Calcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.

The thesis research was designed to study the characteristics of two important physiological processes in smooth muscle: Ca2+ transport mediated by the plasmalemmal Ca2+-ATPase and muscarinic receptor-G protein interactions. In resting smooth muscle, several Ca2+ extrusion or sequestration processes offset the passive inward leak of Ca2+. Although biochemical evidence suggests that the plasmalemmal Ca2+ pump plays a key role in this process, the precise role of this enzyme could not be proven until a reliable estimate of the inward Ca2+ leak was measured. Recent studies using dispersed smooth muscle cells from the toad stomach provided an estimate of the basal transmembrane Ca2+ flux rate; thus, we examined the transport capacity of the plasmalemmal Ca2+pump in this tissue. Gastric smooth muscle tissue was disrupted by homogenization and nitrogen cavitation. Membranes enriched 20 fold for plasma membrane markers were obtained using differential centrifugation and purification by flotation on discontinuous sucrose gradients. The membrane vesicles exhibited an ATP-dependent 45Ca uptake that was insensitive to azide or oxalate but sensitive to stimulation by calmodulin or inhibition by orthovanadate and the calmodulin antagonists trifluoperazine (TFP) or calmidazolium (CMZ). 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by agents that stimulate Ca2+ release from the sarcoplasmic rettculum (caffeine, inositol trisphosphate, GTP). However, both CMZ and TFP evoked a Ca2+ release that was comparable to that observed in the presence of Ca2+ ionophore, suggesting that these compounds have profound effects on membrane Ca2+permeability. 45Ca transport exhibited a high affinity for Ca2+ (KD 0.2 μM) and a high transport capacity, producing a > 12,000-fold gradient for Ca2+and a transmembrane flux rate at least 3-fold greater than that observed in resting smooth muscle cells. As a first step toward understanding the biochemical basis for the diversity of muscarinic cholinergic actions on smooth muscle, we examined the distribution of muscarinic receptor subtypes and coupling to guantne nucleotide-binding (G) proteins in airway and gastric smooth muscle. Receptor subtypes were classified in membranes prepared from bovine trachea and toad stomach based on the relative abilities of the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3) to displace the binding of nonselective antagonist [3H]QNB (quinuclidinyl benzilate). Based on the binding profiles for these antagonists, it was concluded that both smooth muscle types contain a mixture of M2 and M3 subtypes. In trachea the majority of receptors (86%) were M2, whereas in stomach the majority of receptors (88%) were M3. The displacement of [3H]QNB binding by the agonist oxotremorine indicated a mixed population of high affinity (KD = 4 nM) and low affinity (KD = 2-4 μM) binding sites. The addition of GTPγS abolished all high affinity agonist binding, suggesting that coupling of the receptors to G proteins may confer high affinity. Reaction of membranes with pertussis toxin in the presence of [32P]NAD caused the [32P]-labelling of a ~ 41 kD protein in both gastric and tracheal smooth musc1e. Pretreatment of the membranes with pertussis toxin and NAD completely abolished high affinity agonist binding in gastric smooth muscle, but produced little if any decrease in high affinity agonist binding in trachea. We conclude that, although muscarinic receptor activation leads to the elevation of intracellular Ca2+ and to contraction of both airway and gastric smooth muscle, the dissimilar distributions of receptor subtypes and distinct patterns of coupling to G proteins may indicate that each smooth muscle type uses different receptor-G protein interactions to regulate intracellular signalling pathways.

The chicken hepatoma cell line, LMH, was evaluated with respect to its usefulness for studies of the regulation of heme metabolism. Levels of δ-aminolevulinate synthase mRNA arid accumulation of porphyrins were used to evaluate the heme biosynthetic pathway. Regulation of heme oxygenase-1 by known inducers was used as a measure of heme degradation. The induction of heme oxygenase-1 by sodium arsenite was characterized. AP-1 transcription factor elements and MAP kinase signal transduction pathways that modulate expression of endogenous heme oxygenase-1 and transfected heme oxygenase-1 reporter gene constructs in response to arsenite were delineated. In initial studies, the drug glutethimide was used alone or in combination with ferric nitrilotriacetate to induce δ-aminolevulinate synthase mRNA. Levels of porphyrins, intermediates in the heme biosynthetic pathway, and levels of δ-aminolevulinate synthase mRNA were increased by these treatments in a manner similar to those previously observed in the widely used model system, primary chick embryo liver cells. The iron chelator, deferoxamine, gave a characteristic shift in the glutethimide induced porphyrin accumulation in primary hepatocytes, but was found to have no, effect on LMH cells. Heme mediated repression of δ-aminolevulinate synthase mRNA levels was similar among primary hepatocytes and LMH cells. Heme oxygenase-1 was regulated by heme, metals, heat shock, and oxidative stress-inducing chemicals in LMH cells. Heat shock induction of heme oxygenase-1 mRNA levels was observed for the first time in primary chick embryo liver cells. These data supported the further use of LMH cells to elucidate mechanisms responsible for modulating heme oxygenase-1 gene expression in response to inducers. The remainder of the studies focused on the role of heme oxygenase-1 as a stress response protein. The oxidative stress inducer, sodium arsenite was used to probe the cellular mechanisms that control the expression of heme oxygenase-1. A series of promoter-reporter constructs were used to search the heme oxygenase-1 promoter for arsenite responsive elements. Several activator protein-1 (AP-1) transcription factor binding elements were identified by computer sequence analysis. Three of these sites, located at -1578, -3656, and -4597 base pairs upstream of the transcription start site, were mutated. The arsenite responsiveness of the reporter constructs containing mutated AP-1 elements was less than that of the same constructs containing wild type AP-1 elements. At least part of the arsenite-mediated induction of heme oxygenase-1 required the activity of AP-1 transcriptional elements. The MAP kinase signal transduction pathways and heme oxygenase-1 are activated by similar stimuli, including cellular stress. MAP kinases have been shown to exert control over gene expression through effects on the AP-1 family of transcription factors. The MAP kinases ERK, JNK, and p38 were activated by arsenite in LMH cells. Constitutively activated components of the ERK and p38 pathways increased expression of heme oxygenase-1 promoter-luciferase reporter constructs. Arsenite-mediated induction of heme oxygenase-1 was blocked by dominant negative ERK or p38 pathway components, and by specific inhibitors of MEK (upstream ERK kinase) or p38. In contrast, reporter gene expression was unchanged in the presence of constitutively activated JNK pathway components. Dominant negative JNK pathway components had no effect on arsenite induced heme oxygenase-1 gene activity. In summary, LMH cells were characterized as a new model system for the study of heme metabolism. This cell line was then used to delineate promoter elements and signaling pathways involved in the arsenite responsiveness of heme oxygenase-1 gene expression. Three AP-1 transcription factor binding sites in the heme oxygenase-1 promoter region were required for responsiveness to arsenite. The MAP kinases ERK and p38 were shown to play an integral role in arsenite-mediated induction of heme oxygenase-1. These studies elucidate one facet of heme oxygenase-1 regulation, and provide tools that will be useful in delineating additional regulatory mechanisms.

Nitric oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzymatic conversion of arginine to citrulline, NO, has known roles in circulatory, immune and nervous tissues. In the nervous system nitric oxide has been implicated in long-term potentiation, neurotransmitter release, channel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitric oxide in cells, namely, neuronal differentiation. During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To deternine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53 and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells. In further studies of this pathway, we have examined the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In NGF-treated PC12 cells, we find that nNOS is induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks nNOS induction and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB 203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we determine that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0l26 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction. The activation of soluble guanylate cyclase and the production of cyclic GMP is one of the best characterized modes of NO action. Having shown that inhibition of NOS blocks PC12 cell differentiation we tested whether nitric oxide acts through soluble guanylate cyclase to lead to cell cycle arrest and neuronal differentiation. Unlike NOS inhibition, the inhibition of soluble guanylate cylcase does not block the induction of neuronal markers. Moreover, treatment of NGF-treated, NOS-inhibited PC12 cells with a soluble analog of cyclic GMP was unable to restore differentiation of those cells. Hence, cGMP is not a component of this pathway and we had to consider other mechanisms of NO action. It has become increasingly evident that another manner by which NO may exert its effects is by S-nitrosylation of cysteine residues. We tested, in vitro whether nitric oxide may control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, the first step in Hdm2 regulation of p53. The presence of cysteine or DTT blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl-sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies in order to disrupt Hdm2-p53 binding. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation.

This thesis examines the effects of nerve growth factor (NGF) and lithium on the regulation of neurotensin/neuromedin N (NT/N) gene expression in PC12 pheochromocytoma cells. In PC12 cells, the expression of the rat NT/N gene is strictly dependent on simultaneous exposure to combinations of NGF, glucocorticoids, activators of adenylate cyclase, and lithium. Transient transfection experiments indicated that a consensus AP-1 site located within the NT/N promoter is the principal target of NGF and lithium action. NGF rapidly, but transiently, induces the expression of several AP-1 genes in PC12 cells, suggesting that the effect of NGF on NT/N gene expression results from increased AP-1 activity. These results led to the prediction that the induction of NT/N gene expression should be rapid, transient and dependent on de novoprotein synthesis. These experiments also suggested that the NT/N gene is principally regulated through the initiation of transcription. However, post-transcriptional mechanisms may also be involved. Experiments in this thesis were designed to examine the regulatory mechanisms responsible for increased NT production in PC12 cells when treated with different inducer combinations and whether AP-1 factors could act as mediators in responses to NGF and lithium. Results described in this thesis indicate that the principal mechanism by which NGF and lithium regulate NT biosynthesis is by activating NT/N gene transcription. Comparison of NT/N mRNA, pro NT/N synthetic rates, proNT/N proteins and mature NT levels in induced PC12 cells, demonstrated that NGF and lithium had no effect on the translation of NT/N mRNA and had only a modest effect on post-translational processing. Nuclear run-on assays showed that NT/N transcription is transicntly activated in maximally induced cells. A rapid RNase protection assay was developed to examine both the kinetics of NT/N gene activation and whether activation requires newly synthesized proteins. Quantitation of nuclear NT/N precursor RNA. using a probe spanning the junction between exon onc and intron one, provides a sensitive measure of NT/N gene activity and by several criteria provides an accurate measure of NT/N transcription. When either NGF or lithium was combined with dexamethasone and forskolin, nuclear NT/N precursor RNA transiently accumulated, although each inducer displayed different kinetics, rapid and delayed, respectively. De novo protein synthesis was not required for activating NT/N transcription when NGF was used as the permissive agent, although newly synthesized proteins secm to be needed for subsequent down-regulation. The response to lithium displayed a marked requirement for new protein synthesis, consistent with the involvement of newly synthesized AP-1 factors. RNA blot analysis showed that lithium either alone or in combination with dexamethasone and forskolin induced c-jun and fra-1 gene expression with delayed kinetics, consistent with c-Jun/Fra-1 complexes mediating the effects of lithium on NT/N gene transcription. The pathway identified by lithium does not activate or require protein kinase C. This pathway is also active in neuronally-differentiated PC12 cells suggesting that it could be involved in the regulation of NT/N gcne exprcssion in the intact nervous system. These results and order of addition experiments demonstrate that NGF and lithium activate distinct pathways required for NT/N gene induction.

BK channels are well studied targets of acute ethanol action. They play a prominent role in neuronal excitability and have been shown to play a significant role in behavioral ethanol tolerance in invertebrates. The focus of my work centers on the effects of alcohol on the BK channel and comprises studies that examine how subcellular location affects acute ethanol sensitivity and how duration of acute alcohol exposure impacts the development of rapid tolerance. My results also provide potential mechanisms which underlie acute sensitivity and rapid tolerance. I first explore BK channel sensitivity to ethanol in the three compartments (dendrite, cell body, and nerve terminal) of magnocellular neurons in the rat hypothalamic-neurohypophysial (HNS) system. The HNS system provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins because the cell bodies are physically separated from the nerve terminals. Using electrophysiological and immunohistochemical techniques I characterize the BK channel in each of the three primary compartments and find that dendritic BK channels, similar to somatic channels, but in contrast to nerve terminal channels, are insensitive to alcohol. Furthermore, the gating kinetics, calcium sensitivity, and iberiotoxin sensitivity of channels in the dendrite are similar to somatic channels but sharply contrast terminal channels. The biophysical and pharmacological properties of somatodendritic vs. nerve terminal channels are consistent with the characteristics of exogenously expressed αβ1 vs. αβ4 channels, respectively. Therefore, one possible explanation for my findings is a selective distribution of β1 subunits to the somatodendritic compartment and β4 subunits to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate β1 or β4 channel clusters in the membrane of somatodendritic or nerve terminal compartments, respectively. In conclusion, I found that alcohol sensitivity of BK channels within the HNS system is dependent on subcellular location and postulate that β-subunits modulate ethanol sensitivity of HNS BK channels. In the second and primary focus of my thesis I explore tolerance development in the striatum, a brain region heavily implicated in addiction. Numerous studies have demonstrated that duration of drug exposure influences tolerance development and drug dependence. To further elucidate the mechanisms underlying behavioral tolerance I examined if BK channel tolerance was dependent on duration of alcohol exposure using patch clamp techniques in cultured striatal neurons from P8 rats. I found that persistence of rapid tolerance is indeed a function of exposure time and find it lasts surprisingly long. For example, after a 6 hr exposure to 20 mM ethanol, acute sensitivity was still suppressed at 24 hrs withdrawal. However, after a 1 or 3 hr exposure period, sensitivity had returned after only 4 hrs. I also found that during withdrawal from a 6 hr but not a 3 hr exposure the biophysical properties of BK channels change and that this change is correlated with an increase in mRNA levels of the alcohol insensitive STREX splice variant. Furthermore, BK channel properties during withdrawal from a 6 hr exposure to alcohol closely parallel the properties of STREX channels exogenously expressed in HEK293 cells. In conclusion I have established that BK channels develop rapid tolerance in striatal neurons, that rapid tolerance is dependent upon exposure protocol, and is surprisingly persistent. These findings present another mechanism underlying BK channel tolerance and possibly behavioral tolerance. Since these phenomena are dependent on duration of drug exposure my results may find relevance in explaining how drinking patterns impact the development of alcohol dependence in humans.

É crescente a demanda por materiais que atendam as exigências para construção de barreiras selantes (liners) em obras para disposição de resíduos. A utilização de solos tropicais para esta finalidade só não é maior por falta de conhecimento sobre muitas de suas características técnicas, principalmente a migração de contaminantes. Tentativas de melhoria das qualidades de solos supostamente inadequados também são importantes. Esta tese apresenta estudos sobre a migração dos íons \'CD POT.2+\', \'K POT.+\', \'CL POT.1-\' e \'F POT.-\' em latossolos residuais das formações Botucatu e Serra Geral e três diferentes misturas destes solos, com ênfase nos processos de sorção e difusão molecular. O potencial de retenção dos íons é avaliado através da construção de 33 isotermas de sorção resultantes de ensaios de equilíbrio em lote. Seus parâmetros são obtidos da adequação dos modelos linear, Freundlich e Langmuir as isotermas. O potencial de transporte dos íons é avaliado através de ensaios de difusão em colunas estanques, onde curvas teóricas produzidas no programa POLLUTE são ajustadas aos dados de concentração para a estimativa dos parâmetros de difusão. Os resultados dos estudos de retenção mostram que o \'CL POT.-\' experimenta sorção sob certas condições, o \'K POT.+\' e \'F POT.-\' possuem taxas semelhantes de retenção, e que a sorção do \'CD POT.2+\' se mostrou altamente dependente da composição da solução contaminante. Os ensaios de difusão resultaram em vários valores para o coeficiente de difusão efetiva (D*), mostrando que o fluxo difusivo dos íons diminui para solos mais argilosos. / There is a growing demand for materials that can be used in lining systems for waste disposal facilities. The use of tropical soils for this purpose is restricted due to the lack of technical knowledge about this type of material. Attempts to improve the quality of supposed unsuitable soils play also an important role. This thesis presents studies concerning the migration of the ions \'CD POT.2+\', \'K POT.+\', \'CL POT.-\' and \'F POT.-\' in latosoils from the Botucatu and Serra Geral formations and three different mixtures of these soils, with emphasis on sorption and molecular diffusion. Batch equilibrium tests were conduced to examine the retention capacity of the soils, and the sorption parameters are estimated by fitting the linear, Freundlich and Langmuir models to the experimental isotherm plots. The potential for these soils to transport ions is evaluated by diffusion tests, where theoretical curves produced in the POLLUTE code are used to estimate the effective diffusion coefficients (D*). The results show that the \'CL POT.-\' is sorbed under specific conditions and, therefore, it can not always be used as a conservative ion (i.e. nonreactive ion), \'K POT.+\' and \'F POT.-\' are sorbed in similar amounts, and that the \'CD POT.2+\' sorption is highly dependent on the contaminant solution composition. Several D* values are reported, showing that the diffusion rates are decreased for soils with more clay.

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.

Membrane excitability, gene expression, and neurotransmitter release are all controlled by voltage-gated L-type Ca2+ (L- )channels. In turn, Ca2+ channels are highly regulated by signal transduction cascades initiated by G protein-coupled receptor (GPCR) activation. In medium spiny neurons of the striatum, both the muscarinic M1 receptors (M1R) and dopaminergic D2 receptors (D2R) specifically inhibit the Cav1.3 L-channel. In Chapters III and IV, the pathways downstream of M1Rs and D2Rs are examined to determine whether an overlap or intersection in inhibition of Cav1.3 occurs by these two receptors. Transient transfection of Cav1.3 channels in HEK 293 cells, stably transfected with the M1R, and in ST14A cells were used as model systems. While a further characterization of ST14A cells determined that they exhibit a striatal profile, D2Rs or M1Rs did not inhibit Cav1.3. Lack of current inhibition may be due to the finding of no detectable expression of phospholipase Cβ-1 protein in ST14A cells. Ca2+ channels are multiprotein complexes comprised of α1, β, and α2δ subunits. While the actions of arachidonic acid (AA) have been shown to mimic M1R inhibition of L-current in superior cervical ganglion neurons, the precise identity of the L-channel in these neurons -either Cav1.2 or Cav1.3 or both- is not known. The transfected model systems allowed for the analysis of whole-cells currents with different β subunit combinations as well as the study of only Cav1.3 channels. In Chapter III, I show that activation of M1Rs with the agonist Oxo-M inhibited Cav1.3 channels coexpressed with either β1b, β2a, β3, or β4 subunits. Surprisingly, the magnitude of Cav1.3, β2a currents was inhibited less than Cav1.3 currents with other β subunits. In Chapter V, AA is shown to mimic the profile of M1R stimulation on Cav1.3 currents. The magnitude of Cav1.3, β2a currents was inhibited less than Cav1.3 currents with other β subunits by AA. This discovery points to a novel role for accessory β subunits in altering the magnitude of AA inhibition and kinetic changes of Cav1.3. Arachidonic acid (AA) inhibits Ca2+ channels by an unknown mechanism at an unknown site. In Chapter V, I found that Cavl.3 inhibition by AA was state-dependent and most likely stabilizes a closed channel conformation. The finding that the Ca2+ channel accessory β subunit alters the magnitude of AA inhibition and kinetic changes of Cav1.3 suggests that AA could alter processes which rely on L-channels such as Ca2+-dependent gene expression, secretion and membrane excitability.

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.