No abstract

Phosphatidylinositol-4,5-bisphosphate (PIP) is a membrane phosphoinositide that regulates the activity of many ion channels. Influx of calcium primarily through voltage-gated calcium (Ca) channels promotes insulin secretion in pancreatic β-cells. However, whether Ca channels are regulated by PIP, as is the case for some non-insulin-secreting cells, is unknown. The purpose of this study was to investigate whether Ca channels are regulated by PIP depletion in pancreatic β-cells through activation of a muscarinic pathway induced by oxotremorine methiodide (Oxo-M). Ca channel currents were recorded by the patch-clamp technique. The Ca current amplitude was reduced by activation of the muscarinic receptor 1 (MR) in the absence of kinetic changes. The Oxo-M-induced inhibition exhibited the hallmarks of voltage-independent regulation and did not involve PKC activation. A small fraction of the Oxo-M-induced Ca inhibition was diminished by a high concentration of Ca chelator, whereas ≥50% of this inhibition was prevented by diC8-PIP dialysis. Localization of PIP in the plasma membrane was examined by transfecting INS-1 cells with PH-PLCδ1, which revealed a close temporal association between PIP hydrolysis and Ca channel inhibition. Furthermore, the depletion of PIP by a voltage-sensitive phosphatase reduced Ca currents in a way similar to that observed following MR activation. These results indicate that activation of the MR pathway inhibits the Ca channel via PIP depletion by a Ca-dependent mechanism in pancreatic β- and INS-1 cells and thereby support the hypothesis that membrane phospholipids regulate ion channel activity by interacting with ion channels.

The plasma membrane Ca(2+)-ATPase (PMCA) located in the hepatocyte is a controversial molecule in itself since it displays different features to those regarded as canonical for P-type Ca(2+)-ATPases, and from which transcript expression as well as catalytic activity continues to be under active investigation. Our aim in this study was to explore at a first glance, pmca isoform distribution using isolated parenchymal and non-parenchymal cells from rat liver tissue. Expression of pmca transcripts was analyzed in fresh or cell-enriched culture preparations, confirming pmca1 and pmca4 as the housekeeping isoforms in all cell types studied (hepatocytes, Kupffer cells, and stellate cells). However, for the first time we show expression of pmca3 transcripts edited at two different sites in both hepatocytes and non-parenchymal cells. Interestingly, employing non-parenchymal cells we demonstrate the specific expression of pmca3e transcripts previously considered nearly exclusive of excitable tissues. Real-time PCR quantification shows a significant decrease of pmca3 transcripts in cultured Kupffer and hepatic stellate cells in comparison with fresh cells. The presence of pmca2 along with pmca3 in all liver cell types studied suggests that high affinity isoforms are relevant to the adequate management of calcium in liver tissue, particularly when hepatic cells become activated by diverse stimuli.

Disruption of calcium homeostasis in epileptic cells is characterized by both short- and long-term perturbations of Ca(2+) buffering systems. Along with the Na(+)/Ca(2+) exchanger, the plasma membrane Ca(2+)-ATPase (PMCA) plays an important role in excitable cells. The involvement of PMCAs in epileptogenesis has primarily been studied in brief intervals after various stimuli; however, the specific contribution of this molecule to epileptogenesis is not yet fully understood. Our aim has been to investigate whether PMCA expression in the chronic stages of epilepsy is altered. Through an interdisciplinary approach, involving whole-cell recordings and real-time reverse transcriptase-polymerase chain reaction, we have shown that epileptic neurons in our preparation consistently show changes in electrical properties during the period of chronic epilepsy. These changes included increased spike frequency, altered resting membrane potential and changes in passive membrane properties. Following these observations, which indicate an altered excitability in the epileptic cells studied, PMCA mRNA transcripts were studied. It was found that while PMCA1 transcripts are significantly increased one month following the pilocarpine epileptogenic stimulus, PMCA3, an isoform important in excitable tissues, was significantly, decreased. These findings suggest that, in the long-term, a slow PMCA (PMCA1) plays a role in the reestablishment of a new calcium homeostasis attained by epileptic cells. Overall, this phenomenon points out the fact that in seizure disorders, changes that take place in the balance of the different molecules and their isoforms in charge of maintaining neuronal calcium homeostasis, are fundamental in the survival of affected cells.

The precise control for maintenance of a normal intracellular calcium concentration in eukaryote cells is accomplished by several systems located at the plasma membrane, as well as several internal membrane systems. Neurons are especially sensitive to changes in these control systems, since when fail and calcium homeostasis disturbed, the cell's metabolism is immediately modified and a pathological condition emerges. Such a condition has been associated with epileptogenesis, and especially to those mechanisms associated to calcium entrance or ON mechanisms. On the other hand, calcium extrusion mechanisms or OFF mechanisms, have been investigated to a lesser extent and therefore remain much less understood. Here, we present a review of these calcium extrusion systems located at the plasma membrane considered to be critical in the process of epileptogenesis; first of all the plasma membrane calcium ATPase (PMCA) as the catalytic moiety of the enzyme that moves calcium outwards in an energy-dependent fashion, and the Na + /Ca 2+ exchanger (NCX) coupled to the (Na + /K + )-ATPase. Based on present knowledge considering the wide range of isoforms found for PMCA and NCX and their specific kinetic characteristics, a hypothesis for their participation on the OFF mechanisms related to the genesis of epilepsy is discussed.