1Department of Biology, Indiana University-Purdue University at Indianapolis; 2Division of Nephrology, Indiana University School of Medicine; and 3Richard Roudebusch Veterans Affairs Medical Center, Indianapolis, Indiana 46202
Submitted 7 May 2004 ; accepted in final form 21 July 2004
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ABSTRACT |
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phosphatidylinositol 3,4,5-trisphosphate; insulin-stimulated Na+ transport; metabolic syndrome; real-time confocal imaging
Clinical studies indicate a causal link between the compensatory hyperinsulinemia and the development of hypertension and suggest that insulin resistance may precede the increase in blood pressure (1, 10, 18, 29). There is a positive correlation between plasma insulin levels and blood pressure in humans as well as in some rodent models of hypertension (10, 24, 26). In addition, insulin resistance and salt sensitivity in blood pressure also are correlated in individuals who are normotensive (1, 18, 26, 32).
Insulin stimulates an increase in renal Na+ reabsorption, thus contributing to the salt and water retention that is the hallmark of salt-sensitive hypertension (14). The primary site of insulin's action is the epithelial Na+ channel (ENaC) found in salt-absorbing tissues, including the principal cells of the distal nephron (36, 27). The channel is composed of three subunits: ,
, and
(8).
Clinically, mutations in the COOH-terminal region of the - or
-subunit can cause Liddle syndrome, a severe form of hypertension resulting from constitutive activation of the ENaC (19, 34). Naturally occurring loss of function mutations in all three subunits have also been described. These result in pseudohypoaldosteronism type 1, characterized by renal salt wasting and hyperkalemic metabolic acidosis (12, 17, 24). These clinical observations support the principle that the ENaC is crucial for the regulation of salt and water homeostasis and the fact that mutations in the channel cause serious disturbances in the maintenance of this delicate balance.
In response to insulin stimulation, the ENaC is translocated from a diffuse intracellular pool into the apical plasma membrane (5, 6). The pathway linking the insulin receptor on the basolateral membrane to the ENaC on the apical membrane is complex. Investigators at our laboratory have identified the phosphoinositide (PI) signaling cascade as a major component of insulin-stimulated Na+ reabsorption in an amphibian cell model (A6) of the distal nephron (5, 16, 27). Phosphatidylinositol 3-kinase (PI3-kinase), the first committed step of the PI pathway, catalyzes the phosphorylation of the 3 position of the head group of phosphatidylinositol. One of the products formed by this reaction is phosphatidylinositol 3,4,5-trisphosphate (PIP3). Recently, investigators at our laboratory have shown that apical membrane targeting of the ENaC is dependent on the activity of PI3-kinase. In A6 cells, insulin causes an increase in PIP3 within 1 min (27). Insulin-stimulated Na+ flux and the formation of PIP3 are inhibited by LY-294002, a specific inhibitor of PI3-kinase (5, 27).
Within 1 min after insulin stimulation, the ENaC is colocalized with PI3-kinase, predominately along the lateral surfaces of the epithelial cells (5). Although the interaction of the two proteins is necessary for ENaC insertion into the apical membrane, the colocalization is no longer present once the ENaC has reached the apical membrane. It is unlikely that the recruitment of the ENaC to the area of the lateral membrane is indicative of ENaC insertion into this membrane, because transmembrane, multisubunit channel proteins are unable to cross the tight junction. While the actual mechanism is unknown, we hypothesized that one of the key components for channel insertion may be the formation of PIP3 and its diffusion into the apical membrane. Lipids of the cytoplasmic leaflet have been shown to cross the junctional complex (15, 37). PIP3 would be an ideal candidate for this type of signaling, because in many cell types it is produced exclusively at the cell membrane (25), and it is often found in cells only after stimulation of PI3-kinase. The results of the present study confirmed our hypothesis and elucidated an alternate pathway for propagation of a basolateral stimulus to the apical membrane via lipid intermediates.
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METHODS |
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Electrophysiology.
In polarized epithelial cells, insulin binds to a receptor on the basal membrane to initiate a signal transduction cascade, resulting in numerical and functional increases in apical membrane ENaC (36, 27). Short-circuit current (Isc) measurements were used to monitor the functional ion transport response to insulin stimulation in the A6 cell line (21). Nucleopore filters (4.7 cm2) containing confluent A6 cells (14 days after seeding) were removed from the Transwell chambers and clamped between the halves of an Ussing chamber. Each half of the chamber contained a tapered fluid compartment with fittings for voltage electrodes and current electrodes. The fluid chamber was water jacketed to maintain a constant temperature (27°C). The cells were bathed in serum-free, nonsupplemented medium. The medium was circulated in the chambers and oxygenated by means of a 5% CO2-95% O2 gas lift. The electrodes were connected to a current-voltage clamp for measurement of net ion flux, which was monitored as Isc during a zero-voltage clamp (21). Transepithelial resistance was calculated every 2 min by applying a 2-mV pulse across the epithelium and measuring the resultant deflection in Isc. Cultures were used only if the resistance was
1,000
/cm2. The cultures were incubated in the chambers until a steady baseline was achieved (0.51 h). Subsequently, insulin was added to the basolateral bathing medium. After 10 min, amiloride (105 M) was added to the apical bathing medium to determine the proportion of the Isc that was due to net Na+ movement in an absorptive direction. Statistical significance was determined using Student's t-test.
Confocal fluorescence microscopy and image acquisition.
Clonal lines of A6 cells stably expressing green fluorescent protein (GFP)-ENaC (3) were grown to confluence on membrane filter supports and scanned using a PerkinElmer Ultraview spinning disk confocal microscope (PerkinElmer, Boston, MA) equipped with He-Ne, Kr-Ar, and Ar lasers. The scan head is attached to a Nikon Diaphot inverted microscope (Nikon USA, Melville, NY). Membrane filters were excised from the supports and placed on a no. 1.5 coverslip with 500 µl of medium. In experiments in which we used GFP, the labeled cells were imaged at 488-nm excitation and 525-nm emission wavelengths. Images were collected at a rate of 32 frames/s. One entire z-stack was collected before insulin was applied to the basal sides of the cells. z-Stacks were collected with a displacement along the z-axis of 0.2 µm. Sequential z-stacks were acquired for 25 min after insulin stimulation.
For experiments using cells labeled with GFP and N-(3-triethylammoniumpropyl)-4-{6-[4-(diethylamino)phenyl]hexatrienyl}pyridinium dibromide (FM 4-64; Molecular Probes, Eugene, OR), imaging was conducted using 488- and 568-nm excitation wavelengths, respectively. Emissions were collected with band-pass filters at 525 and 600 nm. Images were collected at a rate of 5 frames/s.
Image processing and analysis. Three-dimensional (3-D) reconstructions were made using a modified version of Voxx (13) running on a Dell Optiplex computer (Dell Computer, Round Rock, TX) equipped with an nVidia GeForce 4 video card (nVidia, Santa Clara, CA). ENaC membrane insertion was analyzed by creating image stacks from the first six apical images collected at each time point. Extended focused images were produced from the apical z-stacks using the Metamorph software package (Universal Imaging, Downington, PA) on a Dell Optiplex computer. Average intensity values integrated over the area of the apical membrane were measured for each time point. Average intensities were compared with baseline intensities obtained from images recorded before insulin stimulation. Statistical significance was determined using Student's t-test.
Extended focus images generated with the Metamorph software program were converted to tagged image file format and imported into Adobe Photoshop, version 6.0, software files (Adobe, Seattle, WA). In all figures, equivalent changes in contrast and brightness were applied to all photomicrographs to ensure that the images were directly comparable.
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RESULTS |
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The close association of a portion of the ENaC with the lateral membrane in this and a previous study (5) raised the question whether the ENaC enters this membrane before its ultimate destination. To address this question, A6 cell plasma membranes were labeled with vital membrane dye FM 4-64. As shown in Fig. 2B, along the basolateral membrane, GFP-tagged ENaC remained constrained within the margins delineated by the plasma membrane marker FM 4-64 and did not appear to enter the lateral membrane. However, the ENaC was clearly integrated into the apical membrane.
Investigators at our laboratory have shown that immediately after insulin stimulation, PI3-kinase colocalized in the vicinity of the ENaC along the lateral membrane (5). The product of PI3-kinase, PIP3, is a very low abundance lipid. Previous confocal studies in single cells indicated that PIP3 production is largely restricted to the plasma membrane (25). In accordance with these previous studies, we were unable to detect labeling in endosomes, suggesting that the PIP3 was generated in the plasma membrane. In the A6 cell line, there is a measurable increase in PIP3 as early as 1 min after insulin stimulation. Furthermore, inhibition of PI3-kinase abolishes insulin-stimulated Na+ transport (27). These observations suggest that formation of PIP3 plays a crucial role in the natriferic action of insulin. Insulin-stimulated production and diffusion of PIP3 was followed using a PIP3-specific biosensor containing the enhanced GFP-tagged PH domain of Grp1 (EGFP-PH/Grp1) (25, 38).
Within 10 s of insulin application, stimulation of PI3-kinase mediated an increase in PIP3 initiated on the basolateral plasma membrane (Fig. 3). PIP3 rapidly diffused along the lateral membrane, crossed the tight junction, and diffused into the apical membrane. During this period, A6 cells swelled and a dramatic shift in PIP3 probe from a nuclear/perinuclear compartment to the lateral membrane was observed. Cell swelling continued during the first 30 s of insulin stimulation. The timing of PIP3 movement correlated with ENaC-GFP movement along the lateral membrane and preceded delivery of channels to the apical membrane (Figs. 2 and 3).
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DISCUSSION |
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Insulin-stimulated Na+ transport increased by 4 min after insulin application and reached a plateau by 10 min (Fig. 1). Intravital imaging of A6 cells expressing GFP-tagged ENaC -subunits revealed a dramatic redistribution of this protein in response to insulin. In resting, unstimulated cells, a significant amount of GFP-ENaC resided in an intracellular distribution. Within 30 s of insulin stimulation, cell volume increased twofold and GFP tagged ENaC aligned along the lateral membrane (Fig. 2A and movies in Supplementary Material). ENaC does not insert into the lateral membrane as evidenced by the lack of colocalization with the vital membrane-specific probe, FM 4-64 (Fig. 2B). By 30 s of insulin stimulation, increased ENaC is observed in the apical membrane.
The rapidity of ENaC delivery to the apical membrane was somewhat surprising and led us to question how an epithelial cell can transmit a basal signal to the apical surface within the time period defined by our intravital studies. Prior work at our laboratory has shown that the PI3-kinase inhibitor LY-294002 not only inhibits insulin-stimulated PIP3 production and Na+ transport but also blocks ENaC redistribution to the lateral membrane in response to insulin (5, 27). These observations underscored the critical requirements for PI3-kinase activation as a prerequisite for insulin-stimulated ENaC membrane assembly and led us to examine the dynamics of PIP3 generation after insulin stimulation of A6 cells. We used a chimeric biosensor for PIP3 to take advantage of the fact that the PH domain of Grp1 specifically binds to PIP3 (38). EGFP-PH/Grp1 was transiently transfected into A6 cells. This transfection had no functional effect on insulin-stimulated Na+ transport as measured by Isc analysis. In resting, unstimulated cells, EGFP-PH/Grp1 was observed in a perinuclear and perhaps intranuclear location. This distribution has been observed in other cell types (25, 38). When insulin was applied to the basal membrane, EGFP-PH/Grp1 moved from its perinuclear location to the lateral membrane within 10 s of stimulation (Fig. 3). From the lateral membrane, the PIP3 probe moved up along the lateral membrane, past the tight junction, and collected in the apical membrane (Fig. 3).
Previous studies have demonstrated that lipids formed in the inner leaflet of the plasma membrane can diffuse across the junctional complexes (15, 37). The formation of PIP3 within the inner leaflet permits rapid deployment of this intermediate to other regions of the cell membrane, where biological responses can be coordinated. The resultant localized changes in membrane composition may alter the apical membrane environment favoring channel insertion.
Contemporaneously with the rapid change in lipid and channel movements, there was a notable increase in cell size (Fig. 2 and 3). The increase was maximal at 1 min and then started to diminish, returning to prestimulated size by 5 min. Cell volume is regulated in response to many extracellular factors and has been postulated to act as a second messenger in the transmission of some hormonal signals (22). Although cell swelling has not been described as part of the insulin-stimulated natriferic response, there is a precedent for such a phenomenon as an integral part of insulin's action in other cells. This effect is best described in hepatocytes, in which insulin stimulates a PI3-kinase-dependent cell swelling that in turn regulates signaling cascades, leading to antiproteolytic effects as well as stimulation of hepatic glycogen synthesis. The action has been shown to be initiated by insulin-mediated K+ accumulation via activation of Na+-K+-2Cl cotransporter, Na+/H+ exchanger, and Na+-K+-ATPase (31).
The increase in cell volume in response to insulin stimulation in A6 cells was not a secondary consequence of the overexpression of ENaC as GFP-ENaC, because it occurred to the same extent in cells that expressed high and low amounts of GFP-ENaC as it did in those cells that did not express additional ENaC over the endogenous level (Fig. 3). The dramatic change in cell volume may be caused by a rapid influx of Na+ and water. The change in cell size also implies that membrane surface area increases. It is possible that the increase in cell volume is a normal part of cellular responses to natriferic stimuli that has been captured only with the advent of confocal microscopes and imaging software programs capable of 3-D imaging in real time. The nature of this effect requires further studies.
A working model of insulin signaling in polarized epithelia is shown in Fig. 4. Pathway B depicts the classic pathway in which intermediates are envisioned diffusing within the cytoplasmic space. In contrast, the model delineated in Pathway A illustrates the membrane delimited signaling pathway suggested by the data produced in the present study. In epithelial cells, PIP3 is able to bypass the tight junction, while the junctional complex restricts lipids in the outer leaflet of the plasma membrane as well as integral membrane proteins (15, 37). The diffusion of signaling components in Pathway B scales as a ratio of the target size in the apical region to the apical-basal distance. In contrast, diffusion in Pathway A occurs in a planar membrane and scales as the log of the apical target size and apical-basal distance. At the scale of an epithelial cell, diffusion in Pathway A can transmit a signal three times faster than Pathway B. Pathway A, while demonstrated here with regard to insulin-stimulated Na+ transport, may be more generally applicable to other signaling systems in which diffusion through the entire cytoplasmic space would constrain rapid transcellular signal transduction. For example, neuronal cells with extended cell processes could also use membrane delimited signaling to transmit signals rapidly between axons and dendrites.
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PIP3 may also have a function in the activation of ENaC. Tong et al. (36) recently showed that both PIP3 and PI3,4P2 increase the open probability of ENaC heterologously expressed in Chinese hamster ovary cells, and Markadieu et al. (23) demonstrated permeant derivatives of PIP3 and PI3,4P2, but not PI4,5P2, stimulated Na+ transport in A6 cells. Furthermore, the latter investigators also showed that transfection with PTEN, a phosphatase and tensin homolog that reduces PIP3 to PI4,5P2, decreased both insulin and PIP3-stimulated Na+ transport (23). Thus the lipid products of PI3-kinase may be important in targeting ENaC to the cell membrane and, subsequently, may also play a role in the activation of the apically resident channel.
Understanding insulin signaling in polarized epithelial cells provides an important link between the findings of salt-sensitive hypertension in people with metabolic syndromes and associated hyperinsulinemia (1, 2, 10, 14, 18, 24, 26, 29, 32, 33). Diurnal cyclic hyperinsulinemic episodes would increase the number of active ENaC, leading to a state of chronic increased Na+ reabsorption in distal nephron segments. Because increased renal Na+ reabsorption is causally linked to increased blood pressure (19, 30, 34), it follows that hyperinsulinemia contributes to hypertension. The correlation between insulin resistance, hyperinsulinemia, and hypertension is independent of age, sex, and degree of obesity (1, 2, 18, 24, 26, 29, 32, 33). The causal correlation between hyperinsulinemia and hypertension can have important implications for the choice of pharmaceutical intervention to manage hypertension in patients with metabolic syndrome and remains, in clinical practice, one of the underappreciated aspects of this disease.
In summary, signaling from insulin receptors on basal membranes stimulates ENaC insertion into apical membranes via a lipid-mediated alteration in plasma membranes. Lipids formed in the inner leaflet of bilayers are uniquely positioned to act as signaling components. They can diffuse rapidly in the plane of the membrane and, unlike proteins, can cross junctional complexes of high-resistance, polarized epithelial cells (7, 15). This lipid-based, planar membrane, transcellular signaling mechanism bypasses three-dimensional diffusion restrictions, resulting in rapid signaling kinetics, and may have wider applicability to other polarized signaling pathways.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00226.2004/DC1.
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