Background/Rationale: In addition to their established cardioprotective role, β-adrenergic antagonists also increase the risk of stroke and mortality. We propose that a vascular mechanism could contribute to cerebral tissue ischemia in β-blocked patients. Methods: Cardiac output (CO), mean arterial pressure (MAP) and microvascular brain oxygen tension (PBrmvO2) were measured in anesthesized mice treated with metoprolol (3mg•kg-1, i.v.). Dose-response curves (DRCs) for adrenergic-agonists were generated in mesenteric resistance arteries (MRAs; isoproterenol, clenbuterol) and posterior cerebral arteries (PCAs; phenylephrine, isoproterenol) before and after metoprolol treatment. Results: Metoprolol reduced CO, maintained MAP and increased systemic vascular resistance (SVR) resulting in a decreased PBrmvO2 in mice. Metoprolol attenuated β-adrenergic mediated vasodilation in both MRAs and PCAs. Conclusions: Metoprolol reduced brain perfusion in mice. A decrease in CO contributed however, metoprolol also inhibited β-adrenergic vasodilation of mesenteric and cerebral arteries. This provides evidence in support of a vascular mechanism for cerebral ischemia in β-blocked patients.

Thesis (M.S.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Secretin and VIP stimulate cardiac adenylyl cyclase activity and exert a positive inotropic action in several mammalian species. This study examined positive inotropic activity and cAMP levels in rat papillary muscle. Isoproterenol and secretin increased contractions by 150 ± 31% and 129 ± 27%, respectively. VIP increased contraction by 30 ± 21% only at 10 μM. Isoproterenol significantly increased cAMP levels by 82%, whereas increases by secretin (58%) and VIP (56%) were not significant. These results are consistent with reports that secretin and VIP stimulate cardiac adenylyl cyclase in the rat, but suggest that cAMP tissue levels cannot totally explain the positive inotropic responses to secretin and VIP.

The effect of papaverine on the albumin permeability-surface area product (PS), reflection coefficient (σ), and capillary filtration coefficient (K(f)) was examined in isolated rabbit lungs. Because PS and K(f) are functions of vascular surface area and permeability, we also compared papaverine with two other means of maximizing lung surface area: isoproterenol (1 x 10-7 M) and a mild increase in vascular pressure. Only lungs perfused with 0.1 mg/ml papaverine were significantly different from control. PS increased from control (2.80 ± 0.16 to 5.53 ± 0.20 ml · min-1 · g dry lung-1 x 10-2), whereas σ decreased from control (0.92 ± 0.01 to 0.78 ± 0.03). K(f) after papaverine was significantly lower than baseline predrug K(f) (5.60 ± 0.78 to 4.56 ± 0.53 ml · s-1 · cmH2O-1 · g dry lung-1 x 10-3). However, this group's predrug K(f) was higher than that of any other group. Our results indicate that papaverine increases albumin permeability and decreases endothelial selectivity. The isolated perfused lung appears fully recruited, because K(f) and PS did not increase with isoproterenol or increased vascular pressure. Papaverine should be used with caution in the Ringer-perfused lung.

The scope of cardiac pathophysiology in sepsis has not been fully defined. Accordingly, we evaluated the effects of sepsis on heart rate (HR), HR variability, and conduction parameters in a murine model of sepsis. Electrocardiograms were recorded noninvasively from conscious mice before and after cecal ligation and puncture (CLP) or sham surgery. Responses of isolated atria to tyramine and isoproterenol were quantified to assess the functional state of sympathetic nerves and postjunctional sensitivity to adrenergic stimulation. Cecal ligation and puncture mice had lower HR compared with sham at 16 to 18 h postsurgery (sham, 741 ± 7 beats/min; CLP, 557 ± 31 beats/min; n = 6/group; P < 0.001), and there was significant prolongation of the PR, QRS, and QTc intervals. Slowing of HR and conduction developed within 4 to 6 h after CLP and were preceded by a decrease in HR variability. Treatment of CLP mice with isoproterenol (5 mg/kg, intraperitoneally) at 25 h after surgery failed to increase HR or decrease conduction intervals. The lack of in vivo response to isoproterenol cannot be attributed to hypothermia because robust chronotropic and inotropic responses to isoproterenol were evoked from isolated atria at 25°C and 30°C. These findings demonstrate that impaired regulation of HR (i.e., reduced HR variability) develops before the onset of overt cardiac rate and conduction changes in septic mice. Subsequent time-dependent decreases in HR and cardiac conduction can be attributed to hypothermia and would contribute to decreased cardiac output and organ perfusion. Because isolated atria from septic mice showed normal responsiveness to adrenergic stimulation, we conclude that impaired effectiveness of isoproterenol in vivo can be attributed to reversible effects of systemic factors on adrenergic receptors and/or postreceptor signaling.

Ozone, being an unstable molecule, is believed to be one of the strongest oxidant agents known to man. Rapid growth in the application of ozone — both as disinfectant and as form of alternative medicine — led to questions about the effects of uncontrolled ozone exposure and inhalation, whether intentional or unintentional, on the human body. This study specifically focussed on examining, identifying and substantiating the respiratory effect of acute exposure (10 min or less) to considerably higher ozone concentrations than reported on before (19.5 ± 0.5 ppm). Respiratory tissue of rodents (Duncan–Hartley guinea pigs of both sexes and Male Wistar rats) was subjected to ozone by utilising three distinctly diverse models of ozone introduction: (a) in vitro exposure, (b) in vivo exposure, and (c) ex vivo by employing an isolated lung perfusion model which allows for real–time, breath–by–breath data acquisition of ozone’s effect on respiratory mechanics. The effect of ozone on the isolated trachea in the presence of various drugs with well–known effects, including methacholine, isoproterenol and ascorbic acid was also examined. The results found in this study identified two direct effects on the isolated trachea due to ozone exposure: (1) a definite contraction of the isolated trachea immediately after exposure to ozone, and (2) a clearly visible and significant hyper responsiveness of the isolated trachea to irritants, e.g. methacholine. Although ozone has a negative effect on the trachea, it was concluded that ozone has no adverse effect on muscarinic acetylcholine receptors. An apparent EC50 value of ozone on the trachea was established by two different methods as (2.77 ± 0.02) x 10–3 M and (2.10 ± 0.03) x 10–3 M, respectively. Ozone furthermore displayed an attenuation of the beneficial pharmacological response of –sympathomimetic drugs (i.e. isoproterenol), while isoproterenol itself has a relaxing effect on the ozone–induced contraction of the isolated trachea. Indomethacin pre–treatment of isolated tracheal tissue significantly (77%) reduced the ozone–induced contraction of tracheal smooth muscle, suggesting that COXproducts of arachidonic acid play a prominent role in the development of pulmonary function decrements consequent to acute high–dose ozone exposure. Ascorbic acid exhibited a meaningful prophylactic effect on ozone–induced contraction of both isolated tracheal tissue and in the isolated lung perfusion model, emphasising the major role antioxidants play in both the epithelium lining fluid (ELF) of the respiratory system and in plasma throughout the body in protecting against the destructive effects of ozone. Surprisingly, pre–treatment with ascorbic acid did not prevent hyper responsiveness of isolated tracheal preparations to methacholine after a 10 min ozone (19.5 ± 0.5 ppm) exposure. In the lung perfusion model, the presence of ascorbic acid in the perfusion medium did, however, significantly reduce the magnitude and rate of decline in lung compliance after ozone exposure (46% decline with ascorbic acid versus 96% in the control study without ascorbic acid). Examination of a lung perfusion model exposed to ozone (19.5 ± 0.5 ppm O3; 5 seconds) presented a significant decline in lung compliance (95.6% within 2 min), tidal volume (70%) and maximum inspiratory flow (71.2%), with an ensuing reduction in lung elasticity and severely hampered breathing pattern. Microscopic examination after acute high–dose inhalation studies did not display any significant cellular damage, oedema or inflammation after acute high–dose ozone exposure. This suggests that significant cellular injury and inflammation is possibly not the causative factor of early breathing difficulty experienced after acute high–dose ozone inhalation, as these symptoms and particularly the result of inflammatory precursors, is believed to probably only set in at a later stage. Although the potential advantages of ozone in certain fields of medicine are not disputed, ozone, depending on its concentration and cumulative dose, can be either therapeutic or toxic. Observations in this study emphasised that even short bursts of high–dose ozone inhalation have deleterious effects on respiratory health and care should be taken not to jump to conclusions regarding ozone’s medical application without relevant scientific evidence. It must be stressed that high–dose inhalation of ozone should be avoided at all cost – especially by those with existing airway diseases. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.

Ozone, being an unstable molecule, is believed to be one of the strongest oxidant agents known to man. Rapid growth in the application of ozone — both as disinfectant and as form of alternative medicine — led to questions about the effects of uncontrolled ozone exposure and inhalation, whether intentional or unintentional, on the human body. This study specifically focussed on examining, identifying and substantiating the respiratory effect of acute exposure (10 min or less) to considerably higher ozone concentrations than reported on before (19.5 ± 0.5 ppm). Respiratory tissue of rodents (Duncan–Hartley guinea pigs of both sexes and Male Wistar rats) was subjected to ozone by utilising three distinctly diverse models of ozone introduction: (a) in vitro exposure, (b) in vivo exposure, and (c) ex vivo by employing an isolated lung perfusion model which allows for real–time, breath–by–breath data acquisition of ozone’s effect on respiratory mechanics. The effect of ozone on the isolated trachea in the presence of various drugs with well–known effects, including methacholine, isoproterenol and ascorbic acid was also examined. The results found in this study identified two direct effects on the isolated trachea due to ozone exposure: (1) a definite contraction of the isolated trachea immediately after exposure to ozone, and (2) a clearly visible and significant hyper responsiveness of the isolated trachea to irritants, e.g. methacholine. Although ozone has a negative effect on the trachea, it was concluded that ozone has no adverse effect on muscarinic acetylcholine receptors. An apparent EC50 value of ozone on the trachea was established by two different methods as (2.77 ± 0.02) x 10–3 M and (2.10 ± 0.03) x 10–3 M, respectively. Ozone furthermore displayed an attenuation of the beneficial pharmacological response of –sympathomimetic drugs (i.e. isoproterenol), while isoproterenol itself has a relaxing effect on the ozone–induced contraction of the isolated trachea. Indomethacin pre–treatment of isolated tracheal tissue significantly (77%) reduced the ozone–induced contraction of tracheal smooth muscle, suggesting that COXproducts of arachidonic acid play a prominent role in the development of pulmonary function decrements consequent to acute high–dose ozone exposure. Ascorbic acid exhibited a meaningful prophylactic effect on ozone–induced contraction of both isolated tracheal tissue and in the isolated lung perfusion model, emphasising the major role antioxidants play in both the epithelium lining fluid (ELF) of the respiratory system and in plasma throughout the body in protecting against the destructive effects of ozone. Surprisingly, pre–treatment with ascorbic acid did not prevent hyper responsiveness of isolated tracheal preparations to methacholine after a 10 min ozone (19.5 ± 0.5 ppm) exposure. In the lung perfusion model, the presence of ascorbic acid in the perfusion medium did, however, significantly reduce the magnitude and rate of decline in lung compliance after ozone exposure (46% decline with ascorbic acid versus 96% in the control study without ascorbic acid). Examination of a lung perfusion model exposed to ozone (19.5 ± 0.5 ppm O3; 5 seconds) presented a significant decline in lung compliance (95.6% within 2 min), tidal volume (70%) and maximum inspiratory flow (71.2%), with an ensuing reduction in lung elasticity and severely hampered breathing pattern. Microscopic examination after acute high–dose inhalation studies did not display any significant cellular damage, oedema or inflammation after acute high–dose ozone exposure. This suggests that significant cellular injury and inflammation is possibly not the causative factor of early breathing difficulty experienced after acute high–dose ozone inhalation, as these symptoms and particularly the result of inflammatory precursors, is believed to probably only set in at a later stage. Although the potential advantages of ozone in certain fields of medicine are not disputed, ozone, depending on its concentration and cumulative dose, can be either therapeutic or toxic. Observations in this study emphasised that even short bursts of high–dose ozone inhalation have deleterious effects on respiratory health and care should be taken not to jump to conclusions regarding ozone’s medical application without relevant scientific evidence. It must be stressed that high–dose inhalation of ozone should be avoided at all cost – especially by those with existing airway diseases. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Please see fulltext for abstract. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming.

SLMAP is a tail anchored membrane protein that alternatively splices to generate three isoforms, SLMAP1, SLMAP2 and SLMAP3. Previous studies in our lab have shown that the postnatal cardiac-specific overexpression of SLMAP1 results in intracellular vesicle expansion and enhanced endosomal recycling. I generated a postnatal cardiac-specific knockout model using the Cre-Lox system to nullify all three SLMAP isoforms and further evaluate its role in the mouse myocardium. SLMAP knockdown and knockout mouse hearts were analyzed with western blotting and qPCR. I found that only SLMAP3 was nullified and phenotypic evaluation through echocardiography indicated that young and old SLMAP3 knockout animals showed no remarkable changes in cardiac function. Furthermore, challenge with stressor isoproterenol had a similar response to wildtype and knockout mice in cardiac structure and function. Surprisingly the level of expression of SLMAP1 and SLMAP2 was maintained in the myocardium from SLMAP3 deficient mice. Interestingly the machinery involved in endosomal recycling was not impacted by the loss of SLMAP3. These data indicate that loss of SLMAP3 does not alter cardiac structure and function in the postnatal myocardium in the presence of SLMAP1 and SLMAP2.

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.