Modulation of Neuroscretory Granule Mobilization and Neuropeptide Release by Ryanodine Receptors in Neurohypophysial Terminals

Abstract

The neuropeptides oxytocin (OT) and vasopressin (AVP) are contained in large dense core vesicles (LDCV) and are released at the neurohypophysis (NH). The mobilization and translocation of vesicles to exocytotic release sites is modulated by cytosolic Ca2+. Intracellular structures and organelles able to store and release Ca2+ are significant contributors of cytosolic Ca2+. The presence of ryanodine receptors (RyR) in LDCV of NH terminals, coupled with the demonstration that pharmacological activation of these receptors can induce spontaneous focal Ca2+ transients, make them ideal modulators of cytosolic levels of Ca2+, and therefore, vesicle mobilization and subsequent neuropeptide (NP) release.To test this hypothesis, the association of LDCV in an area within 0.45 μm of the plasma membrane was assessed using immunolabeling of Neurophysin I (OT) and II (AVP) along with high stringency deconvolution techniques in isolated NH terminals. We found that the total amount of membrane associated NP-immunoreactivity varies significantly between terminal type; significantly higher in OT than in AVP terminals. This membrane associated distribution pattern can be enhanced by exposure to agonist concentrations (1-5 μM) of ryanodine in OT terminals only. RyR antagonists, 8-Br-cADPR or higher concentrations (>10 μM) of ryanodine had the opposite effect; significantly reducing the amount of OT associated with the membrane area.Additionally, Ca2+-evoked NP release from permeabilized terminals was increased by agonist concentrations of ryanodine and conversely, decreased by antagonist concentrations of this drug. Agonist concentrations of ryanodine were also able to increase the asynchronous phase of low frequency electrically stimulated capacitance increases from isolated NH terminals. Thus, the ryanodine-sensitive mobilization of secretory granules seems to have a functional role in modulating secretion of neuropeptides from NH terminals. [Supported by UMass Grant P60037094900000 (SOM) and NIH Grant NS29470 (JRL)]