Vesicular L-Glutamate Transporter in Microvesicles from Bovine Pineal Glands

Abstract

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl ؊ concentration, being negligible in the absence of Cl ؊ and maximum at 2-5 mM and decreasing gradually at 20 -100 mM. The membrane potential (inside positive) was maximum at 0 -10 mM Cl ؊ and then decreased gradually depending on the Cl ؊ concentration, whereas a pH gradient was practically absent without Cl ؊ and increased gradually up to 100 mM Cl ؊ . Ammonium acetate or nigericin plus K ؉ , a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K ؉ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP-and Cl ؊ -dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H ؉ -ATPase being affected. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, similarly inhibited the glutamate uptake in a Cl ؊ protectable manner. Furthermore, ATP-and glutamate-dependent acidification of MVs was observed when 4 mM Cl ؊ was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl ؊ regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.Pinealocytes are parenchymal endocrine cells of pineal glands that synthesize and secrete melatonin into the blood (1-3). At least two kinds of secretory vesicle-like organelles have been identified in pinealocytes: dense core granules, which are speculated to be involved in the storage and secretion of melatonin and some neuropeptides such as arginine vasotocin (3, 4), and a large number of synaptic-like microvesicles (MVs) 1 containing synaptophysin (5). Although histochemical evidence indicated that MVs are distinct from neuronal synaptic vesicles in their lack of synapsin I, the possible participation of MVs in some secretory pathways in pinealocytes was also speculated...

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

“…The addition of either L-aspartate or D-aspartate at 1.0 mM during the assay had a little effect on the ATP-dependent glutamate uptake, indicating that the glutamate transporter does not recognize L-aspartate or D-aspartate as a substrate, one of the characteristics of vesicular glutamate transporters (25)(26)(27). The inability of DNPI to transport D-aspartate was also confirmed by the direct uptake assay (Fig.…”

“…In the earlier stage of studies, vesicular glutamate transporter was defined as the ATP-dependent proton conductor-sensitive glutamate transport activity in synaptic vesicles, but little was known about the protein nature of the transporter at the molecular level (25)(26)(27)39). In the last year, two groups have independently reported that BNPI is vesicular glutamate transporter itself and that BNPI is a potential tool for substantial studies on vesicular glutamate transporter.…”

“…Mammalian pinealocytes accumulate L-glutamate in SLMVs, counterparts of synaptic vesicles in endocrine cells, and secrete it to the extracellular space through exocytosis (28,29). Vesicular glutamate transporter is responsible for the storage of L-glutamate in pineal SLMVs (27,30). To determine whether or not DNPI is expressed in pinealocytes, expression of the DNPI gene in pineal gland was examined by RT-PCR using specific DNA probes.…”

d-Aspartate Is Stored in Secretory Granules and Released through a Ca2+-dependent Pathway in a Subset of Rat Pheochromocytoma PC12 Cells

“…7). The involvement of the brain-specific Na ϩ -dependent inorganic phosphate transporter (BNPI), recently identified as a vesicular glutamate transporter in neuronal cells (23,35), in the accumulation of D-aspartate is unlikely because a vesicular glutamate transporter does not recognize D-aspartate as a substrate (36,37) and is not expressed in PC12 cells (35). The absence of BNPI in PC12 cells was confirmed by immunoblotting using the sitespecific polyclonal antibodies against BNPI (data not shown).…”

Immunoreactivity of Vacuolar H+-ATPase in Human Tissues. Using Polyclonal Rabbit Antibodies against V-ATPase Subunits.

“…The V1 domain, containing the ATP catalytic site, is composed of a hexamer of three subunit A (65-77 kDa) and three subunit B (55-60 kDa) proteins plus accessory subunit C (40-45 kDa), D (33-34 kDa) and E (26)(27)(28)(29)(30)(31)(32)(33). The VO domain, as a proton transporter, is composed of four subunits of 16 kDa, one subunit of 20 kDa and one subunit of 115 kDa.…”