positioner placed near the cell under study, which has an average response time of 2–3 s (25). Data analysis During data acquisition and analysis, the pClamp suite (Axon Instruments) and Origin 4.1 (Microcal Inc., Northampton, Mass.) software were routinely used. The firing frequency was computed by means of the ‘Peak’ routine in Origin 4.1. The peaks were considered spikes when they were higher than ⫺30 mV. The time of the successive peaks was used to calculate the instantaneous frequency, which was then averaged (‘running’ average over at least 50 points) and plotted (see Figs. 5 and 6). The data are expressed as mean values ⫾ se. To evaluate the total predicted insulin release from the membrane potential (VM) recordings during each glucose application, it is necessary to calculate the Ca2⫹ currents (ICa) from the voltage-dependent activation curves and, given the roughly linear relationship between ICa and exocytosis (26, 27), evaluate the time integral of the instantaneous release. To compute this integral, we calculated the hypothetical ICa, at 2 ms intervals, as the product of the voltage-dependent activation Boltzmann curve 1/(1⫹e-(VM-V ⁄ )/k) (where V1/2 and k are the half activation and the slope factor in mV) multiplied by the driving force term (VM ⫺80), assuming that the Nernst potential for Ca2⫹ is ⫹80 mV. The time integral of this variable is shown in Figs. 5–7. To investigate the importance of setting the correct activation curve, we made preliminary calculations using theoretical curves derived from published data (4, 28). The Boltzmann curve parameters V1/2 and k of these two models had the values (mV) of ⫺12, 11, and ⫹9, 15. The results of these calculations, expressed as the percentage increase in release (with the HERG blocking drug) with respect to control, were 88% and 74% (see Fig. 6C). On the whole, the similar results obtained using two theoretical models with reasonably different V1/2 values led us to conclude that the overall calculation of total insulin release is relatively weakly dependent on the absolute properties of the ICa activation curve, and we finally decided to use the model of Kelly et al. (28) for plotting the data shown in Figs. 5–7. 12

ments. The herg1 transcript was analyzed using the RNAase Protection Assay with a probe consisting of a 240 bp-long fragment belonging to the herg1 sequence (accession number NM000238.1 KCNH2). The results obtained from two different pancreatic islet samples can be seen in Fig. 1, which shows that a protected herg1 transcript is detectable in human brain (used as a positive control) and, at the same or higher intensity, in the two islet samples. K(ATP) and HERG channels coexist in human ␤-cells Patch-clamp electrophysiology experiments were performed on 35 human ␤-cells identified as being spontaneously silent (in low glucose) and responding to tolbutamide, including 25 that also responded to high levels of glucose (only one responded under nonperforated patch conditions). IHERG was detected in all of these cells, and we occasionally detected spontaneously firing cells that did not respond to glucose (probably ␦-cells) or cells that became silent in high glucose levels and did not respond to tolbutamide (probably ␣-cells). These findings agree with those of Nadal et al. (11) in intact mouse Langerhans islets. In a typical experiment (see Fig. 2A), a cell perfused under normal glucose concentrations (3 mM) was tested in current-clamp mode using two concentrations (10 and 50 ␮M) of tolbutamide, a reversible blocker of K(ATP) channels, in order to verify that some of these channels are blocked and that the cell consequently

RESULTS The herg1 gene is present in human pancreatic islets Of the three known erg genes (29), only the human herg1 gene has a completely known sequence. To demonstrate the presence of this gene in pancreatic ␤ cells, its transcript was first checked in RNA extracted from the cell culture used in the patch-clamp experiINSULIN RELEASE AND HERG K⫹ CHANNELS

Figure 1. Presence of herg1 transcript in human pancreatic islets. The RNA extracted from brain and two different preparations of human pancreatic islets were probed with the human herg1 clone. Hyman cyclophillin (Ambion) was used as an internal control and yeast tRNA as a negative control for the probe self-protection bands. 2603

Figure 2. K(ATP) and HERG channels coexist in single human ␤-cells and the HERG blocker WAY does not affect K(ATP) channels. A) Recordings of tolbutamide-induced depolarizations at two different concentrations of 50 and 10 ␮M. [K⫹]o ⫽ 5 mM in the same currentclamped human ␤-cells. B, C) Voltageclamp recordings of currents elicited with the protocol shown in panel G, in the same cell as that in panel A under control conditions (B), and during perfusion with 50 ␮M tolbutamide (C). D) The result of subtracting traces panel C from those in panel B, the so-called ‘tolbutamide-sensitive’ currents. E, F) Voltage-clamp recordings in insulin-secreting cells RINm5F, of tolbutamide-sensitive and WAY-sensitive currents obtained by subtracting traces during drug (tolbutamide and WAY) application from traces in control. Test potentials were 0/⫺160 mV; WAY-123,398 5 ␮M; tolbutamide 50 ␮M; [K⫹]o ⫽ 40 mM. G) The protocol used in recordings B—D. E, F) The holding potential was set at ⫺60 mV and test potentials reach ⫺160 mV.

depolarizes. After this test establishing that the cell is a ␤-cell, we switched to the voltage-clamp mode in which we used the relatively high [K⫹]o (40 mM), useful to record consistent HERG K⫹ currents (HERG dependence on [K⫹]o; see refs 14 –18, 23). From a holding potential (VH) of 0 mV, the protocol in Fig. 2G was used to elicit deactivating tail currents under control conditions (Fig. 2B) and during the perfusion of 50 ␮M tolbutamide (Fig. 2C). Tolbutamide produced a decrease in current that clearly revealed a transient current which completely disappeared in the presence of 1 ␮M of the antiarrhythmic drug WAY-123,398 (WAY), a specific blocker of HERG channels (18; not shown). The recordings of the tolbutamide-sensitive current (obtained as recordings B minus C) are shown in Fig. 2D.

We used WAY either for isolating IHERG (see Fig. 3) or during the experiments in current-clamp (Fig. 6), but the blocker was also used in the insulin release experiments (Table 1) at relatively high doses. To exclude a blocking action of WAY on the K(ATP) channels, we performed a specific test that is shown in Fig. 3E, F. For this purpose, we used an insulin-secreting cell clone RINm5F (30), known to express K(ATP) channels, and did experiments both at normal [K⫹]o of 5 mM (n⫽5) and high [K⫹]o of 40 mM (n⫽5) at a maximal concentration of 5 ␮M of WAY. Figure 2E, F shows the tolbutamide-sensitive (IK(ATP)) and WAY-sensitive currents ([K⫹]o⫽40 mM) elicited by the protocol shown in Fig. 2G, but from a holding of ⫺60 mV used for the purpose of not activating ERG channels (recordings are the

Figure 3. Biophysical properties of WAY123,398-sensitive currents. A) Steady-state activation curve obtained from recordings similar to those shown in panel B. The symbols are means ⫾ se (n⫽10); the dashed line is the best fit to the data according to a Boltzmann curve with V1/2, ⫽ ⫺29.5 ⫾ 1.1 and slope ⫽ 9.9 ⫾ 0.9. B) Tail currents elicited according to the protocol shown at the bottom of the panel. Preconditioning time 15 s; holding potential 0 mV; inter-episode time, 20 s. C) Voltage-dependent steady-state inactivation obtained from the recordings shown in panel D. The experimental points (open squares) were obtained as normalized chord conductances. The currents were obtained by extrapolating the deactivating currents to ⫹1 ms after the onset of the test pulse. The dashed line is the best fit to the data according to a Boltzmann curve with V1/2, ⫺42 ⫾ 1.6 and slope 17.4 ⫾ 1.8. D) Inward currents recorded according to the protocol shown at the bottom of the panel. The holding potential 0 mV and the inter-episode time 10 s [K⫹]o ⫽ 40 mM. 2604

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TABLE 1. Insulin secretion by human islets stimulated by various glucose concentrations with or without 20 mM arginine in the absence or presence of 1, 5 or 10 ␮M WAYa Glucose (mM)

WAY-123,398 (␮M)

Arginine (mM)

Insulin secretion (␮U/ml)

n

1.1 1.1 1.1 1.1 5.5 5.5 5.5 5.5 11.1 11.1 11.1 11.1 1.1 1.1 5.5 5.5

0 1 5 10 0 1 5 10 0 1 5 10 0 5 0 5

0 0 0 0 0 0 0 0 0 0 0 0 20 20 20 20

5.3 ⫾ 1.4 10.4 ⫾ 3.9 12.7 ⫾ 5 # 15.1 ⫾ 6.2§,## 10.1 ⫾ 1.8 13.2 ⫾ 6 13.2 ⫾ 6.1 12.2 ⫾ 2.3 12.5 ⫾ 5.4 13.4 ⫾ 4.9 15.6 ⫾ 6.1 16 ⫾ 2.7 9.1 ⫾ 2.5 27.1 ⫾ 13.7* 12.1 ⫾ 4.4 22.8 ⫾ 4.4*

7 8 9 9 7 8 6 8 8 7 9 8 6 6 5 6

a Statistical analysis was performed by using the ANOVA test (plus the Bonferroni correction) to test differences in each group of data for any given glucose concentration (1.1, 5.5 and 11.1 mmol/l); the results obtained in the arginine experiments were analyzed by the unpaired, two-tailed Student’s t test. Values are expressed as (␮U/ ml) and represent means ⫾ sd. § P ⫽ 0.02 by the ANOVA test in this group; # P ⬍ 0.05 by the Bonferroni test vs. 1.1 mmol/l glucose alone; ## P ⬍ 0.01 by the Bonferroni test vs. 1.1 mmol/l glucose alone; * P ⬍ 0.01 by the Student’s t test vs. values without WAY.

result of subtracting traces in the presence of drug from control traces). It can be seen that in the same cell in which a large IK(ATP) current can be recorded, the application of WAY did not produce any measurable effect, thus excluding that the HERG channels blocker could contribute through the K(ATP) channels to the effects shown in Fig. 6 and Table 1. In all other experiments (n⫽10) the results were similar.

obtained by evaluating the normalized peak cord conductance (18) from recordings similar to those shown in Fig. 3D, which were obtained after applying the protocol shown at the bottom of the panel. These traces describe the same phenomenon (described before) of removal of inactivation and succeeding deactivation but tested at different membrane potentials. The experimental points fitted a Boltzmann relationship with the following parameters (mV): V1/2, ⫺42 ⫾ 1.6, slope 17.4 ⫾ 1.8. On the whole, the voltage-dependent properties of the currents found in human ␤-cells are in line with those described in the literature (14, 15, 17, 18). B cell electrophysiological identification with tolbutamide, glucose, and arginine The ␤-cells were identified by means of perforatedpatch current-clamp experiments using tolbutamide-, arginine-, or glucose-induced electrical depolarization as shown in Fig. 4. The typical membrane resistance and resting potential respectively ranged from 1.5 to 4 G⍀ (mean 1.8⫾0.2) and from ⫺49 to ⫺65 mV (mean ⫺57⫾4 mV (n⫽35)). The application of different concentrations of tolbutamide led to various depolarizing responses (see also Figs. 5 and 6), but the application of the highest concentration (50 ␮M) generally induced strong firing very similar to that obtained using the highest

The biophysical properties of HERG channels in ␤-cells The experiments designed to characterize the properties of the HERG currents were performed in high [K⫹]o extracellular solution (40 mM) and high [ATP]i (5 mM). Figure 3A shows the results of experiments carried out on 10 cells to determine the voltage dependence of the activation curve. The recordings from a typical cell are shown in Fig. 3B (protocols shown at the bottom of Fig. 3B) and were obtained as WAY-sensitive currents (see Materials and Methods). The traces are tail currents from various steady-state preconditioning levels, and represent a first and fast (7– 8 ms) phase of removal of inactivation and a second phase of deactivation lasting ⬃100 ms. The data in Fig. 3A fitted a Boltzmann relationship with the following parameters (mV): V1/2, ⫺29.5 ⫾ 1.1, slope 9.9 ⫾ 0.9. The voltage dependence of the inactivation curve (Fig. 3C) was INSULIN RELEASE AND HERG K⫹ CHANNELS

Figure 4. Pharmacological identification of human ␤-cells and the properties of tolbutamide-, arginine-, and glucose-induced responses. A) The perforated current-clamp recordings of the response of a single ␤-cell during the application of 50 ␮M tolbutamide. B) The same experiment as in panel A, but obtained during the manual injection of the hyperpolarizing current necessary to clamp the cell at a membrane potential of ⫺71 mV. C) The recording of the response of a single ␤-cell during the application of 20 mM arginine. The recordings in panels A–C were obtained using repetitive injections of a 1.4 s, 4 pA hyperpolarizing current pulse in order to monitor membrane resistance. D) The response of a single ␤-cell during a 3.5 min application of 15 mM glucose and subsequent brief puffs of 40 mM KCl or 50 ␮M tolbutamide; the inset shows a 5 s record of the first two action potentials of the glucose-induced response. 2605

glucose (15 mM) or arginine (20 mM) concentrations. In some cells (2 out of 15), the highest dose of glucose did not reach the firing level whereas 20 mM arginine almost always (8 out of 9) led to maximal depolarization. The delay in the onset of the depolarizing wave after the glucose application varied from 1.5 to 6 min (Fig. 4D). To verify that the tolbutamide-induced depolarization was sustained by an increase in membrane resistance, we applied short (1.4 s) hyperpolarizing current pulses every 15 s to test the voltage response (Fig. 4A): the application of tolbutamide led to a strong depolarization and a large increase in resistance. The same experiment was repeated also at a constant voltage (⫺71 mV) by manually injecting a hyperpolarizing steady-state current in the presence of the drug (Fig. 4B). The increases in membrane resistance were similar in both experiments and are consistent with the hypothesis that ATP-dependent K⫹ channels are closed by the drug (7). Durability and stability of the glucose-induced response in single human ␤-cells Given the lack of consistent data concerning the reproducibility of successive glucose-evoked responses in the same human ␤-cell, we analyzed the variability of the responses to various applications of glucose using two different criteria of analysis during glucose-evoked electrical activity (Fig. 5A): 1) average firing frequency (Fig. 5B) and 2) predicted cumulative insulin release (Fig. 5C), assuming that it directly correlates with the inflow of Ca2⫹ (see section on Data analysis for details). The results of one of the four experiments are shown in Fig. 5B, C. The experiments showed that the variability in glu-

cose responses after consecutive perfusions and washouts was relatively small. The mean frequency in Hz (averaged on the number of action potentials, n) was 2.97 (n⫽256), 2.76 (n⫽227), and 2.88 (n⫽297) in the three applications; the predicted cumulative release was 0.75, 0.56, and 0.53 arbitrary units. The results obtained in the four cells from which we could obtain consecutive responses fluctuated around the mean, with an se of ⫾0.04 for the average frequency and ⫾0.12 for the cumulative predicted release. On the whole, these data indicate that single human ␤-cells are capable of surviving under good conditions for more than 30 min and producing physiological responses to high glucose concentrations. Given that these responses are reproducible, any manipulation interfering with electrical activity during insulin secretion can be detected. In our experiments, we observed a variable latency and a strong initial phase of activity, but never found any evidence of oscillatory phases because we used much shorter glucose applications than those used by other laboratories. Hyperexcitability during glucose-induced response in the presence of a HERG channel blocker The threshold of the activation curve for HERG channels is around ⫺60 mV, which is the usual level of the resting potential of these cells. It is therefore reasonable to suppose that HERG channels blockade would be ineffective in obtaining depolarizations at rest, i.e., when the K(ATP) channels are still open. To verify whether the presence of HERG channels is capable of regulating the firing and consequent insulin release during glucose applications, we per-

Figure 5. The reproducibility of the glucose-induced excitability response in human ␤-cells. A) A 30 min perforated patch recording from a ␤-cell: three 15 mM glucose applications (⬃4.5 min). The first response was due to a brief application of tolbutamide. B) The computed average frequency of the action potentials during the three glucose applications. The dotted line refers to the mean frequency of each application. See text. C) The cumulative predicted insulin release in arbitrary units. See details in Materials and Methods: data analysis. Notice the small variability of the three results.

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formed experiments in which we added the WAY123,398 blocker (1 ␮M) to extracellular solutions with a high glucose concentration (5.5 min application). One of the five experiments of this type is shown in Fig. 6. We could repeat the glucose⫹WAY application twice; the same type of analysis was made as in the control experiment shown in Fig. 5. Voltage-clamp experiments (not shown) revealed that this concentration of the blocker effectively inhibits 95% of IHERG in ⬃50 – 60 s and that 90% recovery from the drug takes place in ⬃500 –700 s (18, 25). In the presence of the drug we did not find any significant differences in either the resting potential or the properties of the spikes during firing (16), but both the firing frequency and the predicted cumulative release were greatly enhanced during the HERG channel blockade. The frequency changed from 1.65 Hz in the control to 2.58 (⫹56%) and 2.26 (⫹37%) during the HERG blockade; the number of action potentials was 102, 319, and 299, respectively; the cumulative release increased by ⬃88% and 83% in comparison with the control value measured during the first glucose application without WAY. Overall, in the five cells tested under control conditions and during HERG channel blockade, the firing frequency increased by 32 ⫾ 5% and the predicted release by 77 ⫾ 8%. Hyperexcitability during arginine-induced response in the presence of HERG channel blocker As shown in Fig. 4, the human ␤-cells responded to a high concentration of arginine. Only a few cells responded to 5 mM (one out of five) and 10 mM of arginine (three out of five); eight out of nine

promptly, reversibly, and repeatedly responded to 20 mM arginine, thus suggesting that the response time is as short as in the case of tolbutamide and much faster than that induced by glucose. Using arginine instead of glucose, we performed the same set of experiments as that shown in Fig. 6, in which we blocked the HERG channel with the specific blocker. The results of one of six successful experiments are shown in Fig. 7. Figure 7A shows the recording of a 5 min application of 20 mM arginine without and with, after washout, the addition of 1 ␮M WAY123,398. Figure 7B, C shows the instantaneous frequency and predicted insulin release calculated according to the procedures used in Figs. 5 and 6, which revealed a fourfold increase in the average frequency (from 0.22 to 1 Hz, dotted line) and a doubling of release after the HERG channels blockade. Overall, in the six cells tested under control conditions and during HERG channel blockade, the predicted release increased by 89 ⫾ 6%. Insulin release from intact human islets To evaluate whether the findings reported above are paralleled by changes in insulin release from intact human islets, we tested the insulin secretion in response to varying glucose concentrations (from 1.1 to 11.1 mM) and/or 20 mM arginine with or without the addition of 1, 5, or 10 ␮M WAY-123,398, during static incubation experiments (see Table 1). At the low glucose concentration (1.1 mM), insulin release clearly increased in a dose-dependent manner after the addition of the HERG channel blocker. This effect was not found at the higher glucose concentrations (5.5 and 11.1 mM), suggesting that the

Figure 6. The effect of a HERG channel blocker produces hyperexcitability during high glucose application. In a different ␤-cell but using the same procedures for data analysis as in Fig. 5. During the last two applications of glucose, 1 ␮M of WAY-123,398 was added to the perfusate. Notice the much higher firing frequency and predicted release. See text for details.

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Figure 7. The effect of a HERG channel blocker produces hyperexcitability during high arginine application. A) A 20 min perforated patch recording from a ␤-cell. One application of 20 mM arginine (⬃5 min) was followed by a second application of arginine⫹1 ␮M WAY. The small negative jumps are due to the injection of 2 pA current pulses, before and during washout, for the purpose of monitoring membrane resistance (⬃3 G⍀). B) The computed average frequency of action potentials during the two drug applications. The dotted line refers to the mean frequency of each application. See text. C) The cumulative predicted insulin release in arbitrary units. See details in Materials and Methods: data analysis. Notice the much higher release (100%) in the presence of the HERG channel blocker.

HERG channel may play an important role in reducing insulin secretion only at low glucose levels. Blocking the HERG channels definitely increased arginine-potentiated insulin release at both low (1.1 mM) and medium (5.5 mM) glucose concentrations, but the present data do not allow any conclusion as to whether this was due to a direct effect of the blocker on the ␤-cells or to the action of the amino acid on the ␣-cells, with the subsequent action of arginine-stimulated glucagon on insulin secretion. Taken together, these data reinforce the evidence (shown in Figs. 6 and 7) that blocking HERG channels induces a hyperexcitable condition in ␤-cells that leads to more pronounced islets insulin secretion than under normal conditions.

DISCUSSION The availability of human pancreatic islets is a chance event, which induced us to undertake experiments using different techniques. In this paper, we describe some of the previously unknown aspects of glucose- and arginine-induced activity in human ␤-cells by comparing the results obtained, by means of intact single cell electrophysiological recordings and islet insulin secretion experiments, under normal conditions and after HERG channels blockade. This study was carried after discovering that human ␤-cells constitutively express the herg1 gene (Fig. 1) and are endowed with HERG currents (Fig. 3) whose properties are very similar to those already described in other tissues. Recordings of glucoseevoked electrical activity in human ␤-cells are rela2608

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tively rare (4 – 6), and we wanted to examine and analyze some of their behavioral properties using appropriate tools. This procedure was necessary in order to be able to compare the responses of single cells in which the HERG channels were normal or blocked; as far as we know, no other experiments of this type in human ␤-cells have been documented. Our single cell electrophysiological recordings and the correlated analyses (Figs. 6 and 7 for the glucoseand arginine-induced responses, respectively) indicate that the blockade of HERG channels is sufficient to cause unequivocal hyperexcitability during the application of insulin releasing drugs. A number of hypotheses have been proposed to clarify the processes that underlie the ␤-cells bursting pattern (for review see 31). Very recently Go¨pel et al. (32) suggested, in mouse ␤-cells, a novel IK(Ca) current. They show that an apamin-, charybdotoxin-, and tolbutamide-insensitive but Cd2⫹- and nifedipine-sensitive current is activated during a simulated firing command. Our group (17) used a similar type of command to explain the role of HERG currents in firing accommodation and the deactivation time constants found by Go¨pel et al. are perfectly in line with those of HERG channels (17). Moreover, Cd2⫹ produces (33), a remarkable activation right-shift that is probably sufficient to virtually block IHERG at ⫺40 mV; some Ca2⫹ channel antagonists (we tested also nifedipine) are also IHERG blockers (34). The results of the parallel islet experiments qualitatively agree with the single cell data, but revealed a different pattern at low glucose concentrations because of the basically different environmental conditions of the ␤-cells. In the Petri dish used during

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the electrophysiological experiments, the single patched ␤-cell is surrounded by only a few other cells, which prevents any paracrine signaling, whereas the ‘integrated’ system represented by the islets (in which the various endocrine cells can affect their mutual functions) means that the single ␤-cells are subject to a complex orchestration of stimuli. No major change in glucose-stimulated insulin secretion was induced in the islet preparation with higher glucose levels by the presence of WAY-123,398, thus suggesting that other signaling pathways are likely to predominate in human ␤-cells under our experimental conditions. Nesidioblastosis (also called persistent hyperinsulinemic hypoglycemia of infancy) and insulinomas are syndromes that cause severe hypoglycemia because of the excessive release of insulin, which may lead to seizures and epileptic events. Standard pancreatectomy and/or treatment with diazoxide (a K⫹ ATP-dependent channel opener) are the usual therapies, but although it has been discovered that some forms of nesidioblastosis are linked to mutations in the sulfonylurea receptor or the Kir6.2 ATP-dependent K⫹ channel (35, 36), the origin of others described in the literature (37–39) is not related to either. It has been reported (40) that islets prepared from an adult subject with nesidioblastosis showed an increase in basal insulin release that was not further enhanced by higher glucose levels. These findings resemble the data of the present study, which show that insulin release increases at low glucose levels after HERG channels blockade, and suggest the possibility that alterations in HERG channel function may play a role in the altered insulin release associated with some hyperinsulinemic conditions. It has been shown in mouse that the role of various types of amino acids is to shift the usual glucose dose response curve toward the left in such a way that a smaller glucose concentration is necessary to produce insulin secretion and electrical activity (41). The application of 20 mM of arginine in the presence of a normal glucose concentration led to a marked increase in single ␤-cell electrical activity and in islets insulin release. The HERG channel blocker considerably increased this effect in both preparations, thus suggesting that although arginine acts through a different signaling pathway, it can still depolarize ␤-cells in such a way that the HERG channels retain an inhibitory role that is eliminated when they are blocked. Furthermore, hypoglycemic conditions associated with hyperinsulinemia may be induced by amino acid feeding (42), which again suggests that HERG channels may play a role in regulating insulin release under certain circumstances. To conclude, the present study, while revealing INSULIN RELEASE AND HERG K⫹ CHANNELS

another task entrusted to the multifaceted activity of HERG channels, discloses a new branching of the complex signaling network that controls the insulin secretion in human ␤-cells. We thank Dr. D. Cuccuru, E. Redaelli, G. Curia, and Miss R. Restano Cassulini for the cell cultures, and Mr. G. Mostacciuolo for technical improvements. This work was supported by grants from the Comitato Telethon Fondazione ONLUS project 1046 and the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST-COFIN 1997–98, No. 9705157384) to E.W., the Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST-COFIN 1997–98) to M.O., the Associazione Italiana per la Ricerca sul Cancro and the Consiglio Nazionale delle Ricerche (Finalized Project ACRO) to M.O., and the Associazione Italiana contro le Leucemie (Florence) to A.A. M.L. is a Ph.D. student at Milano-Bicocca University’s Department of Biotechnology and Biosciences. O.C. is a Ph.D. student at the Department of Experimental Pathology and Oncology of Florence University.

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Received for publication February 21, 2000. Revised for publication April 24, 2000.

ROSATI ET AL.