Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner

Myo-inositol transport into endothelial cells derived from nervous system microvessels

Myo-inositol, the precursor in the biosynthesis of inositol phospholipids and inositol phosphates, is found in many tissues at concentrations well above its concentration in the plasma, but the highest concentrations are found in the central nervous system and the neuroretina. We describe an active, sodium gradient-dependent transport of myo-inositol into cultured endothelial cells derived from bovine retinal microvessels. Transport is inhibited by cytochalasin B, and phloridzin > phloretin. Mannitol, sorbitol, and fructose do not inhibit uptake, but -galactose inhibits uptake > -glucose > -glucose. The apparent Km of this transport system is 311 +/- 47 (S.D.) [mu]M and the apparent Vmax is 40.8 +/- 2.8 (S.D.) pmol[middle dot]mg protein-1[middle dot]min-1. This transport system may be a key in the maintenance of high tissue concentrations as it could concentrate myo-inositol from the plasma into the extracellular spaces of the eye and central nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28672/1/0000489.pd

2,4,6-trinitrotoluene detection using photoluminescence response of diatom biosilica functionalized with single-chain variable fragment

A label-free, photoluminescence (PL) based biosensor for 2,4,6-trinitrotoluene (TNT) detection was developed by functionalizing diatom biosiilica with a TNT-specific single-chain variable fragment (scFv). The scFv loading was estimated to be 0.040±0.003 (μg scFv/μg biosilica). In saturated concentration, TNT binding to scFvbiosilica quenched about 13% of its PL. Dose response follows Langmuir isotherm with half saturated concentration at 3.66×10⁻⁸ M. The method detection limit (MDL) was estimated to be 3.87×10⁻⁸ M. No interference was observed in the presence of non-complementary explosives. Limitations like small signal intensity and high background signal can be address by optimizing combination of transducer and bioreceptor. In addition, the biosensor system has the potential to be self-assembled by genetically modified diatom, eliminating the whole chemical functionalization steps. This biosensor shows potential for low-cost, high-sensitivity, on-site and selective TNT detection.Keywords: label-free, scFv, photoluminescence, TNT detection, diatom biosilic

Androgens in Men During Illness, Exercise and Psychological Stress

My early clinical experience had demonstrated that abnormalities of endocrine testing particularly with regards to thyroid function are common in ill patients. Interest in endocrine effects of illness was further stimulated by my study of hormone levels of uraemic patients undergoing differing forms of dialysis therapy. Chronic renal failure resulted in abnormal hypothalamic-pituitary-testicular and hypothalamic-pituitary-thyroid function irrespective of the nature of dialysis. Evidence for low levels of adrenal androgen in serum of uraemic patients was also found. As it was well known that abnormalities of thyroid function occurred in a variety of illnesses so it appeared possible that abnormalities of adrenal androgen status and hypothalamic-pituitary-testicular function might also be non-specific consequences of illness. To investigate this hypothesis these endocrine parameters were measured in several groups of patients:- those ill in a general medical ward, in diabetic men with ketoacidosis, during recovery from burns injury and before and after surgery. Serum levels of the most abundant adrenal androgen dehydroepiandrosterone sulphate (DHAS) were low in patients with a variety of non-endocrine medical illnesses only if they had been unwell for more than two weeks while serum androstenedione concentrations were higher than in controls irrespective of the duration of illness. Testosterone levels were also low but this was not related to the duration or severity of illness nor could it be accounted for by changes in binding proteins, hyperprolactinaemia nor on the basis of classical primary or secondary testicular failure. A study of men during an episode of diabetic ketoacidosis revealed that this complication of diabetes mellitus is also accompanied by depression of serum testosterone concentrations. However serum testosterone concentrations of non-ketotic diabetic men at routine review were not found to be related to diabetic control as assessed by glycosylated haemoglobin estimation. There was some limited evidence in the literature to suggest that adrenal androgen levels in diabetic men might be low and that this might have a deleterious effect on islet cell function but no abnormality of these hormones could be demonstrated in this group of patients. 19 burned men were followed sequentially for several weeks. Profound depression of serum testosterone concentrations for several weeks was found with levels falling into the range found in healthy females in the majority of patients studied. This could not be explained on the basis of binding protein changes nor by alterations in the concentrations of prolactin or gonadotrophins. DHAS levels although normal within 24 hours of admission fell to subnormal levels and remained low for several weeks. Serum androstenedione levels were high following admission and tended to remain so for several weeks. These changes in adrenal androgen concentrations were reminiscent of my observations during medical illness. Serum gonadotrophin levels of post-menopausal females were studied following cholecystectomy. Unlike the situation in medically ill men this operation led to a marked reduction in gonadotrophin levels sometimes down to the pre-menopausal range. Serum DHAS levels fell to below control values four days after surgery while serum androstenedione concentrations rose transiently post-operatively. The situation was however complicated by the administration of several drugs in particular parenteral opioids which might have affected gonadotrophin levels. As such profound endocrine changes had been observed with illness, it was of interest to discover whether these were specific to illness or whether other forms of stress such as the physical stress associated with exercise or the psychological stress accompanying academic examinations might result in similar changes. Following marathon running there was a small fall in serum testosterone concentrations. The situation differed from that following illness in that there was a massive elevation of serum cortisol as well as a fall in luteinising hormone (LH) levels. On the other hand veteran athletes who had run at least 25 miles a week for many years and who are therefore exposed to recurring and chronic physical stress had normal serum testosterone levels. Neither veteran athletes nor marathon runners showed evidence of a reduction in serum concentrations of adrenal androgens. The stress of physical exercise has a short-lived adverse effect on testicular function but unlike illness there are no changes in adrenal androgen levels. Review of the literature suggested that psychological stress can adversely affect gonadal function. (Abstract shortened by ProQuest.)

Neutrophils and vascular reactivity in ischaemia/reperfusion

SIGLEAvailable from British Library Document Supply Centre- DSC:DX180584 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Canine Vasculature: A Study of alpha1- Adrenoceptors and Heart Failure

Heart disease is an important cause of morbidity and mortality in the canine population, with the two most common causes of acquired heart disease being dilated cardiomyopathy and endocardiosis. In human patients with heart disease, it has been noted that the clinical signs and symptoms are often of greater severity than expected from the degree of left ventricular dysfunction, suggesting that additional factors contribute to the syndrome. It has now been shown in several human and experimental animal studies, that blood flow to exercising skeletal muscle is reduced in heart failure and that this is not due to an inability to increase cardiac output. This suggests an inability of the vasculature to accommodate the increased blood flow required by the exercising muscle, leading to an early switch over to anaerobic metabolism and premature fatigue. While the neurohumoral aspects of cardiac failure have been well characterised at a systemic level, the local vascular effects have not. To gain further knowledge of the local effects and their role in the pathophysiology of cardiac disease, it is necessary to characterise normal vessels, in addition to examining vessels from heart failure animals. With these goals in mind, the primary aim of this project was to characterise both the dog saphenous vein and the dog subcutaneous resistance arteries, in relation to their functional alpha1-adrenoceptor population. Findings are discussed in detail in Chapter 3 and Chapter 4. The functional effects of five competitive reversible antagonists and the irreversible alkylating agent CEC, on noradrenaline mediated contractions of dog saphenous vein and dog subcutaneous resistance arteries, were analyzed. In both vessels the alpha1-adrenoceptors appeared to have a low affinity for the alpha1-adrenergic antagonist prazosin, necessitating their classification as alpha1L-adrenoceptors. In addition, in both vessels, there was evidence for the involvement of another subtype in the noradrenaline response. This receptor, despite a low affinity for prazosin had some characteristics of the alpha1B-adrenoceptor. Chapter 5 describes the cloning and sequencing of an 891 bp fragment of the canine alpha1a-adrenergic receptor cDNA. This subtype was chosen because of the mounting evidence that it is responsible for the alpha1L-pharmacology. The fragment was initially isolated using reverse-transcription polymerase chain reaction (RT-PCR) and primers designed from areas of high homology in the alpha1a-adrenoceptor of the human and bovine alpha1a-adrenergic sequence. This fragment, together with a canine partial alpha1b-sequence was used to probe cell lines expressing the human alpha1a- and alpha1b-adrenergic receptors, as well as canine prostate and brain RNA. The canine alpha1a-probe failed to detect message in any of the samples and while the alpha1b-probe hybridized to the alpha1b-expressing cell line RNA, there was evidence for a lack of subtype specificity of this probe. Finally, Chapter 6 describes experiments comparing isolated femoral artery, saphenous vein and subcutaneous resistance arteries from dogs with naturally occurring heart failure, and controls. The findings suggest that in heart failure there is a decrease in sensitivity to exogenous noradrenaline in both the saphenous vein and the femoral artery, but not in the resistance arteries. In addition, vasorelaxations to acetylcholine were examined in all vessels and no significant differences were found between vessels from control and heart failure dogs. Interestingly, on examination of the case details from the heart failure dogs used, it was found that in the cohort of patients used for the large vessel studies, the majority of animals had received no treatment, (seven out of eight animals were not treated). This was in contrast to the cohort used in the resistance artery group, where four out of five animals had received treatment for their cardiac disease. The relevance of this and possible effects are discussed in this chapter

GABAergic activity influencing cerebral function in the rat

Currently available evidence suggests that gamma-aminobutyric acid is the major inhibitory neurotransmitter in the mammalian central nervous system. As such, a fuller knowledge of the functional role played by GABAergic neurotransmission would increase our understanding of the complexities of brain activity. Three approaches were employed to analyse GABAergic influences in cerebral function; 1) systemic administration of GABA agonists (muscimol and THIP) or related compounds (benzodiazepines), 2) specific lesioning of a known GABAergic pathway (from caudate nucleus) with subsequent pharmacological challenge and 3) local intracerebral injection of a GABA agonist (muscimol) into a region with known GABA synaptic mechanisms (caudate nucleus). All experiments were performed on restrained, conscious animals, using principally the 2-deoxyglucose quantitative autoradiographic approach to measure cerebral function (as it is reflected in rates of glucose utilisation). The intravenous administration of muscimol and THIP resulted in a heterogeneous pattern of significantly reduced glucose utilisation in the CNS. The regional hierarchy of changes in glucose utilisation was similar for both muscimol and THIP in all regions (with the exception of the superior colliculus), with muscimol being approximately six times more potent in all regions investigated. The regions in which glucose utilisation was extremely sensitive to change, displaying reductions of approximately 40% following muscimol (1.5 mg/kg) or THIP (10 mg/kg), included the neocortex and thalamic areas. In contrast, in a large number of areas, including cerebellum and related motor nuclei, hypothalamus, lateral habenula and amygdala, there were only minimal reductions in glucose use following muscimol and THIP. Regions displaying moderate reductions in glucose utilisation included most extrapyramidal regions and a number of cortical and subcortical limbic areas. In no region of the 60 CNS areas measured was a significant increase in glucose utilisation observed with any concentration of either muscimol or THIP. The regional distribution of alterations in glucose utilisation following muscimol and THIP, which do not correspond to the known topography of GABAergic neurons and receptors, provided a comprehensive description of the functional alterations, as reflected in rates of glucose utilisation, which occur in conscious rats following systemic administration of these two putative GABAergic agonists. Although benzodiazepines are thought to be active at the GABA receptor site, the response in terms of glucose use following diazepam was markedly different from that; following GABA agonists. In particular, the degree of heterogeneity of depression in glucose use was less and the hierarchy of responsiveness in diverse brain areas was different, suggesting that the responses to GABA agonists were not merely a ftinction of susceptibility to depression in general. The relationship between local cerebral blood flow (measured with iodoantipyrine) and glucose use was analysed in two groups of rats. Both blood flow and glucose use decreased in parallel in the 38 areas of the brain analysed following muscimol administration, thus maintaining the relationship observed in control rats. These studies offered no evidence for a direct vasodilatatory action for muscimol (reported by others in vitro) in the intact, conscious rat, but pointed to underlying metabolic activity as the primary determinant of cerebral blood flow. (Abstract shortened by ProQuest.)