Fermentation product butane 2,3-diol induces Ca2+ transients in E. coli through activation of lanthanum-sensitive Ca2+ channels

The results here are the first demonstration of a physiological agonist opening Ca2+ channels in bacteria. Bacteria in the gut ferment glucose and other substrates, producing alcohols, diols, ketones and acids, that play a key role in lactose intolerance, through the activation of Ca2+ and other ion channels in host cells and neighbouring bacteria. Here we show butane 2,3-diol (5-200mM; half maximum 25mM) activates Ca2+ transients in E. coli, monitored by aequorin. Ca2+-transient magnitude depended on external Ca2+ (0.1-10mM). meso-Butane 2,3-diol was approximately twice as potent as 2R,3R (-) and 2S,3S (+) butane 2,3-diol. There were no detectable effects on cytosolic free Ca2+ of butane 1,3-diol, butane 1,4-diol and ethylene glycol. The glycerol fermentation product propane 1,3-diol only induced significant Ca2+ transients in 10mM external Ca2. Ca2+ butane 2,3-diol Ca2+ transients were due to activation of Ca2+ influx, followed by activation of Ca2+ efflux. The effect of butane 2,3-diol was abolished by La3+, and markedly reduced as a function of growth phase. These results were consistent with butane 2,3-diol activating a novel La3+-sensitive Ca2+ channel. They have important implications for the role of butane 2,3-diol and Ca2+ in bacterial-host cell signalling.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca²⁺]_i, Ca²⁺ transients and membrane Ca²⁺ current, I_Ca, in cultured murine HL-1 cardiomyocytes. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca²⁺]_i, Ca²⁺ transients and I_Ca. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca²⁺]_i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca²⁺]_i, and inhibited Ca²⁺ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca²⁺]_i, and Ca²⁺ transients and I_Ca. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca²⁺]_i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca²⁺]_i required for excitation-contraction coupling in cardiomyoctyes.     

Verapamil, a Ca2+ channel inhibitor acts as a local anesthetic and induces the sigma E dependent extra-cytoplasmic stress response in E. coli

Verapamil is used clinically as a Ca(2+) channel inhibitor for the treatment of various disorders such as angina, hypertension and cardiac arrhythmia. Here we study the effect of verapamil on the bacterium Escherichia coli. The drug was shown to inhibit cell division at growth sub inhibitory concentrations, independently of the SOS response. We show verapamil is a membrane active drug, with similar effects to dibucaine, a local anesthetic. Thus, both verapamil and dibucaine abolish the proton motive force and decrease the intracellular ATP concentration. This is accompanied by induction of degP expression, as a result of the activation of the RpoE (SigmaE) extra-cytoplasmic stress response, and activation of the psp operon. Such effects of verapamil, as a membrane active compound, could explain its general toxicity in eukaryotic cells.     

Roles of Ca2+ in hyphal and yeast-form growth inCandida albicans. Growth regulation by altered extracellular and intracellular free Ca2+ concentrations

The dimorphic fungusCandida albicans has both a yeast form and a hyphal form. When yeast-form cells were starved and then transferred to aN-acetylglucosamine medium, the formation of true hyphae from the unbudded yeast-form cells was induced. Removal of Ca²⁺ from the medium with EGTA inhibited hyphal formation by 50%, resulting in only thin and short hyphae. Externally applied excess Ca²⁺ (>10⁻²M) also affected the hyphal formation, resulting in formation of pseudohyphae. This effect required a high concentration of Ca²⁺ but was Ca²⁺-specific. Deprivation of Ca²⁺ also inhibited yeast-form growth. Interestingly, such cells had abnormally wide bud necks and became defective in cell separation. To measure cytosolic free Ca²⁺, fura-2 was introduced into hyphal cells by electroporation. Its normal value was estimated to be about 100 nM. The electroporation caused transient elevation of cytosolic free Ca²⁺ concentration and transient cessation of hyphal growth. There was a close correlation between the timing of recovery of Ca²⁺ concentration and that of the resumption of hyphal growth. Our results demonstrate the importance of extracellular and intracellular free Ca²⁺ for the growth ofC. albicans.     

Dimethylaminochalcone, an indicator of the structural changes in the cellular envelop of E. coli under the action of Ca2+ cations and tris buffer]

An attempt was made to detect possible structural changes in E. coli cell envelope induced by Ca2+ treatment with the help of an uncharged fluorescent probe 4-dimethylaminochalcon (DMC). The effects of the treatment with tris buffer (0.01 M) at 0 degrees C and other agents (Mg2+ and EDTA) were also studied for the purpose of comparison. It is shown that Ca2+ treatment of E. coli cells results in structural changes in the cell envelope surface, whick differ from those induced by tris-buffer at 0 degrees C, Mg2+ and EDTA. DMC can be used successfully as a suitable probe for monitoring structural changes in biomembranes.     

Leukotriene D4 and E4 induce transmembrane signaling in human epithelial cells. Single cell analysis reveals diverse pathways at the G-protein level for the influx and the intracellular mobilization of Ca2+

Leukotrienes are recognized as important mediators of the inflammatory process. Recently, increasing attention has been paid to the role of noninflammatory cells in the regulation of the inflammatory process. To further increase our knowledge of this matter we have, in the present study, investigated leukotriene-induced Ca2+ signaling, using a single cell technique in a human epithelial cell line, Intestine 407. It was evident that both LTD4 and LTE4, at physiological concentrations (10 nM), triggered rapid and pronounced cytosolic free Ca2+ transients, due to both influx across the plasma membrane and intracellular mobilization. Preincubation with pertussis toxin (1200 ng/ml) decreased the level of agonist-induced Ca2+ transients to an extent similar to that caused by depletion of extracellular Ca2+, suggesting that the toxin affected the influx but not the intracellular mobilization of Ca2+. Indeed, by using the Mn2+ quenching technique, it could be shown that pertussis toxin totally inhibited the influx of Ca2+. The fact that, even after pertussis toxin treatment, direct G-protein activation by AIF4- was still able to trigger a cytosolic free Ca2+ transient, indicates that, in these cells, G-proteins (GTP-binding proteins) that are insensitive to pertussis toxin are capable of mediating a Ca2+ signal. In order to test the idea that such G-proteins regulate mobilization of intracellular Ca2+ induced by LTD4 and LTE4, we electropermeabilized and preincubated the Intestine 407 cells with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), let them reseal, and, after loading with fura2, investigated the effects on agonist-stimulated Ca2+ signaling. Electropermeabiization and resealing alone did not significantly affect the Ca2+ responses triggered by LTD4 or LTE4. Addition of GDP beta S, in the presence of extracellular Ca2+, reduced the Ca2+ responses by approximately 60-70%. In Ca2(+)-depleted medium, GDP beta S also impaired the LTD4-induced response by 65%, however, it had no effect on the Ca2+ response induced by LTE4. In conclusion, LTD4 and LTE4 trigger cytosolic free Ca2+ signaling in a human epithelial cell line by causing both an influx of Ca2+ and mobilization of intracellular Ca2+. The Ca2(+)-signaling mechanism appears to consist of dual pathways, since the influx is regulated by a pertussis toxin-sensitive G-protein, but, the mobilization of Ca2+ is not. Furthermore, our data suggest that the LTD4-induced mobilization is regulated by a pertussis toxin-insensitive G-protein whereas the LTE4-induced mobilization is relatively insensitive to both pertussis toxin and GDP beta S.