Intracellular components involved in parathyroid sensing of extracellular calcium

Sammanfattning: Parathyroid glands are the primary regulator of extracellular calcium ([Ca2+]e). Ca2+ plays a key role in many fundamental biological processes and is also an essential structural component of the skeleton. The parathyroid chief cells detect small changes in [Ca2+]e and respond by altering the secretion of parathyroid hormone (PTH). This thesis details studies towards the identification of intracellular components involved in the parathyroid sensing mechanism of [Ca2+]e.Src family members c-Src, c-Yes, Fyn, Lyn, Lck and PKC isozymes (α, βI, βII, ε, ζ and ι) were found to be expressed in bovine parathyroid cells. Increase in [Ca2+]e to 3.0 mM caused activation of c-Src and c-Yes that was maximal at 5 min of incubation of the parathyroid cells. This activation was still evident after 30 min. Parathyroid caveolin along with 62, 38, 36, and 32 kDa proteins were found to be phosphorylated in high [Ca2+]e. Inhibition of Src kinases blocked tyrosine phosphorylation of caveolin and the 38 kDa protein and uncoupled the inhibition of PTH secretion seen at high [Ca2+]e. ERK was found to be strongly activated at 3 mM and also, but more weakly at 0.5 mM compared to at 1.25 mM [Ca2+]e. Inhibition of ERK blocked PTH secretion at low [Ca2+]e.Increase of [Ca2+]e to 3.0 mM also elicited translocation of PKCβI, -ε, -ζ and -ι to the cell periphery. In contrast, at 0.5 mM [Ca2+]e PKCα was translocated to the cell periphery and PI 3-kinase was found to be activated. Inhibition of PI 3-kinase activity blocked the dissociation of the cortical actin ring of parathymid cells evident at low [Ca2+]e and inhibited the subcellular translocation of PKCε, -ζ, and -ι in cells exposed to high [Ca2+]e. Moreover, the secretion of PTH induced at low [Ca2+]e was blocked by inhibition of PI 3-kinase. These results suggestthat c-Src, c-Yes, ERK, PI 3-kinase and several PKC isozymes are involved in the mechanism that couples the [Ca2+]e stimulus to secretion in the parathyroid cell.