Calmodulin Activity Research Articles

Evaluation of the Effects of Schisandra chinensis on the Myocardium of Rats with Hyperthyroid Heart Disease by Using Velocity Vector Imaging Combined with the Estimation of p53 Expression and Calmodulin Activity.

Schisandra chinensis (SC) is reported to improve myocardial ischemia. Velocity vector imaging (VVI) is a noninvasive technique for evaluating myocardial function in humans, while few reported on the application in animals. In this study, we aimed to evaluate the improved effects of SC on the myocardium of Sprague Dawley rats having hyperthyroid heart disease (HHD) using VVI technique. HHD models were established by injecting daily with subcutaneous levothyroxine (0.5 mg/kg). Then, the SC group was administered the aqueous extract of SC (2 g/kg) once daily, while the HHD and control (CON) groups were administered the same amount of distilled water daily. All the rats were provided the same amount of food and water daily, and the intervention was stopped after 28 days. The efficacy of SC in HHD rats was evaluated by ultrasound VVI. The serum total triiodothyronine level, total thyroxine level, N-terminal pro-brain natriuretic peptide expression, p53 expression, and calmodulin (CaM) activity were assessed by western blotting, Hematoxylin-Eosin and Masson staining, and electron microscopy. The results indicated that SC significantly improved the systolic velocity, diastolic velocity, strain, systolic strain rate, and diastolic strain rate of the heart by significantly reducing p53 expression and CaM activity (P < 0.05), improving myocardial fibrosis in HHD rats. Also, VVI can be a valuable tool for the evaluation of myocardial function in HHD rats.

Role of calcium in regulating anthocyanin accumulation in ‘Manicure Finger’ grape berries

The objective of this study was to clarify the effects of calcium treatments on the anthocyanin accumulation of ‘Manicure Finger’ grape berries. Calcium treatments increased the calcium contents and enhanced the calmodulin activities in grapevine leaves and berries, upregulated the expression of the sugar transporter genes SUC12 and SUC27 in leaves, promoted the transport of photosynthetic products to berries, and increased the expression of anthocyanin biosynthesis-related genes in the grape berry skin. These results indicate that the application of exogenous calcium enhances anthocyanin accumulation in ‘Manicure Finger’ grape berries in the following two ways: (1) calcium promotes the transport of photosynthetic products to berries, thereby providing the basic material required for anthocyanin biosynthesis; (2) calcium induces anthocyanin biosynthesis by regulating the expression of relevant structural and regulatory genes. Our results will help researchers characterize the mechanism responsible for the calcium-mediated promotion of anthocyanin biosynthesis in grapevine and related plant species.

Elfn1-Induced Constitutive Activation of mGluR7 Determines Frequency-Dependent Recruitment of Somatostatin Interneurons.

Excitatory synapses onto somatostatin (SOM) interneurons show robust short-term facilitation. This hallmark feature of SOM interneurons arises from a low initial release probability that regulates the recruitment of interneurons in response to trains of action potentials. Previous work has shown that Elfn1 (extracellular leucine rich repeat and fibronectin Type III domain containing 1) is necessary to generate facilitating synapses onto SOM neurons by recruitment of two separate presynaptic components: mGluR7 (metabotropic glutamate receptor 7) and GluK2-KARs (kainate receptors containing glutamate receptor, ionotropic, kainate 2). Here, we identify how a transsynaptic interaction between Elfn1 and mGluR7 constitutively reduces initial release probability onto mouse cortical SOM neurons. Elfn1 produces glutamate-independent activation of mGluR7 via presynaptic clustering, resulting in a divergence from the canonical "autoreceptor" role of Type III mGluRs, and substantially altering synaptic pharmacology. This structurally induced determination of initial release probability is present at both layer 2/3 and layer 5 synapses. In layer 2/3 SOM neurons, synaptic facilitation in response to spike trains is also dependent on presynaptic GluK2-KARs. In contrast, layer 5 SOM neurons do not exhibit presynaptic GluK2-KAR activity at baseline and show reduced facilitation. GluK2-KAR engagement at synapses onto layer 5 SOM neurons can be induced by calmodulin activation, suggesting that synaptic function can be dynamically regulated. Thus, synaptic facilitation onto SOM interneurons is mediated both by constitutive mGluR7 recruitment by Elfn1 and regulated GluK2-KAR recruitment, which determines the extent of interneuron recruitment in different cortical layers.SIGNIFICANCE STATEMENT This study identifies a novel mechanism for generating constitutive GPCR activity through a transsynaptic Elfn1/mGluR7 structural interaction. The resulting tonic suppression of synaptic release probability deviates from canonical autoreceptor function. Constitutive suppression delays the activation of somatostatin interneurons in circuits, necessitating high-frequency activity for somatostatin interneuron recruitment. Furthermore, variations in the synaptic proteome generate layer-specific differences in facilitation at pyr → SOM synapses. The presence of GluK2 kainate receptors in L2/3 enhances synaptic transmission during prolonged activity. Thus, layer-specific synaptic properties onto somatostatin interneurons are mediated by both constitutive mGluR7 recruitment and regulated GluK2 kainate receptor recruitment, revealing a mechanism that generates diversity in physiological responses of interneurons.

Structural Investigation of Melatonin Metabolites as Calmodulin Antagonists and potential Nitric Oxide Synthase Inhibitors

Nitric oxide synthase (NOS) is a flavo‐hemoprotein that catalyzes the conversion of L‐arginine to L‐citrulline in the production nitric oxide (NO). Mammalian NOS exists in three isoforms: endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). Each isoform requires calmodulin (CaM) for activation as CaM binding induces a conformational change, which facilitates electron transfer within the NOS peptide via a highly dynamic process. NO overproduction is implicated in several neurodegenerative and inflammatory conditions including septic shock, epilepsy, myocarditis and Alzheimer's, thus the development of a NOS isoform‐specific inhibitor is of clinical significance. Previous research has shown that N1‐acetyl‐5‐methoxykynuramine (AMK), a metabolite of melatonin (aMT), is a CaM‐dependant inhibitor of nNOS in rat striatum1,2. Despite attenuation of nNOS activity demonstrated both in vitro and in vivo, there has yet to be a comprehensive structural analysis of AMK‐CaM‐NOS complexes. To further validate possible therapeutic applications, we conducted an investigation of AMK interactions with CaM and the CaM‐NOS binding domain for all three NOS isoforms. Our results showed CaM‐AMK binding using gel shift assays, while circular dichroism (CD) results displayed the greatest spectral differences when AMK was added to the CaM‐nNOS complex. This indicates potential isoform‐dependent variation in NOS inhibition due to differences in the CaM‐NOS binding domain. These results were substantiated by subsequent multidimensional NMR experiments, in which the largest differences in chemical shifts occurred when inhibitors were added to the CaM‐nNOS sample, primarily amongst residues found in the N‐lobe of CaM. Our investigation provides significant insight into the potential of AMK as an isoform‐specific NOS inhibitor.Support or Funding InformationThis work was funded by the NSERC of Canada.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Attenuation of calmodulin regulation evokes Ca2+ oscillations: evidence for the involvement of intracellular arachidonate-activated channels and connexons

Intracellular Са2+ controls its own level by regulation of Ca2+ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca2+ concentration in different cells. We have found persistent Са2+ oscillations in cultured white adipocytes in response to calmidazolium (CMZ). They appeared at [CMZ] > 1μM as repetitive sharp spikes mainly superimposed on a transient or elevated baseline. Similar oscillations were observed when we used trifluoperazine. Oscillations evoked by 5μM CMZ resulted from the release of stored Ca2+ and were supported by Са2+ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A2 inhibited by AACOCF3 was responsible for initial Ca2+ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA2 by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са2+ stores engaged by CMZ were entirely dependent on thapsigargin. The oscillations existed in the presence of inhibitors of ryanodine or inositol 1,4,5-trisphosphate receptors, or antagonists of Ca2+ transport by lysosome-like acidic stores. Carbenoxolone or octanol, blockers of hemichannels (connexons), when applied for two hours, prevented oscillations but did not affect the initial Са2+ release. Incubation with La3+ for 2 or 24h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca2+ rise. These results suggest that Ca2+-CaM regulation suppresses La3+-sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca2+ oscillations, and connexons are intimately implicated in their generation mechanism.

Developmental Coordination during Olfactory Circuit Remodeling in Drosophila

Developmental neuronal remodeling is crucial for proper wiring of the adult nervous system. While remodeling of individual neuronal populations has been studied, how neuronal circuits remodel-and whether remodeling of synaptic partners is coordinated-is unknown. We found that the Drosophila anterior paired lateral (APL) neuron undergoes stereotypic remodeling during metamorphosis in a similar time frame as the mushroom body (MB) ɣ-neurons, with whom it forms a functional circuit. By simultaneously manipulating both neuronal populations, we found that cell-autonomous inhibition of ɣ-neuron pruning resulted in the inhibition of APL pruning in a process that is mediated, at least in part, by Ca2+-Calmodulin and neuronal activity dependent interaction. Finally, ectopic unpruned MB ɣ axons display ectopic connections with the APL, as well as with other neurons, at the adult, suggesting that inhibiting remodeling of one neuronal type can affect the functional wiring of the entire micro-circuit.

Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6.

Calcium (Ca2+) plays a major role in numerous physiological processes. Ca2+ homeostasis is tightly controlled by ion channels, the aberrant regulation of which results in various diseases including cancers. Calmodulin (CaM)-mediated Ca2+-induced inactivation is an ion channel regulatory mechanism that protects cells against the toxic effects of Ca2+ overload. We used cryo-electron microscopy to capture the epithelial calcium channel TRPV6 (transient receptor potential vanilloid subfamily member 6) inactivated by CaM. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM carboxyl-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the pore's intracellular entrance. We propose a mechanism of CaM-mediated Ca2+-induced inactivation that can be explored for therapeutic design.

Calcineurin and Its Role in Synaptic Transmission.

Calcineurin (CaN) is a serine/threonine phosphatase widely expressed in different cell types and structures including neurons and synapses. The most studied role of CaN is its involvement in the functioning of postsynaptic structures of central synapses. The role of CaN in the presynaptic structures of central and peripheral synapses is less understood, although it has generated a considerable interest and is a subject of a growing number of studies. The regulatory role of CaN in synaptic vesicle endocytosis in the synapse terminals is actively studied. In recent years, new targets of CaN have been identified and its role in the regulation of enzymes and neurotransmitter secretion in peripheral neuromuscular junctions has been revealed. CaN is the only phosphatase that requires calcium and calmodulin for activation. In this review, we present details of CaN molecular structure and give a detailed description of possible mechanisms of CaN activation involving calcium, enzymes, and endogenous and exogenous inhibitors. Known and newly discovered CaN targets at pre- and postsynaptic levels are described. CaN activity in synaptic structures is discussed in terms of functional involvement of this phosphatase in synaptic transmission and neurotransmitter release.

Calmodulin-induced Conformational Control and Allostery Underlying Neuronal Nitric Oxide Synthase Activation

Nitric oxide synthase (NOS) is the primary generator of nitric oxide signals controlling diverse physiological processes such as neurotransmission and vasodilation. NOS activation is contingent on Ca2+/calmodulin binding at a linker between its oxygenase and reductase domains to induce large conformational changes that orchestrate inter-domain electron transfer. However, the structural dynamics underlying activation of full-length NOS remain ambiguous. Employing hydrogen–deuterium exchange mass spectrometry, we reveal mechanisms underlying neuronal NOS activation by calmodulin and regulation by phosphorylation. We demonstrate that calmodulin binding orders the junction between reductase and oxygenase domains, exposes the FMN subdomain, and elicits a more dynamic oxygenase active site. Furthermore, we demonstrate that phosphorylation partially mimics calmodulin activation to modulate neuronal NOS activity via long-range allostery. Calmodulin binding and phosphorylation ultimately promote a more dynamic holoenzyme while coordinating inter-domain communication and electron transfer.

Structural Dynamics of Calmodulin in Regulation of Calcium Release in Health and Disease

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) spectroscopy to study the structural changes in calmodulin (CaM) that are relevant to regulation of the skeletal muscle calcium release channel, the ryanodine receptor (RyR1 isoform). Regulation of RyR1 by CaM is disrupted by oxidation and disease-causing mutations. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the correlation between structure and function in physiologically relevant conditions is largely unknown. To test the hypothesis that the modulatory action of CaM on RyR1 is caused by structural changes in the CaM-RyR1 complex, we used TR-FRET between two fluorescent probes covalently bound to each lobe (N and C) of CaM to measure Ca2+-dependent changes in the distance between lobes when CaM is bound to RyR1 in sarcoplasmic reticulum membranes, isolated RyR1, and a peptide corresponding to a CaM-binding domain of RyR1 (RyRp; RyR residues 3614-3643). [3H]-ryanodine binding measurements were performed to ensure functional integrity of labeled CaM constructs, and to directly correlate the FRET structural readout with the CaM-RyR functional interaction. At low Ca2+, we find that CaM adopts a different structure when bound to full-length RyR1 compared with binding to RyRp. However, this structural difference, as reflected by the distance between CaM's lobes, is only apparent at low Ca2+. This suggests that CaM-RyRp binding may only be representative of CaM binding to full-length RyR1 at high, not low Ca2+. Ongoing studies utilize this CaM biosensor to determine the structural changes that contribute to regulation of the cardiac RyR isoform (RyR2). This work was supported by NIH grants AG26160 (DDT), HL092097 (RLC) and 1F31AG052329-01A1 (MRM) and an American Heart Association Predoctoral Fellowship 15PRE25700131 (MRM).

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels

Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2+-dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46-CaM interaction and attenuated its abolishment by Ca2+ depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2+-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2+ sensitivity. The data suggest that the G143R-induced enhancement of the CaM-Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.

The Role of Calmodulin Methionine Oxidation in Regulating Conformational Change

We have used molecular dynamics simulations to understand the role of calmodulin (CaM) methionine (Met) oxidation in disrupting calcium-induced conformational changes associated with CaM's regulation of its target proteins. Previous work suggests that C-terminal CaM methionines, particularly M109 and M124, are critical for functional interaction with the major calcium release channel in muscle, the ryanodine receptor (RyR). Methionine to glutamine (Gln) substitutions at M109 or M124 designed to mimic oxidation of Met to Met sulfoxide block CaM activation of RyR at low calcium levels and inhibition of RyR at high calcium levels (Journal of Biological Chemistry 278(18) 2003). Spectroscopic distance measurements across CaM's lobes suggest that these same Met to Gln substitutions altered the equilibrium between key CaM conformations (Biochemical and biophysical research communications 456(2) 2015). We carried out microsecond molecular dynamics simulations of the open and closed CaM structural states with Met to Gln substitutions at M109 or M124 to gain an atomic-level understanding of (1) the role of methionines in stabilizing CaM structure, and (2) how Met oxidation might influence conformational equilibria and regulatory interactions. We found that every CaM C-terminal Met participates in a Met-aromatic interaction with nearby Phe residues with interaction energies of ∼4-10 kcal/mol. M109 stably interacts with F92, but only in the open structure, and M124 stably interacts with F141, but only in the closed structure. Met to Gln substitution at M109 or M124 alters interaction energies with these nearby Phe and alters the stability of the open and closed structures. We expect that these results will be applicable to understanding the general mechanism through which Met oxidation orchestrates redox cell signaling.

Changes in Activities of Key Enzymes in Sugarcane Stem at Different Growing Stages

Sugarcane is the most important sugar crop in China, which mainly focuses on the upper stem of the harvested land. The proper regulation of the proportion of internode elongation is the key to determine the yield and sugar. Therefore, it is of great significance to study the mechanism of the dynamic change of elongation between cane joints in order to improve the yield of sugar cane and sucrose. To investigate the biochemical mechanism of stem elongation in sugarcane, stem samples were collected at the pre-elongation stage (9-10 leaves) (Ls1), early elongation stage (12-13 leaves) (Ls2) and rapid elongation stage (15-16 leaves) (Ls3). The change trends in the activities of NAD kinase (NADK), calcium-dependent protein kinase (CDPKs), α-mannosidase, α-galactosidase, β-glucosidase, cellulase, xyloglucan endo-transglycosylase/hydrolase (XTH) and catalase (CAT) were completely consistent, showing rapid elongation stage > early elongation stage > pre-elongation stage, while the activities of β-glucosidase, peroxidase (POD) and α-glucosidase were in opposite, and the activities of calmodulin and β-mannosidase showed the same single-peak trend, and the peak was at early elongation stage. The enzyme activities of β-galactosidase and pectinase did not show significant difference at different stages. The results indicate that the elongation of internodes was closely related to the complex physiological metabolism of sugarcane, and the key enzymes play roles at different time but β-galactosidase and pectinase have little effect on internodes elongation in sugarcane.

The Angiotensin II Type 1 Receptor Antagonist Losartan Affects NHE1-Dependent Melanoma Cell Behavior

Background/Aims: The peptide hormone angiotensin II (ATII) plays a prominent role in regulating vasoconstriction and blood pressure. Its primary target is the angiotensin II receptor type 1 (AT1), the stimulation of which induces an increase in cytosolic [Ca2+] and calmodulin activation. Ca2+-bound (activated) calmodulin stimulates the activity of the Na+/ H+ exchanger isoform 1 (NHE1); and increased NHE1 activity is known to promote melanoma cell motility. The competitive AT1 receptor inhibitor losartan is often used to lower blood pressure in hypertensive patients. Since AT1 mediates ATII-stimulated NHE1 activity, we set out to investigate whether ATII and losartan have an impact on NHE1-dependent behavior of human melanoma (MV3) cells. Methods: ATII receptor expression was verified by PCR, F-actin was visualized using fluorescently labeled phalloidin, and cytosolic [Ca2+] and pH were determined ratiometrically using Fura-2 and BCECF, respectively. MV3 cell behavior was analyzed using migration, adhesion, invasion and proliferation assays. Results: MV3 cells express both AT1 and the angiotensin II receptor type 2 (AT2). Stimulation of MV3 cells with ATII increased NHE1 activity which could be counteracted by both losartan and the Ca2+/ calmodulin inhibitor ophiobolin-A. ATII stimulation induced a decrease in MV3 cell migration and a more spherical cell morphology accompanied by an increase in the density of F-actin. Independently of the presence of ATII, both NHE1 and migratory activity were reduced when AT1 was blocked by losartan. On the other hand, losartan clearly increased cell adhesion to, and the invasion of, a collagen type I substrate. The AT2 inhibitor PD123319 did not affect NHE1 activity, proliferation and migration, but increased adhesion and invasion. Conclusion: Losartan inhibits NHE1 activity and the migration of human melanoma cells. At the same time, losartan promotes MV3 cell adhesion and invasion. The therapeutic use of AT1 antagonists (sartans) in hypertensive cancer patients should therefore be given critical consideration.

Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4,5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca2+. These findings motivate our core hypothesis that non-selective inward Ca2+ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca2+ load, which thereby magnifies intracellular Ca2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca2+ dysregulation, including whether enhanced SL Ca2+ influx is sufficient to elevate cytosolic Ca2+ load [6], and if so, how might amplified Ca2+ transients perturb Ca2+-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca2+ loading of the SR and that greater fractional release may contribute to the Ca2+-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca2+ in the SR and dyadic space collectively drive greater fractional SR Ca2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca2+ dysregulation in pre-diabetes.

Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin.

Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α-helical content to CaMWt, and only modest differences in α-helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C-terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N-terminal CaM and CyaA-ACD.

Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism.

Human aquaporin 4 (AQP4) is the primary water channel protein in brain astrocytes. Hypothermia is known to cause astrocyte swelling in culture, but the precise role of AQP4 in this process is unknown. Primary human cortical astrocytes were cultured under hypothermic (32 °C) or normothermic (37 °C) conditions. AQP4 transcript, total protein and surface‐localized protein were quantified using RT‐qPCR, sandwich ELISA with whole cell lysates or cell surface biotinylation, followed by ELISA analysis of the surface‐localized protein, respectively. Four‐hour mild hypothermic treatment increased the surface localization of AQP4 in human astrocytes to 155 ± 4% of normothermic controls, despite no change in total protein expression levels. The hypothermia‐mediated increase in AQP4 surface abundance on human astrocytes was blocked using either calmodulin antagonist (trifluoperazine, TFP); TRPV4 antagonist, HC‐067047 or calcium chelation using EGTA‐AM. The TRPV4 agonist (GSK1016790A) mimicked the effect of hypothermia compared with untreated normothermic astrocytes. Hypothermia led to an increase in surface localization of AQP4 in human astrocytes through a mechanism likely dependent on the TRPV4 calcium channel and calmodulin activation. Understanding the effects of hypothermia on astrocytic AQP4 cell surface expression may help develop new treatments for brain swelling based on an in‐depth mechanistic understanding of AQP4 translocation.

Population Effects of Calcium Phosphate Nanoparticles in Drosophila melanogaster: The Effects of Phase Composition, Crystallinity, and the Pathway of Formation.

Unpredictable biological response due to the finest nanostructural variations is one of the hallmarks of nanoparticles. Because of this erratic behavior of nanoparticles in living systems, thorough analyses of biosafety must precede the analyses of the pharmacotherapeutic efficacy and simple animal models are ideal for such purposes. Drosophila melanogaster, the common fruit fly, is an animal model capable of giving a fast, high-throughput response as to the safety and efficacy of drug delivery carriers and other pharmacological agents, while minimizing the suffering imposed onto animals in more complex in vivo models. Here we studied the effects on the viability and fertility of D. melanogaster due to variations in phase composition, crystallinity, and the pathway of formation of four different calcium phosphate (CP) nanopowders consumed orally. To minimize the effect of other nanostructural variables, CP nanopowders were made to possess highly similar particle sizes and morphologies. The composition of CP affected the fecundity of flies, but so did crystallinity and the pathway of formation. Both the total number of eclosed viable flies and pupae in populations challenged with hydroxyapatite (HAP) greatly exceeded those in control populations. Viability was adversely affected by the only pyrophosphate tested (CPP) and by the metastable and the most active of all CP nanopowders analyzed: the amorphous CP (ACP). The pupation peak was delayed and the viable fly to-pupa ratio increased in all the CP-challenged populations. F1 CPP population, whose viability was most adversely affected by the CP consumption, when crossed, produced the largest number of F2 progeny under regular conditions, possibly pointing to stress as a positive evolutionary drive. The positive effect of HAP on fertility of fruit flies may be due to its slow absorption and the activation of calmodulin during the transit of oocytes through the reproductive tract of fertilized females. Exerted in the prepupation stage, the effect of CP is thus traceable beyond the instar larval stage and to the oogenesis stage of the Drosophila lifecycle.

Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

Physiological vs. pharmacological signalling to myosin phosphorylation in airway smooth muscle.

Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction. Ca2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1SM+/+ , MYPT1SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction.