Novel pituitary actions of TAC3 gene products in fish model: Receptor specificity and signal transduction for prolactin and somatolactin α regulation by neurokinin B (NKB) and NKB-related peptide in carp pituitary cells

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.postprin

Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

BACKGROUND: Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. RESULTS: In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with K(d )values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca(2+ )substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail) each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C) and under low-Ca(2+ )conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca(2+ )dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. CONCLUSIONS: We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca(2+ )is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca(2+ )elevation, all calmodulin except that bound to IQ3 should dissociate

Pathway and Network Analysis in Primary Open Angle Glaucoma

Glaucoma, a group of multifactor ocular diseases, is the second leading cause of blindness worldwide. Primary open angle (POA) is the most common type of glaucoma, characterized by progressive optic nerve degeneration. Numerous genes and proteins have been revealed to be associated with POAG, but the pathologic mechanisms of the disease are still poorly understood. Proteomics, the collective study of proteins in an organism at a given condition, has extensively been used for the high-throughput identification of proteins related to POAG. A significant obstacle in proteomics studies is the data variability which makes it hard to interpret the results. Pathway analysis and network topological information can help address the challenge and provide a greater appreciation of the disease mechanism and progression. The purpose of this paper is to determine POAG biological and network information to further understand the mechanisms associated with POAG. PANTHER classification system was used, including classification with gene ontology, protein class and pathway. 474 gene/protein IDs were extracted from previous proteomic studies. Among pathways found by PANTHER classification, apoptosis signaling pathway was the most significant pathway (with the p-value of 5.54E-12). Other PANTHER categories results demonstrated that developmental processes, receptor binding, extracellular region and extracellular matrix proteins were the most significant biological process, molecular function, cellular component and protein class respectively. Pathway analysis aids to find probable mechanisms involved in POAG. A network analysis on proteins was also performed using STRING database and cytoscape software. From network analysis, candidate biomarkers for the disease were introduced.

A Selective PMCA Inhibitor Does Not Prolong the Electroolfactogram in Mouse

Within the cilia of vertebrate olfactory receptor neurons, Ca(2+) accumulates during odor transduction. Termination of the odor response requires removal of this Ca(2+), and prior evidence suggests that both Na(+)/Ca(2+) exchange and plasma membrane Ca(2+)-ATPase (PMCA) contribute to this removal.In intact mouse olfactory epithelium, we measured the time course of termination of the odor-induced field potential. Replacement of mucosal Na(+) with Li(+), which reduces the ability of Na(+)/Ca(2+) exchange to expel Ca(2+), prolonged the termination as expected. However, treating the epithelium with the specific PMCA inhibitor caloxin 1b1 caused no significant increase in the time course of response termination.Under these experimental conditions, PMCA does not contribute detectably to the termination of the odor response

Dynamics of Ca2+-Calmodulin–dependent Inhibition of Rod Cyclic Nucleotide-gated Channels Measured by Patch-clamp Fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca2+ ions. Rod CNG channels are directly inhibited by Ca2+-calmodulin (Ca2+/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca2+/CaM-dependent modulation. The molecular basis for Ca2+/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca2+/CaM to a site in the NH2-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca2+/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca2+/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH2- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca2+/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH2- and COOH-terminal regions underlies Ca2+/CaM-dependent inhibition

Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro

Calreticulin inhibits commitment to adipocyte differentiation

Calreticulin, an endoplasmic reticulum (ER) resident protein, affects many critical cellular functions, including protein folding and calcium homeostasis. Using embryonic stem cells and 3T3-L1 preadipocytes, we show that calreticulin modulates adipogenesis. We find that calreticulin-deficient cells show increased potency for adipogenesis when compared with wild-type or calreticulin-overexpressing cells. In the highly adipogenic crt−/− cells, the ER lumenal calcium concentration was reduced. Increasing the ER lumenal calcium concentration led to a decrease in adipogenesis. In calreticulin-deficient cells, the calmodulin–Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathway was up-regulated, and inhibition of CaMKII reduced adipogenesis. Calreticulin inhibits adipogenesis via a negative feedback mechanism whereby the expression of calreticulin is initially up-regulated by peroxisome proliferator–activated receptor γ (PPARγ). This abundance of calreticulin subsequently negatively regulates the expression of PPARγ, lipoprotein lipase, CCAAT enhancer–binding protein α, and aP2. Thus, calreticulin appears to function as a Ca2+-dependent molecular switch that regulates commitment to adipocyte differentiation by preventing the expression and transcriptional activation of critical proadipogenic transcription factors

Ca2+/Calmodulin-Dependent Kinase Kinase α Is Expressed by Monocytic Cells and Regulates the Activation Profile

Macrophages are capable of assuming numerous phenotypes in order to adapt to endogenous and exogenous challenges but many of the factors that regulate this process are still unknown. We report that Ca2+/calmodulin-dependent kinase kinase α (CaMKKα) is expressed in human monocytic cells and demonstrate that its inhibition blocks type-II monocytic cell activation and promotes classical activation. Affinity chromatography with paramagnetic beads isolated an approximately 50 kDa protein from nuclear lysates of U937 human monocytic cells activated with phorbol-12-myristate-13-acetate (PMA). This protein was identified as CaMKKα by mass spectrometry and Western analysis. The function of CaMKKα in monocyte activation was examined using the CaMKKα inhibitors (STO-609 and forskolin) and siRNA knockdown. Inhibition of CaMKKα, enhanced PMA-dependent CD86 expression and reduced CD11b expression. In addition, inhibition was associated with decreased translocation of CaMKKα to the nucleus. Finally, to further examine monocyte activation profiles, TNFα and IL-10 secretion were studied. CaMKKα inhibition attenuated PMA-dependent IL-10 production and enhanced TNFα production indicating a shift from type-II to classical monocyte activation. Taken together, these findings indicate an important new role for CaMKKα in the differentiation of monocytic cells