New insights in parathyroid hormone secretion : With focus on exocytosis and calcium signaling

Sammanfattning: The parathyroid is distinguished from other endocrine systems since its primary stimuli inhibit hormone secretion. It is generally considered that extracellular Ca2+ binds to a membrane receptor (calcium sensing receptor, or CaSR), and via second messengers modulate parathyroid hormone (PTH) secretion. However, the intracellular signaling pathway is not completely characterized, but intracellular Ca2+ concentration (Ca2+i) is set aside as a centre mediator for this regulation. In regulated hormone secretion, or exocytosis, synaptosomal-associated proteins (i.e. SNAPs and others) are one of the key-players in hormone vesicle and membrane fusion. These proteins also refered to as SNARE-proteins are widly expressed in neurons and neuroendocrine cells. In paper I, immunohistochemistry and Western blot were used to investigate expression of SNARE-proteins, i.e. SNAP-25, SNAP-23, Syntaxin1 and VAMP in normal and pathological human parathyroid tissues. SNAP-25 and Syntaxin1 were absent in normal parathyroid, but expressed in 20% of chief cell adenoma and 45% of parathyroid carcinomas. SNAP-23 and VAMP were expressed in all parathyroid samples, indicating that SNAP-23 and VAMP, rather than SNAP-25 and Syntaxin1, play a central role in exocytosis in human parathyroid cell. Ca2+ i has a pivotal role in the stimulation-secretion coupling in parathyroid cell, and study II and III focuse on this process. Increased levels of extracellular Ca2+ triggers Ca2+ influx in parathyroid cells, but the mechanism for this influx is not completely understood. Using patch-clamp technique and Ca2+ i measurements (Fura-2), we show the presence of store operated calcium current in human parathyroid and that this is constituted by TRPC1, STIM1 and Orai1 ternary complex (Paper II). In addition, calmodulin and calmodulin dependent protein kinase II (CaMKII), two important proteins in Ca2+ i handling, are demonstrated in human parathyroid cells. Blocking of calmodulin and CaMKII regulated activity results in an increase of PTH secretion that was disassociated from Ca2+ i. Our findings suggest that calmodulin and CaMKII are involved in PTH secretion (Paper III). Interesting from a clinical perspective is that a negative correlation was seen between serum calcium and phosphorylated CaMKII protein level. Finaly, in paper IV, we show that at least two types of Ca2+-activated K+ channels, i.e. KSK and KBK channel, are expressed in the parathyroid cell. These channels are sensitive to specific peptide toxin blockers (apamin, charybdotoxin) and activator (NS-11021). Modulation of these channels affects the membrane potential, and hence the PTH secretion. In summary, the stimulation-secretion coupling in the parathyroid cell is complex and in some aspects unique. This thesis characterizes different parts of this process, from Ca2+ i handling and influx, ion channel events to exocytosis. In vivo, all parts may influcence each other and regulate PTH secretion.