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In sympathetic neurons, it is well-established that the neurotransmitters, norepmephrine (NE), neuropeptide Y (NPY), and ATP are differentially coreleased from the same neurons. In this study, we determined whether synaptotagmin (syt) I, the primary [Ca.sup.2+] sensor for regulated release, could function as the protein that differentially regulates release of these neurotransmitters. Plasmid-based RNA interference was used to specifically and stably silence expression of syt I in a model secretory cell line. Whereas stimulated release of NPY and purines was abolished, stimulated catecholamine (CA) release was only reduced by ~50%. Although expression levels of tyrosine hydroxylase, the rate-limiting enzyme in the dopamine synthesis pathway, was unaffected, expression of the vesicular monoamine transporter 1 was reduced by 50%. To evaluate whether NPY and CAs are found within the same vesicles and whether syt I is found localized to each of these NPY- and CA-containing vesicles, we used immunocytochemistry to determine that syt I colocalized with large dense core vesicles, with NPY, and with CAs. Furthermore, both CAs and NPY colocalized with one another and with large dense core vesicles. Electron micrographs show that large dense core vesicles are synthesized and available for release in cells that lack syt I. These results are consistent with syt I regulating differential release of transmitters. neuropeptide Y; catecholamines; adenosine 5'-triphosphate; dopamine; norepinephrine

Synaptotagmin (syt) I is a [Ca.sup.2+]-binding protein that is well accepted as a major sensor for [Ca.sup.2+]-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as [Ca.sup.2+] sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting [Ca.sup.2+] and stimulated [Ca.sup.2+] influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC 12 cells, and another protein likely has a role as a [Ca.sup.2+] sensor for regulated release of transmitter. RNA interference; amperometry; exocytosis doi:10.1152/ajpcell.00539.2005

Presynaptic N-type [Ca.sup.2+] channels ([Ca.sub.v]2.2, [[alpha].sub.1B]) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the [alpha]1B subunit. Whether binding of syntaxin to the N-type [Ca.sup.2+] channels is required for coupling [Ca.sup.2+] ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant [[alpha].sub.1B] [Ca.sup.2+] channel subunit and transiently transfected either the wild-type [[alpha].sub 1B] or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent [Ca.sup.2+] channels. Secretion was elicited by activation of exogenously transfected [Ca.sup.2+] channel subunits. The current-voltage relationship was similar for the wild-type and mutant [[alpha].sub.1B]-containing [Ca.sup.2+] channels. Although total [Ca.sup.2+] entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when [Ca.sup.2+] entry was normalized to cell size and limited to cells with similar [Ca.sup.2+] entry ([approximately equal to] 150 x 106 [Ca.sup.2+] ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the [alpha].sub.1B] synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of [Ca.sup.2+] influx through [alpha].sub.1B]-containing [Ca.sup.2+] channels to exocytosis.

The effect of nicotinic acetylcholine receptors (nAChRs) on calcium channel-dependent exocytosis in rat pheochromocytoma (PC12) cells was investigated. Single cells were patch-clamped in the whole-cell environment and stimulated with either voltage steps to activate voltage-dependent calcium ion channels or brief pulses of nicotine to activate the nAChRs. Results showed that activation of nAChRs serves as a significant calcium ion entry pathway underlying calcium ion-mediated cellular processes such as exocytosis.

Abstract Background The function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I. Results To test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect. Conclusions These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.

Bioactive glasses have recently been shown to promote regeneration of soft tissues by positively influencing tissue remodeling during wound healing. We were interested to determine whether bioactive glasses have the potential for use in the treatment of peripheral nerve injury. In these experiments, degradable bioactive borate glass was fabricated into rods and microfibers. To study the compatibility with neurons, embryonic chick dorsal root ganglia (DRG) were cultured with different forms of bioactive borate glass. Cell viability was measured with no media exchange (static condition) or routine media exchange (transient condition). Neurite extension was measured within fibrin scaffolds with embedded glass microfibers or aligned rod sheets. Mixed cultures of neurons, glia, and fibroblasts growing in static conditions with glass rods and microfibers resulted in decreased cell viability. However, the percentage of neurons compared with all cell types increased by the end of the culture protocol compared with culture without glass. Furthermore, bioactive glass and fibrin composite scaffolds promoted neurite extension similar to that of control fibrin scaffolds, suggesting that glass does not have a significant detrimental effect on neuronal health. Aligned glass scaffolds guided neurite extension in an oriented manner. Together these findings suggest that bioactive glass can provide alignment to support directed axon growth. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 2767-2775, 2014. [ABSTRACT FROM AUTHOR]

Abstract: Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca2+, although the absolute magnitude of the Ca2+ current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca2+ to total current through native and recombinant ASIC1a receptors. We found that acidification of the extracellular medium evoked amiloride and psalmotoxin 1-sensitive currents in isolated chick dorsal root ganglion neurons and human embryonic kidney cells, but did not alter fura-2 fluorescence when the bath concentration of Ca2+ was close to that found in normal physiological conditions. Further, activation of recombinant ASIC1a receptors also failed to produce measurable changes in fluorescence despite of the fact that the total cation current through the over-expressed receptor was ten-fold larger than that of the native channels. Finally, we imaged a field of intact DRG neurons loaded with the Ca2+-sensing dye Fluo-4, and found that acidification increased [Ca2+]i in a small population of cells. Thus, although our whole-field imaging data agree with previous studies that activation of ASIC1a receptors can potentially cause elevations in intracellular free Ca2+, our single cell data strongly challenges the view that Ca2+ entry through the ASIC1a receptor itself contributes to this response. [Copyright &y& Elsevier]

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference, Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt 1 knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter. [ABSTRACT FROM AUTHOR]

Presyhaptic N-type Ca2+ channels (Cav2.2, α1B are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and Ill of the α1B subunit Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant α1B Ca2+ channel subunit and transiently transfected either the wild-type α1B or the synprint deletion mutant into mouse pheochotiocytoma (MPQ cell line 9/3L a cell line that has the machinery required for rapid stimulated exocytosis' but lacks endogenous voltage-dependent Ca2+ channels; Secretion was elicited by activation of exogenously transfected Ca2+ channel' subunits. The current-voltage relationship was similar for the wild-type and mutant α1B-containing Ca2+ channels. Although total Ca2+ entry was slightly larger,for the synprint deletion channel, compared with the wild-type channel, when Ca2+ entry was normalized to cell size and limited to cells with similar Ca2+ entry (≈150 times; 106 Ca2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly-reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels Furthermore, the amount of endocytosis was significantly reduced in cells with the am synprint deletion mutant, compared with the wild-type subunit These results suggest that the synprint site is necessary for efficient coupling of Ca2+ influx through α1B influx through α1B-containing Ca2+ channels to exocytosis. [ABSTRACT FROM AUTHOR]

Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca2+ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca2+ and stimulated Ca2+ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca2+ sensor for regulated release of transmitter.