Molecular Mechanisms of Neuropeptide Secretion from Neurohypophysial Terminals: a Dissertation

A clear definition of the mechanisms involved in synaptic transmission is of paramount importance for the understanding of the processes governing synaptic efficacy. Despite decades of intense study, these mechanisms remain poorly understood. The work contained in this thesis examines several such mechanisms using the hypothalamic-neurohypophysial system (HNS), a classical preparation for the study of Ca2+-dependent neuropeptide release. The first portion of this thesis is comprised of my efforts to define the cellular machinery essential for the exocytosis of secretory granules isolated from peptidergic neurohypophysial terminals of the HNS. Here, using the planar lipid bilayer model system, I have been able to show that syntaxin alone in the target membrane is sufficient to elicit fusion of modified neurohypophysial secretory granules. Surprisingly, SNAP-25 does not appear to be necessary for this process. This suggests that syntaxin may be able to substitute for SNAP-25 to form functional non-cognate fusion complexes. Additionally, the coupling of amperometric detection with the planar lipid bilayer system has allowed me to confirm these results using native, unmodified secretory granules, and also provides some insight into the kinetics of release in this reconstituted system. This model system should provide a convenient means for the study of additional regulatory factors believed to be involved in secretory vesicle exocytosis. The second and third sections of this thesis involve my examination of the role of presynaptic Ca2+ stores in neuropeptide secretion from isolated peptidergic neurohypophysial terminals (NHT). I initially examined the source of recently discovered ryanodine-sensitive Ca2+ stores in this system. Using Immuno-electron microscopy I have found that ryanodine receptor (RyR) labeling appears to co-localize with large dense core granules. Additionally, I have shown that a large conductance cation channel, with similarities to the RyR, found in the membrane of these granules has the same characteristic response to pharmacological agents specific for the RyR. Further, application of RyR agonists modulates basal neuropeptide release from NHT. These results suggest that the large dense core granules of NHT serve as the source of a functional ryanodine-sensitive Ca2+store. Recent work has revealed that spark-like Ca2+ transients, termed syntillas, can be observed in NHT. These syntillas arise from ryanodine-sensitive intracellular stores. In other neuronal preparations, similar Ca2+ transients have been suggested to affect spontaneous transmitter release. However, such a role for syntillas had yet to be examined. To assess if syntillas could directly trigger spontaneous release from NHT, I used simultaneous Ca2+imaging along with amperometric detection of release. Amperometry was adapted to this system via a novel method of false-transmitter loading. Using this approach I have found no apparent correlation between these two events, indicating that syntillas are unable to directly elicit spontaneous transmitter release. As this finding did not rule out an indirect modulatory role of syntillas on release, I additionally present some preliminary studies examining the ability of ryanodine-sensitive Ca2+ release to modulate vesicular priming. Using immunocytochemistry, I have shown that RyR agonist treatment shifts the distribution of neuropeptides toward the plasma membrane in oxytocinergic NHT, but not in vasopressinergic NHT. RyR antagonists have the opposite affect, again only in oxytocinergic NHT. Further, I have found that application of RyR agonists result in a facilitation of elicited release in NHT using membrane capacitance recording. This facilitation appears to be due primarily to an increase in recruitment of vesicles to the readily-releasable pool. These findings suggest that ryanodine-sensitive Ca2+stores may be involved in vesicular priming in NHTs. Taken together, the work presented in this thesis provides some new and interesting insights into the underlying mechanisms and modulation of transmitter release in both the HNS and other CNS terminals

Mitochondria and chromaffin cell function

Producción CientíficaChromaffin cells are an excellent model for stimulus– secretion coupling. Ca2+ entry through plasma membrane voltage-operated Ca2+ channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca2+ signal. The endoplasmic reticulum amplifies the cytosolic Ca2+ ([Ca2+]C) signal by Ca2+- induced Ca2+ release (CICR) and helps generation of microdomains with high [Ca2+]C (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca2+ from HCMD and stop progression of the Ca2+ wave towards the cell core. On the other hand, the increase of [Ca2+] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca2+]C decreases rapidly and mitochondria release the Ca2+ accumulated in the matrix through the Na+/Ca2+ exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus–secretion coupling while avoiding propagation of the Ca2+ signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders

Role of Internal Calcium Stores in Exocytosis and Neurotransmission: A Dissertation

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca2+] in the vicinity of plasmalemmal Ca2+ channels due to Ca2+ influx, elicits exocytosis. This dissertation examines the effect on both spontaneous and elicited exocytosis of a rise in focal cytosolic [Ca2+] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca2+ syntillas. Ca2+ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (Scintilla, Latin for spark, found in nerve terminals, normally synaptic structures.) We have also observed Ca2+ syntillas in mouse adrenal chromaffin cells (ACCs). Here the effect of Ca2+syntillas on exocytosis is examined in ACCs, which are widely used as model cells for the study of neurosecretion. Elicited exocytosis employs two sources of Ca2+, one due to influx from the cell exterior through voltage-gated Ca2+ channels (VGCCs) and another due to release from intracellular stores. To eliminate complications arising from Ca2+ influx, the first part of this dissertation examines spontaneous exocytosis where influx is not activated. We report that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine after which syntillas were decreased. We conclude that Ca2+syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas. The second part of this dissertation examines the role of syntillas in elicited exocytosis whereby Ca2+ influx is activated by physiologically relevant levels of stimulation. Catecholamine and neuropeptide release from ACCs into the circulation is controlled by the sympathetic division of the Autonomic Nervous System. To ensure proper homeostasis tightly controlled exocytic mechanisms must exist both in resting conditions, where minimal output is desirable and under stress, where maximal, but not total release is necessary. It is thought that sympathetic discharge accomplishes this task by regulating the frequency of Ca2+ influx through VGCCs, which serves as a direct trigger for exocytosis. But our studies on spontaneous release in ACCs revealed the presence of Ca2+ syntillas, which had the opposite effect of inhibiting release. Therefore, assuming Ca2+-induced Ca2+ release (CICR) via RYRs due to Ca2+ influx through VGCCs, we are confronted with a contradiction. Sympathetic discharge should increase syntilla frequency and that in turn should decreaseexocytosis, a paradox. A simple “explanation” might be that the increase in syntillas would act as a brake to prevent an overly great exocytic release. But upon investigation of this question a different finding emerged. We examined the role of syntillas under varying levels of physiologic stimulation in ACCs using simulated action potentials (sAPs) designed to mimic native input at frequencies associated with stress, 15 Hz, and the basal sympathetic tone, 0.5 Hz. Surprisingly, we found that sAPs delivered at 15 Hz or 0.5 Hz were able to completely abolish Ca2+ syntillas within a time frame of two minutes. This was not expected. Further, a single sAP is all that was necessary to initiate suppression of syntillas. Syntillas remained inhibited after 0.5 Hz stimulation but were only temporarily suppressed (for 2 minutes) by 15 Hz stimulation, where global [Ca2+]i was raised to 1 – 2 μM. Thus we propose that CICR, if present in these cells, is overridden by other processes. Hence it appears that inhibition of syntillas by action potentials in ACCs is due to a new process which is the opposite of CICR. This process needs to be investigated, and that will be one of the very next steps in the future. Finally we conclude that syntilla suppression by action potentials is part of the mechanism for elicited exocytosis, resolving the paradox. In the last chapter speculation is discussed into the mechanisms by which physiologic input in the form of an action potential can inhibit Ca2+ syntillas and furthermore, how the Ca2+ syntilla can inhibit exocytic output

Electromagnetic Analysis of Exposure Systems – Potential Factors Underlying the Variation in Duration of Nanosecond Electric Pulse-Evoked Changes in Intracellular Calcium Level in Adrenal Chromaffin Cells

Studying the effects of the nanosecond electric pulses (NEPs) on excitable cells, especially on bovine adrenal chromaffin cells, has been of great interest to our research group. Chromaffin cells undergo a process of exocytosis to release catecholamines from their secretory granules. Exocytosis is triggered when the cells are stimulated with the physiological stimulus that activates acetylcholine nicotinic receptor. This activation leads to an influx of Ca2+ into the cell, which can be imaged using the fluorescence microscopy technique. Imaging reveals that in response to the application of physiological stimulus the cells undergo an initial rapid increase in intracellular calcium concentration ([Ca2+]i) followed by a decrease, resulting in a short-lived Ca2+ response. Similar to nicotinic receptor activation, a single 5 ns electric pulse triggers a rapid increase in [Ca2+]i. However, the return of [Ca2+]i to pre-stimulus level is slower than that triggered by the physiological stimulus. In addition, it was found that the duration of NEP-induced Ca2+ responses varied according to the manner in which the cells were exposed to the 5 ns pulse. In our research, the first NEP delivery system that was used consisted of a gold strip chamber. Cells exposed to a 5 ns, 5 MV/m pulse with this exposure system resulted in short-lived (< 10 s) Ca2+ responses, similar to that evoked by physiological stimulus (5 - 6 s). However, when chromaffin cells were stimulated by a 5 ns, 5 MV/m pulse using a pointy rod electrode exposure system, Ca2+ responses were more prolonged, in some cases such that [Ca2+]i never returned to pre-stimulus levels during the monitoring period. Because the basis of this prolonged rise in [Ca2+]i is not yet known, the main objective of this project is to understand the potential reasons for the variable duration and prolonged increase in [Ca2+]i by the pointy rod electrode exposure system. The starting premise is that specific electrical and/or the geometrical configuration of the exposure systems are factors. Because NEPs (the source for the exposure systems) are electromagnetic waves, analytical and 3D computational electromagnetics studies of the two exposure systems were performed, and the detailed interactions of the electric field (E-field) with cells in each exposure chamber were analyzed. The first objective was to analyze the time and frequency responses of the pointy rod electrode using the Finite Difference Time Domain (FDTD) method. This was necessary to determine if the geometry of the pointy rod electrode acts as a high- or low-pass filter that could distort the NEP signal at the location of the cell and induce a different type of stimulus than that induced by the gold strip chamber. The simulation results confirmed that the pointy rod electrode delivers the original pulse shape to the location of the cell without distortion. We also carried out simulations and measurements in which we determined that factors related to the fabrication of the pointy rod electrodes (for example, slightly different electrode lengths and types of resistors) also do not significantly distort the pulse shape and frequency spectrum at the cell location. The next major consideration was related to the E-field propagation aspect in the two exposure systems. For the pointy rod electrodes, NEP reflection may occur on the surface of the glass cell dish where the cells are attached, and the scattered NEP can re-radiate to the cell, leading to a higher stimulatory effect of the NEP. To access this possibility, an electrically equivalent round cell, referred to as a spherical capacitor model (SCM), was created in the FDTD software and used to compute the E-field distribution at the cell location. The results indicated that with the pointy rod electrode model the reflected E-field caused a high E-field at the bottom membrane of SCM. In contrast, there was no reflection of the NEP for the gold strip chamber. This result may explain the reason for the longer duration of Ca2+ responses in cells exposed to a NEP using the pointy rod electrode. That is, the cells are exposed to the initial E-field then re-exposed by the reflected E-field, which results in stimulation of the cells with a higher E-field. The E-field distribution of the pointy rod electrode obtained from the numerical modeling revealed an unexpected, localized E-field spike that occurs between the cell membrane and the glass surface on which the cell is attached in the dish. That is, the E-field is concentrated in the narrow gap between the round cell membrane and the flat glass dish, a situation that is not present in the gold strip chamber where cells are in suspension and not attached to a glass surface. This E-field increase at the bottom of the cell can be another source of a higher E-field to which a cell is exposed. In addition, the level of attachment of individual cells to the glass surface can vary such that the cell membrane is flattened to different extents. This results in cells having different elliptical shapes due to the level of attachment to the glass surface. To replicate a realistic cell geometry for this condition in an FDTD model, an elliptical capacitor model (ECM) was modeled, and the resulting E-field distribution was compared with that of the spherical cells (SCM). The results showed that there is a larger area of a localized high E-field in the ECM than in the SCM, indicating that the shape of the cells contributes to the variation of the E-field in the cell membrane. Since attached cells will have different shapes, this could explain why the Ca2+ responses vary even for cells in the same dish. Based on the numerical modeling results described above, several electrodes intended to reduce the E-field reflections were designed, fabricated and tested in Ca2+ imaging experiments. These included a coupler shaped electrode, a flat copper-based electrode and a series of pointy rod electrodes with various incident angles (20°, 45°, and 70°). Unfortunately, the new electrode models did not achieve the shorter duration Ca2+ responses in chromaffin cells observed in cells stimulated with 5 ns pulse in the gold strip chambers. In summary, this study identified multiple factors that could underlie the variations in Ca2+ responses in chromaffin cells evoked by pointy rod electrode exposure system that are not associated with the gold strip chamber exposure system. The study also eliminated some potential factors, such as inconsistencies in electrode fabrication and the selection of electrical components, as contributing to Ca2+ variability. Future work of the project includes investigating the effect of 5 ns pulses with different rise times as the gold strip chamber and pointy rod electrodes were fed by 5 ns pulses with different rise and fall times

Pituitary adenylate cyclase-activating polypeptide mediates differential signaling through PAC1 receptor splice variants and activates non-canonical cAMP dependent gene induction in the nervous system - Implications for homeostatic stress-responding

Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. PAC1 occurs in different splice variants of the third intracellular loop, designated PAC1null, hop or hip, affecting second messenger generation as shown in non-neural cells. At the splanchnico-adrenomedullary synapse, PACAP is required for prolonged catecholamine secretion from chromaffin cells to restore homeostasis during prolonged periods of stress. In the central nervous system, PACAP is neuroprotective in neurodegenerative conditions associated with e.g., stroke. In the present study, PAC1 splice variant-specific second messenger production and activation of homeostatic responses were investigated in neuroendocrine and neural cells. Heterologous expression of the major PAC1 splice variant of adrenomedullary chromaffin cells, PAC1hop, in PC12-G cells reconstituted a PACAP-mediated Ca2+ and prolonged secretory response similar to the one observed in primary chromaffin cells. The Ca2+ response mediated by PAC1null was somewhat smaller and PAC1hip failed to couple to Ca2+. Neither variant conferred prolonged catecholamine release, suggesting that expression of the hop cassette in the third intracellular loop of the receptor is required for sustained catecholamine release from neuroendocrine cells. In NG108-15 cells, heterologous expression of the PAC1hop, null and hip receptor conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca2+]i occurred efficiently in PAC1hop- and to a lesser extent in PAC1null-expressing cells. Expression of PAC1hip did not confer an intracellular Ca2+ response, indicating that PAC1hop is the receptor variant most efficiently coupled to combinatorial signaling through cAMP and Ca2+. PAC1hop-mediated signaling activated the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases 1 and 2 (ERK1/2). Signaling to ERK proceeded through cAMP independently of the cAMP dependent protein kinase (PKA). PACAP induced transcription of the gene encoding the putative neuroprotectant stanniocalcin 1 (STC1), which has previously been implicated in neuronal resistance to hypoxic/ ischemic insult; gene induction proceeded through ERK but not PKA. Cyclic AMP generation by forskolin did not activate ERK in NG108-15 cells, but rather induced STC1 mRNA elevation through the canonical PKA dependent pathway. This suggests that activation of non-canonical cAMP signaling, mediating ERK-dependent gene induction, requires additional signaling through Ca2+ via PAC1hop in these cells. Primary rat cortical neurons expressed predominantly the PAC1hop and null variants. Exposure of cortical neurons to PACAP resulted in elevation of the two second messengers cAMP and Ca2+, activation of ERK1/2, and induction of STC1 gene transcription. PACAP-mediated ERK activation proceeded through cAMP but not PKA, and STC1 was induced via ERK but not PKA. Pharmacological stimulation of adenylate cyclases by forskolin also resulted in increased ERK phosphorylation and STC1 mRNA elevation independently of PKA. These results indicate that cAMP production alone is sufficient to activate ERK in differentiated cortical neurons, unlike in the less differentiated NG108-15 cell line. Induction of another PACAP target gene, brain-derived neurotrophic factor (BDNF), occurred through the canonical cAMP/PKA pathway. PACAP has been shown by our laboratory and others to be neuroprotective against ischemia in rodent stroke models. To begin to define the mechanism of this neuroprotection, we employed two cell culture stroke models. Rat cortical neurons subjected to either oxygen-glucose-deprivation or glutamate-induced excitotoxicity underwent cell death as expected. However, treatment with PACAP did not increase neuronal survival in either of the two models, and STC1 over-expression also failed to increase resistance to neuronal cell death during glutamate-induced excitotoxicity. These data suggest that the protective effects of the neurotrophic peptide PACAP and the putative neuroprotectant STC1 during neurodegenerative conditions in vivo are mediated through cells absent in cultures of cortical neurons, such as glial cells. In conclusion, the present study has demonstrated that expression of different PAC1 splice variants determines the degree of activation of two different second messenger pathways that may mediate different functional outcomes during stress-responding. PACAP mediates ERK activation and STC1 induction via non-canonical cAMP signaling. The selective pharmacological activation of this potentially neuroprotective pathway, which is different from the cAMP/PKA pathway critical for learning and memory, could have therapeutic implications for neuroprotection in vivo

Novel Mechanisms In The Sorting Of Proglucagon To The Secretory Granules Of The Regulated Secretory Pathway

The prohormone proglucagon encodes for multiple peptide hormones, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, produced through tissue-specific processing by prohormone convertase (PC) 1/3 and PC2. In alpha cells, PC2 yields glucagon, the major counter-regulatory hormone to insulin, which together, control glucose homeostasis. In contrast, GLP-1 and GLP2 are mainly produced in intestinal L-cells by PC1/3. GLP-1 stimulates insulin secretion following a meal, and therefore has opposing function to glucagon regulating glucose homeostasis; in contrast, GLP-2 enhances gut nutrient absorption. Efficient sorting of proglucagon to secretory granules is required for nutrient-regulated secretion. The aim of this thesis is to discover the molecular mechanisms by which proglucagon is targeted to secretory granules, which ensures that proglucagon is correctly processed to mature hormones, and is necessary for prompt physiologic response to nutrient status. In this thesis, we identify several sorting signals within the hormone domains of proglucagon that encode targeting information. Using quantitative immunofluorescence microscopy and co-localization analyses, I was able to determine the molecular nature by which glucagon and GLP-1 enter granules. Despite these two hormones sharing a large degree of structural homology, it is their particular alpha-helix structures that enable the sorting of proglucagon. Further, I provide evidence that proglucagon is first sorted to granules prior to being processed to active hormones. Furthermore, I have identified carboxypeptidase E in the mechanism by which glucagon sorts within alpha cells. Together, each hormone carries with it a unique sorting “signature” to efficiently reach its destination, and allows alpha and L-cells to tightly regulate nutrient homeostasis

The role of renalase in catecholamine biosynthesis: a new protective mechanism against hypertension?

Renalase is a secretory protein that may protect against hypertension by reducing levels of circulating catecholamines (CAs). Renalase knockout mice are hypertensive with elevated CA levels. There are no known physiological mechanisms that fully describe renalase’s influence on plasma CAs. The adrenal medulla is a major site for CA regulation, however, the role of renalase in this tissue has not been investigated. The purpose of this study was to determine a potential role of renalase in this tissue using three objectives: 1) Examine the regulation of renalase by signal activators of CA biosynthesis; 2) Determine the effects of renalase on the regulation of CA-biosynthesizing enzymes; 3) Examine the expression of renalase in hypertensive rats. For objective 1, renalase expression was analyzed in pheochromocytoma-derived PC12 cells after treatment with 10 signal activators (e.g. phorbol 12-myristate 13-acetate (PMA), forskolin, cobalt chloride (CoCl2), and dexamethasone (Dex)). For objective 2, PC12 cells were treated with recombinant renalase and mRNA levels of CA-biosynthesizing enzymes were quantitated. For objective 3, renalase expression was examined in adrenal glands from spontaneously hypertensive rats (SHRs) and compared with normotensive Wistar-Kyoto rats (WKYs). RT-PCR and western blotting were performed to quantify mRNA and protein levels. Three signal activators significantly altered renalase expression; changes in renalase expression were observed after PMA, CoCl2, and Dex treatment. Recombinant renalase treatment did not change the expression of CA-biosynthesizing enzymes. SHRs compared to WKYs had significantly higher levels of renalase mRNA, but not protein. Overall, this study is a first step to determine if renalase’s role in the adrenal medulla is to regulate CA biosynthesis and protect against hypertension.Master of Science (MSc) in Biolog

Nature of Large Vesicle Exocytosis in Pancreatic β-cells: Release of ATP and GABA

Several high resolution electrophysiological techniques such as measurements of cell membrane capacitance and amperometry are used to detect exocytotic events. A novel method was developed in this study based on the expression of P2X2 receptor channel in secreting cells. The presence of ATP sensitive channel on the cell membrane permitted the electrical detection of minute (amoles) quantities of ATP and thus created a possibility to measure ATP released from a single insulin granule. The release of peptides and low molecular weight substances was explored by combining the detection of exocytotic events by P2X2 receptor method with imaging of secretory granules. It was found that in rat insulinoma cells these substances were released independently and with different time course: nucleotides - 280 ms and granular peptide marker IAPP - 2.2 s after the membrane depolarization. In 72% of all exocytotic events nucleotide release was not followed by the discharge of peptide cargo. The chief inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is also found in pancreatic β-cells though its role in coordinating processes in the pancreas is unknown. Sub-cellular localization has not been fully resolved too. Electrical measurements of ATP, GABA and 5-HT release in rat pancreatic β-cells allowed us to demonstrate that at least 75% of GABA release events were attributable to the exocytosis of large dense core vesicles in these cells and its selective release was regulated by the size of the fusion pore. The combination of the cell capacitance measurements, the electrical measurements of ATP release and amperometric detection of 5-HT release demonstrated that in rat pancreatic β-cells ATP is stored and released by large dense core vesicles. The contribution of small synaptic like vesicles is below the detection levels of methods used. The possibility of multi-vesicular exocytosis in pancreatic β-cells was explored by the cell-attached capacitance measurements, the total internal reflection microscopy and by electrical ATP release measurements. It was found that global increase in [Ca2+]i promotes the formation of complexes of interconnected granules within cell cytoplasm that subsequently undergo exocytosis as one unit. This may lead to exocytosis of up to 15 granules simultaneously (compound exocytosis)