Immunological Identification of Vesicular Nucleotide Transporter in Intestinal L Cells

Abstract

It is well established that vesicular nucleotide transporter (VNUT) is responsible for vesicular storage of nucleotides such as ATP, and that VNUT-expressing cells can secrete nucleotides upon exocytosis, playing an important role in purinergic chemical transmission. In the present study, we show that VNUT is expressed in intestinal L cells. Immunohistochemical evidence indicated that VNUT is present in glucagon-like peptide 1 (GLP-1) containing cells in rat intestine. VNUT immunoreactivity is not co-localized with GLP-1, a marker for secretory granules, and synaptophysin, a marker for synaptic-like microvesicles (SLMVs). Essentially the same results were obtained for GLUTag clonal L cells. Sucrose density gradient analysis confirmed that VNUT is present the light fraction, unlike secretory granules. These results demonstrate that intestinal L cells express VNUT in either the unidentified organelles at light density other than secretory granules and SLMVs or a subpopulation of SLMVs, and suggest that L cells are purinergic in nature and secrete nucleotides independent of GLP-1 secretion.

During the purinergic chemical transmission, neurons, neuroendocrine cells, glial cells and other types of cells secrete ATP, and communicate with each other through purinoceptors.¹⁾ All purinoceptors have been identified and characterized so far, and the overall features and mechanisms of intracellular signaling cascades upon stimulation of purinoceptors are thus well characterized.¹⁾ In contrast, how ATP secretion initiates purinergic chemical transmission, i.e., where, when and how ATP is secreted, is less understood. In neurons and neuroendocrine cells, ATP is stored in secretory vesicles and then exocytosed upon stimulation.¹⁾ Vesicular nucleotide transporter (VNUT) is the last identified member of the SLC17 anion transporter family, and transports nucleotides such as ATP and ADP at the expense of the electrochemical gradient of protons across the membranes, which is established by vacuolar H⁺-ATPase.^2–4) Recent evidence indicated that VNUT is responsible for the vesicular storage of ATP and plays an essential role in the secretion of ATP, since VNUT is expressed and localized with ATP-storing secretory vesicles such as adrenal chromaffin granules, insulin granules of islet β cells, and glutamate-containing synaptic vesicles of hippocampal neurons, and its suppression of expression by RNA interference causes decreased secretion of ATP.^2–7) Thus, it is regarded that VNUT is a potential and useful marker for sites of vesicular storage and secretion.

Since information regarding ATP-secreting cells is still limited, it is necessary to identify VNUT-expressing cells and characterize its subcellular localization. Intestinal L cells are neuroendocrine cells that secrete glucagon-like peptide 1 (GLP-1), one of the proglucagon-derived peptides secreted by gut neuroendocrine cells that maintain blood glucose homeostasis through GLP-1 receptor on pancreatic β cells.^8–10) It was reported that many neuroendocrine cells release ATP or express purinoceptors and regulated their function in an autocrine or paracrine manner.¹⁾ Therefore, it is important to reveal that how neuroendocrine cells store and release nucleotides. In the present study, we focused on L cells, intestinal neuroendocrine cells, and investigated whether or not L cells express VNUT in rodent intestine and clonal GLUTag cells.

MATERIALS AND METHODS

Antibodies

The immunological specificities of rabbit polyclonal antibodies against mouse VNUT and V-ATPase rabbit polyclonal antibodies prepared in-house were determined previously.^2,11) The following antibodies were obtained commercially: anti-GLP-1 mouse monoclonal (Sigma, St. Louis, MO, U.S.A.), anti-synaptophysin mouse monoclonal (Sigma), anti-vesicular glutamate transporter 2 (VGLUT2) mouse monoclonal (Abcam, U.K.), anti-GM130 mouse monoclonal (BD, U.S.A.) and anti-LAMP1 mouse monoclonal (StressMarq Biosciences Inc., Canada) antibodies.

Animals and Cell Culture

C57BL/6 male mice and Wistar male rats were obtained from Japan SLC (Shizuoka, Japan). All animal procedures and care were approved by the Institutional Animal Care and Use Committee, and were carried out according to the guidelines of Okayama University. GLUTag cells were cultured in Dulbecco’s modified Eagle’s medium (5.6 mmol/L D-glucose) supplemented with 10% (v/v) fetal bovine serum, 55 mg/L sodium pyruvate, 100 U/mL penicillin and 0.1 mg/mL streptomycin, as described previously.¹²⁾ High Five cells were grown in Express Five SFM medium supplemented with 18 mM L-glutamine and 10 µg/mL gentamicin at 27°C.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis

Mouse total RNAs from brain and small intestine were purchased from UNITECH, Japan. RNeasy mini kit (QIAGEN, Netherlands) was used for GLUTag total RNA extraction according to the manufacturer’s instructions. cDNA was generated from total RNA with a Reverse Transcriptase Kit (TOYOBO, Japan) using 1 µg of total RNA as the template. The RT-PCRs were carried out as previously described.¹²⁾ The primer sets used for detection of mouse VNUT and VGLUT2 were as follows: 5′-GGT CTG CTC CAA GGT GTC TAC-3′ and 5′-GAC TGA TAA GGC GGT CGG AG-3,′ which amplifies 523 bp of VNUT and 5′-AAC ACA TCA ACC AAG CAA GTC-3′ and 5′-AGG TAG TGA ATG GGA GAG CA-3,′ which amplifies 611 bp of VGLUT2.

Immunofluorescence Microscopy

Indirect immunofluorescence microscopy was performed as previously described.¹³⁾ In brief, Wistar male rats were anesthetized with ether and then perfused intracardially with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The organs were isolated, and frozen sections were prepared. In the case of cultured cells, GLUTag cells on poly-L-lysine-coated coverslips were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min. The first antibody treatment was performed with anti-mouse VNUT antibodies diluted 1 : 200 (organs) or 1 : 100 (cells) in PBS containing 0.5% bovine serum albumin (BSA) for 1 h at room temperature. Washing and secondary antibody treatment were performed according to the published procedures.¹³⁾ The specimens were observed under an Olympus FV300 confocal laser microscope.

Western Blot Analysis

For immunoblotting, cells (5×10⁷ cells) or organ were suspended in 20 mL 10 mM MOPS-Tris (pH 7.0) containing 0.3 M sucrose, 5 mM ethylenediaminetetraacetic acid (EDTA), 10 µg/mL pepstatin A and 10 µg/mL leupeptin, and then homogenized with a Dounce homogenizer. The homogenate was centrifuged at 900×g for 10 min. The resulting supernatant was centrifuged at 150000×g for 1 h. The pellet (membrane fraction) was suspended in the same buffer, and then denatured with sample buffer containing 1% sodium dodecyl sulfate (SDS) and 10% β-mercaptoethanol. Polyacrylamide gel electrophoresis and Western blotting were performed as described previously.¹³⁾ Immunoreactivity was visualized by enhanced chemiluminescence (ECL) amplification according to the manufacturer’s manual (Amersham). Protein concentrations were determined with a protein assay kit (Bio-Rad, Hercules, CA, U.S.A.) with bovine serum albumin as a standard.

Subcellular Fractionation by Sucrose Density Gradient Ultracentrifugation

The membrane fraction from GLUTag cells was resuspended in 400 µL buffer consisting of 10 mM MOPS-Tris, pH 7.0, 0.3 M sucrose and 5 mM EDTA. The supension was laid on top of a 4.5 mL continuous sucrose gradient consisting of 0.4–1.4 M sucrose. After centrifugation at 100000×g for 3.5 h, the gradient was divided from the bottom in 10 fractions and stored at −80°C until use.

RESULTS

VNUT Is Localized in L Cells of the Small Intestine

As the first step of the study, we examined whether or not VNUT is expressed in the small intestine. In RT-PCR analyses, a 523-bp VNUT-specific transcript was amplified in small intestine as well as brain (Fig. 1A). Western blot analysis with membrane fraction of scraped intestinal epithelium revealed a VNUT counterpart with apparent molecular mass of 62 kDa, suggesting the presence of a VNUT counterpart in the rat intestine (Fig. 1B). Immunohistochemical analysis further indicated the presence of VNUT-expressing cells (Fig. 2A). The immunoreactivity exhibited a punctate distribution throughout the cells (Fig. 2A). Double labeling experiments showed that VNUT immunoreactivity was present in GLP-1-containing cells (Fig. 2B). Among 27 VNUT-positive cell tested, 21 cells (78%) expressed GLP-1 and among 28 GLP-1-expressing cells tested, 21 cells (75%) contained VNUT in the rat intestinal epithelium. VNUT-expressing cells also expressed synaptophysin, a marker of SLMV (Fig. 2B). As we reported previously,¹²⁾ VGLUT2 is present in L cells and associated with GLP-1 containing secretory granules. As shown in Figure 2B, little VNUT immunoreactivity overlapped GLP-1, VGLUT2 and synaptophysin, suggesting that VNUT-containing organelles are different from GLP-1-containing secretory granules or SLMVs.

Fig. 1. VNUT Is Expressed in Small Intestine

(A) RT-PCR analysis was performed to examine expression of VNUT mRNAs in mouse small intestine (523 bp, top). The PCR product from brain mRNA was shown as positive control. Expression of glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) gene was also shown as a RNA quality control (150 bp, bottom). (B) Western blot analysis with anti-VNUT rabbit polyclonal antibodies. Membrane fractions from insect cells (High Five cells, left column, 10 µg), mouse synaptic vesicles (right column, 10 µg) and small intestine (right column, 50 µg) were examined. Insect cell membrane was used as negative control. The arrow indicates the position of VNUT.

Fig. 2. Expression and Localization of VNUT in Intestinal L Cells

(A) Indirect immunofluorescence microscopy revealed that VNUT was expressed in intestinal endocrine cells. Pictures merged with Nomarski images were also shown. (B) Sections of small intestine were doubly immunostained with antibodies against VNUT (green) and GLP-1 (red), VGLUT2 (red), or synaptophysin (red). Merged pictures are also shown. Bars=10 µm.

Expression and Localization of VNUT in GLUTag Cells

To examine the subcellular localization of VNUT to more details, we used GLUTag cells, which are used as a model system for L cells.^14–16) RT-PCR analysis of an RNA fraction prepared from cultured GLUTag cells yielded amplified fragments of the VNUT and VGLUT2 genes (Fig. 3A). Then, we performed subcellular fractionation by sucrose density gradient ultracentrifugation in GLUTag cells. After fractionation of the organelles prepared from GLUTag cells, aliquots were analysed by immunoblotting with either anti-VNUT antibodies or antibodies for other markers. As shown in Fig. 3B, VNUT immunoreactivity was only present in the light density fraction 7 as a 62 kDa polypeptide. Both V-ATPase and VGLUT2 exhibited as two peaks at heavy and right density. In contrast, synaptophyin distributes broadly but is in particular rich in the fractions 5 and 6. Immunohistochemical analysis with GLUTag cells indicated that VNUT immunoreactivity distributes throughout cells (Fig. 4). Both GLP-1 and VGLUT2 immunoreactivities were rather weak than those in intestinal L cells. Consistent with the results for intestine slices, little VNUT immunoreactivity seems to be co-localized with GLP-1, VGLUT2 and synaptophysin (Fig. 4). Furthermore, VNUT immunoreactivity was not co-localized with LAMP1, a lysosome marker and GM130, a Golgi marker (Fig. 4). Collectively, it is concluded that L-cells express VNUT, but that it is not principally associated with GLP-1 containing secretory granules or SLMVs.

Fig. 3. Expression and Subcellular Distribution of VNUT in GLUTag Cells

(A) RT-PCR analysis was performed to examine expression of VNUT mRNAs in GLUTag cells (523 bp). The PCR product of VGLUT2 was shown as positive control (611 bp). G3PDH gene was also shown as a RNA quality control (150 bp). (B) For subcellular fractionation, a membrane fraction of GLUTag cells was applied on a 0.4–1.4 M discontinuous sucrose gradient, which was then centrifuged at 100000×g for 3.5 h. Sucrose gradient fractions (1–10) were analyzed by Western blotting for the presence or not of the VNUT in GLUTag cells. Antibodies against VNUT, V-ATPase, synaptophysin and VGLUT2 were used for immunoblotting.

Fig. 4. Expression and Localization of VNUT in GLUTag Cells

Cultured GLUTag cells were fixed and doubly immunostained with antibodies against VNUT (green), GLP-1 (red), VGLUT2 (red), synaptophysin (red), LAMP1 (red) and GM130 (red). Merged images were also shown. Bars=10 µm.

DISCUSSION

There are more than 18 types of neuroendocrine cell in the gastropancreointestinal tract.¹⁷⁾ These neuroendocrine cells receive chemical signals upon food intake and transduce them into hormonal signals for target cells. In fact, the purinergic signaling system including the storage and release of ATP in intestinal neuroendocrine cells is not well understood. In this study we demonstrated that VNUT is expressed in intestinal L cells in the rodent small intestine. Since VNUT is responsible for vesicular storage and subsequent exocytosis of nucleotides, our results suggest that L cells may store and secrete nucleotides. Consistently, our preliminary experiments indicated that GLUTag cells can secrete ATP upon the addition of 55 mM KCl (1.9 nmol ATP/mg protein/5 min). However, because ecto-ATPase activity degraded the released ATP rapidly, further quantitative study of ATP secretion is difficult (Y.H. and M.H., unpublished observation).

It has been shown that VNUT functions in the various kinds of secretory vesicles including chromaffin granules, synaptic vesicles, secretory vesicles of microglia, mucin granules, secretory vesicles of biliary epithelial cells and secretory lysosomes of astrocyte by immunohistochemical and VNUT knock down studies.^{2,6,7,18–21)} Therefore, it is important to determine what organelles contain VNUT, since this information will show us about the mechanism how L cells secrete nucleotides. It has been shown that L cells may possess at least two different secretory vesicles: GLP-1-containing secretory granules and SLMVs of unknown function.¹²⁾ Sucrose density gradient centrifugation with membrane fraction of GLUTag cells showed that VNUT is present in the limited fraction at light density. Taking the limited co-localization of VNUT with synaptophysin and VGLUT2 as revealed with immunohistochemistry into consideration, VNUT seems to be present in either a subpopulation of SLMVs or unknown small organelles.

It should be stressed that L cells are glutamatergic in nature and co-secrete glutamate and GLP-1 through exocytosis upon glucose stimulations.¹²⁾ The released glutamate may act as an intercellular messenger to regulate secretion of GLP-1 in a paracrine fashion. In the present study, we could add a novel feature of intestinal L cells: L cells are also purinergic in nature and may secrete nucleotides, but unlike glutamate secretion, nucleotide secretion may be expected to be independent of secretion of GLP-1. Further studies, in particular, regarding identification of VNUT-containing organelles, physiological stimuli to trigger the seceretion of ATP and expression of purinoceptors will be helpful to elucidate the entire feature of the significance of the putative purinergic signaling in the L cells.

Acknowledgment

We wish to thank Drs. Y. Moriyama and H. Omote at Okayama University for their help throughout this study. We are also grateful to Dr. D. J. Drucker for providing GLUTag cells. This work was supported in part by the Program to Disseminate Tenure Tracking System, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and Grants-in-Aid for Young Scientists (B) (No. 25860062) to M.H. M.H. and Y.H. designed and performed the experiments, analysed the data and wrote the paper.

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