Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca²⁺]_i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca²⁺-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca²⁺]_i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.     

Serotonin 2C receptor agonists improve type 2 diabetes via melanocortin-4 receptor signaling pathways

The burden of type 2 diabetes and its associated premature morbidity and mortality is rapidly growing, and the need for novel efficacious treatments is pressing. We report here that serotonin 2C receptor (5-HT(2C)R) agonists, typically investigated for their anorectic properties, significantly improve glucose tolerance and reduce plasma insulin in murine models of obesity and type 2 diabetes. Importantly, 5-HT(2C)R agonist-induced improvements in glucose homeostasis occurred at concentrations of agonist that had no effect on ingestive behavior, energy expenditure, locomotor activity, body weight, or fat mass. We determined that this primary effect on glucose homeostasis requires downstream activation of melanocortin-4 receptors (MC4Rs), but not MC3Rs. These findings suggest that pharmacological targeting of 5-HT(2C)Rs may enhance glucose tolerance independently of alterations in body weight and that this may prove an effective and mechanistically novel strategy in the treatment of type 2 diabetes.     

2-aminoethoxydiphenyl borate stimulates pulmonary C neurons via the activation of TRPV channels

This study was carried out to determine the effect of 2-aminoethoxydiphenyl borate (2-APB), a common activator of transient receptor potential vanilloid (TRPV) type 1, 2, and 3 channels, on cardiorespiratory reflexes, pulmonary C fiber afferents, and isolated pulmonary capsaicin-sensitive neurons. In anesthetized, spontaneously breathing rats, intravenous bolus injection of 2-APB elicited the pulmonary chemoreflex responses, characterized by apnea, bradycardia, and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 2-APB no longer evoked any of these reflex responses. In open-chest and artificially ventilated rats, 2-APB evoked an abrupt and intense discharge in vagal pulmonary C fibers in a dose-dependent manner. The stimulation of C fibers by 2-APB was attenuated but not abolished by capsazepine, a selective antagonist of the TRPV1, which completely blocked the response to capsaicin in these C fiber afferents. In isolated pulmonary capsaicin-sensitive neurons, 2-APB concentration dependently evoked an inward current that was partially inhibited by capsazepine but almost completely abolished by ruthenium red, an effective blocker of all TRPV channels. In conclusion, 2-APB evokes a consistent and distinct stimulatory effect on pulmonary C fibers in vivo and on isolated pulmonary capsaicin-sensitive neurons in vitro. These results establish the functional evidence demonstrating that TRPV1, V2, and V3 channels are expressed on these sensory neurons and their terminals.     

Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G

There are two known phosphorylation-mediated inactivation mechanisms for TRPC3 channels. Protein kinase G (PKG) inactivates TRPC3 by direct phosphorylation on Thr-11 and Ser-263 of the TRPC3 proteins, and protein kinase C (PKC) inactivates TRPC3 by phosphorylation on Ser-712. In the present study, we explored the relationship between these two inactivation mechanisms of TRPC3. HEK cells were first stably transfected with a PKG-expressing construct and then transiently transfected with a TRPC3-expressing construct. Addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG), elicited a TRPC3-mediated [Ca2+]i rise in these cells. This OAG-induced rise in [Ca2+]i could be inhibited by phorbol 12-myristate 13-acetate (PMA), an agonist for PKC, in a dose-dependent manner. Importantly, point mutations at two PKG phosphorylation sites (T11A-S263Q) of TRPC3 markedly reduced the PMA inhibition. Furthermore, inhibition of PKG activity by KT5823 (1 microM) or H8 (10 microM) greatly reduced the PMA inhibition of TRPC3. These data strongly suggest that the inhibitory action of PKC on TRPC3 is partly mediated through PKG in these PKG-overexpressing cells. The importance of this scheme was also tested in vascular endothelial cells, in which PKG plays a pivotal functional role. In these cells, OAG-induced [Ca2+]i rise was inhibited by PMA, which activates PKC, and by 8-BrcGMP and S-nitroso-N-acetylpenicillamine (SNAP), both of which activate PKG. Importantly, the PMA inhibition on OAG-induced [Ca2+]i rise was significantly reduced by PKG inhibitor KT5823 (1 microM) or DT-3 (500 nM), suggesting an important role of PKG in the PMA-induced inhibition of TRPC channels in native endothelial cells.     

Antibody activates cationic channels via second messenger Ca2+

Patch-clamp recordings were used to study single channels permeable to multiple cations in a macrophage cell line. At least three conductance levels were found, consistent with the existence of several types of nonselective cation channels or a single channel with multiple open states. The activity of the channels depended very little on voltage but was affected by internal Ca2+ concentration. Specific subclasses of immunoglobulins (IgG1 and IgG2b) bound to an Fc receptor on the surface of these macrophages. When an IgG2b was applied to the cell exterior after a patch pipette had been sealed in the cell-attached mode, the nonselective cation channels within the patch were activated. Thus, these channels must be modulated by a second messenger. Since antibodies binding to the Fc receptor have been shown to produce a rise in intracellular Ca2+, this cation must be considered a candidate as a second messenger that amplifies the effect of antibody in gating these channels.     

BVT.3531 reduces body weight and activates K(ATP) channels in isolated arcuate neurons in rats

A number of hormones, including leptin, have been shown to inhibit food intake in humans and animals. Analogues of 3-guanidinopropionic acid have also been found to reduce total food intake, but their mechanisms of action have not been well studied. The present study investigated the effects of intracerebroventricular infusion of the analogue BVT.3531 on food intake, meal pattern, and body weight in rats during 7 days. Single channel recordings from arcuate neurons and insulinoma cells were used to determine the effects of BVT.3531 on K(ATP) activity. Data analysis showed that BVT.3531 significantly decreased body weight and food intake, primarily by reducing meal size. BVT.3531 activated K(ATP) channels in cell-attached recordings from insulin-secreting cells and rat arcuate neurons but had no effect on K(ATP) channel activity in inside-out membrane patches from either cell type. BVT.3531 did not alter the firing rate or K(+) channel activity of arcuate neurons devoid of K(ATP). The study suggests that small molecules capable of mimicking the effects of leptin on food intake and body weight may utilize output mechanisms similar to those of leptin to elicit changes in arcuate neuron excitability.     

Activation of human TRPC6 channels by receptor stimulation

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K(0.5) of approximately 10 microm and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30-300 microm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 microm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.     

Contortrostatin activates ERK2 and tyrosine phosphorylation events via distinct pathways

We report that cells adhering to contortrostatin show transient increases in activation of Extracellular signal Regulated Kinase 2 (ERK2). The kinetics and degree of activation are similar to cells adhering to fibronectin or vitronectin. We have recently shown that contortrostatin induces tyrosine phosphorylation in tumor cells. Contortrostatin is shown here to stimulate activation of ERK2 in suspended cells, but this activation follows a different dose-response pattern than contortrostatin-induced tyrosine phosphorylation. Since contortrostatin induces tyrosine phosphorylation via alphavbeta3, we explored the effects of an alphavbeta3-blocking antibody, 7E3, on contortrostatin-stimulated ERK2 activation. While 7E3 completely blocks the effect of contortrostatin on tyrosine phosphorylation, this antibody had no effect on activation of ERK2. In cells lacking expression of alphavbeta3, tyrosine phosphorylation was unaffected by contortrostatin treatment, but ERK2 was activated. This is strong evidence that contortrostatin is regulating tyrosine phosphorylation events and ERK2 activation via separate pathways and through different integrin receptors.     

The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.

Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.     

Rat hypocretin/orexin neurons are maintained in a depolarized state by TRPC channels

In a previous study we proposed that the depolarized state of the wake-promoting hypocretin/orexin (hcrt/orx) neurons was independent of synaptic inputs as it persisted in tetrodotoxin and low calcium/high magnesium solutions. Here we show first that these cells are hyperpolarized when external sodium is lowered, suggesting that non-selective cation channels (NSCCs) could be involved. As canonical transient receptor channels (TRPCs) are known to form NSCCs, we looked for TRPCs subunits using single-cell RT-PCR and found that TRPC6 mRNA was detectable in a small minority, TRPC1, TRPC3 and TRPC7 in a majority and TRPC4 and 5 in the vast majority (～90%) of hcrt/orx neurons. Using intracellular applications of TRPC antibodies against subunits known to form NSCCs, we then found that only TRPC5 antibodies elicited an outward current, together with hyperpolarization and inhibition of the cells. These effects were blocked by co-application of a TRPC5 antigen peptide. Voltage-clamp ramps in the presence or absence of TRPC5 antibodies indicated the presence of a current with a reversal potential close to -15 mV. Application of the non-selective TRPC channel blocker, flufenamic acid, had a similar effect, which could be occluded in cells pre-loaded with TRPC5 antibodies. Finally, using the same TRPC5 antibodies we found that most hcrt/orx cells show immunostaining for the TRPC5 subunit. These results suggest that hcrt/orx neurons are endowed with a constitutively active non-selective cation current which depends on TRPC channels containing the TRPC5 subunit and which is responsible for the depolarized and active state of these cells.     

Serotonin increases excitability of rabbit C-fiber neurons by two distinct mechanisms

Serotonin (5-HT) increases impulse activity in visceral afferent C-fibers in vivo. A 5-HT-induced membrane depolarization may partially account for this effect. Here, we examined the potential contribution of an additional mechanism to the 5-HT-mediated increase in impulse activity. Approximately 40% of rabbit visceral C-fiber neurons exhibit a protracted (greater than 3 s) spike afterhyperpolarization (AHPslow) that is a major determinant of repetitive firing properties in these neurons. Intracellular recording methods were applied to rabbit nodose ganglion neurons in vitro to assess whether 5-HT could increase excitability through effects on the AHPslow. Results revealed a concentration-dependent 5-HT-mediated depression of the AHPslow amplitude and duration that was accompanied by decreased accommodation of action potential firing. Experiments with 5-HT receptor antagonists further showed that this autacoid depressed the AHPslow through a different 5-HT receptor subtype than that subserving the 5-HT-induced depolarization. Thus the AHPslow represents a distinct locus where 5-HT can increase the impulse activity of these visceral C-fiber afferents.     

Affixin activates Rac1 via betaPIX in C2C12 myoblast

Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.     

A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca²⁺ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca²⁺ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca²⁺ influx that may lead to dysregulated cell growth in ADPKD.     

Junctate, an inositol 1,4,5-triphosphate receptor associated protein, is present in rodent sperm and binds TRPC2 and TRPC5 but not TRPC1 channels

The acrosome reaction, the first step of the fertilization, is induced by calcium influx through Canonical Transient Receptor Potential channels (TRPC). The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. In this study, we demonstrate that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, we show by pull-down experiments that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. We focused on the interaction between TRPC2 and junctate, and we show that the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. We show that junctate binds to TRPC2 independently of the calcium concentration and that the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. In the present study, we investigated whether DAG could promote the acrosome reaction. We found that 100 microM OAG, a permeant DAG analogue, was unable to trigger the acrosome reaction. Altogether, these results provide a new hypothesis concerning sperm TRPC2 gating: TRPC2 activation may be due to modifications of its interaction with both junctate and IP3R, induced by depletion of calcium from the acrosomal vesicle.     

The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels

TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.     

The roles of G proteins in the activation of TRPC4 and TRPC5 transient receptor potential channels

TRPC4 and TRPC5 channels are important regulators of electrical excitability in both gastrointestinal myocytes and neurons. Much is known regarding the assembly and function of these channels including TRPC1 as a homotetramer or a heteromultimer and the roles that their interacting proteins play in controlling these events. Further, they are one of the best-studied targets of G protein-coupled receptors and growth factors in general and Gαq protein coupled receptor or epidermal growth factor in particular. However, our understanding of the roles of Gαi/o proteins on TRPC4/5 channels is still rudimentary. We discuss potential roles for Gαi/o proteins in channel activation in addition to their known role in cellular signaling.     

Ghrelin raises [Ca2+]i via AMPK in hypothalamic arcuate nucleus NPY neurons

Ghrelin, an orexigenic hormone, directly activates neuropeptide (NPY) neurons in the hypothalamic arcuate nucleus (ARC), and thereby stimulates food intake. The hypothalamic level of AMP-activated protein kinase (AMPK), an intracellular energy sensor, is activated by peripheral and central administration of ghrelin. We examined whether ghrelin regulates AMPK activity in NPY neurons of the ARC. Single neurons were isolated from the ARC and cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured by fura-2 microfluorometry, followed by immunocytochemical identification of NPY, phospho-AMPK, and phospho-acetyl-CoA carboxylase (ACC). Ghrelin and AICAR, an AMPK activator, increased [Ca²⁺]_i in neurons isolated from the ARC. The ghrelin-responsive neurons highly overlapped with AICAR-responsive neurons. The neurons that responded to both ghrelin and AICAR were primarily NPY-immunoreactive neurons. Treatment with ghrelin increased phosphorylation of AMPK and ACC. An AMPK inhibitor, compound C, suppressed ghrelin-induced [Ca²⁺]_i increases. These results demonstrate that ghrelin increases [Ca²⁺]_i via AMPK-mediated signaling in the ARC NPY neurons.     

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl⁻ and could be reversibly blocked by the Cl⁻ channel blockers 4-acetamido-4′-isothio-cyanatistilbene-2,2′disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl⁻]_i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl⁻ conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K⁺ or Na⁺ ruled out the possibility that the Cl⁻ conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca²⁺ imaging experiments showed that betaine did not affect [Ca²⁺]_i, suggesting that the Cl⁻ current is not Ca²⁺ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons. Accepted: 20 March 1998     

Mechanical stretch activates glycometabolism-related enzymes via estrogen in C2C12 myoblasts

Moderate exercise improves glycometabolic disorder and type 2 diabetes mellitus in menopausal females. So far, the effect of exercise-induced estrogen on muscular glycometabolism is not well defined. The current study was designed to explore the effect of mechanical stretch-induced estrogen on glycometabolism in mouse C₂C₁₂ myoblasts. The mouse C₂C₁₂ myoblasts in vitro were assigned randomly to the control (C), stretch (S), and stretch plus aromatase inhibitor anastrozole (SA) groups. Cells in the S group were stretched by the Flexcell FX-5000™ system (15% magnitude, 1 Hz frequency, and 6-hr duration) whereas those in the SA group were treated with 400 μg/ml anastrozole before the same stretching. Glucose uptake, estradiol levels, PFK-1 levels, and oxygen consumption rate were determined, and the expression of HK, PI3K, p-AKT, AKT, and GLUT4 proteins were semiquantified with western blot analysis. Compared to the control, the estradiol level, oxygen consumption rate, expression of HK, PI3K, and PFK-1 proteins, the ratio of p-AKT to AKT, and the ratio of GLUT4 in the cell membrane to that in the whole cell were higher in the S group. On the other hand, the estradiol level, glucose uptake, expression of PFK-1 and GLUT4 proteins, oxygen consumption rate, expression of HK protein, and the ratio of p-AKT/AKT were lower in the myoblasts in the SA group than those in the S group. The level of estradiol was positively correlated with glucose uptake (p < .01, r = .818). Therefore, mechanical stretch-induced estrogen increased the expression of glycometabolism-related enzymes and proteins in the mouse C₂C₁₂ myoblasts.     

Serotonin 5-HT1A receptors regulate AMPA receptor channels through inhibiting Ca2+/calmodulin-dependent kinase II in prefrontal cortical pyramidal neurons

We have studied the regulation of AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC). Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT(1A) receptor agonists and blocked by 5-HT(1A) antagonists, indicating the mediation by 5-HT(1A) receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT(1A) agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. We concluded that serotonin, by activating 5-HT(1A) receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice.