Despite the extensive use of drugs in the treatment of atherosclerotic coronary heart disease, there are few studies in the literature dealing with the actions of drugs on the coronary circulation in the atherosclerotic state. One of the main physiological characteristics of the coronary circulation is its functional adaptability, i.e. changing to meet the nutritional needs of the heart. [...]

In previous studies from this laboratory, it has been found that certain antisympathomimetic drugs (phenoxybenzamine, piperoxane and chlorpromazine) increase the urinary excretion of adrenaline and noradrenaline and elevate the urinary recovery of intravenously administered adrenaline and noradrenaline (Benfey, Mazurkiewicz and Melville, 1958; and Benfey, Ledoux and Melville, 1958, 1959). This was a surprising result since antisympathomimetic agents have been employed to obtain vasodilatation and reduction of an elevated blood pressure, conditions in which a rise in free adrenaline and noradrenaline in the body are undesired.

Despite extensive laboratory and clinical investigations, the exact role of electrolyte changes in the cardiac action of digitalis is still obscure. While it is well established that potassium and calcium are essential ions for the maintenance of normal heart function, the relationship and possible significance of these ions in the pharmacological and toxicological actions of digitalis have been rather controversial. The primary objective of this investigation was to elucidate, if possible, these potassium-calcium-digitalis interrelationships. [...]

Thesis (PH.D.) -- Syracuse University, 2007. / "Publication number" AAT 3281739

Thesis (PH.D.) -- Syracuse University, 2007. / "Publication number AAT 3281739"

Thesis (Ph. D.)--University of Michigan.

Thesis (Ph. D.)--University of Michigan.

Ischemic cardiovascular disease shows trends of increasing morbidity and mortality in the United States and around the world. Current therapeutic options are limited, but the identification of key disease mechanisms and targets will inform novel therapeutic development to help decrease disease burden. One potential target is the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a key regulator of Ca2+ homeostasis which plays multiple roles in the cardiovascular system. SERCA catalyzes the hydrolysis of ATP and couples it to the translocation of free cytosolic Ca2+ into SR/ER stores. SERCA is redox-regulated, and is susceptible to both stimulatory and inhibitory oxidative post-translational modification. For example, oxidation of SERCA by physiological levels of nitric oxide (NO) causes reversible oxidative modification of SERCA cysteine thiols by introducing glutathione adducts. S-glutathiolation enhances SERCA Ca2+ uptake activity, which results in rapid reductions in cytoplasmic Ca2+ levels, and promotes endothelial angiogenic responses in vitro. S-glutathiolation of SERCA specifically at cysteine-674 (C674) is a key signal regulating SERCA activity under physiological conditions, and a next crucial step is establishing causal relationships between defects in C674 S-glutathiolation and human disease. The following study elucidates the role of redox regulation of the C674 thiol in the mechanisms of vascular disease by employing a SERCA 2 C674S knock-in (SKI) mouse in which the key thiol is lacking in 50% of SERCA, rendering the protein less able to be activated by glutathiolation. Following hind limb ischemia, SKI animals had impaired blood flow recovery, indicating an angiogenic defect. Cultured SKI microvascular endothelial cells showed impaired migration and decreased network formation. Fura-2 Ca2+ signaling studies revealed lower Ca2+ stores and decreased VEGF- and NO-induced Ca2+ influx. Also, hypoxia-induced expression of pro-angiogenic genes (VEGF, VEGF receptor 2 and eNOS) was decreased in SKI endothelial cells. Adenoviral overexpression of calreticulin, a major ER Ca2+ binding protein, enhanced levels of VEGF receptor protein and eNOS phosphorylation. Taken together, these data indicate that impairing normal redox regulation of the C674 thiol via reversible S-glutathiolation interferes with endothelial cell Ca2+ homeostasis and angiogenic gene expression, suggesting mechanisms by which impaired SERCA glutathiolation contributes to worsened angiogenesis during ischemia.

BACKGROUND: Hypertension is a major health concern with a myriad of possible causes. Sodium is a major component of blood pressure regulation implicated in the maintenance of fluid volume. The blood pressure response to changes in salt intake varies considerably among individuals; salt-sensitive individuals exhibit increases in blood pressure parallel to elevated sodium intake, whereas salt-resistant individuals maintain constant blood pressure regardless of variations in sodium intake. Salt-Sensitive Hypertension (SSH) develops due to an impairment of normal mechanisms that react to an elevated sodium load. Aging is another major risk factor for the development of hypertension. The effects of aging have a profound impact on the cardiovascular, renal, and nervous systems, which work together to regulate blood pressure. The development of SSH and impact of aging on blood pressure have been well-established, but the neurophysiological mechanisms implicated in SSH and aging, have only been recently explored. OBJECTIVE: To provide mechanistic insight regarding the integrated roles of the renal and nervous systems in age-dependent hypertension in male Sprague-Dawley (SD) rats. METHODS: Male SD rats of various ages were randomly assigned to various experimental protocols. 3-month-old male SD rats were randomly assigned to receive Renal-Capsaicin surgery (Renal-CAP) to ablate the afferent renal nerve (ARN) or sham surgery, followed by an acute 5% body weight i.v.-isotonic volume expansion protocol (5%-VE) in which natriuretic response and cardiovascular functions (HR/MAP) were continuously monitored and analyzed for the duration of the experimental period. 3/8/16-month-old male SD rats without Renal-CAP surgery were similarly exposed to the 5%-VE protocol. C-fos immunohistochemistry (c-Fos IHC) was performed on brain slides prepared from rats assigned to the 5%-VE protocol to assess PVN parvocellular neuronal activation, as a marker for ARN activity. 3/8/16-month-old male SD rats on normal-salt and high-salt (NS/HS; 0.6%/4% sodium chloride respectively) diets were assigned to an amiloride and hydrochlorothiazide protocol (AM-HCTZ) to evaluate NCC activity, and exposed to i.p. hexamethonium to account for the sympathetic contribution to blood pressure. Renal/plasma NE content was assessed via ELISA to further account for sympathetic tone. Immunoblotting was performed on 3-month-old control saline-infused, s.c.-norepinephrine (NE), and s.c.-norepinephrine+terazosin/propranolol male SD rats to assess NCC, phosphorylated NCC (pNCCT58), SPAK, WNK1, OxSR1, and phosphorylated OxSR1 protein levels, to evaluate the roles of the α1/β-adrenoceptors in the NCC-implicated ARN-mediated sympathoinhibitory pathway. RESULTS: In response to the 5%-VE protocol, the natriuretic response was attenuated in Renal-Cap rats. Renal-CAP rats also experienced a significant increase in MAP in response to 5%-VE, while sham Renal-CAP rats did not. Both Renal-CAP and sham Renal-CAP rats experienced a significant increase in the mean number of PVN Fos-positive cells (Fos+) post-expansion, although the increase in Fos+ cells in Renal-CAP rats was smaller in magnitude in comparison to their sham Renal-CAP counterpart. An increase in MAP and decrease in urinary volume/sodium excretion was observed in male SD rats of increasing age in response to 5%-VE. An increase in the mean number of Fos+ cells was observed post-expansion in all age groups, while 3-month-old rats experienced a larger increase in Fos+ cell count relative to 8-month-old rats. An increase in MAP was observed in rats of increasing age on NS-intake. An increase in MAP was also observed in rats of the same age with an increase in dietary salt intake from a NS to HS-intake. An increase in MAP drop due to hexamethonium was observed in rats on HS-intake and of older age. An increase in renal/plasma NE content was observed in rats of increasing age on NS-intake, and increases in salt intake in the same age group led to a significant increase in NE content. An increase in NCC activity was observed in s.c.-NE/Renal-CAP 3-month-old rats in response to HS-intake, and in rats of increasing age on NS-intake. In response to HS-intake, a decrease in NCC activity occurred in rats treated with terazosin but not propranolol. Furthermore, a decrease in NCC/pNCC/WNK1/pOxSR1 expression was observed in rats treated with terazosin in response to HS-intake, whereas protein levels in rats treated with propranolol varied independently of dietary salt intake. CONCLUSION: This present study has provided abundant evidence regarding the integrated roles of renal sodium handling and the ARN-mediated sympathetic tone in the pathophysiology of age-dependent hypertension in male SD rats. Through the demonstration of a hypertensive mechanism, involving an impairment of fluid and electrolyte homeostasis that implicates the renal, cardiovascular, and nervous systems, this study provides a crucial stepping stone for the development of mechanistic treatments for age-dependent hypertension in elderly individuals with elevated sympathetic tone.

Substandard and counterfeit medicines are major obstacles to the treatment of infectious diseases. Substandard medicines vary from standard drugs in terms of dose, bioavailability, or the presence of impurities. Current methods to identify substandard and counterfeit antimicrobial drugs are either resource intensive or have poor specificity. This dissertation examined two issues related to poor quality antimicrobial medicines: 1) Methods to detect and prevent the consumption of substandard drugs. 2) The relationship between substandard medicines and the evolution of rifampicin resistance. This dissertation advanced two technologies that may aid in the detection of substandard medicines: aptamers and biosensors. Oligonucleotide aptamers may be adapted for drug detection by coupling binding events to changes in fluorescence, luminescence or colorimetric signals. A computational model was developed to discover experimental factors that increase the probability of selecting a high affinity aptamer. Among them are: micromolar drug target concentration, high affinity substrate to partition aptamers, and high aptamer library affinity distribution. Random losses of aptamers due to experimental noise greatly decreased the probability of selecting an aptamer. Experimental parameters to optimize the process of aptamer discovery for small molecules are discussed. Bacterial biosensors are an alternative strategy for the detection of active pharmaceutical ingredients. Here, luciferase-expressing Escherichia coli were used to create profiles of drug interactions for anti-mycobacterial drugs. Drug interactions were tested by the Loewe additivity model. A novel method to differentiate rifamycin drugs from the drug degradation product rifampicin quinone was developed by analyzing each drug’s unique interactions. While subinhibitory drug doses are known to select for antimicrobial resistance in vitro, the role of substandard anti-mycobacterial medicines in the development of rifampicin resistance remains poorly understood. The role of the drug degradation product rifampicin quinone on rifamycin resistance was assessed through in vitro studies of bacteria. Wild type Escherichia coli and Mycobacterium smegmatis cultured in the presence of rifampicin quinone acquired high levels of resistance to rifamycin drugs. Resistance was associated with genetic mutations in the rifampicin resistance cluster of the rpoB gene. The studies presented here demonstrate that substandard medicines can contribute towards rifamycin resistance, and offer methodologies to identify substandard medicines. / 2020-10-24T00:00:00Z