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Endocrinology Vol. 139, No. 7 3227-3231
Copyright © 1998 by The Endocrine Society

ARTICLES

Unmasking of a Periodic Na+ Entry into Glucose-Stimulated Pancreatic ß-Cells after Partial Inhibition of the Na/K Pump1

Eva Grapengiesser

Department of Medical Cell Biology, Uppsala University, S-751 23 Uppsala, Sweden

Address all correspondence and requests for reprints to: Dr. E. Grapengiesser, Department of Medical Cell Biology, Biomedicum, Box 571, S-751 23 Uppsala, Sweden. E-mail: eva.grapengiesser{at}medcellbiol.uu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cytoplasmic concentration of Na+ ([Na+]i) was measured in individual mouse ß-cells using dual wavelength microfluorometry and the indicator sodium-binding benzofuran isophtalate. Under conditions known to induce large amplitude oscillations in cytoplasmic Ca2+ (1.3 mM Ca2+; 11 mM glucose), [Na+]i remained low and stable at 10–14 mM. Partial suppression of the Na/K pump with 50 µM ouabain resulted in oscillations of [Na+]i in 65% of the cells (frequency, 0.13 ± 0.01 min-1; amplitude, 4.4 ± 0.3 mM). The oscillations were unaffected by the presence of 3 µM tetrodotoxin, but disappeared when the medium was depleted of Ca2+ or supplemented with 10 µM methoxyverapamil. The analysis of the ouabain effect was facilitated by replacing extracellular Ca2+ with 5 mM Sr2+. In the Sr2+-containing medium, oscillations of [Na+]i were seen in more than 70% of the ß-cells exposed to 11 mM glucose. Ouabain (50 µM) modified the [Na+]i oscillations by increasing their amplitudes almost 3-fold and reducing the frequency from once every 3 min to once every 10 min. A relationship between oscillations of cytoplasmic Sr2+ and Na+ was apparent both from observations of similar frequencies and for the modifications obtained with ouabain. It is concluded that the glucose-induced oscillations of cytoplasmic Ca2+ result in a rhythmic entry of Na+, usually balanced by the Na/K pump. A resulting periodic consumption of ATP in the Na/K pump might have implications for the release of insulin by affecting ATP-dependent processes associated with the plasma membrane.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SODIUM ion is important for the release of insulin by interacting with other ions in the ß-cells. Examples of such interactions are the extrusion of H+ (1) and Ca2+ (2) in exchange for Na+. A relationship with K+ handling is demonstrated by the presence of an active Na/K pump (3) and by coupled Na+-K+-2Cl-cotransport (4). When discussing the role of Na+ for ß-cell function, attention should also be paid to cotransport with amino acids (5) and to a requirement of Na+ for oxidation of glucose (6). The question of how glucose, the major physiological stimulator of insulin release, affects the Na+ handling of pancreatic islets was initially addressed in studies with radioactive 22Na. The original findings by Kawazu et al. (7) that glucose stimulates both the entry and efflux of Na+ in isolated islets with minor effects on the steady state content of sodium were later confirmed by measurements using integrating flame photometry (8, 9). The understanding of Na+ handling advanced after measuring its cytoplasmic concentration ([Na+]i) in individual ß-cells with dual wavelength microfluorometry (10, 11). Using this approach it was possible to demonstrate that glucose induces oscillations of [Na+]i when the entry of the ion is promoted by veratridine (12).

The significance of the Na/K pump for the ß-cell handling of Na+ is evident from studies of pancreatic islets using radiotracer (13) and electrophysiological (3) techniques as well as from measurements of the Na/K-adenosine triphosphatase (Na/K-ATPase) activity in islet cell membranes (14). There are reasons to believe that diabetes is associated with a defect in the Na/K pump in ß-cells as has been shown in other types of cells in this disease (15). It is also of interest to note that enhanced levels of circulating ouabain or ouabain-like substances have been observed in diabetic patients (16). To date the effects of inhibiting the Na/K pump in individual ß-cells have been analyzed in the presence of millimolar concentrations of ouabain, resulting in a rapid increase in [Na+]i (12). This inhibition was associated with closure of the K-ATP channels, presumably resulting from accumulation of ATP in the submembrane space due to suppression of the energy consumption in the Na/K pump (17).

In the present study the importance of the Na/K pump was analyzed by exposing individual ß-cells to various concentrations of ouabain. It will be shown that partial suppression of the pump activity results in the appearance of glucose-induced oscillations of [Na+]i, probably due to a periodic entry of Na+ related to oscillations of cytoplasmic Ca2+.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Reagents of analytical grade and deionized water were used. Collagenase, HEPES, and BSA (fraction V) were obtained from Boehringer Mannheim (Mannheim, Germany). Pluronic-127 and the acetoxymethyl esters of sodium-binding benzofuran isophtalate (SBFI) and fura-2 were products of Molecular Probes (Eugene, OR). Ouabain and methoxyverapamil were purchased from Sigma Chemical Co. (St. Louis, MO), and gramicidin D was obtained from Fluka Chemie (Buchs, Switzerland). Tetrodotoxin was provided by Calbiochem (San Diego, CA).

Preparation of ß-cells
Islets of Langerhans were isolated from ob/ob mice by collagenase digestion as previously described (17). Aggregates and single cells were prepared by shaking the islets in a Ca2+-deficient medium followed by suspension in RPMI 1640 medium supplemented with 10% (vol/vol) FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 30 µg/ml gentamicin. The cells were allowed to attach to circular 25-mm coverglasses during culture for 24–48 h at 37 C in an atmosphere of 5% CO2 in humidified air.

Measurements of cytoplasmic Na+
Cells attached to the coverglasses were loaded with the Na+ indicator SBFI at 37 C during 1.5- to 2-h exposure to 5 µM of its acetoxymethyl ester added to a modified RPMI 1640 medium containing 3 mM glucose. Uptake of the indicator was facilitated by including 0.02% of the nonionic dispersing agent Pluronic 127 in the loading medium. The coverglasses with the SBFI-loaded cells were used as exchangeable bottoms of a perifusion chamber placed on the stage of an inverted microscope within a climate box maintained at 37 C (18). Subsequent experimental handling was performed with a basal medium containing 125 mM NaCl, 4.0 mM KCl, 1.2 mM MgCl2, 1.3 mM CaCl2 or 5 mM SrCl2, and 25 mM HEPES, adjusted to pH 7.40 with NaOH and supplemented with 0.5 mg/ml BSA. Modifications of the medium are indicated in the figure legends. The microscope was equipped for epifluorescence fluorometry with a 400-nm dichroic mirror and a x40 fluor oil immersion objective. A 75-watt xenon arc lamp combined with 340- and 380-nm interference filters (10- to 13-nm half-band width) were used for excitation. Images were collected through a 30-nm half-band width filter at 510 nm with an intensified CCD camera (Extended ISIS-M, Photonic Science, Robertsbridge, UK). The excitation filter changer was part of a Magiscan image analysis system (Applied Imaging, Gateshead, UK). The cells were illuminated only during capture of the images, and photodamage was minimized with neutral density filters. Images were captured at the two excitation wavelengths every 4.0–8.0 sec, with each image consisting of 16 accumulated video frames divided by 8; the time between the averaged 340 and 380 images was 1.1 sec. The 340/380 nm ratio images were calculated after subtraction of backgrounds. The Tardis program (Applied Imaging) allowed the responses of all individual cells in an image field to be studied separately. Calibration was performed by exposing the cells to different concentrations of Na+ in the presence of 10 µg/ml gramicidin D (10). The identity of the cells was checked at the end of the experiments by immunostaining for insulin using the peroxidase-antiperoxidase technique (19).

The selectivity of the indicator SBFI is sufficiently high to allow accurate measurements of [Na+]i in the presence of physiological variations in other ions, including Ca2+ (20). It was assured that the glucose-induced oscillations of [Sr2+]i did not interfere with the fluorescence excitation ratio of SBFI by clamping [Na+]i with gramicidin and adding up to 1 mM Sr2+ to a medium containing the ionophore Br-A 23187. The latter concentration of Sr2+ far exceeds the oscillatory peak values reported in glucose-stimulated ß-cells (21). Permeabilization of the plasma membrane with digitonin revealed that about 80% of the fluorescence was confined to the cytoplasm. Most of the compartmentalized dye was probably localized in organelles with an acidic milieu known to make SBFI relatively insensitive to changes in Na+ (22). The compartmentalized fluorescence can therefore be regarded as a background signal that remains essentially unchanged despite variations in [Na+]i.

Measurements of cytoplasmic Ca2+ and Sr2+
Cells attached to the coverglasses were loaded with fura-2 by incubation for 30–40 min at 37 C with 0.5 µM of its acetoxymethyl ester added to the basal medium supplemented with 3 mM glucose. Measurements of the cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Sr2+ ([Sr2+]i) were performed following a protocol similar to that used for [Na+]i. [Ca2+]i was calculated from the 340/380 nm fluorescence excitation ratio (18, 23). In view of the uncertainties regarding the Kd value for the Sr2+ complex with fura-2 and possible contributions of remaining [Ca2+]i to the fura-2 signal, the [Sr2+]i data are presented as the 340/380 nm fluorescence excitation ratio (21).

Statistical analysis
Results are expressed as the mean ± SE. Statistical comparisons were made using Student’s t test for paired data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In a great majority (~90%) of the ß-cells [Na+]i remained at a stable low level of 10–14 mM during perifusion with a medium containing 11 mM glucose and 1.3 mM Ca2+. Partial suppression of the Na/K pump with 25–100 µM ouabain resulted in an increase in [Na+]i manifested either as oscillations or as a sustained elevation (Fig. 1Go). The sustained elevation corresponded to a rise of [Na+]i by 2.0 ± 0.4 mM (n = 12), 3.6 ± 0.7 mM (n = 8), and 11.5 ± 2.3 mM (n = 7) at 25, 50, and 100 µM ouabain, respectively. Exposure to 1 mM ouabain resulted in a continuing rise (not shown) equivalent to 31.5 ± 5.9 mM after 10 min (n = 7). The oscillatory response to ouabain required the presence of a stimulatory concentration of glucose, being suppressed when the sugar was lowered from 11 to 3 mM (Fig. 2AGo). Comparing the effects of different concentrations of ouabain, it was evident that the oscillations were more often seen at 50 than at 25 or 100 µM (Table 1Go). The oscillations observed in the presence of 50 µM ouabain (n = 17) had frequencies of 0.13 ± 0.01 min-1 and amplitudes of 4.4 ± 0.3 mM. The rhythmicity was not affected by the Na+ channel inhibitor tetrodotoxin (Fig. 2BGo), but rapidly disappeared when the voltage-dependent Ca2+ channels were blocked with methoxyverapamil (Fig. 2CGo).



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  		Figure 1. Effects of ouabain on cytoplasmic Na+ in individual ß-cells stimulated with glucose. The experiments were performed in medium containing 1.3 mM Ca2+ and 11 mM glucose. Ouabain was added at concentrations of 25, 50, and 100 µM as indicated. The responses are representative for 9 of 14 (A), 22 of 34 (B), and 7 of 9 (C) cells.

 


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  		Figure 2. Effects of lowering the glucose concentration or adding tetrodotoxin (TTX) or methoxyverapamil (MV) on ouabain-induced oscillations of cytoplasmic Na+ in individual ß-cells. The experiments were performed in medium containing 1.3 mM Ca2+, 11 mM glucose, and 50 µM ouabain. The responses are representative for all 6 (A), 8 (b), and 12 (C) cells.

 

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  		Table 1. Alterations of cytoplasmic Na+ oscillations in glucose-stimulated ß-cells exposed to different concentrations of ouabain

 
The analyses of the [Na+]i oscillations were facilitated by replacing extracellular Ca2+ with Sr2+. In the presence of this Ca2+ analog, distinct oscillations of [Na+]i were seen in a majority (>70%) of the ß-cells exposed to 11 mM glucose. Ouabain suppression of the Na/K pump resulted in a prolongation of the [Na+]i oscillations (Fig. 3AGo and Table 2Go). The prolongation was associated with increases in the amplitudes from 4.0 ± 0.2 to 5.9 ± 0.9 (P < 0.025) and 11.4 ± 1.1 (P < 0.001) mM in the presence of 25 or 50 µM ouabain, respectively (n = 12–19). The glucose-induced oscillations of [Na+]i had their counterparts in oscillations of the cytoplasmic concentration of Sr2+ (Fig. 3BGo). The relationship between the two types of oscillations was emphasized from the observation of similar frequencies both with regard to the control medium and in the presence of 25 and 50 µM ouabain (Table 2Go). In the case of Sr2+, the ouabain-induced increase in the oscillatory period included a prolongation of the intervals separating the peaks (Fig. 3BGo).



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  		Figure 3. Effects of ouabain on oscillations of cytoplasmic Na+ and Sr2+ in individual ß-cells stimulated with glucose. The experiments were performed in Ca2+-deficient medium containing 5 mM Sr2+ and 11 mM glucose. Ouabain (50 µM) was added as indicated. In B, the concentration of cytoplasmic Sr2+ is presented as the 340/380 nm fluorescence excitation ratio obtained with the indicator fura-2. Assuming a Kd value of 2.62 µM for the Sr2+ complex with fura-2 and that [Ca2+]i does not interfere with the measurements (21 ), the [Sr2+]i oscillations start from a basal level of about 0.5 µM, reaching peak values of 2–3 µM. The responses are representative for all 12 (A) and 14 (B) cells.

 

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  		Table 2. Oscillatory frequencies of cytoplasmic Sr2+ and Na+ in glucose-stimulated ß-cells in the presence or absence of ouabain

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glucose stimulates both the entry and efflux of Ca2+ in pancreatic ß-cells (24). Another effect of raising glucose is an increase in turnover of the ß-cell content of sodium (11). The glucose-induced elevation of cytoplasmic Ca2+ is usually manifested as large amplitude oscillations with a frequency of 0.1–0.5 min-1 (25). Apparently these oscillations reflect a rhythmic entry of Ca2+ due to periodic depolarization of the ß-cells (26, 27). In the search for a corresponding rhythmicity of [Na+]i in individual ß-cells, it was sometimes possible to demonstrate periodic fluctuations with peak values not exceeding 50% increase above the basal level (12). The observation that the glucose-induced [Na+]i rhythmicity is less pronounced than that of [Ca2+]i raises the question of whether the periodic entry of Na+ is normally balanced by an efficient Na/K pump. If this is true, a partial inhibition of the pump may result in cyclic variations in [Na+]i not otherwise seen. The present study indicates that this is the case.

It has been suggested that a rhythmic production of ATP accounts for the depolarization of the ß-cells resulting in large amplitude oscillations of [Ca2+]i (28, 29). Being a glucose-dependent phenomenon with a rhythmicity similar to that of [Ca2+]i, it seems likely that the oscillations of [Na+]i also reflect alterations of the metabolism. Previous studies of glucose-stimulated ß-cells have shown that the Na+ channel agonist veratridine evokes [Na+]i oscillations that are readily suppressed by tetrodotoxin, a specific blocker of voltage-dependent Na+ channels (12). However, it is obvious that the [Na+]i oscillations now observed depend on other mechanisms, as they are insensitive to tetrodotoxin. Although voltage-dependent Na+ channels resistant to tetrodotoxin have been reported in neurons and muscle cells (30), their existence in ß-cells remains uncertain. When discussing various alternatives for how a glucose-induced depolarization can induce rhythmic entry of Na+ that is insensitive to tetrodotoxin, the Na/Ca countertransport deserves particular attention. It is easy to envisage that each oscillatory cycle of [Ca2+]i is reflected in a stimulated influx of Na+ via the Na/Ca exchanger, which constitutes an important pathway for the Ca2+ extrusion in the ß-cell (2, 31, 32). The potential significance of this mechanism was evident from the observation that the Na+ oscillations disappeared when the extracellular medium was depleted of Ca2+ or the Ca2+ channels were blocked by verapamil. A major role for the Na/Ca countertransport in the establishment of [Na+]i oscillations does not exclude that other Ca2+-activated mechanisms contribute to the entry of Na+. It has, for example, been suggested that Ca2+ stimulates Na+ influx into ß-cells via nonselective cation channels (33). Moreover, it should be kept in mind that oscillatory Ca2+ peaks can stimulate the production of ATP by activating mitochondrial dehydrogenases (34).

Oscillations of [Na+]i appeared only under conditions known to induce oscillations of Ca2+ or Sr2+. Furthermore, the oscillations of [Na+]i became more prominent after replacing Ca2+ with Sr2+. The latter ion is a useful analog for Ca2+, which penetrates the cells via voltage-dependent Ca2+ channels (35) and is expelled by countertransport with Na+ (36). The fact that Sr2+ does not readily enter via the capacitative pathway may explain why the ß-cell oscillations of [Sr2+]i are more stable than those of [Ca2+]i (37). It is possible to use high concentrations of Sr2+ with a minimal positive shift of the gating of the voltage-dependent channels (38). Accordingly, there are reasons to believe that the observed amplitude increase in the [Na+]i oscillations after replacing extracellular Ca2+ with Sr2+ reflects a larger influx of the latter ion.

A periodic increase in cytoplasmic Ca2+/Sr2+ may not only promote the entry of Na+, but also affect the activity of the Na/K pump. Both Ca2+ and Sr2+ inhibit Na/K-ATPase in cell-free systems (39, 40). However, studies of intact cells suggest that Ca2+ can also stimulate the pump by binding to calmodulin (41). Consistent with the idea that Ca2+/Sr2+ promotes the entry of Na+, partial suppression of the Na/K pump with ouabain resulted in oscillations of [Na+]i in the presence of Ca2+ and made the existing oscillations more pronounced in Sr2+-containing media. Rodent tissues contain isoforms of the catalytic subunit of Na/K-ATPase, which are highly resistant to ouabain (42). When raising the concentration of ouabain to 100 µM or 1 mM the ß-cell oscillations of [Ca2+]i are often transformed into sustained elevation (17). In accordance with the idea that the [Na+]i rhythmicity now observed depends on periodic variations in [Ca2+]i, these oscillations also disappeared in the presence of high concentrations of ouabain. Testing the effects of ouabain in the Sr2+-containing medium, it became evident that suppression of the Na/K pump prolonged the periods and increased the amplitudes of both the [Sr2+]i and [Na+]i cycles. The glucose-induced oscillations of [Ca2+]i are known to be modified by ouabain in a similar way (17).

The present data indicate that glucose induction of [Ca2+]i oscillations in pancreatic ß-cells is associated with a periodic entry of Na+, which is usually balanced by fluctuations in the activity of the Na/K pump. The resulting rhythmicity of the Na/K pump is not only important for maintaining steady state [Na+]i, but can also result in a periodic consumption of the membrane-associated ATP regulating the activity of the K-ATP channels and the energy-requiring steps of exocytosis.


   Acknowledgments
 
The skillful technical assistance of Mrs. Heléne Dansk is gratefully acknowledged.


   Footnotes
 
1 This work was supported by grants from the Swedish Medical Research Council (12X-562), the Swedish Diabetes Association, the Novo Nordisk Foundation, the Novo Nordisk Pharma AB, the Åke Wiberg Foundation, and the Family Ernfors Foundation. Back

Received January 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals