Substance P (SP) is associated with metabo- and mechanoreceptor afferent fibers ('ergoreceptors') in skeletal muscle as well as the afferent fibers from carotid sinus baroreceptors. Afferent activity from each of these are at least partially integrated in the nucleus tractus solitarius (NTS). The purpose of this study was to determine whether SP was released from the NTS during acute reflex-induced changes in blood pressure caused by stimulating these receptors. Both the muscle pressor response and the baroreflex were studied in adult cats anaesthetized with α-chloralose. SP antibody-coated microprobes were used to measure the possible release of SP from the NTS. The muscle pressor response caused a release of immunoreactive SP-like substances (irSP) from the rostral medial NTS, as well as the dorsal motor nucleus (DMV) and lateral tegmental field (FTL). This release was not dependent on intact afferent input from the carotid sinus nerve, but was a function of activation of muscle ergoreceptors, since no irSP was released in response to stimulation of the motor nerves after the muscle was paralyzed. There was no detectable release of irSP from the mNTS during carotid artery occlusions (baroreceptor unloading). Baroreceptor activation, induced by the i.v. injection of the vasoconstrictor, phenylephrine, did not cause the release of irSP from the mNTS above resting baseline levels. These data suggest that SP is involved with the mediation of the afferent signal from muscle ergoreceptor fibers in the medial NTS. SP is not involved with the mediation of baroreceptor afferent signaling in the medial NTS. The release of SP in response to ergoreceptors activation may function to excite an inhibitory pathway which inhibits baroreflex signals that would tend to reduce the blood pressure and heart rate during the muscle pressor response.

Substance P (SP) is an undecapeptide whose functions are as varied as its locations. In the nervous system, it is thought to act as a neurotransmitter. In the peripheral vasculature, it has hypotensive effects and it causes contraction in the smooth muscle of the gut. In salivary gland, it is a potent secretagogue and it is how this effect is transduced that is the subject of this dissertation. Activation of the SP receptor in rat submaxillary gland by SP results in the hydrolysis of inositol phospholipids and the mobilization of intracellular Ca2+. These second messengers are then able to activate a pathway(s) which results in the secretion of electrolytes, water and macromolecules. The production of these second messengers, however, is thought to require the participation of a guanine nucleotide binding protein (G protein). The G protein that couples to the SP receptor (Gp), has not yet been identified. Although several investigators have recently reported the purification of G protein α subunits that are capable of activating phospholipase C, it is not known if they couple to receptors in order to activate phospholipase C. In an effort to learn more about the mechanisms of signal transduction by SP in salivary gland, the interactions of the SP receptor with G proteins were studied. In the first study, the question of whether the SP receptor functionally couples to a G protein was investigated. Alkaline treatment was used to deplete membranes containing SP receptors of endogenous G proteins. These membranes were not capable of binding SP with high affinity. High affinity binding capability was restored in those membranes, however, by reconstituting them with exogenous G proteins. Thus, it was concluded that that SP receptor agonist affinity is regulated by a G protein. It was also determined that the G proteins (a Go/Gi mixture) used to reconstitute the membranes may not be those that couple to the SP receptor in vivo, since the reconstituted Go/Gi mixture was inactivated by treatment with pertussis toxin, while Gp was not. The next study was undertaken in an effort to identify other G proteins that are able to interact with the SP receptor. G proteins were chromatographically purified from horse submaxillary gland membranes, and assayed for characteristics that could identify one or more G proteins as potential physiological couplers to the SP receptor. G proteins were identified in fractions by the ability to bind [35S]GTPγS. These GTP-binding proteins were further characterized by testing their susceptibility to ADP- ribosylation catalyzed by pertussis toxin and their ability to restore high affinity agonist binding in membranes containing the SP receptor, but no endogenous G proteins. In addition to identifying G proteins that are substrates for pertussis toxin-catalyzed ADP-ribosylation (e.g. Go and/or Gi), a GTP-binding protein was identified which possesses characteristics that are unlike those of the well-known G proteins, Go, Gi and Gs. This protein elutes from anion exchange resins at a high salt concentration, is not susceptible to ADP- ribosylation catalyzed by pertussis toxin, is able to reconstitute high affinity binding in G protein depleted rat submaxillary gland membranes and is not recognized by antibodies to Go, Gi, Gs or Gz. Finally, a direct characterization of the G protein coupled to the SP receptor in rat submaxillary gland was undertaken. Using photo-affinity labelling techniques in conjunction with chemical crosslinking techniques, a covalent 96 kDa SP receptor complex was identified. The generation of this 96 kDa complex was inhibited by a nonhydrolyzable analog of GTP, but not a nonhydrolyzable analog of ATP. Furthermore, the complex could not be produced in membranes that had been depleted of G proteins by alkaline treatment. Reversal of the chemical crosslink yielded only the 53 kDa SP receptor, showing that the protein crosslinking to the SP receptor possesses a molecular weight of about 43 kDa. This molecular weight is typical of G protein α subunits. It was concluded that the 96 kDa crosslinked receptor complex consisted of the SP receptor, the radioiodinated SP derivative and the α subunit of Gp. The studies show that the SP receptor may be coupled to a novel G protein, whose purification characteristics differ from those of the known G proteins. Although Gp has yet to be identified, comparisons of the results of these investigations with those of several recent articles in which the purification of G protein α subunits that are capable of stimulating phospholipase C is reported, suggests that Gp is similar, if not identical to those proteins. Furthermore, this dissertation describes a unique reconstitution system and crosslinking techniques which should prove useful in the identification of Gp, as well as in the study of other receptor-G protein interactions and perhaps, the reconstitution of the receptor-G protein-phospholipase C signal transduction pathway.

Intravenous injection of substance P (SP) stimulates sympathetic ganglia to evoke a greater increase in renal sympathetic nerve activity, heart rate (HR) and blood pressure (BP) in hypertensive than normotensive rats due to upregulation of the NK1 receptor. These experiments were designed to determine the cellular basis for the enhanced ganglionic responsiveness to NK1 agonists in spontaneously hypertensive rats (SHR) in comparison to their normotensive counterparts, Wistar-Kyoto rats (WKY). Studies were also conducted to determine whether the increased ganglion responsiveness to SP in SHR is causally related to the increased BP or is a unique characteristic of this model of essential hypertension. Nerve recordings were made from the external carotid branch of the superior cervical ganglion (SCG) in pentobarbital anesthetized rats. Animals were treated with the ganglion blocking agent chlorisondamine (10.5 μmol/kg) and pre- and postganglionic SCG nerves were cut. SP (1.0 to 100 nmol/kg) evoked a greater increase in postganglionic nerve firing from the SCG of SHR vs. WKY. Intracellular microelectrode recordings were made from isolated SCG. Membrane properties were similar between strains. Picospritzer application of the NK1 agonist GR-73632 (100 μM, 1 s) caused slow depolarization and increased neuron excitability. Depolarization amplitude and duration were similar between strains, however, a greater percentage of neurons were depolarized by the NK1 agonist in SHR. To determine if the ganglion sensitivity to SP was correlated with blood pressure WKY were made hypertensive by unilateral nephrectomy and deoxycorticosterone acetate (DOCA)/salt treatment. Tail cuff BP was the same in treated WKY and untreated SHR. Increases in sympathetic nerve activity, HR and BP in response to SP (1.0 to 100 nmol/kg) were the same in treated and untreated WKY rats. In conclusion, SHR are more responsive to ganglion stimulation by NK1 agonists due to a greater number of responsive cells within their SCG rather than an enhanced responsiveness of individual neurons. The increased sympathetic nerve responsiveness to SP is an inherent characteristic and not an adaptive response of sympathetic ganglion neurons to hypertension. This enhanced action of SP at sympathetic ganglia may contribute to the elevated sympathetic outflow observed in this model of hypertension.

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Degeneration of nociceptive afferent axons and terminals in the heart is associated with painless sudden cardiac death. However, innervation, distribution and morphological structures of sympathetic cardiac nociceptive afferent axons and terminals have not yet been fully characterized. The aim of the present study is to characterize the density, arrangement, and structural features of differentiated sympathetic afferent axons and terminals in whole-mount FVB mouse atria. FVB mice (3-6 months old) were perfused and the tissues were fixed. The right and left atria were processed with immunohistochemistry. Calcitonin gene-related peptide (CGRP) and substance P (SP) are two neuropeptides which have been widely used to label sympathetic nociceptive afferent axons in many tissues. CGRP (rabbit anti-CGRP) and SP (Goat anti-SP) primary antibodies were applied, followed by Alexa Fluor 594 and 660 conjugated secondary antibodies. Whole-mount preparations of right and left atria were examined using a laser scanning confocal microscope. We found that 1) CGRP immunoreactive (IR) axon bundles innervated the right and left atria including the auricle and entrance area of the superior vena cava, the inferior vena cava, left precaval vein and pulmonary veins. Large axon bundles entered the area from the major veins and bifurcated into smaller axon bundles and single axon fibers to form terminal end-nets and free endings in the epicardium at each region with a similar pattern. In the atrial muscle layer, varicose CGRP-IR axons had close contacts with muscle fibers. In addition, CGRP-IR axons iv terminated in the intrinsic cardiac ganglia (ICGs) with varicosities surrounding individual ganglionic principle neurons (PNs). In the aortic arch, the CGRP-IR fibers exhibited similar terminal structures to those seen in the atria. 2) SP-IR axons also projected to the right and left atria and aorta. Similar to CGRP-IR axons, these SP-IR axons also formed end-nets and free endings in these areas. In cardiac ganglia, SP-IR axons formed varicose endings around many individual PNs. However, a salient difference was found: There appeared to be fewer SPIR axons and terminals than CGRP-IR axons and terminals in the atria. 3) None of the cardiac PNs in ICG were CGRP-IR or SP-IR. 4) Many SP-IR axon terminals around PNs within ICGs and atrial muscles were found to have colocalized expression of CGRP-IR. Collectively, our data for the first time documented the distribution patterns and morphology of sympathetic afferent axons and terminals in each region of the atria in the mouse model. This will provide a foundation for future analysis of the pathological changes of sympathetic afferent nerves in the atria in different disease models (e.g., diabetes, sleep apnea, and aging). This study was supported by NIH R01 HL- 79636.

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<p>Based on the concept that the tactile hypersensitivity and the central sensitization observed in animal models of peripheral neuropathy are maintained by peripheral drive from primary sensory neurons, the present project measured the changes in electrophysiological, immunohistochemical, and pharmacological properties of the dorsal root ganglia (DRG) neurons induced by a peripheral neuropathy. The aim of this study was to make a systematic survey and a unique understanding of changes that occur in primary sensory neurons that can sustain peripheral drive in this model. The data of this study indicate a prominent role of large diameter Aβ-fibers, including low threshold mechanoreceptors in peripheral neuropathy.</p> / Doctor of Philosophy (Medical Science)

INTRODUCTION: HIV- associated neurocognitive disorder (HAND) and pathology are common manifestations of HIV-infection, and often persist in spite of controlled peripheral viremia. Severity of HAND can range from loss of concentration and psychological changes to frank dementia. Inflammatory host-immune responses and chemotaxis of immune cells into the CNS are thought to be integral to development of NeuroAIDS and HAND. OBJECTIVES: This studies' primary aim was to determine if significant differences existed between Substance P and NK1R expression in brain tissue samples of HIV-infected individuals with neurocognitive disorder or pathology. The secondary aim was to determine whether expression of HIV viral entry receptors CCR5 and CXCR4 correlate with expression of Substance P or NK1R. The tertiary aim of this study was to determine if age at death, CNS penetration-effectiveness of antiretroviral therapy, diagnosis before HAART, average plasma CD4, or abnormal alcohol or drug use increased prevalence of neurocognitive disease. STUDY DESIGN: Cross-sectional study of HIV-infected individuals (n=60) from the larger National NeuroAIDS Tissue Consortium Cohort. Pre-death demographic data, neurocognitive assessment, alcohol and drug use, ART regimens, date of diagnosis and death, and plasma CD4 levels, as well as pathology findings at autopsy and brain tissue samples were provided by the NNTC; expression levels of Substance P, NK1R, CCR5, and CXCR4 from brain samples were provided by Dr. Steven Douglas of The Children's Hospital of Philadelphia. RESULTS: In this sample of HIV-infected individuals, Substance P expression was significantly less in the cingulate cortex of individuals with (p=0.003). Within-subject expression patterns of CCR5 and truncated-NK1R in the cingulate cortex and cerebellum were both significantly altered by neuropathology and cannabis use; CCR5 expression was also significantly affected by opiate use. CCR5 and CXCR4 expression correlated strongly with truncated-NK1R expression. No between-subject factors significantly altered prevalence of neurocognitive impairment in this HIV-infected population. CONCLUSIONS: The study found significant changes in Substance P, NK1R, and CCR5 expression associated with neuropathology. Furthermore, in heterogeneous populations, expression patterns may be more important than overall level of expression in identifying risk factors for NeuroAIDS and other chronic diseases. / Epidemiology