75 research outputs found
Arabidopsis inositol phosphate kinases, IPK1 and ITPK1, constitute a metabolic pathway in maintaining phosphate homeostasis
Emerging studies have implicated a close link between inositol phosphate (InsP) metabolism and cellular phosphate (Pi) homeostasis in eukaryotes; however, whether a common InsP species is deployed as an evolutionarily conserved metabolic messenger to mediate Pi signaling remains unknown. Here, using genetics and InsP profiling combined with Pi starvation response (PSR) analysis in Arabidopsis thaliana, we showed that the kinase activity of inositol pentakisphosphate 2âkinase (IPK1), an enzyme required for phytate (inositol hexakisphosphates; InsP6) synthesis, is indispensable for maintaining Pi homeostasis under Piâreplete conditions, and inositol 1,3,4âtrisphosphate 5/6âkinase 1 (ITPK1) plays an equivalent role. Although both ipk1â1 and itpk1 mutants exhibited decreased levels of InsP6 and diphosphoinositol pentakisphosphate (PPâInsP5; InsP7), disruption of another ITPK family enzyme, ITPK4, which correspondingly caused depletion of InsP6 and InsP7, did not display similar Piârelated phenotypes, which precludes these InsP species as effectors. Notably, the level of D/LâIns(3,4,5,6)P4 was concurrently elevated in both ipk1â1 and itpk1 mutants, which showed a specific correlation to the misregulated Pi phenotypes. However, the level of D/LâIns(3,4,5,6)P4 is not responsive to Pi starvation that instead manifests a shootâspecific increase in InsP7 level. This study demonstrates a more nuanced picture of the intersection of InsP metabolism and Pi homeostasis and PSR than has previously been elaborated and additionally establishes intermediate steps to phytate biosynthesis in plant vegetative tissues
Epac1 mediates protein kinase Aâindependent mechanism of forskolin-activated intestinal chloride secretion
Intestinal Clâ secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Clâ secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Clâ secretion. FSK-stimulated Clâ secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 ”M), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,Nâ,Nâ-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 ”M). Both FSK and the Epac activator 8-pCPT-2â-O-Me-cAMP (50 ”M) elevated [Ca2+]i, activated Ras-related protein 2, and induced Clâ secretion in intact or basolateral membraneâpermeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2â-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2â-O-Me-cAMP on Clâ secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2â-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Clâ conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Clâ>Brâ>Iâ permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Clâ secretion, which is carried by a novel, previously undescribed Clâ channel
Static Magnetic Field Exposure Reproduces Cellular Effects of the Parkinson's Disease Drug Candidate ZM241385
This study was inspired by coalescing evidence that magnetic therapy may be a viable treatment option for certain diseases. This premise is based on the ability of moderate strength fields (i.e., 0.1 to 1 Tesla) to alter the biophysical properties of lipid bilayers and in turn modulate cellular signaling pathways. In particular, previous results from our laboratory (Wang et al., BMC Genomics, 10, 356 (2009)) established that moderate strength static magnetic field (SMF) exposure altered cellular endpoints associated with neuronal function and differentiation. Building on this background, the current paper investigated SMF by focusing on the adenosine A(2A) receptor (A(2A)R) in the PC12 rat adrenal pheochromocytoma cell line that displays metabolic features of Parkinson's disease (PD).SMF reproduced several responses elicited by ZM241385, a selective A(2A)R antagonist, in PC12 cells including altered calcium flux, increased ATP levels, reduced cAMP levels, reduced nitric oxide production, reduced p44/42 MAPK phosphorylation, inhibited proliferation, and reduced iron uptake. SMF also counteracted several PD-relevant endpoints exacerbated by A(2A)R agonist CGS21680 in a manner similar to ZM241385; these include reduction of increased expression of A(2A)R, reversal of altered calcium efflux, dampening of increased adenosine production, reduction of enhanced proliferation and associated p44/42 MAPK phosphorylation, and inhibition of neurite outgrowth.When measured against multiple endpoints, SMF elicited qualitatively similar responses as ZM241385, a PD drug candidate. Provided that the in vitro results presented in this paper apply in vivo, SMF holds promise as an intriguing non-invasive approach to treat PD and potentially other neurological disorders
Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.
Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell-permeant analogue of cAMP synergistically enhanced secretory responses to calcium-mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses
Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation
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