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J. Biol. Chem., Vol. 281, Issue 14, 9361-9372, April 7, 2006

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The Zn²⁺-transporting Pathways in Pancreatic -Cells

A ROLE FOR THE L-TYPE VOLTAGE-GATED Ca²⁺ CHANNEL^*

Armen V. Gyulkhandanyan, Simon C. Lee, George Bikopoulos¹, Feihan Dai, and Michael B. Wheeler, Supported by a CIHR Investigator Award²

From the Departments of Physiology and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, M5S 1A8 Canada

Received for publication, August 3, 2005 , and in revised form, December 19, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In pancreatic -cells Zn²⁺ is crucial for insulin biosynthesis and exocytosis. Despite this, little is known about mechanisms of Zn²⁺ transport into -cells or the regulation and compartmentalization of Zn²⁺ within this cell type. Evidence suggests that Zn²⁺ in part enters neurons and myocytes through specific voltage-gated calcium channels (VGCC). Using a Zn²⁺-selective fluorescent dye with high affinity and quantum yield, FluoZin-3 AM and the plasma membrane potential dye DiBAC₄(3) we applied fluorescent microscopy techniques for analysis of Zn²⁺-accumulating pathways in mouse islets, dispersed islet cells, and -cell lines (MIN6 and -TC6f7 cells). Because the stimulation of insulin secretion is associated with cell depolarization, Zn²⁺ (5-10 µM) uptake was analyzed under basal (1 mM glucose) and stimulatory (10-20 mM glucose, tolbutamide, tetraethylammonium, and high K⁺) conditions. Under both basal and depolarized states, -cells were capable of Zn²⁺ uptake, and switching from basal to depolarizing conditions resulted in a marked increase in the rate of Zn²⁺ accumulation. Importantly, L-type VGCC (L-VGCC) blockers (verapamil, nitrendipine, and nifedipine) as well as nonspecific inhibitors of Ca²⁺ channels, Gd³⁺ and La³⁺, inhibited Zn²⁺ uptake in -cells under stimulatory conditions with little or no change in Zn²⁺ accumulation under low glucose conditions. To determine the mechanism of VGCC-independent Zn²⁺ uptake the expression of a number of ZIP family Zn²⁺ transporter mRNAs in islets and -cells was investigated. In conclusion, we demonstrate for the first time that, in part, Zn²⁺ transport into -cells takes place through the L-VGCC. Our investigation demonstrates direct Zn²⁺ accumulation in insulin-secreting cells by two pathways and suggests that the rate of Zn²⁺ transport across the plasma membrane is dependent upon the metabolic status of the cell.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Zinc is an important trace element in living organisms, and it plays a key role in many biological processes (1, 2). Under normal conditions the concentration of free Zn²⁺ in cells is extremely low (3). However, the total intracellular Zn²⁺ concentration can reach 150-200 µM (4, 5). A low concentration of Zn²⁺ in the cytoplasm is maintained by Zn²⁺-buffering systems such as metallothioneins and by compartmentalization into intracellular vesicles (6, 7). Regulation of Zn²⁺ metabolism in cells can also include plasma membrane Zn²⁺ transporters that can have opposite roles in cellular Zn²⁺ homeostasis. The Zip (Zrt/Irt-related protein) family of metal ion transporters facilitates Zn²⁺ uptake from the extracellular space or the lumen of intracellular organelles into the cytoplasm, whereas the ZnT (zinc transporter) family of transporters promotes Zn²⁺ efflux from cells or into various intracellular compartments (8-10).

In pancreatic -cells a fraction of the intracellular Zn²⁺ pool is stored with insulin in vesicles as a complex of Zn²⁺-insulin with a stoichiometry of 2:1 (3). During exocytosis Zn²⁺ is released together with insulin into the extracellular medium. The concentration of Zn²⁺ in insulin-containing vesicles of -cells is 20 mM (11, 12). Thus during exocytosis Zn²⁺ is released from vesicles into the extracellular space and, because of the increase of Zn²⁺, it can be transported back into the host cell or into neighboring cells (13). Although there is a significant amount of literature regarding transport of Zn²⁺ into various types of cells (14-24), surprisingly, little is known about transport of Zn²⁺ into -cells. Studies conducted several years ago, demonstrated the possibility of differential mechanisms of Zn²⁺ uptake into rat pancreatic islets under basal and stimulatory conditions and suggested that Zn²⁺ may be required for metabolic processes in addition to insulin crystallization (25, 26). The precise subcellular localization and distribution of Zn²⁺ in islets is also under debate. Histological data in fixed cells have shown mainly granular localization of Zn²⁺ (27, 28), whereas experiments with fractionation of islets have demonstrated that only 20-30% of the total islet Zn²⁺ was associated with the granular fraction (26, 29). Given the significance of Zn²⁺ for proinsulin biosynthesis and exocytosis, understanding the mechanisms of its uptake into islet cells is important. Dys-regulation of the Zn²⁺ transport system may result in secretory defects and contribute to -cell dysfunction associated with type-2 diabetes (30). It is also known that Zn²⁺ can induce oxidative damage (31-33), mitochondrial depolarization, opening of the mitochondrial permeability transition pore (34, 35), and function as a regulator of apoptosis (36). In addition, it has been suggested that Zn²⁺ can act as a paracrine factor involved in pancreatic cell death (37) and in the suppression of glucagon secretion from -cells (13, 38).

Electrical excitability of pancreatic -cells that controls the triggering phase of insulin secretion (39) depends on the coordinated activity of specific ion channels including the L-type VGCC³ (L-VGCC) (40, 41) and ATP-regulated potassium channels (K_ATP channels) (42). Here glucose metabolism increases the ATP/ADP ratio resulting in the closure of K_ATP channels, which in turn depolarizes the plasma membrane and opens L-VGCCs. This event leads to an acceleration of Ca²⁺ influx through the L-VGCCs (43, 44). Insulin stored in secretory vesicles is released in response to an elevation of intracellular Ca²⁺. Plasma membrane depolarization and increase in intracellular Ca²⁺ also result in the opening of voltage-dependent K⁺ (K_V) and Ca²⁺-sensitive voltage-dependent K⁺ (K_Ca) channels, which restore an outward flow of K⁺, thereby repolarizing of the cell membrane and closing the VGCC (39, 45).

It has been demonstrated that Zn²⁺ entry into heart cells can take place via dihydropyridine-sensitive Ca²⁺ channels (20), or through the N-methyl-D-aspartate- and -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate-activated Ca²⁺-permeable channels in neurons (18, 19). Routinely, the transport of Zn²⁺ into cells is investigated by radioactive methods (16, 17, 25, 26), Ca²⁺ fluorescent dyes (20, 21, 32), or the Zn²⁺-sensitive fluorescent dye Newport Green (22-24). Recently a new Zn²⁺-selective fluorescent dye, FluoZin-3, was introduced as a probe for Zn²⁺ detection (46). FluoZin-3 binds Zn²⁺ with very high affinity (K_D = 15 nM), and it has much higher quantum yield than other Zn²⁺-sensitive dyes previously used (46). We employed a cell-permeant form of the dye, FluoZin-3 AM, to elucidate the Zn²⁺-transporting pathways in the -cell lines MIN6 and -TC6f7 (-TC), mouse islets, and dispersed islet cells. We observed that Zn²⁺ uptake into -cells occurred through two distinct pathways, one involving the plasma membrane Zn²⁺ transporter under basal (low glucose/hyperpolarized) conditions and another through L-VGCC during the depolarization of the plasma membrane. Our data suggest that Zn²⁺ entry into -cells is linked to the metabolic status of the cell. Under stimulatory conditions, Zn²⁺ entry is mediated through the L-type VGCC, whereas under basal conditions its uptake is independent of this channel and possibly dependent on the ZIP family of transporters. To this end we demonstrate by real time PCR analysis the expression of Zip1, Zip2, Zip3, Zip4, Zip5, Zip8, Zip9, Zip10, and Zip14 mRNA in MIN6 cells and mouse islets, with Zip1 and Zip8 being the most abundantly expressed transcripts in MIN6 cells and Zip1 in islets.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents—Fluorescent dyes FluoZin-3 AM, bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC₄(3)), tetramethylrhodamine methyl ester (TMRM), Hoechst 33342 were obtained from Molecular Probes (Eugene, OR). Dispase II (neutral protease) was from Roche Diagnostics. Pyrithione, N,N,N',N',-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), verapamil, nifedipine, nitrendipine, tolbutamide, tetraethylammonium (TEA), Gd³⁺, La³⁺, collagenase, collagen, poly(L-lysine), and poly(L-ornithine) were from Sigma-Aldrich.

MIN6 and -TC Cells—MIN6 insulinoma cells (passages 35-45), a gift from Dr. S. Seino (Chiba University), were cultured in high glucose Dulbecco's modified Eagle's medium with 10% bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 µl/500 ml -mercaptoethanol at 37 °C and 5% CO₂-95% air. -TC insulinoma cells (passages 40-45), a gift from Dr. S. Efrat (Albert Einstein University), were cultured in RPMI 1640 medium with 10% bovine serum, 1% L-glutamine, 5.6 mM glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, pH 7.4. Culture medium in both cell lines was changed every 48 h, and cells were grown in monolayer to 80-90% confluence. For fluorescent measurements confluent cell cultures were lifted using treatment by EDTA (0.5 mM)/trypsin (0.05%) solution. The harvested cells were centrifuged, and the pellet was diluted in culture medium and plated on glass coverslips. For experiments coverslips with MIN6 cells were maintained in culture medium for 1-3 days. Experiments with -TC cells were performed typically 1-2 days after plating.

Pancreatic Islets—Islets were isolated from C57BL/6 mice as described previously (47, 48) with minor modifications. Briefly, mice were anesthetized, and the pancreatic duct was perfused with RPMI 1640 containing type V collagenase (0.8 mg/ml) and bovine serum albumin (2%). The pancreas was then removed and digested for 12 min at 37 °C. Islets were subsequently picked by hand, transferred to fresh medium, and maintained at 37 °C. The islets were cultured in RPMI 1640 supplemented with 10% bovine serum, 11.1 mM glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, pH 7.4. The islets were plated on glass coverslips coated with collagen, poly(L-lysine) or poly(L-ornithine) and maintained in culture medium for 1-2 days.

Dispersed Islet Cells—To obtain dispersed cells, isolated islets were incubated for 10 min in Ca²⁺-free phosphate-buffered solution, supplemented with 2 mM EGTA, 3 mM glucose, 100 units/ml penicillin, and 100 µg/ml streptomycin. Islets were then centrifuged and incubated with dispase II for 10 min at 37 °C for digestion, followed by the addition of RPMI 1640 medium with 11.1 mM glucose, 10% bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES, pH 7.4. The suspension was centrifuged, and the pellet resuspended in the same medium. The cells were plated on glass coverslips coated with poly(L-lysine) and maintained in culture for 1-3 days.

Conventional Fluorescent Measurements—For fluorescent measurements the incubation and perfusion buffer had the following composition: 130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM NaHCO₃, 200 nM TPEN, 10 mM HEPES, pH 7.4, or the same medium without Ca²⁺. In selected experiments, to depolarize the cells, 50 mM NaCl was replaced with 50 mM KCl.

Fluorescent experiments were carried out using an Olympus BX51W1 fluorescent microscope fitted with 20x/0.95 water immersion objective and cooled CCD camera equipped with magnification changer (U-TVCAC, Olympus). For excitation a xenon lamp-based DeltaRam high speed monochromator from Photon Technology International (PTI, Lawrenceville, NJ) was used. For the control of the monochromator and videocamera, as well as for fluorescent imaging and collection of the data, ImageMaster 3.0 software (PTI) was used.

For experiments, the cells or islets were transferred to an open chamber, placed on the microscope stage, and perfused at 1 ml/min. All time-dependent experiments were performed at 36-37 °C using TC-324B Heater Controller (Warner Instruments, Hamden, CT). In static experiments a Delta T culture dish controller (Bioptechs, Butler, PA) was used for heating. 1-30 cells or 1-2 islets were analyzed per coverslip.

Measurement of Zn²⁺ and Plasma Membrane Potential (_P)—For Zn²⁺ measurements coverslips with MIN6, -TC, dispersed islet cells, or islets were loaded with 2 µM FluoZin-3 AM for 50 min in incubation buffer in the presence of low glucose (1 mM) at 37 °C and 5% CO₂-95% air. The cells or islets were subsequently incubated for 10 min in the same conditions without dye. The fluorescence of FluoZin-3 AM was excited at 480 nm and emission measured with 525 nm band pass filter using 505 nm beam splitter.

We examined the response profile of dispersed islet cells, loaded with FluoZin-3 AM and exposed to sequential perfusion with Zn²⁺. Tominimize background Zn²⁺ level in these experiments all chemicals used for the buffer were of highest purity, and the water was of HPLS grade (Sigma). However, perfusion of cells with the Zn²⁺ ionophore pyrithione in the absence of Zn²⁺ led to a considerable increase of fluorescence, due to the presence of Zn²⁺ traces. To avoid this initial pyrithione-induced increase in fluorescence 200-300 nM Zn²⁺ chelator TPEN was added in the buffer. In this case, perfusion with pyrithione in the absence of Zn²⁺ led to a slight increase of fluorescence. Experiments showed that perfusion even with 100 nM Zn²⁺ resulted in a significant increase of fluorescence (supplemental Fig. 1).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Fluorescent Zn2+ detection in MIN6 cells. A coverslip with cells in incubation medium containing 1 mM glucose was loaded with 2 µM FluoZin-3 AM for 50 min at 37 °C and washed twice with the same medium without dye and glucose. The coverslip was then transferred to the chamber with incubation medium at 37 °C and after treatments the corresponding images were captured. A, DIC image of MIN6 cell. B, fluorescent image of the same cell incubated with 1 mM glucose before Zn2+ treatment. C,10 µM Zn2+ was added to incubation medium, and the image was acquired after incubation for 30 min. D, 250 µM KATP channel inhibitor tolbutamide was added to C, and the image was acquired after 15 min. Under the fluorescent images the corresponding line profiles of FluoZin-3 fluorescence intensity along the line drawn in A are shown. Fluorescence was normalized to background (100%).

Confocal Fluorescent Measurements—Confocal imaging was performed using a Zeiss LSM510 laser scanning microscope. Coverslips with islets, dispersed cells, or MIN6 cells loaded with FluoZin-3 AM (2 µM), or co-loaded with FluoZin-3 AM and TMRM (100 nM), were transferred into the chamber and plated on the stage of the microscope fitted with a 40 x 0.75 water immersion objective. For both FluoZin-3 AM and TMRM excitation the 488 nm argon laser line was used, and emissions were acquired using fluorescein isothiocyanate and TRITC set of filters, respectively. The temperature was kept at 37 °C by warming the closed box surrounding the microscope stage with air. Samples were subjected to optical sectioning by moving the focal plane along the vertical (z) axis. The images were analyzed using Zeiss LSM Image Browser software.

Deconvolution Fluorescent Microscopy—Coverslips with MIN6 cells co-loaded with FluoZin-3 AM (2 µM) and the DNA staining reagent Hoechst 33342 (1 µM) were transferred into the chamber and plated on the stage of the Axioplan 2 microscope fitted with a 40 x 0.75 water immersion objective. For Hoechst 33342 and FluoZin-3 AM excitation and emission, 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate set of filters were used, respectively. The Zeiss Axiovision 3.0 software was used for deconvolution.

Quantitative PCR for Zinc Transporters—Total RNA was isolated from MIN6 cells, mouse islets, and whole mouse brain using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Following extraction, total RNA was treated with rDNase I (Ambion, Houston, TX), and first strand synthesis of cDNA was carried out using Superscript II RNase H reverse transcriptase (Invitrogen) and oligo(dT) primers (Invitrogen) according to the manufacturer's instructions. The resulting cDNA (10 ng/reaction or 2.5 ng/µl) was used for amplification in quantitative real-time PCR (qPCR). Serial dilutions of mouse genomic DNA were used for the generation of a standard curve (9, 3, 1, 0.33, and 0.11 ng and a nontemplate negative control). Briefly, genomic DNA or cDNA (4 µl/well) was added to a qPCR mixture (6 µl/well) containing the following components: 3.5 µl of water, 1 µl of 10x PCR buffer, 0.6 µl of 50 mM MgCl₂, 0.2 µl of 50 µM Primer Mix (or 0.1 µl of forward and 0.1 µl of reverse), 0.2 µl of 10 mM dNTP mixture, 0.2 µl of ROX reference dye, 0.3 µl of SYBR green I (stock-diluted 1:1000 in water) and 0.025 µl (or 125 units) of platinum Taq polymerase (Invitrogen). For PCR amplification the following general protocol was employed: 95 °C (3 min); 40 cycles of PCR: 95 °C (10 s), 65 °C (15 s), 72 °C (20 s); 95 °C (15 s), 60 °C (15 s), 95 °C (15 s). qPCR was performed in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Streetsville, ON, Canada). Gene-specific oligonucleotide primers were designed using the Primer Quest SciTool (Integrated DNA Technologies, Skokie, IL). Primer sequences are indicated in supplemental Table 1. The expression level of various Zip transcripts was calculated using the standard curve method (49). Values were normalized to mouse -actin mRNA and represent the average of three independent experiments.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Time-dependent kinetics of Zn2+ accumulation and P changes in MIN6 cells at basal and depolarizing conditions. A, upper panels depict DIC image and fluorescent images of cells stained with FluoZin-3 AM at different times of Zn2+ accumulation shown in lower panel A. A, Zn2+ uptake by MIN6 cells. As indicated by bars, cells were perfused with 1 mM glucose during the whole experiment. Then 10 µM Zn2+, followed by 250µM tolbutamide and 50 mM KCl were added. As a control a membrane-permeable Zn2+ chelator TPEN was added after KCl. The trace depicts the averaged time-dependent response ± S.E. of 17 separately delineated cells. B, the normalized values of the mean slope ± S.E. of FluoZin-3 fluorescence change after perfusion with corresponding reagents shown in A. For presentation of slope data a linear approximation of corresponding parts of each cell trace was performed and then mean slope ± S.E. was calculated. B, effect of depolarizing compounds on P in MIN6 cells. Upper panels depict fluorescent images of cells loaded with DiBAC4(3) at different times during the perfusion with depolarizing compounds shown in the bottom panel. Cells were loaded with 250 nM DiBAC4(3) for 15 min at 37 °C. Then cells were mounted on perfusion chamber and perfused with incubation buffer containing 1 mM glucose and 250 nM dye. The averaged time-dependent response ± S.E. of 19 cells is shown. Both traces are representative of three experiments. Horizontal bars indicate perfusion with corresponding reagents. As a baseline (100%) here and in subsequent figures, the mean level of fluorescence during the initial 1 min was taken.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Intracellular Zn²⁺ Distribution and Time-dependent Kinetics—In previous studies a cell-impermeable form of the dye FluoZin-3 tetrapotassium salt was used to monitor efflux of Zn²⁺ from the cells (50). In the present study we used the membrane-permeable FluoZin-3 AM to investigate the intracellular distribution and kinetics of Zn²⁺ accumulation into -cell lines and islets. Fig. 1 shows differential interference contrast (DIC) (Fig. 1A) and fluorescent images (Fig. 1, B-D) of a single MIN6 cell stained with 2 µM FluoZin-3 AM. The image in Fig. 1B indicates a visible level of fluorescence after incubation in medium with low glucose and in Zn²⁺-free conditions, demonstrating the presence of endogenous Zn²⁺. Further addition of 10 µM Zn²⁺ to cells strongly increases the fluorescence intensity (Fig. 1C). Following treatment with the membrane-depolarizing agent tolbutamide, an inhibitor of the K_ATP channel, a further increase of fluorescence was observed (Fig. 1D). The corresponding profiles of fluorescence intensity along the line shown in Fig. 1A are presented under the images. Images Fig. 1, B-D and line profiles of fluorescence intensity demonstrate a nonuniform distribution of the fluorescent probe. As seen from the images, after incubation of the cells with Zn²⁺ (Fig. 1C) and then with tolbutamide (Fig. 1D), the whole-cell fluorescence was elevated, and the area of maximal fluorescence intensity was shifted to a more central point in the cell. Similar results were obtained with another insulinoma cell line -TC (supplemental Fig. 2). This shift in fluorescence to the central part of the cell does not indicate that Zn²⁺ penetrates into the nuclear region. Experiments with simultaneous loading of MIN6 cells with FluoZin-3 AM and the DNA staining reagent Hoechst 33342 demonstrated the absence of Zn²⁺ within the nucleus (supplemental Fig. 3A). A series of optical sections of dispersed mouse islet cells incubated with Zn²⁺ for 10 min under depolarized conditions (30 mM KCl) also did not cause Zn²⁺ accumulation in the nucleus (supplemental Fig. 3B). Most likely, the increase in fluorescence after Zn²⁺ treatment (Fig. 1, C and D, and supplemental Fig. 2, C and D) reflects the accumulation of Zn²⁺ in the space surrounding the nucleus. It is noteworthy that a series of optical sections of MIN6 cells (supplemental Fig. 4) stained simultaneously with FluoZin-3 AM (green) and the mitochondrial membrane potential marker TMRM (red) showed a punctuate pattern of fluorescence with evidence of partial co-localization, probably because of the presence of Zn²⁺ within mitochondria. It is interesting to note the prominent location of mitochondria in the perinuclear region. Taking into account the ability of Zn²⁺ to accumulate in mitochondria (51-54) the shift and increase in fluorescence could reflect Zn²⁺ entry into the mitochondria.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. Time-dependent kinetics of Zn2+ accumulation and P changes in islets at basal and depolarizing conditions. A, upper panels depict DIC image and fluorescent images of islet loaded with FluoZin-3 AM at different times during Zn2+ accumulation as shown below in panel A. A,Zn2+ uptake by islet. B, the normalized values of the mean slope ± S.E. of FluoZin-3 fluorescence change after perfusion with corresponding reagents shown in A. Data show the results of three experiments. Scale bar,50 µm. B, effect of depolarizing compounds on P in islet. Upper panels depict fluorescent images of cells loaded with DiBAC4(3) at different times during the perfusion with depolarizing compounds shown in the lower panel. The traces both in A and B show the global response (solid line) and responses from randomly chosen noncontiguous regions from the middle (dotted line) and from periphery (dashed line) of the islet. In both cases these regions are shown in the upper panel by numbers 1 (middle)and3(periphery). In both cases the traces are representative of three independent experiments.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Time-dependent kinetics of Zn2+ accumulation in dispersed islet cells at basal and depolarizing conditions. Upper panels depict DIC and fluorescent images of dispersed islet cells loaded with FluoZin-3 AM at different times during Zn2+ accumulation as shown below in panel A. For measurements the field of view with large cells was chosen. A,Zn2+ uptake by dispersed islet cells. The trace depicts averaged time-dependent response ± S.E. of all cells shown in the upper panel. B, the normalized values of the mean slope ± S.E. of FluoZin-3 fluorescence change after perfusion with corresponding reagents shown in A. The trace is representative of three independent experiments.

To determine that the conditions used in Fig. 2A actually led to cell depolarization we performed experiments with the anionic dye DiBAC₄(3), used for monitoring _P. As seen in Fig. 2B in the presence of 1 mM glucose, MIN6 cells are in a polarized state (low level of fluorescence). Treatment with tolbutamide and subsequently KCl depolarized the plasma membrane (increased level of fluorescence) because of the closure of the K_ATP channel. Upper panels represent fluorescent images of the same cells at corresponding times.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Effect of stimulatory glucose concentration on Zn2+ accumulation in MIN6 cells and islets. A, increase of Zn2+ accumulation by stimulatory amounts of glucose followed by 50 mM KCl in MIN6 cells. The trace depicts mean value ± S.E. of 12 cells. B, effect of high glucose perfusion on P in MIN6 cells. The trace depicts mean value ± S.E. of 16 cells. C, increase of Zn2+ accumulation by stimulatory amounts of glucose followed by 50 mM KCl in islet. D, effect of high glucose perfusion on P in islet. The traces in C and D show the global response (solid line) and responses from randomly chosen noncontiguous regions from the periphery (dashed line) and from the middle (dotted line) of the islet. In all cases the traces are representative of three to five independent experiments.

As seen from Fig. 3A (upper panel) the highest level of fluorescence is localized in the periphery of the islet. It is known that the predominant cell types in islets are -cells and -cells (in general 70-80% and 10-20%, respectively) (38, 55), and in mouse islets -cells mainly occupy the periphery (mantel) of a -cell core (56). To determine the origin of brighter fluorescence intensity in the periphery of the islet we performed a confocal microscopic examination. A series of optical sections through the islet (supplemental Fig. 5, B and C) revealed bright fluorescence not only in the periphery of the islet, but also more centrally where most of the cells are -cells (56). The intensity of fluorescence decreases when approaching the islet center (supplemental Fig. 5, E-G) most likely reflecting a limited diffusion of the dye to the core of the islet.

We also conducted experiments with dispersed islet cells (Fig. 4). Because we measured the FluoZin-3 fluorescence mainly from the large cells (the mean diameter of -cells is more than 20% larger than the -cell diameter (57, 58)), it is highly probable that these data are representative of -cells. Fig. 4 demonstrates that the kinetics of Zn²⁺ accumulation in dispersed cells are similar to these presented in Fig. 2A (MIN6 cells) and Fig. 3A (islet).

It is known that TPEN has very high affinity for Zn²⁺ (10^15.58 M^-1) (59), much higher than its affinity for Ca²⁺ (10^4.4 M^-1) or Mg²⁺ (10^1.7 M^-1) (60). These data strongly suggest that the observed changes in FluoZin-3 fluorescence reflect Zn²⁺ accumulation into cells, rather than an increase in intracellular Ca²⁺ due to Ca²⁺ influx, or a possible indirect effect of Zn²⁺ on intracellular Ca²⁺ stores. To further validate this we investigated the changes in fluorescence of FluoZin-3 AM-loaded MIN6 cells perfused in the absence of Zn²⁺. Our data show that perfusion of cells with 1 mM glucose followed by 20 mM glucose, 50 mM KCl and then by 5 µM Zn²⁺ ionophore pyrithione did not result in an increase of fluorescence (supplemental Fig. 6A). Experiments with islets and dispersed islet cells also demonstrated the absence of a Ca²⁺ effect on the Zn²⁺ dye (supplemental Fig. 6, B and C). As seen from these figures, treatment with high glucose in the absence of Zn²⁺ in the medium resulted in a decrease in intracellular Zn²⁺. This Zn²⁺ release was not necessarily associated solely with insulin granules. It has been previously demonstrated in islets loaded with ⁶⁵Zn that Zn²⁺ release can occur in the absence of detectable insulin secretion (61).

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Effect of TEA treatment on glucose-induced Zn2+ accumulation. TEA-induced acceleration in Zn2+ accumulation (A) and changes in P in MIN6 cells (B). The traces depict mean value ± S.E. of 25 and 31 cells, respectively. TEA-induced acceleration in Zn2+ accumulation (C) and changes in P in islets (D). The traces show the global response (solid line) and responses from randomly chosen noncontiguous regions from the periphery (dashed line) and from the middle (dotted line) of the islet. In all cases the traces are representative of four to six independent experiments.

Glucose-stimulated Zn²⁺ Uptake—Our initial experiments have shown that Zn²⁺ can accumulate in -cells under low glucose (1 mM glucose) and following cell depolarization, using tolbutamide or KCl, which dramatically increased the rate of Zn²⁺ transport into the cytosol. The effect of a more physiological insulinotropic stimulus, glucose (15-20 mM), on FluoZin-3 AM-loaded MIN6 cells and islets, is shown in Fig. 5. Perfusion of MIN6 cells with high glucose led to significant Zn²⁺ accumulation (Fig. 5A). Experiments with DiBAC₄(3) verified that treatment with 20 mM glucose indeed led to the depolarization of the plasma membrane (Fig. 5B). Perfusion of islets with a stimulatory amount of glucose (15 mM) also resulted in Zn²⁺ accumulation (Fig. 5C). Treatment with 15 mM glucose led to depolarization of the plasma membrane in islets as well (Fig. 5D).

The Effect of K_v Channel Inhibition on Glucose-stimulated Zn²⁺ Uptake—It is well known that TEA potentiates glucose-stimulated insulin secretion via the inhibition of the delayed rectifier potassium channel (K_v) causing increased frequency of action potentials (45). We found that combined treatment with 10 mM TEA/high glucose caused a robust increase in the rate of Zn²⁺ accumulation compared with high glucose alone both in MIN6 cells (Fig. 6A) and islets (Fig. 6C). Experiments with DiBAC₄(3) demonstrated the changes in _P after TEA administration in MIN6 cells (Fig. 6B) and in islets (Fig. 6D).

Effect of L-type Ca²⁺ Channel Inhibitors on Zn²⁺ Uptake—It is known that all L-type channels share a common pharmacological profile and are blocked by phenylalkylamines and dihydropyridines (62). For evaluation of the role of L-VGCC in Zn²⁺ transport we used verapamil (phenylalkylamine), nitrendipine, and nifedipine (dihydropyridines), as well as the nonspecific inhibitors of Ca²⁺ transport, Gd³⁺, and La³⁺. In Fig. 7A, curve 1, is a representative trace of the mean value ± S.E. received from control experiments with MIN6 cells. Fig. 7A, curve 2 shows the results of pretreatment with 50 µM nitrendipine on Zn²⁺ uptake. As seen from Fig. 7A (curve 2) nitrendipine did not inhibit Zn²⁺ uptake under nonstimulatory conditions (1 mM glucose). However, it significantly inhibits KCl-induced Zn²⁺ accumulation.

Verapamil, another known L-VGCC inhibitor, also blocked KCl-induced Zn²⁺ uptake in MIN6 and -TC cells (Fig. 7, B and C). Fig. 7D demonstrates the effect of the nonspecific Ca²⁺ channel blocker Gd³⁺ on the kinetics of Zn²⁺ influx during the perfusion of -TC cells with a stimulatory amount of glucose. Curve 1 (Fig. 7D) represents the mean value ± S.E. obtained from control cells. As seen from curve 2 (Fig. 7D) perfusion with 100 µM Gd³⁺ significantly inhibits the increase in Zn²⁺ accumulation caused by 10 mM glucose. Another L-type channel inhibitor (nifedipine 80 µM, not shown) or the nonspecific Ca²⁺ channel blocker La³⁺ (300 µM, not shown) also suppressed depolarization-dependent Zn²⁺ accumulation. Experiments with islets (Fig. 7E) demonstrated that verapamil did not affect Zn²⁺ accumulation under basal (hyperpolarized) conditions but inhibited Zn²⁺ accumulation caused by KCl depolarization as in MIN6 cells (Fig. 7A). These data strongly suggest that depolarization-dependent Zn²⁺ transport is mediated by Zn²⁺ entry through the L-VGCC.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Effect of L-VGCC inhibitors on Zn2+ uptake by MIN6, -TC cells, and islets. A, effect of treatment with nitrendipine on Zn2+ accumulation in MIN6 cells. Curve 1, MIN6 cells were perfused with low amount of glucose followed by 50 mM KCl. Curve 2, MIN6 cells were perfused under same conditions as in curve 1, except that medium additionally contains 50 µM nitrendipine. As seen from comparison of curves 1 and 2, nitrendipine maintains the ability of cells to accumulate Zn2+ in low glucose condition, but inhibits Zn2+ accumulation caused by KCl depolarization. Traces 1 and 2 represent averaged responses ± S.E. of 17 and 18 cells, respectively. B, verapamil-induced inhibition of Zn2+ uptake in MIN6 cells at depolarizing conditions. Traces 1 and 2 represent the averaged responses ± S.E. of 15 and 20 cells, respectively. C, verapamil-induced inhibition of Zn2+ uptake in -TC cells at depolarizing conditions. Traces 1 and 2 represent the averaged responses ± S.E. of 9 and 11 cells, respectively. D, inhibition of Zn2+ accumulation by Gd3 in -TC cells. In both cases traces represent averaged responses ± S.E. of 15 cells. E, inhibition of Zn2+ uptake by verapamil in islets. Verapamil maintains the ability of Zn2+ accumulation in basal hyperpolarized conditions but inhibits Zn2+ accumulation caused by KCl depolarization. The global response of islet is shown. In all cases the traces are representative of three to six independent experiments.

It has long been recognized that Zn²⁺ is an important element in brain function (63, 64). For this reason we used brain tissue as a positive control for the expression of various Zip transcripts. Our experiments in whole brain showed that the most robustly expressed Zn²⁺ transporter transcripts in brain were Zip1, Zip9, Zip10, and Zip14 (Fig. 8B). The high expression of Zip1 mRNA has previously been reported in the human brain (14).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the -cell Zn²⁺ has an important role in the stabilization of insulin molecules and serves as a cofactor for many vital enzymes. It is also possible that Zn²⁺ can regulate Ca²⁺ influx in cells and thereby modulate insulin secretion in an alternative way. This possibility is supported by the observation that Zn²⁺ can regulate Ca²⁺ and K_ATP channels, as demonstrated in pheochromocytoma and kidney cell lines as well as hippocampal mossy fibers (21, 65). The mechanism of Zn²⁺ transport into -cells through the plasma membrane has not been extensively examined. One study suggested that a facilitated mechanism of Zn²⁺ influx exists at low Zn²⁺ (0.5-7 µM) concentrations and a diffusion mechanism at higher Zn²⁺ (10-3000 µM) concentrations (25). Our investigation is the first to demonstrate, both spatially and temporally, direct Zn²⁺ accumulation in insulin-secreting cells. The main finding of our study was that transport of Zn²⁺ into MIN6 and -TC insulinoma cell lines and mouse islets occurs by two pathways, likely via a plasma membrane Zn²⁺ transporter(s) in basal state and through the L-type of Ca²⁺ channels (VGCCs) under depolarizing conditions. These results therefore suggest that the rate of Zn²⁺ transport and accumulation is in part dependent upon the metabolic status of the -cell.

It appears that Zn²⁺ entry into neuronal cells may occur via three pathways: VGCCs, N-methyl-D-aspartate receptor-gated channels, and the Ca²⁺-permeable AMPA-kainate receptor-gated channels (18, 19, 23, 66-68). Zn²⁺ influx through VGCCs is also observed in bovine chromaffin cells (69) and myocytes (20). Activation of VGCCs was caused by high K⁺ solution or AMPA/kainate in neuronal cells (19, 68) and by high K⁺ or 1,1-dimethyl-4-phenylpiperazinium in chromaffin cells (69). In myocytes VGCCs were activated by electrical stimulation (20). In all aforementioned cells Zn²⁺ entry was inhibited by the Ca²⁺ channel antagonist dihydropyridine. Verapamil, another L-VGCC inhibitor had no effect in neuronal cells (68). The unique feature of pancreatic -cells is depolarization of the plasma membrane and activation of VGCCs by stimulatory glucose concentrations (10-20 mM) because of an inhibition of K_ATP channels (42-44). Data presented in Fig. 5 show that perfusion with stimulatory amounts of glucose causes Zn²⁺ entry into -cells. The rate of Zn²⁺ accumulation is significantly enhanced with inhibition of K_V and K_Ca channels (Fig. 6), which repolarize the plasma membrane. Fluorescent images of an individual cell shown in Fig. 1, and the time-dependent kinetics of FluoZin-3 and DiBAC₄(3) fluorescence in insulinoma cells (Fig. 2), islets (Fig. 3), and dispersed islet cells (Fig. 4) also confirm that Zn²⁺ accumulation takes place following depolarization of cells by the K_ATP channel inhibitor tolbutamide or by KCl. Experiments with specific inhibitors of VGCCs (verapamil, nitrendipine, and nifedipine) (Fig. 7) indicate that one of the pathways of Zn²⁺ entry into -cells is the L-type VGCC.

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Real time quantitative PCR analysis for zinc transporter transcript expression in MIN6 cells, islets, and brain. Expression of zinc transporter transcripts in MIN6 cells and islets (A) and brain (B) was calculated using mouse -actin as the normalizing gene. Results represent the average ± S.E. of three to four independent experiments.

The balance between Zn²⁺ accumulation and Zn²⁺ efflux is crucial for cell survival. The zinc efflux transporter (ZnT-5) that is abundantly expressed in the human pancreas was recently cloned and characterized (70). Moreover, a -cell-specific zinc transporter, ZnT-8, localized on insulin secretory granules, was identified and cloned (71). ZnT-8 apparently plays an important role for Zn²⁺ transport into secretory vesicles of pancreatic -cells. However, as stated, little is known about Zn²⁺ influx transporter(s) at the level of the -cell plasma membrane. Detectable levels of Zip1 mRNA were previously demonstrated in human (14), but not mouse, pancreas (72). Two recent studies (17, 73) showed expression of Zip5 mRNA transcripts in the whole mouse pancreas by means of Northern blotting, although in the first case (17) the expression was much higher than in the latter (73). Using immunofluorescent methods the presence of Zip4 in pancreatic -cells was also detected (73).

View larger version (19K): [in this window] [in a new window]   FIGURE 9. Schematic representation of sequence of events resulting in Zn2+ transport into -cells using different pathways. The numbers 1-7 indicate the sequence of events. 1,Zn2+ transport into -cells at basal conditions using transporter(s); 2, increase of ATP/ADP ratio because of stimulation of mitochondrial metabolism by glucose; 3, closure of KATP channel and depolarization of plasma membrane; 4, activation of L-VGCC and Ca2+ entry; 5, release of Zn-insulin granules into extracellular space; 6, Zn2+ entry through L-VGCC; 7, repolarization of plasma membrane by KV channels. The symbols or indicate activation or inhibition, respectively.

The necessity for such a broad spectrum of zinc transporters in -cells is not clear. The mechanism of action of these transporters, as well as which of them plays a predominant role in Zn²⁺ uptake in basal conditions has not been defined. It is possible that accumulation of Zn²⁺ through this transporter(s) is driven by changes in plasma membrane potential. Further studies are needed to clarify the specific localization of these transporters and regulation of their expression. Zip4, which is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency, is an example of zinc transporter gene regulation (73). It has also been demonstrated that in zinc-repleted embryonic kidney cells, Zip1 and Zip3 transporters can rapidly translocate between the plasma membrane and intracellular compartments (74).

It is possible that Zn²⁺ influx into -cells is primarily regulated by the activities of the plasma membrane transporter(s) and the L-VGCCs. Under our model (Fig. 9), during low glucose conditions plasma membrane Zn²⁺ transporter(s) compensate for any possible Zn²⁺ deficiency. However, after glucose-stimulated insulin secretion, when the concentration of Zn²⁺ in the cytoplasm is rapidly depleted, the capacity of Zn²⁺ transporter(s) is insufficient for fast uptake of Zn²⁺ into cells. Under this condition, Zn²⁺ can also accumulate in cells via L-type VGCC. Apparently, switching from one type of transport to another would depend on the metabolic status of the cell.

As we mentioned above, the predominant cell types in pancreatic islets are - and -cells. Because our methods do not distinguish between primary - and -cells we cannot rule out that a similar or related transport system exists in -cells. This possibility is supported by the presence of L-VGCC in -cells (75). In fact, the cells on the periphery of the islet, part of which are -cells (56), do increase Zn²⁺ accumulation after depolarization (Figs. 3A and 5C). Given that Zn²⁺ release from -cells may influence glucagon secretion from -cells (13, 38), such putative transport mechanisms warrant further study in this cell type.

	FOOTNOTES

 
^* This work was supported in part by an operating grant (MOP-49521) from the Canadian Institutes of Health Research (CIHR) (to M. B. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

¹ Supported by a Banting and Best Diabetes Center (BBDC) Studentship.

² To whom correspondence should be addressed: Dept. of Physiology, University of Toronto, 1 King's College Circle, Rm. 3352 Toronto, Ontario, M5S 1A8 Canada. Tel.: 416-978-6737; Fax: 416-978-4940; E-mail: michael.wheeler{at}utoronto.ca.

³ The abbreviations used are: VGCC, voltage-gated calcium channel; L-VGCC, L-type VGCC; K_ATP, ATP regulated K⁺ channel; K_V, voltage-dependent K⁺ channel; K_Ca, Ca²⁺-sensitive voltage-dependent K⁺ channel; _P, plasma membrane potential; DiBAC₄(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol; TMRM, tetramethylrhodamine methyl ester; TEA, tetraethylammonium; TPEN, N,N,N',N',-tetrakis-(2-pyridylmethyl)ethylenediamine; TRITC, tetramethylrhodamine isothiocyanate; qPCR, quantitative real time PCR; DIC, differential interference contrast; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid.

	ACKNOWLEDGMENTS

 
Imaging equipment was purchased through funding opportunities from the BBDC and CIHR. We thank Dr. J. Manning Fox for careful review of the manuscript.

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