Microbiology and Physiological Systems
First Thesis Advisor
Dr. José R. Lemos
Calcium Channels, Neurons, Hypothalamus, Receptors, Opioid, mu, Nerve Endings, Neuropeptides, Receptors, Opioid, kappa
This thesis details my examination of several mechanisms for modulation of neuropeptide release via voltage-dependent and voltage-independent intraterminal signaling in isolated neurohypophysial terminals. The first part of this work characterizes depolarization-induced neuropeptide release in the absence of extracellular calcium. The goal of this project was to examine the relationship between depolarization-induced release of intracellular calcium stores and depolarization-secretion coupling of neuropeptides. We demonstrate that depolarization in the absence of extracellular calcium induced by either High K+ or electrical stimulation induces a rise in [Ca2+]i and subsequent neuropeptide release from Hypothalamic Neurohypophysial System (HNS) terminals. A portion of extracellular calcium-independent neuropeptide release is due to intraterminal calcium, but the remaining depolarization-induced release may be due to calcium-independent voltage-dependent (CIVD) release (Zhang and Zhou, 2002; Zhang et al., 2004; Yang et al., 2005). Nevertheless, our results clearly show that extracellular calcium is notnecessary for depolarization-induced neuropeptide secretion from these CNS terminals.
In addition, I investigated the role of internal calcium stores in mediating μ-opioid inhibition of voltage-gated calcium channels (VGCCs). Inhibition of VGCCs via μ-opioid agonists has been shown to reduce neuropeptide release in response to High K+ stimulation of isolated terminals (Bicknell et al., 1985b; Russell et al., 1993; van Wimersma Greidanus and van de Heijning, 1993; Munro et al., 1994; Ortiz-Miranda et al., 2003; Russell et al., 2003; Ortiz-Miranda et al., 2005). My findings show μ-opioid inhibition, of VGCC and High K+-mediated rise in [Ca2+]i, are via a voltage-independent diffusible second-messenger targeting release of calcium from ryanodine-sensitive stores, possibly mediated via the cyclic ADP ribose signaling pathway.
Furthermore, I detail a different intracellular messenger pathway mediating the κ-opioid inhibition of VGCC and High K+-mediated rise in [Ca2+]ii. In contrast to the μ-opioid inhibition, κ-receptor activation is coupled to a voltage-dependent membrane-delimited pathway. Inhibition of neuropeptide release via both endogenous and exogenous κ-opioid agonists has been extensively studied (Bicknell et al., 1985a; Nordmann et al., 1986a; Wammack and Racke, 1988; Munro et al., 1994; Ingram et al., 1996; Rusin et al., 1997a). My investigation shows that the κ-inhibition of VGCC is voltage-dependent and is furthermore, relieved within the context of a physiological burst of action potentials (APs). This physiologically-evoked, activity-dependent modulation of VGCC and subsequent release, represents an important mechanism for short-term synaptic plasticity at the level of the terminals. Given the ubiquitous nature of voltage-dependent G-protein signaling in the CNS, our results may prove important in understanding modulatory effects of specific bursting patterns throughout the CNS. In the last 30 years the neurohypophysial system has proven to be an excellent system to study the complexities of depolarization-secretion coupling (DSC). There have been many advances in our understanding of the underlying mechanisms involved and their physiological implications. The current work focuses on two important features of DSC; voltage and calcium. Although in many ways these two are intrinsically linked through VGCC activation, we have found that in isolated HNS terminals that is not always the case. We have further found that when voltage and calcium influx are linked during DSC, modulation by opioids may or may not be linked to activity-dependent relief depending on the opioid receptor activated. This finding has important implications in neuropeptide release during patterned stimulation in vivo. As I will discuss further, many factors play into the complexities of the regulatory mechanisms involving release. As investigations into this remarkable field continue, I hope to have contributed a valuable piece to the puzzle.
Velazquez-Marrero, CM. Modulation of Neuropeptide Release via Voltage-Dependent and -Independent Signaling in Isolated Neurohypophysial Terminals: a Dissertation. (2008). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 367. DOI: 10.13028/tgm2-3725. https://escholarship.umassmed.edu/gsbs_diss/367
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