Differential Effects of Gq Protein-Coupled Uridine Receptor Stimulation on IL-8 Production in 1321N1 Human Astrocytoma Cells.

G-protein-coupled receptors (GPCRs) trigger various physiological functions. GPCR-mediated effects largely depend on the receptor-associated G-protein subtypes. However, compelling evidence suggests that single receptor proteins activate multiple G-protein subtypes to induce diverse physiological responses. This study compared responses mediated by three different Gq-binding uridine nucleotide receptors, P2Y2, P2Y4, and P2Y6, by measuring Ca2+ signaling and interleukin (IL)-8 production. In 1321N1 human astrocytoma cells stably expressing these receptors, agonist stimulation evoked concentration-dependent intracellular Ca2+ elevation to a similar extent. In contrast, agonist-induced IL-8 production was prominent in P2Y6-expressing cells, but not in P2Y2- and P2Y4-expressing cells. In addition to inhibition of Gq signaling, G12 signal blockade attenuated uridine 5'-diphosphate (UDP)-induced IL-8 production, suggesting the involvement of a small G-protein pathway. The Rac inhibitor EHop-16 prevented UDP-induced IL-8 release. The P2Y6-triggered IL-8 production was also inhibited by extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) inhibitors. These results suggest that P2Y6 receptor-induced IL-8 production requires Gq-mediated Ca2+ signaling as well as G12-mediated activation of Rac. The results also suggest the importance of considering the involvement of multiple G proteins in understanding GPCR-mediated functions.

Vesicular nucleotide transporter is involved in ATP storage of secretory lysosomes in astrocytes.

Recent studies have suggested that astrocytes release gliotransmitters (i.e., ATP, L-glutamate, D-serine, and peptide hormones) and participate actively in synaptic functioning. Although ATP release from astrocytes modulates the activity of neurons, the mechanisms regulating the ATP release from astrocytes and the source of ATP in astrocytes are not well understood. Recently a vesicular nucleotide transporter (VNUT)/solute carrier family 17, member 9 (SLC17A9) has been identified as a mediator of the active accumulation of ATP into vesicles. Here we show by immunocytochemical analysis under confocal microscope and live cell imaging under total internal reflection fluorescence microscope that lysosome-associated VNUT is responsible for ATP release in astrocytes. VNUT was expressed in both primary cultured cortical astrocytes and glioma cell line C6 cells, and mainly localized on lysosome in the cells. We found that VNUT-associated secretory lysosomes do not fully collapse into the plasma membrane after lysosomal exocytosis. We also found that inhibition of VNUT function by Evans Blue decreased ATP uptake into secretory lysosomes. Depletion and inhibition of endogenous VNUT by small interference RNA and Evans Blue, respectively decreased the amount of ATP release from the cells, whereas overexpression of VNUT increased it. Taken together, these findings indicate that the participation of VNUT in ATP storage in secretory lysosomes during lysosomal exocytosis of ATP from astrocytes.

Duration of fusion pore opening and the amount of hormone released are regulated by myosin II during kiss-and-run exocytosis.

Since the fusion pore of the secretory vesicle is resealed before complete dilation during 'kiss-and-run' exocytosis, their cargoes are not completely released. Although the transient fusion pore is kept open for several seconds, the precise mechanisms that control fusion pore maintenance, and their physiological significance, are not well understood. Using dual-colour TIRF (total internal reflection fluorescence) microscopy in neuroendocrine PC12 cells, we show that myosin II regulates the fusion pore dynamics during kiss-and-run exocytosis. The release kinetics of mRFP (monomeric red fluorescent protein)-tagged tPA (tissue plasminogen activator) and Venus-tagged BDNF (brain-derived neurotrophic factor), which show slower release kinetics than NPY (neuropeptide Y)-mRFP and insulin-mRFP, were prolonged by the overexpression of a wild-type form of the RLC (myosin II regulatory light chain). In contrast, overexpression of a dominant-negative form of RLC shortened the release kinetics. Using spH (synapto-pHluorin), a green fluorescent protein-based pH sensor inside the vesicles, we confirmed that the modulation of the release kinetics by myosin II is due to changes in the duration of fusion pore opening. In addition, we revealed that the amount of hormone released into the extracellular space upon stimulation was increased by overexpression of wild-type RLC. We propose that the duration of fusion pore opening is regulated by myosin II to control the amount of hormone released from a single vesicle.

Role of the polybasic sequence in the Doc2alpha C2B domain in dense-core vesicle exocytosis in PC12 cells.

The double C2 (Doc2) family is characterized by an N-terminal Munc13-1-interacting domain and C-terminal tandem C2 domains, and it comprises three isoforms, Doc2alpha, Doc2beta, and Doc2gamma, in humans and mice. Doc2alpha, the best-characterized, brain-specific isoform, exhibits Ca(2+)-dependent phospholipid-binding activity through its C2A domain, and the Ca(2+)-binding activity is thought to be important for the regulation of Ca(2+)-dependent exocytosis. In contrast to the C2A domain, however, nothing is known about the physiological functions of the C2B domain in regulated exocytosis. In this study, we demonstrated by a mutation analysis that the polybasic sequence in the C2B domain of Doc2alpha (306 KKSKHKTCVKKK 317) is required for binding of syntaxin-1a/synaptosome-associated protein of 25 kDa (SNAP-25) heterodimer. We also investigated the effect of Lys-to-Gln (named KQ) mutations in the polybasic sequence of the C2B domain on vesicle dynamics by total internal reflection fluorescence microscopy in PC12 cells. A Doc2alpha(KQ) mutant, which lacks binding activity toward syntaxin-1a/SNAP-25 heterodimer, significantly decreased the number of plasma membrane-docked vesicles before stimulation and strongly inhibited high-KCl-induced exocytosis from the plasma membrane-docked vesicles. These results indicate that the polybasic sequence in the C2B domain functions as a binding site for syntaxin-1a/SNAP-25 heterodimer and controls the number of 'readily releasable' vesicles in neuroendocrine cells.