Leydig cells are the main site of production and secretion of testosterone in males. Its synthesis is mainly controlled by the Luteinizing Hormone (LH) whose action on membrane receptors leads to an increase in both cAMP and intracellular calcium concentrations. As a result, the expression of STAR (Steroidogenic Acute Regulatory protein), an essential protein involved in the transport of cholesterol across the internal membrane of the mitochondria, is also increased. Data from our laboratory have shown that calcium entry in Leydig cells can be accomplished by two systems: 1) through ATP activated ion channels of the P2X subtype, known for their high calcium permeability; and 2) through T-type calcium channels present in their plasma membrane, which can be activated by LH. Although our data points to this last system as the main player involved in the intracellular calcium concentration transients observed when the cells are stimulated to secrete by LH, there are indications that calcium depletion of the endoplasmic reticulum also activates a capacitive calcium entry known as SOCE (Store Operated Calcium Entry). Only recently the molecular identity of this entry pathway was associated to two distinct proteins: a) ORAI, which seems to work as an ion channel and b) STIM, which couples the signal from the endoplasmic reticulum to ORAI. Therefore, this project aims at identifying and characterizing such calcium entry pathway in Leydig cells. From an experimental point of view, two approaches will be used: 1) calcium current measurements with the whole cell configuration of the patch clamp technique. Here, the cells will be incubated in the absence of calcium and the endoplasmic reticulum depleted by including IP3 in the pipette solution. SOCE will be recorded by application of voltage ramps every two seconds; and 2) measuring the intracellular calcium ion activity with fluorescent calcium indicators and confocal microscopy, under conditions of calcium depletion of the endoplasmic reticulum. With these experiments we hope to characterize SOCE and correlate it with the effects of LH on mice Leydig cells.
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