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Basic Fibroblast Growth Factor Stimulates Surface Expression and Activity of Na+/H+ Exchanger NHE3 via Mechanism Involving Phosphatidylinositol 3-Kinase*

  • Andrzej J. Janecki
    Correspondence
    To whom correspondence should be addressed: Div. of Gastroenterology, Hepatology and Nutrition, University of Texas Medical School, 6431 Fannin, 4.234 MSB, Houston, TX 77030. Tel.: 713-500-6649; Fax: 713-500-6699
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  • Maria Janecki
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  • Shafinaz Akhter
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  • Mark Donowitz
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  • Author Footnotes
    * This work was supported by National Institutes of Health NIDDK Grants K08DK02557, RO1DK26523, PO1DK44484, R29DK43778, and T32DK0763205, and by the Meyerhoff Digestive Diseases Center for Epithelial Disorders. Part of this work was presented at the 100th Annual Meeting of the American Gastroenterological Association, Orlando, FL, May 16–19, 1999 (Abstract 3877), and was published in abstract form (Janecki, A. J., Janecki, M., Akhter, S., and Donowitz, M. (1999) Gastroenterology 116, G3877).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.
Open AccessPublished:March 17, 2000DOI:https://doi.org/10.1074/jbc.275.11.8133
      Na+/H+ exchanger NHE3 is a plasma membrane (PM) protein, which contributes to Na+ absorption in the intestine. Growth factors stimulate NHE3 via phosphatidylinositol 3-kinase (PI3-K), but mechanism of this process is not clear. To examine the hypothesis that growth factors stimulate NHE3 by modulating NHE3 recycling, and that PI3-K participates in this mechanism, we used PS120 fibroblasts expressing a fusion protein of NHE3 and green fluorescent protein. At steady state, ∼25% of cellular NHE3 content was expressed at PM. Inhibition of PI3-K decreased PM expression of NHE3, which correlated with retention of the exchanger in recycling endosomal compartment. In contrast, basic fibroblast growth factor (bFGF) increased PM expression of NHE3, which was associated with a 2-fold increase in rate constant for exit of the exchanger from the recycling compartment. Qualitatively similar effects of bFGF were observed in cells pretreated with PI3-K inhibitors, but their magnitude was only ∼50% of that in intact cells. These data suggest that: (i) bFGF stimulates NHE3 by increasing PM expression of the exchanger; (ii) PI3-K mediates PM expression of NHE3 in both basal and bFGF-stimulated conditions, and (iii) not all of the effects of bFGF on NHE3 expression are mediated by PI3-K, suggesting additional regulatory mechanisms.
      NHE3
      Na+/H+ exchanger isoform 3
      bFGF
      basic fibroblast growth factor
      eGFP
      red-shifted variant of green fluorescent protein
      EGF
      epidermal growth factor
      GFP
      green fluorescent protein
      JNC
      juxtanuclear cytoplasmic compartment
      PCR
      polymerase chain reaction
      PM
      plasma membrane
      PI 3-K
      phosphatidylinositol 3-kinase
      REC
      recycling endosomal compartment
      Tf
      transferrin
      TfR
      transferrin receptor
      TMA
      tetramethylammonium
      TX-Tf
      Texas Red-conjugated human transferrin
      VSVG
      vesicular stomatitis virus G protein
      In the mammalian intestine, sodium and water are reabsorbed by multiple mechanisms which include the activity of Na+/H+ exchanger NHE3.1 This transmembrane protein is expressed in the epithelium of renal tubules, intestine, gall bladder, and salivary gland, where it was localized to the apical microvillar domain and, at least in the kidney and in the intestine, to an yet undefined cytoplasmic compartment (
      • Biemesderfer D.
      • Rutherford P.A.
      • Nagy T.
      • Pizzonia J.H.
      • Abu-Alfa A.K.
      • Aronson P.S.
      ,
      • Hoogerwerf W.A.
      • Tsao S.C.
      • Devuyst O.
      • Levine S.A.
      • Yun C.H.
      • Yip J.W.
      • Cohen M.E.
      • Wilson P.D.
      • Lazenby A.J.
      • Tse C.M.
      • Donowitz M.
      ,
      • Silviani V.
      • Colombani V.
      • Heyries L.
      • Gerolami A.
      • Cartouzou G.
      • Marteau C.
      ,
      • Park K.
      • Olschowka J.A.
      • Richardson L.A.
      • Bookstein C.
      • Chang E.B.
      • Melvin J.E.
      ). In the small intestine, NHE3 participates in neutral NaCl absorption, and in the increase in Na+ absorption that occurs via neurohormonal stimulation after meals (
      • Donowitz M.
      • Janecki A.
      • Akhtar S.
      • Cavet M.E.
      • Sanchez F.
      • Lamprecht G.
      • Khurana S.
      • Lee-Kwon W.
      • Yun C.H.C.
      • Tse C.M.
      ). The activity of NHE3 is acutely regulated by multiple mechanisms involving growth factors and protein kinases (
      • Donowitz M.
      • Levine S.
      • Yun C.
      • Brant S.
      • Nath S.
      • Yip J.
      • Hoogerwerf S.
      • Pouyssegur J.
      • Tse C.
      ). We and others have shown that stimulation of NHE3 activity by growth factors, okadaic acid, and serum occurs via an increase in the maximal velocity (V max) of the exchange, whereas phorbol ester and carbachol inhibits NHE3 via a decrease inV max (
      • Donowitz M.
      • Levine S.
      • Yun C.
      • Brant S.
      • Nath S.
      • Yip J.
      • Hoogerwerf S.
      • Pouyssegur J.
      • Tse C.
      ). These effects were observed in non-polarized mesenchymal cells as well as in epithelial cells, and they suggested that at least part of the acute regulation might be accomplished by rapid changes in the number of active exchanger molecules at the plasma membrane.
      Over the last few years, a growing body of evidence has indicated that NHE3 might, indeed, be regulated by redistribution of the exchanger molecules between the cytoplasm and the plasma membrane. Thus, recycling of NHE3 has been suggested in kidney epithelial cells based on the results of subcellular fractionation experiments (
      • Hensley C.B.
      • Bradley M.E.
      • Mircheff A.K.
      ,
      • Zhang Y.
      • Mircheff A.K.
      • Hensley C.B.
      • Magyar C.E.
      • Warnock D.G.
      • Chambrey R.
      • Yip K.P.
      • Marsh D.J.
      • Holstein-Rathlou N.H.
      • McDonough A.A.
      ), and on the presence of an intracellular compartment accumulating NHE3 (
      • Biemesderfer D.
      • Rutherford P.A.
      • Nagy T.
      • Pizzonia J.H.
      • Abu-Alfa A.K.
      • Aronson P.S.
      ). Moreover, the protein kinase C-mediated inhibition of endogenous NHE3 in the human colonic adenocarcinoma cell line Caco-2 was reported to involve translocation of the exchanger from brush border into an yet undefined subapical cytoplasmic compartment (
      • Janecki A.J.
      • Montrose M.H.
      • Zimniak P.
      • Zweibaum A.
      • Tse C.M.
      • Khurana S.
      • Donowitz M.
      ). Recently, D'Souza and colleagues (
      • D'Souza S.
      • Garcia-Cabado A., Yu, F.
      • Teter K.
      • Lukacs G.
      • Skorecki K.
      • Moore H.P.
      • Orlowski J.
      • Grinstein S.
      ) provided the first direct evidence for constitutive recycling of NHE3. These investigators used AP-1 cells expressing rat NHE3 to show that the exchanger molecules recycled between plasma membrane and a juxtanuclear cytoplasmic compartment, and that the latter most probably represented the recycling endosomal compartment. The same laboratory also provided evidence that the constitutive recycling of NHE3 in AP-1 cells was dependent on phosphatidylinositol 3-kinase (PI 3-K) activity, and that PI 3-K predominantly controlled the exocytic arm of recycling (
      • Kurashima K.
      • Szabo E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ). These observations suggested some degree of similarity between NHE3 and the family of other membrane proteins whose constitutive recycling has been shown to be controlled by PI 3-K activity. These include the transferrin receptor (
      • Shepherd P.R.
      • Soos M.A.
      • Siddle K.
      ,
      • Spiro D.J.
      • Boll W.
      • Kirchhausen T.
      • Wessling-Resnick M.
      ), glucose transporters GLUT4 and GLUT1 (
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ), and the ATP-dependent canalicular transporters in the liver (
      • Misra S.
      • Ujhazy P.
      • Gatmaitan Z.
      • Varticovski L.
      • Arias I.M.
      ). Some evidence suggest that PI 3-K activity might be essential not only for control of constitutive activity, but also for mediation of the stimulation of NHE3 by growth factors. Our laboratory reported recently that inhibition of PI 3-K with wortmannin eliminated the stimulatory effects of epidermal growth factor (EGF) on the Na+reabsorption in the rabbit ileum, and on Na+/H+exchange in Caco-2 cells (
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ). Based on these results, it was tempting to speculate that the PI 3-K-mediated mechanism by which growth factors stimulate NHE3 activity involves changes in the dynamics of recycling of NHE3, which, in turn, results in an increased expression of the exchanger at the PM. Such mechanism could, theoretically, be based on the same signal transduction pathway(s) as the constitutive regulation of the exchanger. In such situation, regulatory stimulus might simply amplify the existing mechanisms (e.g. by stimulating the activity and/or intracellular redistribution of PI 3-kinase). Alternatively, the growth factor-regulated pathway might also involve specific mechanisms different from those utilized by the constitutive pathway.
      In this report we present data suggesting that bFGF stimulates NHE3 activity by increasing expression of the exchanger at the PM. We also provide evidence suggesting that the increased expression results from increased rate of exit of the exchanger molecules from the recycling endosomal compartment, and that this mechanism is partially dependent on PI 3-K activity. In order to correlate changes in cellular distribution of NHE3 with Na+/H+ exchange rate, we engineered and stably expressed in PS120 fibroblasts a fusion protein of rabbit NHE3 and green fluorescent protein (GFP). The cellular distribution and activity of the fusion protein closely resembled that of NHE3 lacking the GFP tag, confirming usefulness of this model for the intended studies. Changes in the intracellular steady-state distribution and kinetics of recycling of the fusion protein were examined in living cells using laser confocal microscopy and a novel confocal morphometric analysis.

      DISCUSSION

      Until recently, studies on cellular distribution and recycling of membrane proteins utilized indirect methods including subcellular fractionation, immunoelectron microscopy, or immunolocalization of epitope-tagged molecules of interest. Although these methods have been often applied in an elegant and well controlled fashion, they have several limitations. These include the inability to monitor the real-time trafficking of membrane proteins in living cells, the non-linear stoichiometry of antigen-antibody binding, the difficulty in preventing dissociation of labeled antibodies from antigens in the living cells, and the necessity for cell permeabilization to label the intracellular structures. These limitations have been circumvented, to various degrees, by recent introduction of GFP as an in vivoreporter tag. GFP, and especially its recently engineered mutated variants, is brightly fluorescent, relatively resistant to photobleaching, and does not require exogenous cofactors or substrates (
      • Chalfie M.
      • Tu Y.
      • Euskirchen G.
      • Ward W.W.
      • Prasher D.C.
      ,
      • Heim R.
      • Tsien R.Y.
      ). Importantly, in many cases the GFP tag does not significantly affect biological activity, regulation, or intracellular trafficking of the protein of interest (
      • Gerdes H.H.
      • Kaether C.
      ). Results presented in this report indicate that C-terminal fusion of NHE3 with the red-shifted variant of GFP (eGFP) did not significantly alter the investigated properties of the exchanger. As shown in Fig. 2, cellular distribution of NHE3-GFP in stably transfected PS120 cells resembled that of NHE3-VSVG protein lacking the GFP tag. Importantly, the magnitude of response to bFGF and PI 3-K inhibitors was very similar in both cell lines (Fig. 7), indicating that fusion with eGFP did not significantly affect the responsiveness of the NHE3 moiety to these regulatory factors. It remains to be determined whether other properties of NHE3, which have not been investigated in our studies, remained intact following fusion with GFP.
      Growth factors have been previously shown to stimulate NHE3 in rabbit ileum and in non-epithelial as well as epithelial cell lines (
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ,
      • Levine S.A.
      • Montrose M.H.
      • Tse C.M.
      • Donowitz M.
      ,
      • Donowitz M.
      • Montgomery J.L.
      • Walker M.S.
      • Cohen M.E.
      ,
      • Yun C.H.
      • Tse C.M.
      • Donowitz M.
      ). Since stimulation of NHE3 activity occurred via an increase in maximum velocity (V max) of the exchange, it suggested either a rapid increase in the number of NHE3 molecules at the PM or an increase in the turnover number as a putative underlying mechanisms. In this report, we confirmed the redistribution hypothesis by showing that bFGF stimulated NHE3 activity in PS120 cells by increasing expression of the exchanger at the PM. Moreover, practically all of the bFGF-dependent stimulation of Na+/H+ exchange could be accounted for by this increase in surface expression of NHE3. Thus, bFGF stimulated NHE3 activity by ∼60%, and the exchanger's PM expression by ∼50% over control values, respectively. These results effectively ruled out a significant change in the turnover number of the individual NHE3 molecules as the underlying mechanism of stimulation. At this point, three major questions concerning the mechanism of such a rapid increase in the PM expression of the exchanger should be addressed: (i) did the observed effect of bFGF result from altered kinetics of NHE3 recycling and, if so, did it result from an increased rate of insertion of NHE3 molecules into PM, a decreased rate of removal of the exchanger molecules from PM, or both; (ii) what was the intracellular source of molecules being inserted into PM, and; (iii) since PI 3-K has been implicated in mediation of the effects of growth factors on NHE3 activity, what was the role (if any) of this kinase in the effects exerted by bFGF on the recycling of NHE3. In regard to the first question, results of studies presented in this report strongly suggest that bFGF increased the PM expression of NHE3 by a selective stimulation of the rate of exit of the exchanger from REC (Table I). Stimulation of exocytosis by growth factors has previously been described for seemingly heterogeneous group of processes like recycling of TfR in human and mouse fibroblasts (
      • Wiley H.S.
      • Kaplan J.
      ,
      • Davis R.J.
      • Czech M.P.
      ), acrosomal exocytosis in bull spermatozoa (
      • Lax Y.
      • Rubinstein S.
      • Breitbart H.
      ), movement of exocytic vesicles during formation of membrane ruffles in fibroblasts (
      • Bretscher M.S.
      • Aguado-Velasco C.
      ), or an EGF-induced acute increase in brush border surface area in rabbit jejunal epithelium (
      • Hardin J.A.
      • Buret A.
      • Meddings J.B.
      • Gall D.G.
      ). It is not clear at this moment whether all of the above processes, including bFGF-stimulated exocytosis of NHE3, share a common regulatory pathway or whether the regulation is protein- or process-specific. One mechanism leading to higher specificity of regulation might be directing the traffic of regulated pool of the protein away from the constitutive bulk membrane flow. In respect to GLUT4, it has been suggested that only ∼40% of the intracellular GLUT4 molecules shares the same vesicle pool with TfR, whereas the remainder of the transporter molecules is trafficking in a separately regulated vesicle pool (
      • Daro E.
      • van der Sluijs P.
      • Galli T.
      • Mellman I.
      ,
      • Livingstone C.
      • James D.E.
      • Rice J.E.
      • Hanpeter D.
      • Gould G.W.
      ). It has yet to be determined whether similar phenomenon exists in respect to the growth factor-regulated recycling of NHE3.
      In regard to the second question, our data indicated that most, if not all, of the NHE3 molecules arriving at the PM as a result of exposure of PS120-E3G cells to bFGF originated in the JNC (Figs. 5 and 9). In this study, we did not attempt to precisely define the nature of JNC in PS120-E3G cells. However, we did find a high degree of colocalization of NHE3-GFP with the steady-state intracellular accumulation of TX-Tf/TfR complexes. Since TfR is known to accumulate in the recycling endosomal compartment and, therefore, to be a relatively specific marker for this compartment (
      • van Deurs B.
      • Petersen O.W.
      • Olsnes S.
      • Sandvig K.
      ), our findings suggested that JNC corresponded to the recycling endosomal compartment. This is in agreement with conclusions drawn by D'Souza and colleagues (
      • D'Souza S.
      • Garcia-Cabado A., Yu, F.
      • Teter K.
      • Lukacs G.
      • Skorecki K.
      • Moore H.P.
      • Orlowski J.
      • Grinstein S.
      ) in regard to a similar accumulation of NHE3 in the AP-1 cells, another fibroblast cell line. Interestingly, in Caco-2 cells and in the native renal epithelium, NHE3 has been recently shown to accumulate within a subapical intracytoplasmic compartment (
      • Biemesderfer D.
      • Rutherford P.A.
      • Nagy T.
      • Pizzonia J.H.
      • Abu-Alfa A.K.
      • Aronson P.S.
      ,
      • Janecki A.J.
      • Montrose M.H.
      • Zimniak P.
      • Zweibaum A.
      • Tse C.M.
      • Khurana S.
      • Donowitz M.
      ). Some evidence suggest that, at least in Caco-2 cells, this subapical compartment may correspond to the recycling endosomal compartment accumulating NHE3 in non-polarized AP-1 and PS120 cells (
      • Knight A.
      • Hughson E.
      • Hopkins C.R.
      • Cutler D.F.
      ), implicating some important similarities between the pathways of intracellular recycling of NHE3 in non-polarized mesenchymal cells and in polarized epithelial cells.
      The answer to the third question formulated above is related to a more general question raised by our findings, and namely whether the processes of constitutive and bFGF-regulated recycling of NHE3 are controlled by common or separate signaling mechanisms. One recently emerging candidate for a common denominator for both processes is a family of PI 3-kinases. The most abundant product of the PI 3-K activity in mammalian cells is phosphatidylinositol (
      • Silviani V.
      • Colombani V.
      • Heyries L.
      • Gerolami A.
      • Cartouzou G.
      • Marteau C.
      )P, which is also believed to play an important role in vesicle trafficking (
      • Corvera S.
      • Czech M.P.
      ). PI 3-K was shown to be involved in the regulation of constitutive recycling of GLUT4 in adipose cells (
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ), and of TfR in K562 cells (
      • Spiro D.J.
      • Boll W.
      • Kirchhausen T.
      • Wessling-Resnick M.
      ). Recently, Kurashima and colleagues reported involvement of PI 3-K in the constitutive recycling of NHE3 in AP-1 fibroblasts (
      • Kurashima K.
      • Szabo E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ). Our results complemented these findings by showing that NHE3-GFP is constitutively recycling also in PS120 cells, and that PI 3-K activity is required for the regulation of this process. Similarly to AP-1 cells, in PS120 cells inhibition of PI 3-K affected both endo- and exocytic arms of NHE3 recycling, although the effect on k ex was much stronger than that on k in (Table I). Similar effect of PI 3-K inhibitors on both arms of recycling was reported for GLUT4 and for TfR (
      • Spiro D.J.
      • Boll W.
      • Kirchhausen T.
      • Wessling-Resnick M.
      ,
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ). These data implicate that either PI 3-K separately controls exo- and endocytic arm of constitutive recycling of NHE3, or it regulates a step common for both pathways. The latter mechanism has been suggested for recycling of GLUT4, where PI 3-K was shown to inhibit homologous vesicle fusion, a process common for both arms of recycling (
      • Spiro D.J.
      • Boll W.
      • Kirchhausen T.
      • Wessling-Resnick M.
      ,
      • Malide D.
      • Cushman S.W.
      ). Finally, wortmannin as well as LY294002 could theoretically exert diverse effects on recycling of NHE3 by simultaneous inhibition of kinases other than PI 3-K. However, we do not think this was the case. Although wortmannin has been shown to inhibit several kinases including PI 4-kinase (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ), myosin light chain kinase, and protein kinase C (
      • Nakanishi S.
      • Kakita S.
      • Takahashi I.
      • Kawahara K.
      • Tsukuda E.
      • Sano T.
      • Yamada K.
      • Yoshida M.
      • Kase H.
      • Matsuda Y.
      • Hashimoto Y.
      • Nonomura Y.
      ), it is a quite selective PI 3-K inhibitor at the concentration used in this study. Moreover, effects similar to wortmannin were observed when using LY294002, which has the inhibitory mechanism different from wortmannin and, at 50 μm, does not affect protein kinases potentially involved in regulation of NHE3 activity (i.e. PI 4-kinase or protein kinase C) (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ).
      Although dependence of the constitutive recycling of NHE3 on the PI 3-K activity has been reported previously, this is the first report on the involvement of PI 3-K in the bFGF-stimulated up-regulation of the PM expression of the exchanger. Interestingly, our data also suggest participation of a mechanism independent from PI 3-K in this regulation. This conclusion is based on our observation that bFGF stimulated NHE3 activity and PM expression despite inhibition of PI 3-K with wortmannin or LY294002. However, the magnitude of this stimulation was only about 50% of that observed in intact cells, thus suggesting that approximately half of the stimulatory effect of bFGF did not depend on PI 3-K activity. Similar phenomenon of a partial independence from PI 3-K activity was also reported for insulin-stimulated activity of GLUT4 in adipose cells (
      • Yang J.
      • Clarke J.F.
      • Ester C.J.
      • Young P.W.
      • Kasuga M.
      • Holman G.D.
      ). On the other hand, lack of any effect of insulin on GLUT4 activity in wortmannin-pretreated adipose cells has also been reported (
      • Malide D.
      • St-Denis J.F.
      • Keller S.R.
      • Cushman S.W.
      ). Moreover, although PI 3-K has been shown to be involved in the EGF-stimulated activity of NHE3 in the rabbit ileal epithelium and in Caco-2 cells, pretreatment of cells with wortmannin completely abolished the stimulatory effect of the growth factor (
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ). The reason for this discrepancy is not clear.
      Conclusions regarding kinetics of recycling of NHE-GFP presented in this communication need an additional comment, due to certain limitations of the method used in our studies. Calculation ofk in was based on the dynamics of accumulation of vesicles colocalizing eGFP and FM 4–64 within the REC. Theoretically, vesicles containing only FM 4–64 and arriving into the REC could also generate colocalizing pixels, due to the close proximity of these vesicles and an abundance of pre-existing membranes containing eGFP. However, more than 90% of all endocytic vesicles emerging in the peripheral cytoplasm shortly after exposure of cells to FM 4–64 contained both FM 4–64 and eGFP, suggesting that influx of FM 4–64 into REC practically paralleled that of NHE3-GFP. In respect to the apparent k ex value, it should be stressed that it actually reflects the rate of disappearance of FM 4–64 fluorescence (colocalizing with eGFP) from the REC area. Our approach did not let us distinguish between exocytosis and other processes which could possibly result in a decrease in FM 4–64 fluorescence within the REC. These might include sorting of NHE3-GFP molecules away from FM 4–64 within REC, and a homotypic fusion of endocytic vesicles resulting in a significant dilution and subsequent fading of membrane-bound FM 4–64. However, at least two pieces of evidence strongly suggest that the observed decrease in FM 4–64 fluorescence most likely did reflected exit of FM 4–64/NHE3-eGFP vesicles from REC to the PM: (i) disappearance of FM 4–64 fluorescence from REC was associated with appearance of new, yellow-orange particles in the peripheral cytoplasm, suggesting trafficking of REC-derived exocytic vesicles toward the PM (Fig. 5) and (ii) the apparent value of k ex and first order kinetics of disappearance of FM 4–64 from REC closely resembled the rates of exit of TfR and bulk membrane from recycling endosomal compartment reported previously (
      • Mayor S.
      • Presley J.F.
      • Maxfield F.R.
      ,
      • Presley J.F.
      • Mayor S.
      • Dunn K.W.
      • Johnson L.S.
      • McGraw T.E.
      • Maxfield F.R.
      ), thus suggesting similar nature of both processes.
      In conclusion, by studying regulation of the NHE3-eGFP fusion protein expressed in PS120 cells, we have demonstrated that bFGF stimulates the activity of NHE3 by increasing the steady-state expression of the exchanger at the PM. Moreover, we have shown that this effect results from bFGF-stimulated increase in the rate constant for exit of endocytic vesicles containing NHE3 from the juxtanuclear recycling endosomal compartment. We have also shown that at least two mechanisms mediate the effect of bFGF on the recycling of NHE3, and that only one of those mechanisms depends on PI 3-K activity. Finally, we confirmed previous observations that PI 3-K is involved (at least in mesenchymal cells) in the regulation of constitutive recycling of NHE3. However, whereas PI 3-K was apparently involved in the regulation of both endo- and exocytic arms of the constitutive recycling of NHE3, only the exocytic arm of recycling was affected by PI 3-K inhibition during stimulation of the exchanger by bFGF.

      Acknowledgments

      We thank Dr. Shaoyou Chu and Greg Martin for expert advice and help with the confocal microscopy and immunostaining, and David Szent-Gyorgyi from Universal Imaging Corp. for invaluable advice with MetaMorph software.

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