Glibenclamide inhibits NLRP3 inflammasome-mediated IL-1ß secretion in human trophoblasts.

Infection-associated pregnancy complications cause premature delivery. Caspase-1 is involved in the maturation of interleukin (IL)-1ß, which is activated by the NLRP3 inflammasome. To characterize the significance of the NLRP3 inflammasome pathway in the placenta, the effects of activators and inhibitors on NLRP3-related molecules were examined using isolated primary trophoblasts. Caspase-1 and IL-1ß mRNA expression was markedly increased in response to lipopolysaccharide (LPS), a toll-like receptor (TLR)4 ligand. Treatment with the potassium ionophore nigericin significantly increased the level of activated caspase-1. Treatment with either LPS or nigericin stimulated IL-1ß secretion, whereas pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, the Rho-associated coiled-coil kinase inhibitor Y-27632, or a caspase-1 inhibitor significantly decreased nigericin-induced IL-1ß secretion. In addition, dibutyryl-cAMP, which induces trophoblast differentiation, decreased expression of NLRP3, caspase-1, and IL-1ß. These findings suggest that trophoblasts can secrete IL-1ß through the NLRP3/caspase-1 pathway, which is suppressed by glibenclamide, and that the TLR4-mediated NLRP3 inflammasome pathway is more likely to be stimulated in undifferentiated than differentiated trophoblasts. Our data support the hypothesis that inhibition of the NLRP3 inflammasome can suppress placental inflammation-associated disorders.

Role of potassium and pH on the initiation of sperm motility in the European eel.

The role of potassium from the seminal plasma and/or the activation media was examined by selectively removing K+ from this media, and by testing the use of K+ channel inhibitors and a K-ionophore. Sperm motility was measured using a CASA system, intracellular K+ and pH were measured by flow cytometry, and sperm head area was measured by ASMA: Automated Sperm Morphometry Analyses. Sperm motility was notably inhibited by the removal of K+ from the seminal plasma and by treatment with the K+ ionophore valinomycin. This therefore indicates that a reduction of K+ levels in the quiescent stage inhibits further motility. The normal decrease in sperm head area induced by seawater activation was altered by the removal of K+ from the seminal plasma, and an increase in the pHi in the quiescent stage was also induced. Intracellular pH (pHi) was quantitatively measured for the first time in European eel spermatozoa, being 7.2 in the quiescent stage and 7.1 post-activation. Intracellular and external pH levels influenced sperm motility both in the quiescent stage and at activation. The alkalinization of the pHi (by NH4Cl) inhibited sperm motility activation, while acidification (by Na-acetate) did not have any effect. Our results indicate that a pH gradient between the sperm cell and the seminal plasma is necessary for sperm motility activation. The presence of the ion K+ in the seminal plasma (or in the extender medium) is necessary in order to maintain sperm volume, intracellular pH and sperm motility.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.