Abstract
Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca2+-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca2+-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca2+] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH) and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO) to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca2+-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca2+-sensitive release-ready vesicles as determined by slow photo-release of caged Ca2+. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca2+-dependent release of α-MSH.
Highlights
Release of transmitters and peptide hormones from neurons and neuroendocrine cells into the extracellular space is triggered by cytosolic Ca2+ increase
Peptide hormones are packaged in specialized membrane compartments - secretory vesicles, which undergo a series of preparatory steps, including peptide expression, processing, maturation, storage and eventually Ca2+-dependent exocytosis
In our study we used pituitary tissue slice preparation from Rab3a KO mice as a model to assess the role of Rab3a protein in the late steps of stimulus-secretion coupling
Summary
Release of transmitters and peptide hormones from neurons and neuroendocrine cells into the extracellular space is triggered by cytosolic Ca2+ increase. Rab isoforms (a-d) have been assigned heterogeneous functions during the life cycle of a secretory vesicle These include the positive regulation of vesicle docking, priming, fusion and recycling [2,3,4,5]. Loss of Rab3a protein in vitro impairs the secretory output in the pituitary intermediate lobe cells – melanotrophs [7]. These cells primarily produce pro-opiomelanocortin-derived α-melanocyte stimulating hormone (α-MSH) and β-endorphin [8]. Rab3a stimulates secretory output at the step prior to exocytosis, possibly by recruiting vesicles containing a specific pituitary hormone [7]. It remains elusive whether Rab3a affects the kinetics and [Ca2+]i sensitivity of the Ca2+-dependent secretory activity, and the availability of the secreted pituitary hormones in plasma
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