Intracellular Storage Sites Research Articles

S-acylation of SARAF regulates store-operated calcium entry.

The endoplasmic reticulum (ER) is the primary intracellular calcium storage site. Upon cellular stimulation by an agonist, the production of inositol 1,4,5-trisphosphate (IP3) leads to activation of IP3 receptor channels on the ER membrane and calcium efflux from ER lumen. This leads to ER calcium store depletion. Upon store depletion, the ER-resident calcium sensor STIM1 and plasma membrane calcium channel Orai1 bind to each other to form the calcium release-activated calcium (CRAC) channel. These channels lead to calcium entry from extracellular milieu. This entry is known as store-operated calcium entry (SOCE). Previously, we have shown that Orai1 and STIM1 are S-acylated upon store-depletion and this process is crucial for CRAC channel formation and SOCE. TMEM66, also known as SARAF, is a ER transmembrane protein that is known to associate with STIM1 in resting conditions, and subsequently with Orai1 after store-depletion. This binding of SARAF with Orai1 upon store-depletion is important for the inactivation of CRAC currents. We hypothesize S-acylation of SARAF is required for the inactivation of SOCE. Here we show that SARAF is S-acylated in Jurkat T cells upon activation of the T cell receptor (TCR) and subsequent store-depletion. We also show the residue where SARAF undergoes this S-acylation. Importantly, a C320S mutant version of SARAF that cannot undergo S-acylation has deficits in CRAC channel formation and SOCE. Put together, these data suggest SARAF is a crucial component of SOCE.

Artificial intelligence-driven identification of morin analogues acting as CaV1.2 channel blockers: Synthesis and biological evaluation

Morin is a vasorelaxant flavonoid, whose activity is ascribable to CaV1.2 channel blockade that, however, is weak as compared to that of clinically used therapeutic agents. A conventional strategy to circumvent this drawback is to synthesize new derivatives differently decorated and, in this context, morin-derivatives able to interact with CaV1.2 channels were found by employing the potential of PLATO in target fishing and reverse screening. Three different derivatives (5a-c) were selected as promising tools, synthesized, and investigated in in vitro functional studies using rat aorta rings and rat tail artery myocytes. 5a-c were found more effective vasorelaxant agents than the naturally occurring parent compound and antagonized both electro- and pharmaco-mechanical coupling in an endothelium-independent manner. 5a, the series’ most potent, reduced also Ca2+ mobilization from intracellular store sites. Furthermore, 5a≈5c > 5b inhibited Ba2+ current through CaV1.2 channels. However, compound 5a caused also a concentration-dependent inhibition of KCa1.1 channel currents.

Viscosity-Sensitive Solvatochromic Fluorescent Probes for Lipid Droplets Staining.

Lipid droplets (LDs) are simple intracellular storage sites for neutral lipids and exhibit important impact on many physiological processes. For example, the changes in the polar microenvironment inside LDs could affect physiological processes, such as lipid metabolism and storage, protein degradation, signal transduction, and enzyme catalysis. Herein, a new fluorescent chemo-sensor (Couoxo-LD) was formulated by our molecular design strategy. The probe could be applied to effectively label intracellular lipid droplets. Intriguingly, Couoxo-LD demonstrated positive sensitivity to both polarity and viscosity, which might be attributed to its D-π-A structure and the twisted rotational behavior of the carbon–carbon double bond (TICT). Additionally, Couoxo-LD was successfully implemented in cellular imaging due to its excellent selectivity, pH stability, and low biotoxicity. In HeLa cells, the co-localization curve between Couoxo-LD and commercial lipid droplet dyes overlapped at 0.93. The results indicated that the probe could selectively sense LDs in HeLa cells. Meanwhile, Couoxo-LD can be applied for in vivo imaging of zebrafish.

Released ATP Mediates Spermatozoa Chemotaxis Promoted by Uterus-Derived Factor (UDF) in Ascaris suum.

Fertilization requires sperm migration toward oocytes and subsequent fusion. Sperm chemotaxis, a process in which motile sperm are attracted by factors released from oocytes or associated structures, plays a key role in sperm migration to oocytes. Here, we studied sperm chemotaxis in the nematode Ascaris suum. Our data show that uterus-derived factor (UDF), the protein fraction of uterine extracts, can attract spermatozoa. UDF is heat resistant, but its activity is attenuated by certain proteinases. UDF binds to the surface of spermatozoa but not spermatids, and this process is mediated by membranous organelles that fuse with the plasma membrane. UDF induces spermatozoa to release ATP from intracellular storage sites to the extracellular milieu, and extracellular ATP modulates sperm chemotaxis. Moreover, UDF increases protein serine phosphorylation (pS) levels in sperm, which facilitates sperm chemotaxis. Taken together, we revealed that both extracellular ATP and intracellular pS signaling are involved in Ascaris sperm chemotaxis. Our data provide insights into the mechanism of sperm chemotaxis in Ascaris suum.

Co-translational biogenesis of lipid droplet integral membrane proteins.

ABSTRACTMembrane proteins destined for lipid droplets (LDs), a major intracellular storage site for neutral lipids, are inserted into the endoplasmic reticulum (ER) and then trafficked to LDs where they reside in a hairpin loop conformation. Here, we show that LD membrane proteins can be delivered to the ER either co- or post-translationally and that their membrane-embedded region specifies pathway selection. The co-translational route for LD membrane protein biogenesis is insensitive to a small molecule inhibitor of the Sec61 translocon, Ipomoeassin F, and instead relies on the ER membrane protein complex (EMC) for membrane insertion. This route may even result in a transient exposure of the short N termini of some LD membrane proteins to the ER lumen, followed by putative topological rearrangements that would enable their transmembrane segment to form a hairpin loop and N termini to face the cytosol. Our study reveals an unexpected complexity to LD membrane protein biogenesis and identifies a role for the EMC during their co-translational insertion into the ER.

English

The effect of extracellular calcium on isolated uterine muscle contractions stimulated by acetone leaf extract of Anogeissus leiocarpa was investigated in a rat. About 12 m segment of the uterine muscle strip was mounted initially in a thermostatically regulated organ bath (37°C) containing normal Physiological Salt Solution (PSS; De Jalon) and later inphysiological salt solution devoid of CaCl2. The extract contracted the uterine muscle preparation in a concentration-related manner in normal PSS, with 0.53 mg/ml as the lowest active concentration. This contractile response was abolished by the calcium channel blocker, verapamil HCl (2 µg/ml). The extract however, did not evoke any contractile activity on the isolated tissue in a calcium-free media (PSS), even when caffeine (2 mmol) known to release calcium via the calcium induced-calcium release (CICR) mechanism was added to the perfusate. The results demonstrated the requirement for extracellular calcium for the extract mediated contractions and the inability of the extract to access calcium from intracellular storage sites such as the sarcoplasmic reticulum and the mitochondria. Therefore, the influx of Ca2+ into the cell cytosol is a pre-requisite for the extract-mediated uterine muscle contraction. Key word: Anogeissus leiocarpa, thermostatically organ bath, verapamil, caffeine, uterus.

Acid-electrolyzed functional water-induces Interleukin-1α release from Intracellular Storage Sites in Oral Squamous Cell Carcinoma.

The aim of this study was to examine the acid-electrolyzed functional water (FW)-mediated cytokine release in an oral squamous cell carcinoma-derived cell line (OSCC) following treatment with FW. FW is generated by the electrolysis of a sodium chloride solution and accelerate the burn wound healing. To elucidate the underlying mechanisms, the cytokine/chemokine secretion profile of HSC3 cells was examined using a cytokine array. FW treatment significantly induced interleukin (IL)-1α secretion, which was confirmed by enzyme-linked immunosorbent assay. Subsequently, the HSC3 cells were pre-treated with cycloheximide (CHX) for 1 h prior to FW stimulation to determine whether the augmented IL-1α secretion was due to enhanced protein synthesis. CHX pre-treatment did not affect IL-1α secretion suggesting that the secreted IL-1α might have been derived from intracellular storage sites. The amount of IL-1α in the cell lysate of the FW-treated HSC3 cells was significantly lower than that of the non-treated cells. Immunofluorescence staining using a polyclonal antibody against full-length IL-1α revealed a drastic reduction in IL-1α inside the FW- treated cells. IL-1α is synthesized in its precursor form (pIL-1α) and cleaved to produce pro-piece and mature IL-1α (ppIL-1α and mIL-1α) inside the cells. In the present study, only pIL-1α was detected within the HSC3 cells in its resting state. However, FW stimulation resulted in the release of the 33 kDa and two other smaller forms (about 19 kDa) of the protein. These results indicates that FW treatment induces IL-1α secretion, a typical alarmin, from the intracellular storage in OSCC cells.

Extracellular Calcium and Induction of Uterine Muscle Contraction by Aqueous Ethanolic Leaf Extract of <I>Mucuna pruriens</I>

Calcium (Ca2+) serves as an essential signaling molecule in biological systems, regulating a wide range of cellular processes of which uterine smooth muscle contraction is among. The present study was designed to evaluate the involvement of Ca2+ on isolated uterine muscle contraction induced by aqueous ethanolic leaf extract of Mucuna pruriens (M. pruriens). Uterine muscle contraction induced by the extract was concentration-dependent and was completely abolished (100%; P<0.05) in nominally Ca2+ -free physiological salt solution and in solutions containing (EGTA 1.5 mmol), lanthanium chloride (1.5 and 3 mmol), caffeine (3and 4.4 mmol) and verapamil (0.007-0.14 μmol). It is concluded that the inability of the extract to produce contractions in Ca2+ -free media, indicates that it lacks the ability to mobilize calcium from intracellular storage sites. Hence, its uterine stimulatory property is therefore solely dependent on extracellular Ca2+.
 Keywords: Calcium, Mucuna pruriens, Uterus, Contraction.

Effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin-treated microenvironment of A549 cell line

Objective To investigate the effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin(CBP)-treated A549 cell and its possible mechanisms of inhibiting A549 cell line activity. Methods According to whether SonoVue was used or not, and the different dose of CBP, the groups A-F were arranged as the ultrasound(US) group(group A), the ultrasound combined with microbubbles (USMB) group(group B), the low dose CBP (100 μg/ml)+ US group(group C), the low dose CBP+ USMB group(group D), the high dose CBP (200 μg/ml)+ US group (group E) and the high dose CBP+ USMB group(group F). A549 cells were bathed and washed by a calcium-free buffer, loaded with Ca2+ indicator fluo-4 AM. Real-time images were acquired using laser confocal microscopy. The fluorescence intensity of intracellular calcium ion concentration ([Ca2+ ]i) in individual living cell was observed and the calcium overload was analyzed. Results After ultrasound irradiation, the normalized fluorescence intensity of [Ca2+ ]i increased rapidly, then returned to a new homeostasis (selected cells in groups A, B, E, F) or experienced a second calcium oscillation (some cells in group C and D). All the selected cells in group B and some cells in group C and D exhibited superimposed oscillations. The calcium overloading time in group D was longer than those of any other groups. Four cells in group A experienced delayed calcium oscillations. Compared with group A, the selected cells in other groups exhibited a larger amplitude of calcium oscillation(all P<0.05) and the selected cells in group B and D exhibited calcium oscillation for a longer period of time(all P<0.05). Conclusions In the calcium-free buffer, US, USMB, CBP+ US, CBP+ USMB are direct stimuli of calcium overload in A549 cells. SonoVue, CBP, CBP+ SonoVue are all synergistic stimuli of calcium overload in A549 cells irradiated by ultrasound.US, USMB and CBP may synergistically induce calcium release from intracellular store sites in A549 cells. Calcium overload is a possible mechanism of ultrasound combined with microbubbles in assisting CBP chemotherapy. Key words: Sonication; Microbubbles; Carboplatin; A549 cell; Calcium overload; Cavitation effect

Regulation of adult neural progenitor cell functions by purinergic signaling.

Extracellular purines are signaling molecules in the neurogenic niches of the brain and spinal cord, where they activate cell surface purinoceptors at embryonic neural stem cells (NSCs) and adult neural progenitor cells (NPCs). Although mRNA and protein are expressed at NSCs/NPCs for almost all subtypes of the nucleotide-sensitive P2X/P2Y, and the nucleoside-sensitive adenosine receptors, only a few of those have acquired functional significance. ATP is sequentially degraded by ecto-nucleotidases to ADP, AMP, and adenosine with agonistic properties for distinct receptor-classes. Nucleotides/nucleosides facilitate or inhibit NSC/NPC proliferation, migration and differentiation. The most ubiquitous effect of all agonists (especially of ATP and ADP) appears to be the facilitation of cell proliferation, usually through P2Y1Rs and sometimes through P2X7Rs. However, usually P2X7R activation causes necrosis/apoptosis of NPCs. Differentiation can be initiated by P2Y2R-activation or P2X7R-blockade. A key element in the transduction mechanism of either receptor is the increase of the intracellular free Ca2+ concentration, which may arise due to its release from intracellular storage sites (G protein-coupling; P2Y) or due to its passage through the receptor-channel itself from the extracellular space (ATP-gated ion channel; P2X). Further research is needed to clarify how purinergic signaling controls NSC/NPC fate and how the balance between the quiescent and activated states is established with fine and dynamic regulation. GLIA 2017;65:213-230.

Role for RalA downstream of Rac1 in skeletal muscle insulin signalling.

Insulin-stimulated glucose uptake in skeletal muscle is mediated by the translocation of the glucose transporter GLUT4 from intracellular storage sites to the plasma membrane. The small GTPase Rac1 has been implicated in this insulin signalling, but the mechanism whereby Rac1 stimulates GLUT4 translocation remains obscure. In the present study, we examined the role of the small GTPase RalA downstream of Rac1 in skeletal muscle fibres isolated from genetically modified mice. A dominant-negative mutant of RalA, when ectopically overexpressed, significantly reduced GLUT4 translocation in response to insulin or either one of constitutively activated mutants of Rac1 and its upstream regulators, including the guanine-nucleotide-exchange factor FLJ00068, the protein kinase Akt2 and phosphoinositide 3-kinase. Constitutively activated Rac1 also failed to induce GLUT4 translocation in mouse skeletal muscle fibres in which the expression of RalA was abrogated by specific siRNA molecules. Furthermore, we applied a novel approach to detect the activated form of RalA in situ by immunofluorescence microscopy of mouse skeletal muscle fibres, demonstrating that constitutively activated mutants of Rac1 and its upstream regulators as well as insulin indeed cause the activation of RalA. Notably, this RalA activation was remarkably impaired in rac1-deficient skeletal muscle fibres. Taken together, these results provide evidence that RalA is indeed activated and involved in the regulation of GLUT4 translocation in response to insulin downstream of Rac1 in mouse skeletal muscle.

Bone and Calcium Research Update 2015. Role of calcium in the regulation of smooth muscle function

The vascular smooth muscle plays a physiological role in the regulation of the organ blood flow and blood pressure by modulating vascular resistance. The contraction of vascular smooth muscle is regulated by both Ca2+ signal and changes in the sensitivity of the contractile apparatus to Ca2+. The influx of the extracellular Ca2+ and the release of Ca2+ from the intracellular store sites play an important role in generating Ca2+ signal. The regulation of activity of myosin light chain phosphatase plays a critical role in regulating the myofilament Ca2+ sensitivity. It is conceivable that any contractile stimulants or vasorelaxant agents exert their vascular effects by affecting either Ca2+ signal or myofilament Ca2+ sensitivity.

Separate cellular localizations of human T-lymphotropic virus 1 (HTLV-1) Env and glucose transporter type 1 (GLUT1) are required for HTLV-1 Env-mediated fusion and infection.

Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env. The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.

Copper Transport and Trafficking at the Host–Bacterial Pathogen Interface

ConspectusThe human innate immune system has evolved the means to reduce the bioavailability of first-row late d-block transition metal ions to invading microbial pathogens in a process termed “nutritional immunity”. Transition metals from Mn(II) to Zn(II) function as metalloenzyme cofactors in all living cells, and the successful pathogen is capable of mounting an adaptive response to mitigate the effects of host control of transition metal bioavailability. Emerging evidence suggests that Mn, Fe, and Zn are withheld from the pathogen in classically defined nutritional immunity, while Cu is used to kill invading microorganisms. This Account summarizes new molecular-level insights into copper trafficking across cell membranes from studies of a number of important bacterial pathogens and model organisms, including Escherichia coli, Salmonella species, Mycobacterium tuberculosis, and Streptococcus pneumoniae, to illustrate general principles of cellular copper resistance.Recent highlights of copper chemistry at the host–microbial pathogen interface include the first high resolution structures and functional characterization of a Cu(I)-effluxing P1B-ATPase, a new class of bacterial copper chaperone, a fungal Cu-only superoxide dismutase SOD5, and the discovery of a small molecule Cu-bound SOD mimetic. Successful harnessing by the pathogen of host-derived bactericidal Cu to reduce the bacterial load of reactive oxygen species (ROS) is an emerging theme; in addition, recent studies continue to emphasize the importance of short lifetime protein–protein interactions that orchestrate the channeling of Cu(I) from donor to target without dissociation into bulk solution; this, in turn, mitigates the off-pathway effects of Cu(I) toxicity in both the periplasm in Gram negative organisms and in the bacterial cytoplasm. It is unclear as yet, outside of the photosynthetic bacteria, whether Cu(I) is trafficked to other cellular destinations, for example, to cuproenzymes or other intracellular storage sites, or the general degree to which copper chaperones vs copper efflux transporters are essential for bacterial pathogenesis in the vertebrate host.Future studies will be directed toward the identification and structural characterization of other cellular targets of Cu(I) trafficking and resistance, the physical and mechanistic characterization of Cu(I)-transfer intermediates, and elucidation of the mutual dependence of Cu(I) trafficking and cellular redox status on thiol chemistry in the cytoplasm. Crippling bacterial control of Cu(I) sensing, trafficking, and efflux may represent a viable strategy for the development of new antibiotics.

Energy-dependent uptake of benzo[a]pyrene and its cytoskeleton-dependent intracellular transport by the telluric fungus Fusarium solani

In screening indigenous soil filamentous fungi for polycyclic aromatic hydrocarbons (PAHs) degradation, an isolate of the Fusarium solani was found to incorporate benzo[a]pyrene (BaP) into fungal hyphae before degradation and mineralization. The mechanisms involved in BaP uptake and intracellular transport remain unresolved. To address this, the incorporation of two PAHs, BaP, and phenanthrene (PHE) were studied in this fungus. The fungus incorporated more BaP into cells than PHE, despite the 400-fold higher aqueous solubility of PHE compared with BaP, indicating that PAH incorporation is not based on a simple diffusion mechanism. To identify the mechanism of BaP incorporation and transport, microscopic studies were undertaken with the fluorescence probes Congo Red, BODIPY®493/503, and FM®4-64, targeting different cell compartments respectively fungal cell walls, lipids, and endocytosis. The metabolic inhibitor sodium azide at 100 mM totally blocked BaP incorporation into fungal cells indicating an energy-requirement for PAH uptake into the mycelium. Cytochalasins also inhibited BaP uptake by the fungus and probably its intracellular transport into fungal hyphae. The perfect co-localization of BaP and BODIPY reveals that lipid bodies constitute the intracellular storage sites of BaP in F. solani. Our results demonstrate an energy-dependent uptake of BaP and its cytoskeleton-dependent intracellular transport by F. solani.

In This Issue

HomeCirculation ResearchVol. 113, No. 5In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published16 Aug 2013https://doi.org/10.1161/RES.0b013e3182a71457Circulation Research. 2013;113:479Dyssynchrony in Cytosolic Ca2+ Decay (p 527)Download figureDownload PowerPointHeart cells clear up their cytoplasmic calcium somewhat dyssynchronously, say Hohendanner et al, and this dyssynchrony gets worse in failing hearts.Calcium is the major intracellular regulator of cardiomyocyte contraction. It is released from its intracellular storage site, the sarcoplasmic reticulum, into the cytoplasm where it then activates the contractile machinery. But after each contraction, the cell must relax—a process that requires the prompt and efficient removal of calcium from the cytosol. This combined release and clear up of calcium is known as a calcium transient. It has been shown that the release of calcium across the cell is synchronized to ensure coordinated control of the contractile machinery, but less is known about the clean-up, or decay phase. Hohendanner and colleagues now show that calcium removal occurs at different microdomains throughout the cell and that the speed of decay varies from domain to domain. Furthermore, the rate of relaxation at adjacent contractile machinery varies in concordance with the differing decay rates of the domains. They also found that the degree of dyssynchrony is vastly exaggerated in failing human hearts as well as in mice with hypertrophy and pigs with chronic myocardial ischemia. This, increased dyssynchrony slows down the overall rate of relaxation and, the authors suggest that it might contribute to contractile dysfunction associated with cardiac hypertrophy and heart failure.Bone-Derived Stem Cells Repair the Heart (p 539)Download figureDownload PowerPointBelieve it or not, bone stem cells are better than cardiac stem cells for repairing a damaged heart, say Duran et al.While stem cell therapy is a promising approach for repairing damaged hearts, the results obtained to-date have been modest. Part of the problem is that different types of stem cells yield different results and the precise mechanisms underlying the beneficial effects of stem cells remain unclear. Thus, Duran and colleagues examined two mechanisms of repair—differentiation of stem cells into cardiac cell types and secretion of paracrine factors—in two different stem cell types, one from cortical bone and another from the heart. They found that when injected into damaged mouse hearts, the bone cells were actually more adept at differentiating into cardiac cell types—including cardiomyocytes, vascular endothelial cells, and smooth muscle cells—than the heart-derived cells, which only differentiated into immature cardiomyocytes. Cells from the bone also displayed sustained expression of pro-angiogenic paracrine factors, which correlated with better neovascularization of the damaged hearts. Cardiac stem cells, on the other hand, produced similar paracrine factors but only for one day after injection. Importantly, the better performance by the bone cells translated to improved heart function and increased survival for the mice, suggesting that the use of these cells might improve outcomes of cell therapy.IKK2 is a Myosin Light Chain Kinase (p 562)Download figureDownload PowerPointYing et al identify a possible new target for treating hypertension.Constriction of vascular smooth muscle cells (VSMCs), which contributes to hypertension, is controlled in part by phosphorylation of the myosin light chain (MLC). This post-translational modification enables actin and myosin molecules of the contractile machinery to interact and initiate contraction. MLC is known to be phosphorylated by MLCK but recent work suggests that at least one other kinase may be involved. For example, it has been observed that inhibition of MLCK does not lead to the complete dephosphorylation of MLC and that in MLCK-deficient mice, blood vessels remain responsive to vasoconstrictors. In addition, previous work has shown that the kinase IKK2 regulates migration and morphology in mouse embryonic fibroblasts, and that both of these processes depend on MLC phosphorylation. This led Ying and colleagues to ask whether IKK2 could be an MLC kinase in VSMCs. They found that not only was MLC phosphorylated by IKK2 in vitro and but also overexpression of IKK2 in VSMCs led to increased MLC phosphorylation. Conversely, inhibition of IKK2 decreased MLC phosphorylation and attenuated vasoconstrictive responses of isolated rat aortas. Furthermore, mice that lacked IKK2 in their VSMCs showed a decreased hypertensive response to three different vasoconstrictors. These results suggest that IKK2 inhibition could be a novel approach for decreasing hypertension. Previous Back to top Next FiguresReferencesRelatedDetails August 16, 2013Vol 113, Issue 5 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e3182a71457 Originally publishedAugust 16, 2013 PDF download Advertisement

Nexilin, a Cardiomyopathy-Associated F-Actin Binding Protein, Binds and Regulates IRS1 Signaling in Skeletal Muscle Cells

Insulin stimulates glucose uptake through a highly organized and complex process that involves movement of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Previous studies in L6 skeletal muscle cells have shown that insulin-induced activation and assembly of insulin receptor substrate 1 (IRS1) and p85α the regulatory subunit of the Type 1A phosphatidylinositol-3-kinase (PI3K), within remodeled actin-rich membrane structures is critical for downstream signalling mediating the translocation of GLUT4. The mechanism for localization within actin cytoskeletal scaffolds is not known, as direct interaction of IRS1 or p85α with F-actin has not been demonstrated. Here we show that nexilin, a F-actin binding protein implicated in the pathogenesis of familial dilated cardiomyopathies, preferentially binds to IRS1 over IRS2 to influence glucose transport in skeletal muscle cells. Nexilin stably associates with IRS1 under basal conditions in L6 myotubes and this complex is disassembled by insulin. Exposure of L6 myotubes to Latrunculin B disrupts the spatial patterning of nexilin and its transient association with IRS1. Functional silencing of nexilin has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, however it enhances recruitment of p85α to IRS1 resulting in increased PI-3, 4, 5-P3 formation, coincident with enhanced AKT activation and glucose uptake. By contrast, overexpression of nexilin inhibits transmission of IRS1 signals to AKT. Based on these findings we propose that nexilin may tether IRS1 to actin-rich structures under basal conditions, confining IRS1 signaling to specific subcellular locations in the cell. Insulin-elicited release of this constraint may enhance the efficiency of IRS1/PI3K interaction and PI-3, 4, 5-P3 production at localized sites. Moreover, the selective binding of nexilin to IRS1 and not IRS2 may contribute to the differential specificity of IRS isoforms in the modulation of GLUT4 trafficking in skeletal muscle cells.

Cysteine string protein 1 (CSP1) modulates insulin sensitivity by attenuating glucose transporter 4 (GLUT4) vesicle docking with the plasma membrane

Insulin stimulates glucose transporter 4 (GLUT4) vesicle recruitment from its intracellular storage site to the plasma membrane. Cysteine string protein 1 (CSP1) is a SNARE-binding protein involved in the vesicular trafficking of neurotransmitters and other exocytic processes. In this study, we investigated the involvement of CSP1 in insulin-dependent GLUT4 recruitment in 3T3-L1 adipocytes. Over-expression of wild-type CSP1 led to attenuated insulin-stimulated glucose uptake without any change in GLUT4 content in the plasma membrane, rather it inhibits docking by blocking the association of VAMP2 with syntaxin 4. In contrast, knockdown of CSP1 enhanced insulin-stimulated glucose uptake. The mRNA and protein expression of CSP1 was elevated in 3T3-L1 adipocytes in insulin resistant states caused by high levels of palmitate and chronic insulin exposure. Taken together, the results of this study suggest that CSP1 is involved in insulin resistance by interrupting GLUT4 vesicle docking with the plasma membrane.

PS9 - 44. Unravelling the kinetics of insulin signalling in skeletal muscle cells

The insulin signaling pathway, which is of great importance in human energy homeostasis, has been investigated for decades. Skeletal muscle is the most important tissue among all insulin sensitive tissues utilizing the major amount (60-70%) of glucose in plasma. Insulin mediated glucose transport is regulated by the insulin signaling cascade resulting in the translocation of the principal glucose transporter protein, GLUT4, from intracellular storage sites to the plasma membrane.

Mechanism of Action of Methamphetamine within the Catecholamine and Serotonin Areas of the Central Nervous System

Addiction to methamphetamine (METH) is thought to be mediated by dopaminergic effects in the reward pathway in the brain via the A10 dopaminergic pathway. Herein we describe an overview of the results of the basic preclinical science undertaken to provide mechanistic insights into the action of amphetamines in general and METH in particular. A brief history of amphetamine and METH use and abuse is given, and an overview of the relevant chemical aspects of amphetamine as they relate to neurotransmitters in general is made. A review of the methods used to study the biochemical effects of METH is outlined. Finally, a focused analysis of the kinetic mechanisms of action of the amphetamines in general, and METH in particular, at the transmembrane transporters and at the intracellular vesicular storage sites is made. A description of how catecholaminergic and serotonergic nerve signaling may be altered by METH is proposed. Overall, the emphasis here is on differences in effects observed between the striatal (the A9 substantia nigral dopamine pathway) and nucleus accumbens (the A10, ventral tegmental pathway) areas of the brain following acute as well as repeated dosing and withdrawal.