Riyo Morimoto

Differentiation-associated Na+-dependent Inorganic Phosphate Cotransporter (DNPI) Is a Vesicular Glutamate Transporter in Endocrine Glutamatergic Systems

Vesicular glutamate transporter is present in neuronal synaptic vesicles and endocrine synaptic-like microvesicles and is responsible for vesicular storage ofl-glutamate. A brain-specific Na+-dependent inorganic phosphate transporter (BNPI) functions as a vesicular glutamate transporter in synaptic vesicles, and the expression of this BNPI defines the glutamatergic phenotype in the central nervous system (Bellocchio, E. E., Reimer, R. J., Fremeau, R. T., Jr., and Edwards, R. H. (2000) Science 289, 957–960; Takamori, S., Rhee, J. S., Rosenmund, C., and Jahn, R. (2000) Nature 407, 189–194). However, since not all glutamatergic neurons contain BNPI, an additional transporter(s) responsible for vesicular glutamate uptake has been postulated. Here we report that differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI), an isoform of BNPI (Aihara, Y., Mashima, H., Onda, H., Hisano, S., Kasuya, H., Hori, T., Yamada, S., Tomura, H., Yamada, Y., Inoue, I., Kojima, I., and Takeda, J. (2000) J. Neurochem.74, 2622–2625), also transports l-glutamate at the expense of an electrochemical gradient of protons established by the vacuolar proton pump when expressed in COS7 cells. Molecular, biological, and immunohistochemical studies have indicated that besides its presence in neuronal cells DNPI is preferentially expressed in mammalian pinealocytes, αTC6 cells, clonal pancreatic α cells, and α cells of Langerhans islets, these cells being proven to secretel-glutamate through Ca2+-dependent regulated exocytosis followed by its vesicular storage. Pancreatic polypeptide-secreting F cells of Langerhans islets also expressed DNPI. These results constitute evidence that DNPI functions as another vesicular transporter in glutamatergic endocrine cells as well as in neurons.

Secretory granule-mediated co-secretion of L-glutamate and glucagon triggers glutamatergic signal transmission in islets of Langerhans.

l-Glutamate is believed to function as an intercellular transmitter in the islets of Langerhans. However, critical issues, i.e. where, when and howl-glutamate appears, and what happens upon stimulation of glutamate receptors in the islets, remain unresolved. Vesicular glutamate transporter 2 (VGLUT2), an isoform of the vesicular glutamate transporter essential for neuronal storage of l-glutamate, is expressed in α cells (Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro, S., Takeda, J., Yamamoto, A., and Moriyama, Y. (2001) J. Biol. Chem. 276, 43400–43406). Here we show that VGLUT2 is specifically localized in glucagon-containing secretory granules but not in synaptic-like microvesicles in αTC6 cells, clonal α cells, and islet α cells. VGLUT1, another VGLUT isoform, is also expressed and localized in secretory granules in α cells. Low glucose conditions triggered co-secretion of stoichiometric amounts ofl-glutamate and glucagon from αTC6 cells and isolated islets, which is dependent on temperature and Ca2+ and inhibited by phentolamine. Similar co-secretion ofl-glutamate and glucagon from islets was observed upon stimulation of β-adrenergic receptors with isoproterenol. Under low glucose conditions, stimulation of glutamate receptors facilitates secretion of γ-aminobutyric acid from MIN6 m9, clonal β cells, and isolated islets. These results indicate that co-secretion ofl-glutamate and glucagon from α cells under low glucose conditions triggers GABA secretion from β cells and defines the mode of action of l-glutamate as a regulatory molecule for the endocrine function. To our knowledge, this is the first example of secretory granule-mediated glutamatergic signal transmission.

Secretion of L-glutamate from osteoclasts through transcytosis

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L‐glutamate. Osteoclasts secrete L‐glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+‐dependent manner. KCl‐ and ATP‐dependent secretion of L‐glutamate was absent in osteoclasts prepared from VGLUT1−/− knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L‐glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1−/− mice develop osteoporosis. Thus, in bone‐resorbing osteoclasts, L‐glutamate and bone degradation products are secreted through transcytosis and the released L‐glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

Expression and Localization of Vesicular Glutamate Transporters in Pancreatic Islets, Upper Gastrointestinal Tract, and Testis

The wide-ranging expression of glutamate receptors in peripheral tissues suggests an unexpectedly wider role(s) of l-glutamate as an intercellular signaling molecule. However, the peripheral glutamatergic system is poorly understood, partly because the sites of l-glutamate signal appearance are less well characterized. Vesicular glutamate transporters (VGLUTs) are potential probes for the sites of vesicular storage and subsequent secretion of l-glutamate. In this study we raised specific polyclonal antibodies against two VGLUT isoforms, VGLUT1 and VGLUT2, and investigated their localization in peripheral tissues of rat. We detected the expression of either VGLUT1 or VGLUT2, or both, in pancreas, stomach, intestine, and testis. In pancreas, VGLUT1 and VGLUT2 are present in pancreatic polypeptide-containing secretory granules in F-cells in the islets of Langerhans. In stomach, VGLUT2 is abundant in the antrum and pylorus and is present in a subset of pancreatic polypeptide-containing cells. In intestine, VGLUT2 is abundant in the ileum and is co-localized with glucagon-like immunoreactive peptide and polypeptide YY (PYY). In testis, VGLUT2 is expressed and localized in the outer acrosomal membrane of spermatids, where KA1 and GluR5, kainate receptor subunits, are almost always localized. Taken together, these results strongly suggest the occurrence of a peripheral glutamatergic system in the gastroenteropancreatic system and testis.

Co-expression of vesicular glutamate transporters (VGLUT1 and VGLUT2) and their association with synaptic-like microvesicles in rat pinealocytes

A vesicular glutamate transporter (VGLUT) is responsible for the accumulation of l‐glutamate in synaptic vesicles in glutamatergic neurons. Two isoforms, VGLUT1 and VGLUT2, have been identified, which are complementarily expressed in these neurons. Mammalian pinealocytes, endocrine cells for melatonin, are also glutamatergic in nature, accumulate l‐glutamate in synaptic‐like microvesicles (SLMVs), and secrete it through exocytosis. Although the storage of l‐glutamate in SLMVs is mediated through a VGLUT, the molecular nature of the transporter is less understood. We recently observed that VGLUT2 is expressed in pinealocytes. In the present study, we show that pinealocytes also express VGLUT1. RT–PCR and northern blot analyses indicated expression of the VGLUT1 gene in pineal gland. Western blotting with specific antibodies against VGLUT1 indicated the presence of VGLUT1 in pineal gland. Indirect immunofluorescence microscopy with a section of pineal gland and cultured cells indicated that VGLUT1 and VGLUT2 are co‐localized with process terminal regions of pinealocytes. Furthermore, immunoelectronmicroscopy as well as subcellular fractionation studies revealed that both VGLUT1 and VGLUT2 are specifically associated with SLMVs. These results indicate that both VGLUTs are responsible for storage of l‐glutamate in SLMVs in pinealocytes. Pinealocytes are the first exception as to complementary expression of VGLUT1 and VGLUT2.

Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells.

Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage of l‐glutamate, and plays an essential role in glutamate‐mediated intercellular signal transmission in the CNS and in some neuroendocrine cells. Intestinal L cells are the glucose‐responsive neuroendocrine cells responsible for the secretion of glucagon‐like peptide 1 (GLP‐1). We have shown that intestinal L cells express VGLUT2, a VGLUT isoform, which suggests that L cells secrete l‐glutamate. In the present study, we investigated this possibility using GLUTag mouse clonal L cells. RT–PCR and northern blot analyses revealed expression of the VGLUT1 and VGLUT2 genes, but not of the VGLUT3 gene. Western blot analysis revealed immunological counterparts for VGLUT2, whereas an immunological counterpart of VGLUT1 was not detected. Indirect immunofluorescence microscopy revealed a punctate distribution of VGLUT2 immunoreactivity throughout the cells, which co‐localized with GLP‐1. Double‐labeling immunoelectronmicroscopy confirmed the association of VGLUT2 with GLP‐1‐containing secretory granules. The membrane fraction exhibited ATP‐dependent l‐glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive to d,l‐aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts of l‐glutamate and GLP‐1. d‐Glucose and methyl‐α‐d‐glucopyranoside, stimulators of exocytosis of GLP‐1, also triggered the secretion of l‐glutamate. The l‐glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells stored l‐glutamate in secretory granules and secreted it with GLP‐1 by exocytosis. As GLUTag cells and intestinal L cells express kainate receptors and plasma membrane glutamate transporters, these results support the concept of l‐glutamate‐mediated intercellular signaling in the vicinity of intestinal L cells.

Homeoprotein Hex is expressed in mouse developing chondrocytes

Endochondral ossification is a complex process involving the formation of cartilage and the subsequent replacement by mineralized bone. Although the proliferation and differentiation of chondrocytes are strictly regulated, the molecular mechanisms involved are not completely understood. Here, we show that a divergent-type homeobox gene, hematopoietically expressed homeobox gene (HEX), is expressed in mouse chondrogenic cell line ATDC5. The expression of Hex protein drastically increased during differentiation. The chondrogenic differentiation-enhanced expression of Hex protein was also observed in chondrocytes in the tibia of embryonic day 15.5 (E15.5) mouse embryos. The localization of Hex protein in the chondrocytes of the tibia changed in association with maturation; namely, there was Hex protein in the cytoplasm near the endoplasmic reticulum (ER) in resting chondrocytes, which moved to the nucleus in prehypertrophic chondrocytes, and thereafter entered the ER in hypertrophic chondrocytes. These results suggest Hex expression and subcellular localization are associated with chondrocyte maturation.