Estradiol-Mediated Axogenesis of Hypothalamic Neurons Requires ERK1/2 and Ryanodine Receptors-Dependent Intracellular Ca2+ Rise in Male Rats.
ABSTRACT: 17β-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos in vitro, but the mechanism that initiates these events is poorly understood. This study reports the intracellular Ca2+ increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca2+ channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IP3R) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca2+ imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca2+, which often occurs in repetitive Ca2+ oscillations. This response was not observed in the absence of extracellular Ca2+ or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca2+ mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.
Project description:The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment.
Project description:In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics.NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.
Project description:Recent studies have provided evidence that depolarization in the absence of extracellular Ca2+ can trigger Ca2+ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca2+ from intracellular stores in response to depolarization, independent of Ca2+ influx. We measured changes in cytosolic ?F/F0 in individual fluo-4 -loaded sympathetic ganglion neurons in response to maintained K+ depolarization in the presence (2 mM) and absence of extracellular Ca2+ ([Ca2+]e). Progressive elevations in extracellular [K+]e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca2+]e. Peak amplitude of ?F/F0 transients in 2 mM [Ca2+]e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ?F/F0 in 0 mM [Ca2+]e were ~5-10% of those evoked at the same membrane potential in 2 mM [Ca2+]e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ?F/F0 transients in 0 mM [Ca2+]e were slower than those of ?F/F0 transients evoked in 2 mM [Ca2+]e. Rises in ?F/F0 evoked by high [K+]e in the absence of extracellular Ca2+ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, or the inositol 1,4,5-triphosphate (IP3) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd2+, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca2+ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca2+ from IP3-sensitive internal stores in response to membrane depolarization, independent of Ca2+ influx.
Project description:Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) ? and ?. The roles of STRAD? and STRAD? in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions.We find that STRAD? is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRAD?, whereby STRAD? specifically maintains LKB1 protein levels via cytoplasmic compartmentalization.We demonstrate a novel role for STRAD? in axogenesis and also show for the first time in vivo that STRAD?, but not STRAD?, is responsible for LKB1 protein stability.
Project description:Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs.The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal-like cells (ICC-LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo-4.In 1 microM nifedipine, high frequency (10-30 min(-1)) Ca2+ transients and STDs were recorded in ASMCs, while ICC-LCs displayed low frequency (1-3 min(-1)) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2-aminoethoxy-diphenylborate (2-APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine-sensitive Ca2+ release blocked ICC-LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl(-) with isethionate.ASMCs generated Ca2+ transients and cation-selective STDs via mechanisms involving Ca2+ release from IP3-dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine-sensitive stores.
Project description:Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.
Project description:We determined the possible role of large-conductance Ca2+-activated K (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel ?- and ?1-subunits were coexpressed, unitary BK currents were detected, and single-channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca sparks and iberiotoxin-sensitive spontaneous transient outward currents. Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-induced, KCl-induced (20 mM), and BayK8644-induced contraction. Acute inhibition of BK channels with iberiotoxin in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel ?-subunits and L-type Ca2+ channel ?1-C subunits are expressed in murine MV. However, these channels are not functional because murine MV lack nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl, and BayK8644, consistent with our previous studies showing that murine MV do not have BK ?1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone but also BK channels are required for rhythmic oscillations in venous tone.
Project description:The deleterious effects of glutamate excitotoxicity are well described for central nervous system gray matter. Although overactivation of glutamate receptors also contributes to axonal injury, the mechanisms are poorly understood. Our goal was to elucidate the mechanisms of kainate receptor-dependent axonal Ca(2+) deregulation.Dorsal column axons were loaded with a Ca(2+) indicator and imaged in vitro using confocal laser-scanning microscopy.Activation of glutamate receptor 6 (GluR6) kainate receptors promoted a substantial increase in axonal [Ca(2+)]. This Ca(2+) accumulation was due not only to influx from the extracellular space, but a significant component originated from ryanodine-dependent intracellular stores, which, in turn, depended on activation of L-type Ca(2+) channels: ryanodine, nimodipine, or nifedipine blocked the agonist-induced Ca(2+) increase. Also, GluR6 stimulation induced intraaxonal production of nitric oxide (NO), which greatly enhanced the Ca(2+) response: quenching of NO with intraaxonal (but not extracellular) scavengers, or inhibition of neuronal NO synthase with intraaxonal Nomega-nitro-L-arginine methyl ester, blocked the Ca(2+) increase. Loading axons with a peptide that mimics the C-terminal PDZ binding sequence of GluR6, thus interfering with the coupling of GluR6 to downstream effectors, greatly reduced the agonist-induced axonal Ca(2+) increase. Immunohistochemistry showed GluR6/7 clusters on the axolemma colocalized with neuronal NO synthase and Ca(v)1.2.Myelinated spinal axons express functional GluR6-containing kainate receptors, forming part of novel signaling complexes reminiscent of postsynaptic membranes of glutamatergic synapses. The ability of such axonal "nanocomplexes" to release toxic amounts of Ca(2+) may represent a key mechanism of axonal degeneration in disorders such as multiple sclerosis where abnormal accumulation of glutamate and NO are known to occur.
Project description:To characterize mechanisms involved in neurokinin type 1 receptor (NK1R)-mediated emesis, we investigated the brainstem emetic signaling pathways following treating least shrews with the selective NK1R agonist GR73632. In addition to episodes of vomiting over a 30-min observation period, a significant increase in substance P-immunoreactivity in the emetic brainstem dorsal motor nucleus of the vagus (DMNX) occurred at 15?min post an intraperitoneal (i.p.) injection GR73632 (5?mg/kg). In addition, time-dependent upregulation of phosphorylation of several emesis -associated protein kinases occurred in the brainstem. In fact, Western blots demonstrated significant phosphorylations of Ca2+/calmodulin kinase II? (CaMKII?), extracellular signal-regulated protein kinase1/2 (ERK1/2), protein kinase B (Akt) as well as ? and ?II isoforms of protein kinase C (PKC?/?II). Moreover, enhanced phospho-ERK1/2 immunoreactivity was also observed in both brainstem slices containing the dorsal vagal complex emetic nuclei as well as in jejunal sections from the shrew small intestine. Furthermore, our behavioral findings demonstrated that the following agents suppressed vomiting evoked by GR73632 in a dose-dependent manner: i) the NK1R antagonist netupitant (i.p.); ii) the L-type Ca2+ channel (LTCC) antagonist nifedipine (subcutaneous, s.c.); iii) the inositol trisphosphate receptor (IP3R) antagonist 2-APB (i.p.); iv) store-operated Ca2+ entry inhibitors YM-58483 and MRS-1845, (i.p.); v) the ERK1/2 pathway inhibitor U0126 (i.p.); vi) the PKC inhibitor GF109203X (i.p.); and vii) the inhibitor of phosphatidylinositol 3-kinase (PI3K)-Akt pathway LY294002 (i.p.). Moreover, NK1R, LTCC, and IP3R are required for GR73632-evoked CaMKII?, ERK1/2, Akt and PKC?/?II phosphorylation. In addition, evoked ERK1/2 phosphorylation was sensitive to inhibitors of PKC and PI3K. These findings indicate that the LTCC/IP3R-dependent PI3K/PKC?/?II-ERK1/2 signaling pathways are involved in NK1R-mediated vomiting.
Project description:INTRODUCTION: Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs) may display unique structural and functional properties than mature adult cardiomyocytes. METHODS AND RESULTS: Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR) Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM) and full duration at half maximum (FDHM) of spontaneous Ca2+ sparks. CONCLUSIONS: For the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.