Kca3.1 Activation Via P2y2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration.

Disorders in cell signaling mediated by ATP or histamine, activating specific membrane receptors, have been frequently associated with tumorigenesis. Among the elements of response to purinergic (and histaminergic) signaling, ion channel activation controls essential cellular processes in cancer, such as cell proliferation, motility, and death. Here, we studied the effects that ATP had on electrical properties of human ovarian adenocarcinoma cells named SKOV-3. ATP caused increase in intracellular Ca2+ concentration ([Ca2+]i) and, concurrently, it evoked a complex electrical response with a conspicuous outward component. This current was generated through P2Y2 receptor activation and opening of K+ channels, KCa3.1, as indicated by electrophysiological and pharmacological analysis, as well as by immunodetection and specific silencing of P2Y2 or KCa3.1 gene by esiRNA transfection. Low µM ATP concentration increased SKOV-3 cell migration, which was strongly inhibited by KCa3.1 channel blockers and by esiRNA-generated P2Y2 or KCa3.1 downregulation. Finally, in human ovarian tumors, the P2Y2 and KCa3.1 proteins are expressed and co-localized in neoplastic cells. Thus, stimulation of P2Y2 receptors expressed in SKOV-3 cells promotes motility through KCa3.1 activation. Since P2Y2 and KCa3.1 are co-expressed in primary tumors, our findings suggest that they may play a role in cancer progression.

Capacitation and Ca(2+) influx in spermatozoa: role of CNG channels and protein kinase G.

Cyclic guanosine monophosphate (cGMP) has been recently shown to modulate in vitro capacitation of mammalian spermatozoa, but the mechanisms through which it influences sperm functions have not been clarified. There are at least two targets of cGMP, cyclic nucleotide-gated (CNG) channels and cGMP-dependent protein kinase (PKG), involved in several physiological events in mammalian spermatozoa. It has been suggested that CNG channels allow the influx of Ca(2+) to cytoplasm during capacitation, whereas PKG could trigger a phosphorylation pathway which might also, indirectly, mediate calcium entry. Using the patch-clamp technique in whole-cell configuration, we showed how l-cis-Diltiazem (a CNG-channel inhibitor) and KT5823 (a PKG inhibitor) decreased significantly the amplitude of macroscopic ion currents in a dose-response manner, and decreased in vitro capacitation. The inhibition of CNG channels completely abolishes the Ca(2+) influx induced by cyclic nucleotides in mouse spermatozoa. This work suggests that the downstream cGMP pathway is required in mammalian sperm capacitation and the mechanisms involved include CNG channels and PKG, highlighting these molecules as important therapeutic targets for infertility treatments or to develop new male contraceptives.

A thermal study of cellular motility by optical time-resolved correlation.

The study of motor properties of cells under appropriate physical-chemical conditions is a significant problem nowadays. The standard techniques presently used do not allow to evaluate neither large samples nor to control their thermodynamic conditions. In this work, we report a cell motility sensor based on an optical technique with a time-resolved correlation, adapted in a system able to study several samples simultaneously. Image correlation analysis is used to follow their temporal behavior. A wide variety of motile cells, such as archaea, bacteria, spermatozoa, and even contractile cells, can be studied using this technique. Here, we tested our technique with the study of sperm motility. In particular, both the sperm motility and its prevalence are studied under a temperature range from 0 to 37 °C. We found that incubation at 10 °C presents the lengthiest prevalence in motility and observed, for the first time, an interesting thermal reversibility behavior.

A role for cyclic nucleotide-gated channels in the capacitation of mammalian sperm.

In their transit through the female genital tract, mammalian sperm acquire the ability to fertilize the egg in a process called capacitation. During this event the intracellular levels of cAMP and cGMP increase, suggesting that cyclic nucleotide-gated (CNG) channels, which have been identified in mammalian sperm, play a functional role in their physiology. Here we report an electrophysiological characterization of the effect of cyclic nucleotides on mouse sperm. Using the patch-clamp technique in the whole-cell configuration, we show that macroscopic ionic currents are augmented by the addition of both, 8Br-cAMP and 8Br-cGMP to non-capacitated mouse sperm. Although cyclic nucleotide regulates the activity of CNG channels, disparate effects of cyclic nucleotides may also occur. Addition of L-cis-diltiazem (50 microM), a specific inhibitor of CNG channels, partially blocked currents elicited by cGMP, suggesting that CNG channels play a role in the fertilization capability of mammalian sperm.