Browsing by Subject "Nervous System"
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Item Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid(1994-06-01) Taylor, Martin T.; Turner, James; Yorio, Thomas; Wordinger, Robert J.Taylor, Martin T., Anatomical and Biochemical Characterization of the Porcine Spinal Arachnoid. Doctor of Philosophy (Biomedical Sciences), June, 1994. Mast cell products modulate many biologic barrier systems. In the central nervous system (CNS) two such barriers are the blood-brain barrier (B-B-B) and its analogous cerebrospinal fluid-blood barrier (CSF-B-B). Published research has demonstrated that mast cell products increase the permeability of the BBB, but no comparable research has been described for the CSF-B-B. The main goal of this dissertation was to begin to assess the role of mast cell products on the chief component of the CSF-B-B, the arachnoid mater. Specifically, the hypothesis that mast cell products modulate arachnoid function through receptor mediated second messenger system regulation was postulated. Initially, the light and electron microscopic features of the porcine spinal meninges generally and the arachnoid mater specifically were characterized and found to be similar to those of other species and CNS regions. In addition, mast cells were found to be present in the meninges where their product could affect the arachnoid mater. To study the effects of the selected mast products on the arachnoid mater, arachnoid cells were isolated and cultured. Morphologic, immunohistochemical, and physiological studies confirmed the cultured cells were arachnid cells and that they were capable of developing attributes of a barrier membrane in vitro, (e.g. tight junctions, increased transcellular resistance). The effects of mast cell products on arachnoid cells were then assessed biochemically. Arachnoid cells were found to produce cyclic adenosine monophosphate (cAMP) in response to forskolin and prostaglandin D2 (PGD2). Histamine inhibited both forskolin and PGD2 stimulated production of cAMP. Additionally, arachnoid cells produced inositol phosphates (IP) in response to carbachol and histamine via muscarinic and H1-histamine receptors respectively. Since histamine and PGD2 are produced and released by activated mast cells, and since cAMP and IP levels are known to modulate cellular barrier systems, it is concluded that meningeal mast cells and their products may regulate or modulate permeability of the CSF-B-B. An understanding of the specific biochemical actions of mast cell products on the arachnoid may ultimately aid in the understanding of many physiologic and pathologic processes involving the arachnoid such as hydrocephalus, subarachnoid and subdural hemorrhages, cerebral edema, meningitis, and meningiomas.Item Anionic Ligand-Gated Ion Channels: The Convulsive Site and Mechanism of Action(2001-08-01) Dibas, Mohammed I.; Hriday Das; Thomas Yorio; Neeraj AgarwalDibas, Mohammed, Anionic Ligand-Gated Ion channels: The Convulsive Site And Mechanism of Action. Doctor of Philosophy (Biomedical Sciences), August 2001, pp153, 1 table, 24 illustrations, 76 titles. Picrotoxin, a CNS convulsant inhibits all anionic ligand gated ion channels. The mechanism and the binding site for picrotoxin and its related ligands are still undefined. The second transmembrane (TMII) domain of these ligand gated ion channels is found to play a key role in the mechanism of block by picrotoxin. It has been shown that the incorporation of a phenylalanine residue in place of threonine at position 6’ within the TMII domain of B2 subunit conferred high resistance toward picrotoxin in GABAA a3B2(T6’F)y2 receptors. Mediating their blocking effect through the PTX-site, PTZ, TBPS, and U-93631 lost their inhibitory effects due to the same mutation B2(T6’F). Interestingly, this mutation uncovered a low affinity, highly efficacious stimulatory site for PTZ. PTZ seems to mediate its stimulatory effect through a novel distinct site different from that for benzodiazepine. The effect of varying subunit configuration of GABAA receptors dramatically affected the ability of the mutation B2(T6’F) to abolish the inhibitory effect of picrotoxin. While picrotoxin failed to block the current induced by GABA in a3B2(T6’F)y2 receptors, picrotoxin partially blocked the current in a3B2(T6’F)y2 receptors. In B2(T6’F)y2 receptors, picrotoxin restores its full efficacy. When phenylalanine was incorporated at position 6’ in the a1 subunit, picrotoxin completely blocked the current induced by GABA in a1(T6’F)B2y2 receptors. The combined results showed that the ability of (T6’F) mutation to regulate the inhibitory mechanism of picrotoxin as dependent on the subunit configurations and at which subunit is mutated. In addition, picrotoxin is known to inhibit GABAA receptors in use-facilitated mechanism, while it inhibits the glycine receptor in a non-use facilitated fashion. The molecular determinant behind the use-facilitated mechanism was modulated by the nature of the amino acid at position 15’ within the second transmembrane domain. The mutation of serine 15’ to either glutamine or asparagine in the glycine a1 receptors converted picrotoxin from a non-use facilitated blocker to a use-facilitated one. The latter finding suggested that this residue might residue within the PTX binding site or play a key role in the transduction pathway for picrotoxin mechanism. The overall results further support the fact that TMII domain plays a key role in the picrotoxin mechanism.Item Arterial Baroreflex Control of Muscle Sympathetic Nerve Activity(2000-07-01) Fadel, Paul Joseph; Peter B. Raven; Michael Smith; Patricia GwirtzFadel, Paul Joseph, Jr., Arterial Baroreflex Control of Muscle Sympathetic Nerve Activity. Doctor of Philosophy (Biomedical Science), July 2000; 100 pp; 3 tables; 10 figures; bibliography. Arterial baroreflex control of sympathetic nerve activity is dependent on afferent nerve activity emanating from both the aortic and carotid baroreceptors. While several investigations have reported that the aortic baroreceptor reflex dominates in the baroreflex control of heart rate in humans, the role of carotid and the aortic baroreceptors in the control of sympathetic nerve activity remains unclear. In addition, the effect of exercise and long term endurance training on baroreflex-sympathetic nerve activity responses requires further definition. Therefore, the purpose of the investigations described within this dissertation was to: i) describe carotid baroreflex (CBR) control of muscle sympathetic nerve activity (MSNA) at rest and during exercise, ii) examine the relative contribution of the carotid and aortic baroreflexes to the overall arterial baroreflex control of MSNA during acute hypotension, and iii) determine the effect of fitness on arterial baroreflex control of MSNA. In the first investigation, we constructed stimulus-response relationships for CBR control of MSNA at rest and during dynamic arm cycling and demonstrated that carotid baroreflex control of MSNA was reset to function at the higher arterial pressures induced by exercise without a change in reflex sensitivity. Thus, we concluded that the carotid baroreflex control of MSNA was preserved during dynamic exercise. In the second investigation, acute hypotension was induced non-pharmacologically by releasing a unilateral arterial thigh cuff (300 Torr) following nine minutes of resting ischemia under two conditions: control (aortic and carotid baroreflex deactivation) and suction (aortic baroreflex deactivation alone). The application of neck suction to negate the CBR during cuff release caused a significant attenuation of the MSNA response and a greater decrease in mean arterial pressure; thereby signifying the importance of the CBR in the control of MSNA and maintenance of arterial blood pressure. However, when the drop in carotid sinus pressure was counteracted with neck suction a significant MSNA response was noted, indicating the dominance of the aortic baroreflex control of MSNA. Furthermore, a comparison between high-fit (HF) and average fit (AF) subjects indicated that despite an augmented baroreflex control of MSNA, HF subjects exhibited a greater decrease in mean arterial pressure compared to AF subjects. Thus, it appeared that although the arterial baroreflex appropriately increased the MSNA response to hypotension, the regulation of blood pressure remained attenuated in the HF subjects. We contend that an impaired control of vasomotion hinders blood pressure regulation in high-fit subjects.Item Baroreflex Mediated Autonomic Modulation by Acute Pain and Orthostatic Stress(2008-10-01) Raven, Joseph Simon; James Caffrey; Joan Carroll; Robert MalletRaven, Joseph Simon, Baroreflex Mediated Autonomic Modulation by Acute Pain and Orthostatic Stress. Doctor of Philosophy (Integrative Physiology), October 2008, 147 pp.; 23 figures; bibliography; 123 titles. Nociceptive and baroreceptor afferent neurons are implicated as the components responsible for carotid baroreceptor reflex (CBR) resetting. The purpose of this dissertation was to identify the effect of cold induced pain, and cardiopulmonary baroreceptor (CPBR) unloading accompanied by pain, on CBR resetting. First, the relationships between cold induced pain to cardiovascular responses, pain perception, and muscle sympathetic nerve activity (MSNA) were investigated. Questions were addressed through use of the cold pressor test (CPT), finger plethysmography, and microneurography. This study demonstrated perceived pain, MSNA, and blood pressure responses to a cold stimulus were reproducible. Furthermore, graded responses observed in mean arterial pressure (MAP) and MSNA directly correlated to the intensity of the pain stimulus. The next study examined cold induced pain on CBR gain and operational point resetting in healthy normotensive subjects. Using similar experimental methodologies to the previous study, the data demonstrated acute pain shifted the CBR operational point toward the lower limiting value of MSNA. These data also confirmed an upward-rightward shift and increased gain of the CBR function curve during pain. Finally, CBR gain and operational point resetting during simultaneous CPBR unloading and cold induced pain in healthy normotensive subjects was addressed. Using the previous experimental paradigm, this investigation revealed CPBR unloading during acute pain did not abolish the shift of the CBR operational point. Thus, the capacity for hypotensive buffering remained enhanced. This study also determined CPBR unloading during acute pain produced higher prevailing blood pressures compared to periods of CPBR unloading alone. In summary: 1)MSNA and cardiovascular responses were tightly coupled to pain. 2) The CPT was a reliable technique for producing repeated sympathoexcitation within a subject. 3) Acute pain increased CBR gain and induced a shift of the CBR operational point. 4) The CBR operational point shift remained in the presence of CPBR unloading, which precipitated increased MAP during hypotensive stimuli. These findings suggested pain improves blood pressure maintenance during central hypovolemic stress.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Effects of Endurance Training on Aortic and Carotid Baroreflex Function(1999-06-01) Smith, Scott Alan; Peter B. Raven; Michael Smith; Patricia A. GwirtzSmith, Scott Alan, Effects of Endurance Training on Aortic and Carotid Baroreflex Function. Doctor of Philosophy (Biomedical Sciences), June 1999; 122 pp; 8 tables; 10 figures; bibliography, 148 titles. Arterial bareflex control of cardiac function is dependent upon afferent input from both the aortic arch and carotid sinus bareceptors. Extensive research in animals has generated conflicting results as to the range of arterial pressures over which each baroreflex operates. Further, the complex integration of afferent signals within the medullary cardiovascular center, in reference to aortic and carotid baroreceptor input, has been characterized as additive, inhibitory, and facilitatory in nature. Such reports make it difficult to draw definitive conclusions about the behavior or central neural processing within the brainstem. In addition, these relationships have yet to be examined in humans. Therefore, the purpose of the investigations described herein, was to quantify the range of pressures over which the arterial aortic and carotid baroreflexes operate as well as to describe the interactive relationship between the aortic and carotid baroreceptors. In order to investigate these questions, we isolated the arterial, aortic, and carotid-cardiac baroreflexes in volunteer subjects generating sigmoidal stimulus-response curves for each reflex arc. Arterial and aortic baroreflex (ABR) control of heart rate (HR) was assessed by inducing graded increases and decreases in mean arterial pressure (MAP) by bolus infusion of the vasoactive agents phenylephrine (PE) and sodium nitroprusside (SN), respectively. Carotid baroreflex (CBR) function was determined utilizing ramped five second pulses of both pressure and suction applied to the carotid sinus via a neck chamber collar, independent of drug administration. The MAP at which the threshold and saturation were elicited did not differ among the reflexes examined indicating each reflex operated over a similar range of arterial pressures. Further, the simple sum of the independently driven HR response ranges of the CBR and ABR was significantly greater than that produced when both baroreceptor populations were concomitantly stimulated (i.e. arterial baroreflex) suggesting an inhibitory interaction. To investigate differential baroreflex control of HR in response to chronic endurance exercise training, a second investigation was designed implementing the reflex isolation techniques described previously. Stimulus-response relationships were compared between high fit (maximal oxygen uptake, VO2max [greater than] 60ml˖kg-1˖min-1) and average fit (VO2maxml˖kg-1˖min-1) individuals. Interestingly, neither the range of operating pressures for each reflex nor the integrative relationship between the ABR and CBR were altered as a result of aerobic training. However, the HR response range elicited from the aortic baroreceptors as a result of hypotensive and hypertensive insult was markedly attenuated in the aerobically trained population compared to their sedentary counterparts, exclusively causing a requisite reduction in arterial baroreflex sensitivity.Item Elucidation of Mechanism and Molecular Determinants Important in Picrotoxin Action in the 5-Hydroxytryptamine Type 3 Receptor(2003-09-01) Das, Paromita; Basu, Alakananda; Forster, Michael J.; Luedtke, Robert R.Das, Paromita, Elucidation of mechanism and molecular determinants important in picrotoxin action in the 5-hydroxytryptamin type 3 receptor. Doctor of Philosophy (Pharmacology and Neuroscience), September 2003, pp. 192, 3 tables, 26 illustrations, 67 titles. The 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels (LGIC), which mediate fast neurotransmission. Till date, only two subtypes of the receptor i.e. 5-HT3A and 5-HT3B have been investigated. The GABAA receptor antagonist picrotoxin inhibits other anion-selective members of the LGIC. Whether PTX inhibits the cation-selective 5-HT3 receptors was previously unknown. Thus, the primary goal of this study was to elucidate the mechanism of action of PTX and identify the amino acids involved in the action of PTX in 5-HT3 receptors. The overall hypothesis tested was that PTX inhibits the 5-HT3 receptor by interacting in the ion channel. PTX-mediated blockade of the 5-HT3A receptors was non-competitive and use-facilitated similar to GABAA receptors suggesting a conserved site of action of these ligands. The inhibitory effect of PTX was reduced drastically in heteromeric 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in native tissue and recombinant receptor preparations. In anion-selective ion channels, the 2’, 3’ and 6’resides in cytoplasmic aspect of TM2 are known to modulate PTX sensitivity. While mutation of 2’ and 3’ residues in 5-dramatic loss of sensitivity to PTX in 5-HT3A receptors. A converse mutation at 6’ residue in the 5-HT3B subunit caused gain of sensitivity to PTX, suggesting that 6’ is a key determinant of PTX sensitivity. A novel finding was the involvement of 7’ residue in increasing PTX sensitivity in 5-HT3A but not the 5-HT3B subunit. The lack of specific binding by radioligand [3H]EBOB in 5-HT3A receptors suggested that the site of action of convulsants may be different from that anion-selective receptors. The overall results suggest that PTX interacts from that in the anion-selective receptors. The overall results suggest that PTX interact in the ion channel in the 5-HT3 receptors but also underscores the complexity of its interaction with LGICs.Item Endothelin receptor-mediated neurodegeneration in glaucoma(2017-08-01) McGrady, Nolan; Krishnamoorthy, Raghu R.; Yorio, Thomas; Clark, Abbot F.Primary open-angle glaucoma (POAG) is a complex set of optic neuropathies which are characterized by the degeneration of the optic nerve, cupping of the optic disk and loss of retinal ganglion cells (RGCs). There are approximately 3 million Americans who currently suffer from this disease although this is most likely an underestimation since many individuals with glaucoma are unaware that they have the disease. POAG is an age-related disease progressing slowly over the course of several decades and is most commonly associated with an elevation in intraocular pressure (IOP). Currently available treatments for glaucoma, both surgical and pharmacological, are solely focused on the regulation of IOP; nevertheless, some individuals continue to show progressive damage despite being on available therapies. In recent years, there has been increased momentum towards the development of neuroprotective strategies for POAG, particularly in preclinical models of glaucoma. Despite these efforts, there is still no neuroprotective treatment currently available for glaucoma patients. A potential target for the development of a neuroprotective approach is the endothelin system of peptides and their receptors. The endothelin (ET) system is composed of three vasoactive peptides (ET-1, ET-2 and ET-3) which are comprised of 21-amino acids. The peptides bind to two G-protein coupled receptors (ETA and ETB receptors) leading to activation of numerous signal transduction pathways. Although originally described for its role in the vasculature, all components of the ET system has been shown to be expressed in multiple tissues and cell types and are responsible for diverse cellular effects. Clinical studies have demonstrated an increase in ET-1 concentrations both in the aqueous humor and plasma of glaucoma patients. A previous study by our lab, using a rodent model of ocular hypertension, showed that endothelin B (ETB) receptor expression is increased when compared to control eyes and contributes to neurodegeneration (Minton et al., 2012). Preliminary data in the current study, using Brown Norway rats, demonstrated that ETA expression is also increased in the IOP elevated eyes, suggesting the possibility that the ETA receptor might also have a degenerative role during ocular hypertension. We hypothesize that the ETA expression increases following IOP elevation and contributes to the neurodegeneration of retinal ganglion cells and their axons. To test this hypothesis we employed a well-characterized in vivo model of glaucoma as well as multiple cellular and molecular approaches to understand the role of the ETA receptor in glaucomatous degeneration. Our data suggest that overexpression of the ETA receptor promotes cell death in cultured RGCs. Since both ETA and ETB receptors appear to contribute to neurodegeneration, we tested the ability of an FDA approved medication, macitentan, for neuroprotection in the Morrison model of glaucoma in rats and found it to promote RGC survival. Our studies raise the possibility of testing macitentan as a neuroprotective treatment for glaucoma patients.Item Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Identification of Actin Binding Proteins Associated with Cross-Linked Actin Networks(2006-12-01) Mills, Christy E.; Clark, Abbot F.; Yorio, Thomas; Wordinger, Robert J.Mills, Christy E., Identification of Actin Binding Proteins Associated with Cross-Lined Actin Networks. Master of Science (Pharmacology and Neuroscience), December 2006, 95 pp., 9 tables, 16 figures, references, 122 titles. Glucocorticoid therapy can leady to ocular hypertension and glaucoma. The purpose of this study is to examine mechanisms contributing to increased intraocular pressure using tissue culture models of steroid-induced ocular hypertension through identification of specific actin-binding proteins associated with cross-linked actin network (CLANs). Human trabecular meshwork ™ cells were cultured to confluence and treated with dexamethasone or vehicle for 14 days. Total RNA was extracted for gene expression analysis to confirm steroid-induced expression of actin binding proteins in human TM cells. Western blots confirmed expression of actin binding proteins and demonstrated the specificity of selected antibodies. Fluorescence microscopy of treated TM cells showed cytoskeleton rearrangements from linear actin stress fibers to cross-linked actin networks and the position of candidate proteins in relation to CLANs. Dexamethasone treatment of TM cells altered the expression of actin-associated proteins that may be important in the formation of CLANs and increased outflow resistance.Item Influence of the Carotid Baroreflex on Cerebral Blood Flow During Seated Upright Rest(2007-07-01) Eubank, Wendy L.; Peter B. Raven; Robert Mallet; James CaffreyEubank, Wendy L., Influence of Carotid Baroreflex on Cerebral Blood Flow During Seated Upright Rest. Master of Science (Integrative Physiology), July, 2007, 25 pp., 1 table, 4 illustrations, 34 references. This study tested the hypothesis that sympathetic activation via the carotid baroreflex directly influences cerebral vasomotion during seated upright test. This study also examined the effects of pulsatile neck pressure (NP) and neck suction (NS) during seated upright rest in healthy human subjects. Changes in mean arterial pressure (MAP) and mean middle cerebral arterial velocity (MCA V mean), were measured. The power spectral density (PSD) of MAP of 0.1Hz increased during pulsatile NP and NS. The PSD of MCA V mean at 0.1Hz was much greater during NP than that of NS. There were no significant differences between end-tidal CO2 between each condition. These findings suggest that cerebral vasoconstriction during NP was a result of the autoregulatory response to the NP mediated pulsatile changes in arterial pressure and the NP induced sympathetically mediated vasoconstriction.Item Leucine-Enkephalin and Sympathetic Control of Heart Rate(2001-12-01) Stanfill, Amber; Caffrey, James L.; Downey, H. Fred; Shi, XiangrongStanfill, Amber A., Leucine-enkephalin and Sympathetic Control of Heart Rate. Master of Science (Biomedical Sciences), December, 2001, 51 pp., 1 table, 4 figures, references, 48 titles. The following study examined the role of leucine-enkephalin in the sympathetic regulation of the cardiac pacemaker. Leucine-enkephalin (0.3 mM) was administered, by microdialysis into the interstitium of the sinoatrial node in 10 mongrel dogs in conjunction with either sympathetic nerve stimulation or infused norepinephrine. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and stimulated to produce graded (low, 20-30; high 40-50 bpm) increases in heart rate. Once stimulation frequencies were determined, leucine-enkephalin (0.3mM) was added to the dialysis inflow and perfused at 5: 1/min thereafter. The sympathetic stimulations were repeated after 5 and 20 min exposure to leucine-enkephalin. The resulting increases in heart rate during sympathetic stimulation were attenuated at both low (18.2 ±1.3 to 11.4 ±1.4 bpm) and high (45 ±1.5 to 22.8 ±1.5 bpm) frequency stimulation. The degree of inhibition was nearly identical after 20 minutes exposure providing no evidence for a progressively evolving response and for desensitization. Vagal function was also evaluated at 5 and 20 min by stimulating the right cervical vagus at 1 and 3 Hz. Leucine-enkephalin reduced the vagal bradycardia approximately 50% at both time intervals. The administration of the delta-selective opioid antagonist, naltrindole, restored only one third of the sympathetically medicated tachycardia. The same dose of naltrindole completely reversed the coincident vagolytic of leucine-enkephalin. These observations suggested that the sympatholytic effect was either non-opioid or mediated by a different opioid receptor subtype. Study two was conducted to determine if the sympatholytic effect was prejunctional and post-junctional in character. Norepinephrine was added to the dialysis inflow into the SA node in a concentration (6-9 μM) sufficient to produce an intermediate increase in heart rate. The average increase in heart rate was 35.2 ±1.8 bpm. Leucine-enkephalin was then combined with norepinephrine and sympathetic and parasympathetic responses were recorded at 5-min intervals for 20 minutes. The tachycardia mediated by added norepinephrine was unaltered by leucine-enkephalin or the subsequent addition of naltrindole. At the same time intervals, vagal control of heart rate was reduced by more than 50% and then completely restored by naltrindole. When combined with observations in study one, the data support the conclusion that the local nodal sympatholytic effect of leucine-enkephalin was the result of a reduction in the effective interstitial concentration of norepinephrine and not the result of a post-junctional interaction between leucine-enkephalin and norepinephrine.Item Mechanisms of Chemoreflex Control of Muscle Sympathetic Nerve Activity and Blood Pressure in Humans(2004-05-01) Hardisty, Janelle M.; Smith, Michael; Shi, Xiangrong; Clark, MichaelHardisty, Janelle M., Mechanisms of Chemoreflex Control of Muscle Sympathetic Nerve Activity and Blood Pressure in Humans. Doctor of Philosophy (Integrative Physiology), May 2004. The mechanisms linking obstructive sleep apnea (OSA) and cardiovascular disease are not fully understood; however, studies report patients with OSA exhibit chronic elevations in muscle sympathetic nerve activity (MSNA). This appears to be due to altered chemoreflex control of MSNA, mediated primarily by hypoxia. Yet, a correlation between degree of hypoxia and chemoreflex control of MSNA is unknown. Therefore, it was evaluated whether degree of hypoxia occurring during apnea determines the sympathoexcitatory and blood pressure responses, and whether these responses are augmented in OSA patients. Additionally, it was studied whether altered chemoreflex function in OSA patients is predictive of blood pressure response to apnea. In a clinical setting, the blood pressure response to voluntary apnea was determined to evaluate whether this could be used as a non-invasive measure of chemoreflex gain in OSA. Finally, the effect of hyperoxia on MSNA was studied to determine whether 15 min of hyperoxia, following intermittent hypoxic apnea, reverses the elevation of MSNA and altered chemoreflex control of MSNA. Consistent with the hypotheses, a relationship between MSNA responses, blood pressure response and level of hypoxia were determined. MSNA and peak systolic pressure responses were augmented in OSA subjects (p≤0.05 and p≤0.05, respectively), as well as, chemoreflex gain (p≤0.05). Clinically, peak systolic pressure responses to apnea were augmented in OSA patients (p˂0.001). Finally, basal MSNA and chemoreflex control of MSNA, following hyperoxia, was not different from baseline through 180 min of recovery (p=0.940 and p=0.278, respectively). These data support the hypotheses that chemoreflex gain is predicative of the blood pressure response; and furthermore, the MSNA and blood pressure responses to hypoxic apnea are augmented in OSA. Additionally, peak systolic pressure responses to voluntary apnea are augmented in OSA. Additionally, peak systolic pressure responses to voluntary apnea are augmented in OSA patients and could possibly be used as a marker of chemoreflex gain. Moreover, these data support the hypothesis that hyperoxia can reverse basal sympathoexcitation and augmented chemoreflex control of MSNA, associated with hypoxic apnea, supporting that elevations in MSNA are hypoxia mediated.Item Mechanisms of Post-Apneic Symathoinhibition in Humans(2002-08-01) Swift, Nicolette Muenter; Michael Smith; David Barker; John R BurkMuenter Swift, Nicolette, Mechanisms of Post-Apneic Sympathoinhibition in Humans. Doctor of Philosophy (Biomedical Sciences), August, 2002, 110 pp., 14 figures, references. Apnea is accompanied by a concomitant rise in arterial pressure and muscle sympathetic nerve activity (MSNA), the latter primarily due to chemoreflex stimulation and possibly the lack of sympathoinhibitory input from pulmonary stretch receptors. The progressive sympathoexcitation during apnea suggests a possible overriding of arterial baroreflex sympathoinhibitory input to sympathoregulatory centers by apnea-induced sympathoexcitatory mechanisms. Nevertheless, it is unknown whether apnea attenuates baroreflex control of MSNA. Apnea termination is accompanied by a profound and immediate sympathoinhibition, the mechanisms of which are unclear; however, potential mediators include normalization of blood gases (i.e. chemoreflex unloading), the lung inflation reflex, and arterial baroreflex stimulation. Therefore, the purpose of the current studies was to: i) determine the contribution of chemoreflex unloading to post-apneic sympathoinhibition, ii) determine the contribution of the lung inflation reflex to post-apneic sympathoinhibition, and iii) determine whether carotid baroreflex control of MSNA is altered by apnea and its termination. The first study compared MSNA during post-apneic administration of room air versus a gas mixture designed to maintain the subjects’ end-apneic alveolar gas levels. Regardless of post-apneic gas administration, post-apneic MSNA was at or below baseline pre-apneic levels; thus; chemoreflex unloading does not contribute to post-apneic sympathoinhibition. Furthermore, quantification of post-apneic MSNA associated only with the low lung volume phase of respiration, when sympathoinhibitory input from the lung inflation reflex is minimal, demonstrated that post-apneic sympathoinhibition persists even during the low lung volume phase of respiration, when sympathoinhibitory input from the lung inflation reflex is minimal, demonstrated that post-apneic sympathoinhibition persists even during the low lung volume phase of respiration. Therefore, the lung inflation reflex does not appear to be the primary mediator of post-apneic sympathoinhibition. The second study utilized neck suction (NS) and neck pressure (NP) to assess carotid baroreflex function during and following sleep apnea. The sympathoinhibitory response to -60 Torr NS was maintained throughout apnea; conversely, the sympathoexcitatory response to +30 Torr NP was attenuated for nearly one minute post-apnea. Thus, carotid baroreflex control of MSNA is not altered by apnea but is transiently attenuated by apnea termination. We propose that the carotid baroreflex-MSNA function curve resets rightward and upward during apnea. Return of the function curve to baseline upon apnea termination may partly explain the reduced MSNA response to NP post-apnea.Item Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed(2000-08-01) Pearlman, Eric Brian; Barbara Barron; Patricia A. Gwirtz; Michael L. SmithPearlman, Eric B., Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed. Master of Science (Biomedical Science), August, 2000, 37 pp., 3 tables, 11 figures, references, 20 titles. Methionine enkephalin arginine phenylalanine (MERF) has been shown to be co-stored with catecholamines in vesicles. The catecholamines appear to decrease the degradation rate of 3H-MERF in vitro. The aim of this study is to investigate the spillover and metabolism of MERF across the canine heart vascular bed. I hypothesize that 3H-MERF is either degraded in the plasma or taken up and degraded by the heart. I further hypothesize that the exogenous catecholamine, isoproterenol, inhibits or reduces the rate of MERF degradation. Mongrel dogs were anesthetized and instrumented to record cardiovascular parameters, infuse 3H-MERF, and obtain blood samples across the heart. Blood samples were taken before and after stopping 3H-MERF infusion to evaluate kinetics, show steady state, and test the effect of treatments. Steady state concentration of 3H-MERF was observed after 30 min of infusion. Chromatography separated intact from degraded 3H-MERF. Three experimental groups were used: control, propranolol plus isoproterenol, and propranolol only. Blockade of β-receptors was necessary to prevent changes in coronary blood flow. Propranolol bolus (0.2 mg/kg) was administered IV at 50 min. 3 μg/min isoproterenol or 0.5 ml/min normal saline was infused starting at 70 min until the end of sample collection. The 3H-MERF venous-arterial (V-A) difference prior to treatment was negative, indicating degradation in the plasma or uptake and degradation by the heart. The 75 min V-A difference was used to calculate the effect of the infusions on the degradation or uptake of the 3H-MERF; this value was unchanged by any treatment. Spillover of 3H-MERF was significantly lower in the propranolol + isoproterenol dogs (p [less than] 0.05) compared to propranolol only treatment at 75 min. Heart rate was significantly lower for the propranolol only group compared to control. Blood pressure and change in coronary flow were unchanged. In conclusion, isoproterenol does not affect the metabolism of 3H-MERF across the canine heart vascular bed. Propranolol, however, does increase the intact 3H-MERF in the plasma, but additional β adrenergic blockade agents need to be investigated to determine the mechanism by which this takes place.Item Modulation of GABAA Receptor Function by Tyrosine Phosphorylation(1998-05-01) Fang, Mingjun; Glenn Dillon; Thomas Yorio; Eugene E. QuistMingjun, Fang. Modulation of GABAA Receptor Function by Tyrosine Phosphorylation. Master of Science (Biomedical Sciences), May, 1998, 32 pp., 6 illustrations, bibliography, 42 titles. The goal of this study was to determine the modulation of GABAA receptor function by tyrosine kinase phosphorylation, and to detect which subunit is phosphorylated to alter the GABA-induced chloride currents. From previous studies, we suggested that protein tyrosine phosphorylation may maintain GABAA receptor function. Here we tested the hypothesis that tyrosine phosphorylation modulates other GABAA receptor subtypes e.g., α1β2γ2 and α6β2γ2, and subsequently attempted to determine which subunit(s) may be phosphorylated. Our results support the hypothesis that PTK phosphorylation may maintain GABAA receptor function. In addition, we suggest this tyrosine phosphorylation occurs at the γ2 subunit of the receptor.Item Opioid Receptors in Aging and Oxidative Stress(2007-01-01) Raut, Atul M.; Ratka, Anna; Simpkins, James W.; Dillon, GlennRaut, Atul M., Opioid Receptors in Aging and Oxidative Stress. Doctor of Philosophy (Pharmacology and Neuroscience), January 2007, 181 pp, 4 illustrations, 21 figures, 159 titles. Oxidative stress has been implicated in aging and neurodegenerative disorders. Pain sensitivity and responses to opioids change with aging. The effect of aging and oxidative stress on opioid receptor system is not yet well understood. To study the effects of aging on pain sensitivity and opioid-induced antinociception, and to determine the possible association of oxidative stress with these pain parameters, in vivo studies were conducted. To further elucidate the effects of oxidative stress on opioid receptor proteins and their function, in vitro studies were carried out. The effects of aging on pain sensitivity and opioid-induced antinociception were studied in male Fischer 344 rats. Oxidative stress markers in cerebral cortex, hippocampus, striatum and midbrain of these rats were estimated. It was concluded that sensitivity to nociceptive stimulus increases and responses to opioids decrease with aging and age-related oxidative damage is negatively correlated with opioid-induced antinociception. To characterize the effects of oxidative stress on function of opioid receptors, changes in intracellular cyclic adenosine monophosphate (cAMP) was measured in human SK-N-SH neuronal cells under oxidative stress conditions. It was found that oxidative stress decreased the function of mu opioid receptor (MOR) but not that of delta or kappa opioid receptors (DOR and KOR respectively). Antioxidant intervention preserved the function of MOR. Western immunoassays revealed that MOR but not DOR and KOR proteins were decreased under oxidative stress conditions. Thus, these findings show a selective impairment of the MOR function and reduction in MOR protein under conditions of oxidative stress. The results from the in vivo and in vitro projects demonstrate the involvement of aging and oxidative stress in modulation of pass sensitivity, opioid-induced antinociception and opioid receptor function and expression.Item Regulation of Carotid Baroreflex Resetting During Arm Exercise(1999-06-01) Querry, Ross G.; Peter B. Raven; Patricia Gwirtz; Michael SmithQuerry, Ross G., Regulation of Carotid Baroreflex Resetting during Arm Exercise. Doctor of Philosophy (Biomedical Sciences), June 1999, 100 pp., 4 tables, 12 figures, bibliography, 56 titles. Cardiovascular responses to exercise are modulated by the integration of the central nervous system and afferent information from arterial baroreflexes and working skeletal muscle. Investigations have shown that during exercise, the carotid baroreflex (CBR) is reset in proportion to the exercise intensity. The role of the central nervous system contribution to the CBR resetting has not been elucidated. Investigations of CBR function in the animal model consistently report CBR variables such as maximal gain that are different than those reported in humans. These discrepancies may be due in part to methodological limitations in the neck pressure/neck suction (NP-NS) technique used to investigate the isolated CBR function in humans. To accurately examine the internal stimulus from the NP-NS maneuver, subjects were instrumented with a percutaneous catheter to record tissue pressure at the carotid sinus during five-second and rapid pulse NP-NS protocols. Carotid baroreflex function curves were analyzed with and without transmission correction of the carotid sinus pressure (CSP). Results indicated that positive pressure was more fully transmitted (~83%) than negative pressure (~65%) during the five-second-pulse, but not the rapid pulse protocol. Correction of the CSP in either protocol resulted in significant increases in CBR maximal gain and threshold and a reduced saturation pressure. These methodological refinements were then utilized to investigate the role of central command on CBR function during exercise. Subjects performed static and rhythmic handgrip exercise before and after regional anesthesia. Carotid baroreflex curves were analyzed at rest and during exercise before and after blockade at the same absolute workload. Muscle weakness from the blockade required an increased effort to maintain control tension. Heart rate, arterial pressure and perceived exertion during exercise were increased following blockade. During control exercise the CBR function curves were reset upward and rightward compared to rest with a further parallel shift during exercise with blockade. The operating point of the CBR was reset along with the centering point, but did not show the divergence toward the threshold pressure that had been previously described during dynamic exercise. The results support the proposal that central command was a primary mechanism for the resetting of the carotid baroreflex during exercise, but may not be the primary mechanism in the resetting of the operating point of the reflex.Item Regulation of intracellular calcium channels by their associated proteins homer 1 and presenilin 1(2006-05-01) Hwang, Sung-Yong; Koulen, Peter; Dillon, Glenn; Singh, MeharvanSung-Yong, Hwang, Regulation of intracellular calcium channels by their associated proteins homer 1 and presenilin 1. Doctor of Philosophy (Pharmacology and Neuroscience), May, 2006, 184 pp., 4 tables, 20 illustrations, 74 titles. In neurons, Calcium (CA2+) serves as a critical intracellular messenger that regulates a variety of cellular processes such as gene expression, neurotransmitter release, cell death, and synaptic plasticity. Therefore, it is essential for neurons to control their Ca2+ levels tightly. Ca2+ is released within the cell from intracellular stores such as the endoplasmic reticulum by activation of intracellular Ca2+ channels (ICCs) such as the inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs). Each of these two groups of ICC has three isoforms. A number of associated proteins of these two ICCs that were shown to modulate activity of the respective channel have been identified. Homer 1, a synaptic scaffolding protein not only physically associated with IP3R type1 (IP3R1), but also changes the activity of IP3R1, suggesting that Homer 1 is involved in intracellular Ca2+ signaling. Based on the similarity in amino acid sequence and molecular and physiological properties among IP3R isoforms and the fact that IP3R type 3 (IP3R3) contains the proline-rich motif (PPxxFr) that is required for the interaction with Homer, it was hypothesized that Homer 1 associates with IP3R3, leading to changes in the channel activity. Presenilin 1 (PS1) is a transmembrane protein, being expressed in cell body, dendrites, and axon in the neuron. Mutations in PS1 account for most cases of early-onset familial Alzheimer’s disease (AD). PS1 was shown to associate with RyRs and to modulate their channel activity. Therefore, it was hypothesized that specific regions of PS-1 bind to RyR type 2 (RyR2), a major isoform in the brain, resulting in changes in the channel activity. Homer 1c was shown to associate with IP3R3, leading to a decrease in channel activity. A specific region of PS1 that interacts with RyR2 was identified to increase the channel activity of RyR2. Results of the present study contributed to the understanding of the nature of intracellular Ca2+ signaling as well as the mechanisms of action by which ICCs are regulated by their associated proteins. These findings provide the rationale for novel strategies to study neurological disorders including AD and epilepsy that are mediated by Ca2+ dysregulation.Item Role of ΔFosB in nucleus of the solitary tract (NTS) in cardiovascular adaptations to chronic intermittent hypoxia (CIH) in rats(2015-08-01) Wu, Qiong; Mifflin, Steve W.; Cunningham, J. Thomas; Schreihofer, Ann M.Chronic intermittent hypoxia (CIH) rodent model is widely utilized to study obstructive sleep apnea (OSA) associated disease such as hypertension. Arterial chemoreceptor is activated by CIH, and leads to increased sympathetic nerve discharge, resulting in elevated arterial pressure. The central neuronal mechanisms of CIH induced hypertension are barely understood. The nucleus of the solitary tract (NTS) receives the first synaptic inputs from arterial chemoreceptor afferents. Transcription factor ΔFosB is increased in the NTS after a 7 day-CIH exposure. We hypothesize that NTS ΔFosB could mediate neuronal plasticity, contribute to CIH induced hypertension. Three specific aims were addressed. Aim 1: To determine the relationship between NTS ΔFosB and CIH hypertension. Viral constructs were delivered into NTS to functionally block ΔFosB (ΔJunD group). Mean arterial pressure (MAP) was measured in day time when rats were exposed to intermittent hypoxia and night time when they were in normoxia. The increase in MAP observed in ΔJunD and sham groups during day time was dampened in ΔJunD group during night time, indicating the contribution of ΔFosB to the sustained component of CIH associated hypertension. Aim 2: To determine the time-course of induction of ΔFosB immunoreactive NTS neurons during CIH exposure. Rats were separated into normoxia, 1 day, 3, 5, 7 days CIH, and 1 day, 3, 7 days recovery after 7 days CIH groups. ΔFosB immunoreactivity increased within 1 day CIH, and maintained this elevation throughout 7 days of CIH. 1 day recovery was sufficient to reduce ΔFosB immunoreactivity to normoxia level. Therefore, ΔFosB under CIH develops rapidly. Aim 3: To determine the function of ΔFosB in glutamatergic transmission after CIH. Miniature excitatory post-synaptic current (mEPSC) properties of NTS neurons of rats exposed to either different days of CIH or room air were compared. CIH increased mEPSC amplitude but not frequency, suggesting a post-synaptic site of effect. Additionally, functional blockade of NTS ΔFosB with ΔJunD decreased mEPSC amplitude back to normoxia level. Finally, overexpression of NTS ΔFosB increased mEPSC amplitude to similar levels as CIH. These results suggest that ΔFosB in NTS neurons mediates molecular adaptations which might play an important role in CIH associated hypertension.