The role of magnesium sulphate in the control of catecholamine induced cardiovascular disturbances

A dissertation submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg for the degree of Doctor of Philosophy in Medicine Johannesburg, 1988The purpose of this thesis was to investigate the value in clinical situations of the well known in vitro anti-adrenergic effects of magnesium. Research interest in magnesium has been growing rapidly over the last twenty years. However, most of this interest has focused on the clinical consequences of magnesium deficiency states and little attention has been given to magnesium as a therapeutic agent. Despite the wide use of infusions of magnesium sulphate in obstetric practice, there is a dearth of knowledge regarding the actions of magnesium on a cardiovascular system. In laboratory studies, magnesium was shown to inhibit the release of catecholamines from adrenergic nerve terminals, an action which might have great clinical implications.IT201

Dual control of local blood flow by perivascular nerves and endothelial cells: changes in development, atherosclerosis and acrylamide neuropathy

This thesis examines the influence of perivascular nerves and the vascular endothelium on the local control of vascular tone with particular emphasis on how these mechanisms are affected by age and in atherosclerosis. Using the method of in vitro pharmacology, it was shown that ATP is a cotransmitter with noradrenaline (NA) in the hepatic artery of the rabbit and that the response of the vessel to the purine is mediated by ATP acting through post-junctional P2x-purinoceptors. Acetylcholine (ACh) induced a vasodilatation that was entirely dependent on the presence of an intact endothelium whereas adenosine, ATP and 2-methylthio ATP (a selective P2y-purinoceptor agonist) were shown to elicit a relaxation that was independent of the endothelium. The presence of nerve fibres containing substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were demonstrated in the hepatic artery of the rabbit. Furthermore, it was shown that CGRP and VIP mediated a vasodilatation in the absence of endothelium whereas SP produced a relaxation that was endothelium-dependent. Changes in the reactivity of hepatic and saphenous arteries from male and female rabbits (2-36 months) in response to directly acting vasoconstrictor agents (NA, a, methylene ATP (a, meATP), KCl); endothelium-dependent vasodilator agents (ACh, SP); endothelium-independent vasodilator agents (CGRP, VIP, ATP) and transmural nerve stimulation are described. Male and female Watanabe Heritable Hyperlipidaemic (WHHL) rabbits (4-12 months) were used as a model for human homozygous hypercholesterolemia. After an initial reduction in endothelium-dependent vasodilatation, there was an increase in relaxation at 12 months, when plaques were present. Contractions to sympathetic nerve stimulation was also reduced at 12 months. The effects of acrylamide, which is regarded as a model for autonomic neuropathy, on the response of the hepatic, saphenous and basilar arteries are also described

An investigation of the mechanisms underlying hypotension and vascular hyporeactivity in septic shock.

Septic shock is a common condition carrying a high mortality. The clinical features are a high cardiac output circulation with hypotension and vascular hyporeactivity and evidence of organ failure. I developed an organ culture in vitro model and a fluid-resuscitated in vivo model of sepsis to investigate potential underlying mechanisms for the vascular hyporeactivity, namely inducible nitric oxide synthase (iNOS) overproduction and ATP-sensitive K+ (Katp) channel activation. I also studied the phenomenon of vascular supersensitivity to vasopressin/terlipressin in sepsis. I could demonstrate a temporal variation in vascular hyporeactivity to lipopolysaccharide (LPS) in rat mesenteric artery (RMA) in vitro over a 46 h period. This was reversed by a variety of iNOS inhibitors (aminoguanidine, L-NAME, 1400W and GW273629) and the guanylyl cyclase inhibitor, ODQ. Likewise, inhibition of the Katp channel via its pore-forming subunit, rather than its sulphonylurea receptor (SUR), also reversed LPS-induced hyporeactivity in vitro, though vasopressin had no effect In vivo, iNOS inhibition and terlipressin all raised blood pressure in the septic animals over and above the pressor effect achieved in non-septic animals Katp channel inhibition raised blood pressure in shams more than septic animals. However, these agents failed to improve macro- or microcirculatory blood flow, nor did they attenuate the metabolic acidosis. In patients with norepinephrine-resistant septic shock, I reported the first use of terlipressin in reversing hypotension and reducing norepinephrine requirements. Although terlipressin represents a significant advance in our ability to treat septic shock, care should be taken when using it

Prolactin

Prolactin is a polypeptide hormone that is synthesized in and secreted from the lactotrophs of the anterior pituitary gland. We are now aware that synthesis and secretion of prolactin is not restricted to the anterior lobe of the pituitary gland, but other organs and individual cells can also produce it. This book provides the headlines to follow a course of cumulated knowledge on prolactin research during the last two-three decades and it may also help us understand some of the concerns that we face today

The medial amygdala as a key neural centre in maternal aggression: genetic, neural and behavioural analyses

In rodents, as macrosmatic animals, chemosensory processing plays an instrumental role in guiding the expression of social, sexual and maternal behaviours. Social odours and pheromones are processed by the main and the accessory olfactory systems, which information converge mainly in the medial amygdala (Me). The Me, in turn, plays a central role in the vomeronasal–sensorimotor integration that leads to specific behavioural responses such as the above-mentioned social and maternal behaviours. Maternal behaviour comprises physiological and behavioural adaptations that help the dams to successfully raise their offspring. This behaviour is expressed in a wide range of vertebrate species and can be parsed in two main components, namely pup-directed (i.e. maternal care), and non-pup-directed behaviour as maternal aggression. Previous studies have shown that pup-sensitized virgin females, which display all pup-directed behaviours towards pups, do not show maternal aggression towards male intruders. The main goal of this thesis is to investigate the role of Me in the control of maternal aggression and the possible adaptations that this nucleus suffers during lactation, such as changes in gene and protein expression. The results of the experiments inducing a chemogenetic inhibition of Me showed that this nucleus is a key centre in the neural control of maternal aggression. One of the main changes in the Me of dams revealed by our RNA-seq study was an increased PRL expression. Thus, we characterized the prolactinergic (PRLergic) circuits in female brain in different physiological states. The same pattern of PRLergic immunoreactivity was present in pup-sensitized, pup-naïve virgins and lactating female mice. However, we found quantitative differences between dams and the other two groups in the supraoptic nucleus and between dams and virgin naïve females in the suprachiasmatic and the medial preoptic nucleus. Our findings shown a widespread PRL innervation in the female mouse brain, suggesting the existence of important PRLergic pathways. In addition, these pathways suffer some adaptations during lactation. This supports a role for PRL as a neuroactive peptide, in addition to its well-documented endocrine role. In conclusion, hormonal changes linked to pregnancy, parturition and lactation induce modifications of the socio-sexual brain network, including effects on the transcriptomic profile of the Me and the central PRLergic systems. These modifications might contribute to the expression of behaviours specifically linked to motherhood, such as maternal aggression

Deorphanising G protein-coupled receptors : the search for fast steroid receptors

G protein coupled receptors (GPCRs) are the largest family of transmembrane receptors in the genome and are activated by a multitude of ligands including neuropeptides, hormones and sensory signals. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important mediators in homeostatic control. Many modulators of PVN/SON activity, including neurotransmitters and hormones act via GPCRs - in fact over 100 non-chemosensory GPCRs have been detected in either the PVN or SON. The introduction to this thesis begins with a comprehensive summary of GPCR expression within the PVN/SON, with a critique of the detection techniques used within the literature. Also discussed are some aspects of the regulation and known roles of GPCRs in the PVN/SON, as well the possible functional significance of orphan GPCRs. Particular interest is paid to the recently 'deorphanised' G protein-coupled oestrogen (E2) receptor, GPER, which is the first receptor to be acknowledged as a steroid binding GPCR (although there are conflicting studies regarding its affinity for E2) and is expressed in the PVN and SON. Steroids are known to have fast non-genomic effects that are thought to be mediated in-part by membrane-associated forms of the traditional steroid receptors (members of a family of transcription factors). However, the possible discovery of a fast E2 GPCR has raised speculation regarding the existence of other steroid binding GPCRs. Thus the experimental Chapters were undertaken to explore the concept of fast steroid receptors, with particular emphasis on their possible roles in neuroendocrine systems. Firstly, the distribution of the putative E2 receptor was investigated to give further insight into its possible in vivo roles. In the rodent, high levels of GPER gene and protein expression were detected in the oxytocin and vasopressin neurones in the PVN and SON, the anterior and intermediate lobe of the pituitary, adrenal medulla and renal medulla and pelvis, suggesting roles for GPER in multiple functions including hormone release. To clarify the controversy surrounding GPER as an E2 receptor, we investigated GPER function in vitro using a series of cell signalling assays. However, E2 did not stimulate GPER-mediated signalling, suggesting that either GPER remains an orphan GPCR, or the cell lines used in this study lacked a vital component for E2 activation of GPER. As the rapid effects of glucocorticoid have been reported in numerous brain regions (including the PVN and SON), endocrine, and other tissues, the second part of this thesis focussed on the search for a possible fast glucocorticoid receptor. We compared the tissue distribution gene expression profiles of approximately 125 orphan GPCRs common to human and rodent with tissues that are known to exhibit fast effects of steroids (e.g., hippocampus, PVN, SON, thymus, kidney, etc.). Of the 125 orphans,3 GPCRs (GPR108, GPR146, and TMEM87B) had distribution profiles that closely matched the regions/tissues of interest. These orphans were tested for glucocorticoid activation using a universal deorphanisation assay. However, the identity of the fast glucocorticoid receptor remains unknown, as none of the candidate orphan GPCRs responded to glucocorticoids.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Investigating the association between P2X7 receptors, microglia and the actions of morphine

P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing. P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis. Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS. To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia. It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies. Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight. The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation. In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic