<p> The pharmacology of autonomic innervation to the peripheral skin conductance (SC) effector was studied. The drugs used included atropine, bretylium, acetylcholine (ACh), epinephrine (EPI), and amphetamine. Drugs were administered by iontophoresis (IPS) and by local subcutaneous injection. Although several IPS procedures were used, all proved to be inefficient and unreliable. Subsequent experiments using atropine and ACh supported the theory that innervation to the peripheral SC effector was mainly cholinergic. However, results obtained using EPI suggest that an adrenergic component might also be involved. It was concluded though that this component probably had little physiological significance. Experiments using amphetamine and bretylium were inconclusive. A comparison of behavioral and drug induced changes in SC suggested that the psychological relevance of SC might be improved through a range-correction based on pharmacologically determined SC range scores.</p> / Thesis / Master of Arts (MA)

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Physiologic β-adrenergic activation of PKA during the sympathetic “fight-or-flight” response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. The second chapter of this dissertation describes our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Recent data confirm that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites on α1C or β2B subunits. Proximity proteomic labeling methods led us to other potential PKA targets near the CaV1.2 complex, including Rad, a calcium channel inhibitor that changes its position within the calcium channel neighborhood after β-adrenergic stimulation. With expression of α1C, β2B, and Rad in a heterologous expression system, we reconstituted forskolin-PKA regulation of CaV1.2. By mutating potential PKA phosphorylation sites on Rad, we identified specific residues that are critical for this mechanism to occur and validated Rad as the functional PKA target for regulation of CaV1.2. In the third chapter, we probe the contribution of both CaV1.2 α1C and β subunits in β-adrenergic regulation. Previous results have shown that binding between α1C and β subunits is required for adrenergic stimulation of the calcium channel in the heart. Using transgenic mouse models, we demonstrate that this phenomenon requires a rigid IS6-AID linker in the α1C subunit, as introduction of glycines in this region increased flexibility of the linker and abolished a response to adrenergic agonists even though α1C was able to bind to β. The fourth chapter examines the role of the auxiliary β subunit in β-adrenergic regulation of CaV1.2. Binding of Rad to the β subunit is also necessary for this mechanism to occur. Although the β2B isoform is the predominant subunit in the heart, we show that transgenic mice with β3 or β4 replacing β2B in the heart are viable and still have normal β-adrenergic regulation of CaV1.2, indicating that this mechanism is universal to other voltage gated calcium channels that bind to β subunits and RGK proteins. The fifth chapter verifies that Rad is the PKA target in the heart. Using a knock-in mouse model with four PKA phosphorylation sites mutated to alanine, we definitively show that phosphorylation of Rad is necessary for β-adrenergic regulation of CaV1.2 in the heart. We investigate the importance of Rad phosphorylation on many levels. First, we study Rad’s role in regulating the calcium channel. Second, we observe the effect phosphorylation of Rad has on the calcium transient using isolated cardiomyocytes. Third, we examine cardiovascular function in vivo using radiotelemetry and echocardiograms. Finally, we assess the “fight-or-flight” response in an animal model with exercise capacity testing. Together, these findings conclusively show that in the heart, phosphorylation of Rad is the essential mechanism for the sympathetic nervous system control of calcium influx in both atrial and ventricular cardiomyocytes. Additionally, Rad modulates both heart rate and contractility in vivo. In the sixth chapter, we explore the mechanism of Rad modulation of CaV1.2 in depth using a flow-cytometry Förster resonance energy transfer (FRET) two-hybrid assay. We closely examine the roles of phosphorylation sites on both Rad’s N-terminus and C-terminus. By creating phosphomimetic mutations on Rad, we uncover the importance of phosphorylating the C-terminus for release of Rad from both the membrane and the β subunit. Taken together, these findings elucidate the mechanism behind β-adrenergic regulation of CaV1.2 in the heart – a longstanding query for over forty years in the cardiovascular ion channel field. At baseline, Rad “tunes” the amount of calcium influx into the cell by inhibiting a population of channels as a functional reserve. Upon adrenergic stimulation, Rad is phosphorylated, lessening its interaction with the membrane, and releasing inhibition of the calcium channel. The enhanced local calcium influx allows for additional calcium release into the cytoplasm through ryanodine receptors leading to increased contractility of the heart, a notable characteristic of the evolutionary survival mechanism— “fight-or-flight.”

Pneumonic pasteurellosis is a significant disease in beef production medicine. The most recent information suggests that this disease is a $700 million dollar per year economic burden in bovine food animal production The medical and pathological characteristics of this disease are well documented. Many pathological findings associated with pneumonic pasteurellosis may be explained by disruption of the pulmonary vascular adrenergic system. However, only a limited amount of research has addressed the adrenergic system and its relationship to the etiology and pathophysiology of this disease. In an attempt to further investigate the contributions of the vascular adrenergic receptor mechanism to the development of pneumonic pasteurellosis a series of six experiments have been completed. It is to be noted, that in 1999 the organism Pasteurella haemolytica was renamed Mannheimia haemolytica. The name change was based on the taxonomic features of the organism from other closely related organisms, in particular Pasteurella multocida.. The differences noted were identified and described by Dr. Mannheim in 1974. The familiarity of the past nomenclature and the lack of familiarity for the new nomenclature suggests that the more commonly recognized name of Pasteurella haemolytica should be used throughout this document. Scientific evidence suggests that the disruption of the normal homeostatic mechanisms of the pulmonary vasculature to beta adrenergic agents may be part of the etiology of pneumonic pasteurellosis. The dynamics and kinetics of the involvement of the beta receptors, following prophylactic vaccination and in the disease state, has yet to be fully investigated with respect to the events associated with pneumonic pasteurellosis. Evaluation of the time frame of the onset and duration of the events associated with the disruption of pulmonary vascular beta adrenergic receptor mechanisms revealed that an escalating level of dysfunction occurs over the first 24-48 hour period after exposure to parenteral Pasteurella haemolytica and lasts for at least 21 days. A component of P.haemolytica organism or contained in the vaccine using the organism is likely associated with the disruption of vascular beta adrenergic mechanism. This factor is, as yet, not specifically identified, however the likely culprit is the lipid A moiety of the endotoxin. Using the well defined and purified Escherichia coli endotoxin, trials were run to examine the effect of endotoxin on the pharmacological response of vascular associated beta adrenergic receptor mechanisms. The effects of Escherichia coli endotoxin, administered parenterally, on beta adrenergic receptor mechanisms were pharmacologically indistinguishable from those effects following parenterally administered Pasteurella haemolytica. The nature of the disruption in the beta adrenergic receptor remains a mystery. The receptor mechanism involves at least two second messengers to initiate vascular relaxation. Initial activation of the beta adrenergic receptor with a beta selective drug starts a cascade of events involving adenylylate cyclase and cyclic adenylylate monophosphate (cAMP) and nitric oxide. A disruption in the receptor mechanism, as a result of the parenteral administration of Pasteurella haemolytica, which is "upstream" of adenylyl cyclase, would result in a diminished amount of cAMP when compared to the unvaccinated negative controls. An investigation of cAMP accumulation, at the receptor level was inconclusive. The assessment of some previously used vaccines has demonstrated that there is an, as yet unidentified virulence factor, associated with these vaccines that results in the pharmacological disruption of beta adrenergic receptor mechanisms. Two newer vaccines, Once PMH® and One Shot® have been evaluated and there is evidence to suggest that these currently used vaccines also have the ability to disrupt beta adrenergic receptor mechanisms in rats. The effects of parenteral P. haemolytica on the alpha-2 adrenergic receptor mechanism, is described. The alpha-2 receptor mechanism, unlike the beta receptor mechanism appears to increase the amount of vasoconstriction. The possibility that the alpha-2 adrenergic receptor could also mediate vasorelaxation under certain conditions was investigated. The evidence suggests that in the presence of high alpha-1 mediated vascular tone, the alpha -2 receptor can cause vasorelaxation. Evidence, from other scientists active in this area of investigation, suggests that a vasorelaxation response may be mediated by nitric oxide. Elimination of the nitric oxide mediated relaxation may offer an explanation for the increased vasoconstriction noted with alpha-2 selective drugs after exposure to parenteral P. haemolytica. Finally, the importance of the beta adrenergic receptor to the disease process is addressed by elucidation of one of the mechanisms by which Micotil 300® (tilmicosin phosphate) acts to improve cattle with symptomatic pneumonic pasteurellosis. The rapid improvement of animals on Micotil 300®, with-in 24 hours suggests that there is a mechanism beyond the antimicrobial effect of the drug that mediates the clinical improvement. Evaluation of the effect of Micotil 300® demonstrates a pharmacologically measurable amount of beta adrenergic activity with respect to the bovine pulmonary artery and vein. Based on the conclusions drawn as a result of these experiments, the adrenergic system in general, and the beta adrenergic system in particular are important to the development of pneumonic pasteurellosis in cattle. The beta adrenergic system is affected by endotoxin. Further, these receptors maybe responsible for the mediation of the pathological and clinical signs associated with pneumonic pasteurellosis. In conclusion, these investigations have suggested, that it is likely that a disruption in the homeostatic mechanisms mediated by the beta and alpha-2 adrenergic receptors are intimately involved in the development of post vaccination receptor failure as well as the pathophysiology associated with pneumonic pasteurellosis in cattle. / Ph. D.

Yes / The Sphingosine-1-phosphate receptor 1 (S1PR1) and β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCRs) expressed in the heart. These two GPCRs have opposing actions on adenylyl cyclase due to differential G protein-coupling. Importantly, both of these receptors can be regulated by the actions of GPCR kinase-2 (GRK2), which triggers desensitization and down-regulation processes. Although, classical signaling paradigms suggest that simultaneous activation of β1ARs and S1PR1s in a myocyte would simply be opposing action on cAMP production, in this report we have uncovered a direct interaction between these two receptors with a regulatory involvement of GRK2. In HEK293 cells overexpressing both β1AR and S1PR1, we demonstrate that β1AR down-regulation can occur after sphingosine 1-phosphate (S1PR1 agonist) stimulation while S1PR1 down-regulation can be triggered by isoproterenol (βAR agonist) treatment. This cross-talk between these two distinct GPCRs appears to have physiological significance since they interact and show reciprocal regulation in mouse hearts undergoing chronic βAR stimulation and also in a rat model of post-ischemic heart failure (HF). We demonstrate that restoring cardiac plasma membrane levels of S1PR1 produce beneficial effects counterbalancing deleterious β1AR overstimulation in HF.

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.