Monoclonal antibody (mAb) 1C8 was raised against growth cone-enriched fractions (GCF) of postnatal day 0, 7, and 14 rat brains, and its immunoreactivity was characterized. A Western blot analysis of the rat brain membrane fraction showed that mAb 1C8 recognized 23 and 50 kDa proteins (designated GP23 and GP50). After various glycosidase treatments, immunoreactivity to both GP23 and GP50 disappeared with the use of sialidase from Arthrobacter ureafaciens alone. Reactivity with mAb 1C8 was found, not only on neurons in the central nervous system, but also on exocrine goblet cells in rat colon. Immunoelectron microscopic analyses of colon goblet cells demonstrated that this immunoreactivity was restricted to the limiting membranes of secretory granules. These findings indicate that mAb 1C8 recognizes an epitope common to neural and exocrine cells, including sialic acid residue(s). MAb 1C8 may thus be useful for examining the changes in glycosylation during neural development as well as the relationships between neurons and goblet cells.
Recent studies have revealed the involvement of SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins in exocytotic release in mast cells as in the release of neurotransmitters. This suggests that the exocytotic machinery in mast cells is similar to that in neuronal cells. In the present study, we examined the possibility that mast cells have an active zone-like structure that regulates the efficiency and the site of membrane fusion between secretory granules and the plasma membrane. We found the expression of Munc13-1, one of the well-described active zone proteins, in mast cells. Exocytosis was impaired by knockdown of Munc13-1. Furthermore, when Munc13 was targeted to the raft domain, the exocytotic release was observed to become significantly enhanced, whereas when targeted to the non-raft domain, exocytosis was not affected. These results suggest that Munc13-1 regulates exocytosis positively in mast cells, and that it functions in a raft-like structure in the plasma membrane.
The mechanisms of mechanical stress-induced local Ca2+ influx through mechanosensitive (MS) channels, e.g., Ca2+ spots, which are enhanced by lysophosphatidic acid (LPA), were examined by real-time confocal microscopy in cultured bovine lens epithelial cells loaded with a fluorescent Ca2+ indicator, fluo-4. The starting region of Ca2+ spots was located predominantly on the end of actin filaments stained with phalloidin-oregon green 488. Pretreatment with C3 ADP ribosyl-transferase, which inactivates rho protein, inhibited the formation of actin stress fibers; however, the density of LPA-induced Ca2+ spots was unaffected. Neither an inhibitor of rho-associated protein kinase (Y-27632), nor an inhibitor of actin polymerization (cytochalasin D), affected the density of LPA-induced Ca2+ spots, suggesting that rho-related cytoskeletal changes are not involved in the generation of LPA-induced Ca2+ spots. The pretreatment of extracellular matrix proteins with an inhibitor of integrin binding (GRGDNP) significantly inhibited LPA-induced Ca2+ spots. In contrast, similar treatment with an inactive control peptide (GRGESP) did not. Immunoreactivity with anti-integrin β1 chain antibody was observed at the starting regions of Ca2+ spots. These findings demonstrate the involvement of the mechanotransducer function of integrins, but not the formation of actin stress fibers, in LPA-induced Ca2+ spots, which are a result of mechanical stress-induced local [Ca2+]i influx. LPA may sensitize MS channels via activation of this integrin function. In addition, these results strongly suggest that LPA functions as an endogenous mechanosensitizer, which enhances the mechanical stress-induced pathogenesis, including cataract formation.
N-methyl-D-aspartate (NMDA) stimulation is usually used to investigate a series of neuronal mechanisms involved in NMDA receptor-mediated Ca2+ signal. We here examined by using Ca2+ imaging technique whether the NMDA-induced Ca2+ signal is due to the exclusive activation of NMDA receptors or activation of voltage-dependent calcium channels (VDCCs) together with NMDA receptors in mouse hippocampal slices. In order to isolate each component of the Ca2+ signal, we used sodium channel antagonist tetrodotoxin (TTX), which prevents dendritic spikes and the following activation of VDCCs, and L-type VDCC antagonist nicardipine. The results showed that in the CA1 region, almost a half of the NMDA-induced Ca2+ signal was the TTX-sensitive component involved in indirect Ca2+ influx via VDCCs, while the other half was the TTX-insensitive component involved in direct Ca2+ influx via NMDA receptors. In addition, the TTX-sensitive component should be almost via L-type VDCCs. We further examined the effects of corticosterone (CORT), which is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress, on each component. It has been reported that CORT acutely (nongenomically) suppress the NMDA-induced Ca2+ signal in the CA1 region. The present results, however, led us to a new finding that CORT enhanced the TTX-insensitive component and the suppressive effect of CORT reported so far should be attributed to the effect on the TTX-sensitive component.