The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. Miniature endplate potentials (MEPPs) were recorded in a high K + solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and var s p (spatial variance in p ). Administration of CaM, but not heat‐inactivated CaM, encapsulated in liposomes (1000 units ml −1 ) produced an increase in m (25%) that was due to an increase in n . MEPP amplitude was not altered by CaM. Administration of antiCaM, but not heat‐inactivated antiCaM, in liposomes (50 μl ml −1 ) produced a progressive decrease in m (40%) that was associated with decreases in n and p . MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. Bath application of the membrane‐permeable CaM antagonist W7 (28 μ M ) produced a gradual decrease in m (25%) that was associated with a decrease in n . W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m . The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal. British Journal of Pharmacology (2002) 137 , 719–727. doi: 10.1038/sj.bjp.0704923
Abstract ID 100107Poster Board 529 Dopamine receptor stimulation facilitates phosphatidylinositol resynthesis, thus amplifying subsequent responses to activation of phospholipase C-coupled receptors. Phosphatidylinositol synthesis critically depends on the nucleolipid CDP-diacylglycerol. Dopamine robustly increases microsomal CDP-diacylglycerol biosynthesis through stimulation of D1-like receptors, particularly the D5 subtype the majority of which are intracellularly localized. Here, we explored the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol biosynthesis. Dopamine concentration-dependently stimulated CDP-diacylglycerol synthesis in organotypic and primary neuronal cultures devoid of the presynaptic dopamine transporter. Dopamine was saturably transported into cortical primary neurons or B35 neuroblastoma cells expressing wild-type plasmalemmal monoamine transporter (PMAT), which is known to be the principal component of classical Uptake2 transporters in the forebrain. Dopamine uptake and CDP-diacylglycerol biosynthesis in brain slices or cultured cells were inhibited by microtubule disrupters which block cytoskeletal transport, and by decynium-22 which blocks Uptake2-like transporters. Dopamine effects were selectively mimicked by D1-like agonists SKF38393 and SKF83959, competitively inhibited by D1-like antagonist SCH23390, and unaffected by D2-like agonist or antagonist. These observations indicate that dopamine is actively internalized by Uptake2 into postsynaptic-type cells where the monoamine can stimulate its intracellular D5-type receptors to increase CDP-diacylglycerol production. This finding counters the conventional notion that postsynaptic-type cells internalize supra-threshold levels of synaptic dopamine only to inactivate the transmitter. Given the critical involvement of CDP-diacylglycerol in phospholipase C and phosphatidylinositol-3-kinase signaling systems, our findings imply that intracellular dopamine could play an important role in cellular responses and adaptation to high levels of extracellular dopamine. Supported in part by NIH/NIDA Grant R01DA017614 to ASU; NIH/NINDS Grant R16NS129675 to ASU; the New York State Spinal Cord Injury Research Board Grants C32247GG; C37715GG to ASU.
Inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine diphosphate-ribose (cADPR) are second messengers that enhance neurosecretion by inducing Ca(2+) release from smooth endoplasmic reticulum (SER). The putative intracellular messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), releases Ca(2+) from stores that are distinct from SER. Evidence is presented here that NAADP causes a concentration-dependent increase in quantal output that is associated with an increase in probability of transmitter release at the frog neuromuscular junction. This effect is mimicked by A23187, a Ca ionophore that promotes Ca(2+) entry at the plasmalemma. The response to NAADP is potentiated by IP(3) but antagonized by cADPR. Thapsigargin completely blocks IP(3) and cADPR responses and decreases but does not prevent the response to NAADP. We conclude that NAADP, whose receptors are widely distributed in the brain, enhances neurosecretion by releasing Ca(2+) from an internal store near the plasmalemma, possibly from synaptic vesicles in the releasable pool. These data also support the hypothesis of a two-pool model for Ca(2+) oscillations at the presynaptic site.
Membrane contact sites (MCSs) between endosomes and the endoplasmic reticulum (ER) are thought to act as specialized trigger zones for Ca2+ signaling, where local Ca2+ released via endolysosomal ion channels is amplified by ER Ca2+-sensitive Ca2+ channels into global Ca2+ signals. Such amplification is integral to the action of the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, functional regulators of inter-organellar Ca2+ crosstalk between endosomes and the ER remain poorly defined. Here, we identify progesterone receptor membrane component 1 (PGRMC1), an ER transmembrane protein that undergoes a unique heme-dependent dimerization, as an interactor of the endosomal two pore channel, TPC1. NAADP-dependent Ca2+ signals were potentiated by PGRMC1 overexpression through enhanced functional coupling between endosomal and ER Ca2+ stores and inhibited upon PGRMC1 knockdown. Point mutants in PGMRC1 or pharmacological manipulations that reduced its interaction with TPC1 were without effect. PGRMC1 therefore serves as a TPC1 interactor that regulates ER-endosomal coupling with functional implications for cellular Ca2+ dynamics and potentially the distribution of heme.
NAADP (nicotinic acid-adenine dinucleotide phosphate) is an unusual second messenger thought to mobilize acidic Ca(2+) stores, such as lysosomes or lysosome-like organelles, that are functionally coupled to the ER (endoplasmic reticulum). Although NAADP-sensitive Ca(2+) stores have been described in neurons, the physiological cues that recruit them are not known. Here we show that in both hippocampal neurons and glia, extracellular application of glutamate, in the absence of external Ca(2+), evoked cytosolic Ca(2+) signals that were inhibited by preventing organelle acidification or following osmotic bursting of lysosomes. The sensitivity of both cell types to glutamate correlated well with lysosomal Ca(2+) content. However, interfering with acidic compartments was largely without effect on the Ca(2+) content of the ER or Ca(2+) signals in response to ATP. Glutamate but not ATP elevated cellular NAADP levels. Our results provide evidence for the agonist-specific recruitment of NAADP-sensitive Ca(2+) stores by glutamate. This links the actions of NAADP to a major neurotransmitter in the brain.
Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders. Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders. Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders.Journal of Biological ChemistryVol. 288Issue 39PreviewVOLUME 286 (2011) PAGES 34976–34985 Full-Text PDF Open AccessDeregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders.Journal of Biological ChemistryVol. 288Issue 12PreviewVOLUME 286 (2011) PAGES 34976–34985 Full-Text PDF Open Access
Carbon-based nanoprobes are attractive for minimally invasive cell interrogation but their application in cell physiology has thus far been limited. We have developed carbon nanopipettes (CNPs) with nanoscopic tips and used them to inject calcium-mobilizing messengers into cells without compromising cell viability. We identify pathways sensitive to cyclic adenosine diphosphate ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP) in breast carcinoma cells. Our findings demonstrate the superior utility of CNPs for intracellular delivery of impermeant molecules and, more generally, for cell physiology studies. The CNPs do not appear to cause any lasting damage to cells. Their advantages over commonly used glass pipettes include smaller size, breakage and clogging resistance, and potential for multifunctionality such as in concurrent injection and electrical measurements.
The angiotensin II (AngII) type 1 receptor (AT1) plays a critical role in hypertrophy of vascular smooth muscle cells (VSMCs). Although it is well known that Gq is the major G protein activated by the AT1 receptor, the requirement of Gq for AngII-induced VSMC hypertrophy remains unclear. By using cultured VSMCs, this study examined the requirement of Gq for the epidermal growth factor receptor (EGFR) pathway, the Rho-kinase (ROCK) pathway, and subsequent hypertrophy. AngII-induced intracellular Ca2+ elevation was completely inhibited by a pharmacological Gq inhibitor as well as by adenovirus encoding a Gq inhibitory minigene. AngII (100nm)-induced EGFR transactivation was almost completely inhibited by these inhibitors, whereas these inhibitors only partially inhibited AngII (100nm)-induced phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1. Stimulation of VSMCs with AngII resulted in an increase of cellular protein and cell volume but not in cell number. The Gq inhibitors completely blocked these hypertrophic responses, whereas a G protein-independent AT1 agonist did not stimulate these hypertrophic responses. In conclusion, Gq appears to play a major role in the EGFR pathway, leading to vascular hypertrophy induced by AngII. Vascular Gq seems to be a critical target of intervention against cardiovascular diseases associated with the enhanced renin-angiotensin system.
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